Republic of Iraq
Ministry of Education
And Scientific Research
Baghdad University
Science College
Identification of Trichophyton rubrum isolates by
using traditional methods and
RAPD- PCR
A thesis
Submitted to the college of Science, University of Baghdad as
a partial fulfillment of the requirements for the Degree of
Master of Science in Biotechnology
By
ZainbAnas Salman
B.Sc. Biotechnology-College of Science
University of Basrah
2005
Supervised By
Dr. Abdul Kareem J. Hashim
Dr. Jasim M. Karhoot
Assistant Professor
Professor
July-2013 A.C.
Shaban-1434
Summary
Fifty samples of skin scrapings, nail and hair from patients were
obtained during the period beginning January/2011 till end April/2011
in Al-Yarmuk Hospital. All specimens were subjected to direct KOH
(10%) mount smear and culture on Sabourauds dextrose agar media to
determine the dermatophyte species.
The direct KOH (10%) mount smear showed the presence of
fungal elements in 40 (80%) of 50 human cases, while the growth of
dermatophyte were positive in 40(80%) cases. The main causative
agents of human dermatophytes were as follow: Trichophyton rubrum
17(42.5%),
Trichophyton
mentagrophyte
13(32.5%),
and
Microsporum canis 10 (25%).
Identification
of
dermatophytes
species
depended
on
macroscopic colonial morphology and microscopically findings as
well.
The PCR-based technique of randomly amplified polymorphic
DNA (RAPD) was used to fingerprint for seventeen isolates of the
fungus Trichophyton rubrum. Genomic DNA of each isolate was
extracted at final concentration of (750-1360) µg/ 1-2g of dry
mycelium, and the purity of (1.3-1.6). Each DNA sample was
amplified with each 15 primers and the products were resolved
electrophoretically on 1.2% agarose gel, stained with ethidium
bromide and photographed under UV light.
I
Two primers failed to support amplification but remaining 13
primers produced a total of (195) bands, of these bands 68%(126)
bands were polymorphic. The primers (A08 and GS04) were gave the
highest number of polymorphic bands (15), while primers (E02 and
A013) were gave the lowest number of polymorphic bands (6).
The genetic polymorphism value of each primer was determined
and ranged between (46-85%), primer R03 produced the highest
percent of genetic polymorphism compared with primer A013.
It was also possible to find the DNA fingerprinting of sixteen of
T. rubrum isolates through the appearance of number of bands that
were unique to each isolate. That may be used in the future to design
the detection primer for these isolates directly.
II
Introduction
Introduction
The dermatophytes are a group of closely related fungi that have
capacity to invade keratinized tissues (skin, hair, and nail) of humans
and other animals to cause an infection.
Dermatophytosis, commonly referred to as ringworm. Infection
is generally cutaneous and restricted to the nonliving cornified layers
because of the inability of fungi to penetrate the deeper tissue or
organs of immunocompentent host (Dei Cas and Vernes, 1986).
Reactions to dermatophyte infection may range from mild to severe as
a consequence of the host reactions to the metabolic products of the
fungus, the virulence of the infecting strain or species, the anatomic
location of the infection, and local environmental factors.
Trichophyton rubrum is one of the most commonly encountered
dermatophytes that infect human keratinized tissue such as skin, nail,
and possibly hair. This pathogen causes well characterized superficial
infections, and also produced skin infections in unusual parts of the
body in immunodepressed patients (Cervelatti etal., 2004).
In this study, Two general method for identification and
differentatiation of Trichophyton rubrum isolates were used,
identification of the fungus on the basis of phenotype differences
(conventional method) and differentatiation of its isolates on the basis
of molecular differences.
Identification of dermatophytes species by conventional method
requires the examination of colony, microscopic morphological
structures ( Faggi etal., 2001). But the molecular methods are
1
Introduction
regarded as useful in the exact and rapid recognition of dermatophyes
isolates, one of these methods is The RAPD-PCR which used single
primers as short as, about 10 nucleotides long, decamers with random
sequences were used to prime on both strands, producing a diverse
array of PCR products (Sobral and Honeycutt, 1993).
The RAPD-PCR is very suitable method in differentiating
between species and strain (Kanbe et al., 2003; Li et al., 2007).
So the aim of present study is using of RAPD-PCR for
differentiation between T. rubrum isolates, and to achieve this aim the
below steps were followed:
1. Isolation of dermatophyte from skin, hair infectious.
2. Identification the fungi isolates by traditional method
3. Differentiated between T. rubrum isolates by using molecular
method (RAPD-PCR).
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Chapter One
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1.1. HistoricalReview:
The history of human medical mycology started with the
discovery of Remak 1837,while noticed arthrospores and filaments
resembling molds from patients with tinea favosa (Kwon-Chung and
Bennett, 1992;Martin and Kobayashi, 1999). Malmsten erected the
genus Trichophyton and described T. tonsurans, while Charls Robin in
(1847) definedT. mentgrophytes(Ajello, 1977). By 1910 Sabouraud’s
included a classification system recognizing the three genera of
dermatophytes, Microsporum, Trichophyton, and Epidermatophyton,
which were based on clinical rather than botanical observation.
ChesterEmmon’s(1934),redefined the dermtophytes according to the
botanical rules of nomenclature and taxonomy. He included all the
known dermatophytes in three genera (Emmons et al., 1974).The
dermatophytes that causes human infection comprise 42 species of
fungi which belong to three genera of imperfect fungi namely:
1-
Microsporum
2-
Trichophyton
3-
Epidermaphyton
(Rippon, 1985; Jagdish, 1995; Larone, 1995; Anaisse et al., 2003)
Generally, Microsporum spp. infects hair and skin not nail;
Trichophyton spp. Infects skin, hair and nails and Epidermatophytes
spp. infects skin, nails, but not hair (Howard et al., 1987).
At present, the use of physiological characteristics, mating types and
critical antigenic analysis, in addition to the classical methods of
3
Chapter One
Literature Review
descriptive morphology had placed the taxonomy of dermatophytes
and other pathogenic fungi on a firm of scientific basis (Abanmi et al.,
2008; Ameen, 2010; Lakshmipathy and Kannabrian, 2010).
1.2.Mycology of dermatophytes:
Dermatophytes are known to grow best in warm and humid
environment and area, therefore, they are more common in tropical
and subtropical regions and this probably explains why they are very
common in Africa. For instance, same species of dermatophytes such
as T. mentagrophyte var. interdigitale, M. canis, E. floccosum and T.
rubrum are distributed all over the world. However, other species
probably have partial geographic restriction. For example, T.
schoenlenii is found in Africa and Eurasia while T. soudanense is also
restricted within Africa (Weitzman and Summerbell, 1995). Others T.
violaceum are associated to Asia, Africa and Europe and T.
concentricum is known to be common in the Fareast, India and the
Pacifics (Ameen, 2010).
Dermatophytes which comprise a group of closely related
fungimade up of three genera; Trichophyton, Microsprum, and
Epidermatophytes (Weitzman and Summerbell,1995 ; Emmonsʼ,1934)
they have the ability to invade the stratum corenum of the epidermis
and keratinized tissues derived from it, such as skin,hair,and nail of
humans and other animals(Weitzman and Summerbell,1995). It is one
of
the
most
common
coetaneous
infections
all
over
the
world(Ameen,2010; Nweze and Okafor, 2005).They cause superficial
4
Chapter One
Literature Review
fungal infections that pose public health problems to man and animals
(Havliekova et al.,2008). Dermatophytes infections can be disfiguring
and recurrent and generally need long-term treatment with antifungal
agents (Nweze etal., 2007).
1.2.1.Trichophyton:
The genus Trichophyton included 24 species. The colonies on
agar media are powdery, velvety or waxy. The predominant spore type
is microconidia with sparse macroconidia (Jagdish, 1995). Reverse
side pigmentation is characteristics of the species and is used for the
identification of the species within genus(Larone, 1995; Wanger and
Sohnle, 1995).The macroconidia are thin-walled with smooth surface
and variable shape, Some of the Trichophyton species are fastidious in
their requirement for amino acid as nitrogen source; T. tonsurans
require ornithine, citrul-lime and arginine whereas T. mentagrophytes
require methonine.This nutritional specificity has been used by many
authors in the identification of Trichophyton species (Philpot, 1977).
1.2.2.Microsporum:
The genus Microsporum includes 16 species. The colony
morphology of Microsporum species on agar plate surface is either
velvety or powdery with white to brown pigmentation (Jagdish,
1995).Both macro and microconidia are produced but the predominant
conidial structures are macroconidia, microconidia are less abundant.
The macroconidia are multiseptate with thick wall and rough
surface(Emmon’s, 1934).Rarely some species produce neither micro
5
Chapter One
Literature Review
nor macroconidia and they do not have any special nutritional
requirement (Chen and Friedlander, 2001).
1.2.3.Epidermatophyton:
The genus Epidermatophyton includes only two species. The
colonies are slow – growing, powdery and unique brownish yellow in
color. This genus is devoid of microconidia, macroconidia are
abundant
and
produced
in
clusters
(Jagdish,
1995).
These
macroconidia are thin-walled with smooth surface (Emmon’s, 1934).
1.3.Ecological classification:
In the course of evolution these pathogens have developed host
specificity. This host specificity described the differences in the
composition of keratin (Rippon, 1988). Based on their host specificity,
dermatophytes are classified into three ecological groups;
1.3.1.Anthropophilic:
The primary hosts of anthropophilic species are human being but
they may also cause infection in animals. Transmission of infection is
from human to human (Georg, 1960). Anthropophiles are predominant
in the central and northern European countries (Buchvald and
Simalijkova, 1995; Korstanje and Saats, 1995; Nowicki, 1996;
Havillickova et al., 2008). Examples include: T. rubrum, T. tonsurans.
6
Chapter One
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1.3.2.Zoophilic:
These are pathogens with only one animal host and grow as
saprophytes on animals materials.Zoophiles are also reported to infect
human beings. Human beings acquire the infection from infected
animals (English, 1972; Cahtterjee et al., 1980; Starova et al., 2010).
Examples include; T. simi (monkey), M. canis (cats and dogs), M.
nanum (pigs), T. mentagrophyte (bovine and sheep).
1.3.3.Geophilic:
These are generally saprophytes and drive nutrients from
keratinous substrates. Rarely these pathogens cause infection in
animals and human (Connole, 1990). Examples include: T. ajello, M.
cookie and M. gypsum.
1.4.Geographical distribution of dermatophytes species:
Although all dermatophytes are botanically closely related, each
species has certain characteristics in its geographic distribution
(Kwon-Chung and Bennett, 1992).The most prevalent species of
dermatophytes may vary strikingly from one geographic locality to
another.While many species are cosmopolitan, others have very
limited geographic ranges. The reason for these differences is
unknown;however,it is clear that changing patterns of prevalence
occur, and are caused by several factors,including migration of labor,
troopmovement, emigration and other travel, changing world patterns
of animals husbandry, evolution of new genotypes and the transfer or
adaptation of species indigenous to wild animal populations to
7
Chapter One
Literature Review
parasitism in man,and recent therapeutic advances, played important
roles in speeding these fungi(Mackenzie etal .,1986).
The prevalence of the cosmopolitan anthropophilic species in
each country is influenced by the constant change of the
environment(Kwon-Chung and Bennett, 1992). The rapid transit from
continent to continent, and the increasing mobility of people; agents of
disease are no longer geographically restricted.
Disease contracted half way across the world may become
manifest in a country in which the pathogen is not normally found
(Philpot, 1978). Dermatophytosis of scalp (tinea capitis) due to M.
audounii, T. tonsurans, T. violaceum, and T. schoenleinii was highly
prevalent in Western Europe during the early 1900s, and then tinea
capitis due to the four species has been disappearing since World War
II from that region, excluding the Mediterranean area. Sporadic out
breaks of tinea capitis still occur but they are usually due to M. canis
(Kwon-Chung and Bennett, 1992). Geographic dermatophytes are
more significant as agents of ringworm in hotter,drier climates, while
zoophilic fungi usually are more important in colder climates where
they may represent over 80% of human infections and this directly
related to techniques in farming and animals husbandry in colder are
as (Verma, 1978).
The high humidity, warmth, and low standard of living, poor
hygienic conditions of living as well as customs and traditions are
etiological factors causing the high incidence of dermatophytes in a
8
Chapter One
Literature Review
tropical country (Verma, 1978; Kamalom et al., 1981). The incidence
and type of mycoses vary according to age,geographical distribution
of the organism, and the epidemicity of the prevalent species(Ali,
1990).
1.5. Clinical manifestation of dermatophytes:
Dermatophytes typically do not affect the mucus membranes but
rather affect the keratinized tissues. They grow on nails,hairs and the
outer layer of the skin of both man and other animals. Although,the
clinical signs of dermatophytoses may vary depending on the affected
region of the body, puritiesare the most common symptom in humans.
The lesions on the skin are often characterized by inflammation
severity of the lesions is often obvious at the edges. Scaling,
erythematic and sometimes, blister formation are evident. This results
in clinical ringworm formation as seen in tinea corporis often
resembling a central clearance. Hair loss often results, especially on
the facial hair and the scalp (Weitzman and Summerbell, 1995). The
infections caused by dermatophytes are commonly referred to as
"tinea" or "ringworm" due to the characteristics ringed lesions
(Theodore et al., 2008). Clinical types are:
9
Chapter One
Literature Review
1.5.1. Tinea capitis (ringworm of scalp):
It is a dermatophytic infection of the hair and the scalp and
begins with a small papule which spreads to form irregular and scaly
forms of alopecia. Typical cases mostly result in the enlargement of
cervical and occipital lymph nodes. Sometimes, a boggy inflammatory
mass known as a kerion is formed. This is common in children
worldwide especially in African countries (Macura, 1993).The
predominant cause of tinea capitis in U.S.A is now Trichophyton spp.
particularly T. tonsurans, and in Africa, Middle East is T. violaceum,
while in Iraq is M. canis followed by T. verrucosum, T. violaceum and
T. mentagrophytes (Ali, 1990; Kwon-Chung and Bennett, 1992; Ohst
et al., 2004).
1.5.2.Tinea corporis (glaborous region of the body):
It is often referred to as ringworm, is characterized by single or
sometimes multiple scaly lesions, and occurs on the trunk, extremities
and face of human. It is more common in children than in adults and
occurs most frequently is hot climates similar to that found in many
African countries(Macura, 1993).
1.5.3. Tinea pedis (athlete's foot):
It is an infection of the foot, characterized by fissures, scales and
maceration in the toe web,or scaling of the soles and lateral surface of
the feet. It is more common in those who wear occlusive shoes
(Macura, 1993). In majority of cases, vesicles, erytherma, pustules and
bullaen may also be present. Anthropophilic dermatophytes are the
10
Chapter One
Literature Review
major cause of tinea pedis. Most common agents are T. rubrum, T.
mentagrophytes var interdigitale and E. floccosum.
1.5.4. Tinea unguium (onychomycosis, nail):
Tinea ungium, a dermatophyte infection of the nail, is usually
characterized by thickened; broken and discolored nails. It is often
referred to as onychomycosis and may result in the separation of the
nail plate from the nail bed. Both anthropophilic and zoophilic
dermatophytes can cause tinea ungium. T. rubrum and T.
mentasgrophytes var mentagrophytes are the most common agents
(Nenoff et al., 2007).
1.5.5. Tinea manuum (hands):
Tinea manuum is a fungal infection of one or occasionally, both
hands.It often occurs in patients with tinea pedis. The palmar surface
is diffusely dry and hyperkeratosis. When the fingernails are involved
vesicles and scant scaling may be present (Goldstein etal., 2000).
1.5.6. Tinea barbae (barbaesitch; bearded region of face and
nick):
It is an infection of the skin and hair in the beard and mustache
area.It is more common in adult men and hirsute women. Because the
usual cause is zoophilic organism, farm workers are most often
affected. The lesions may include erythematic, scaling and follicular
pustules (Elewski, 1999).
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Chapter One
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1.5.7. Tinea cruris (ringworm of the groin):
Tinea cruris, an infection of the groin. This is occurs when
ambient temperature and humidity are high. Occlusion from wet or
tight-fitting clothing provides an optimal environment for infection.
The dermatophytosis is more common in men than in women
due to spread of the fungus from feet. Tinea cruris affects the
proximal medial thighs and may extend to the buttocks and abdomen.
Thus the usual causative agents are T. rubrum and T. interdigitale
(Hainer, 2003).
1.5.8. Tinea faciei (ringworm of the face):
Tinea faciei is seen on the face especially on the non–
breadedareathe lesions are mostly pruitic with itching and with
burning sensation exposure to sunlight could make infected cases
worse due to itching and burning. However, red area may be
indistinct, especially on pigmented skin, and lesions may have little or
no scaling or raisededges. Because of the subtle appearance, this
dermatophytosis is sometimes known as tinea "incognito"(Zuber and
Baddam, 2001).
1.6. Trichophyton rubrum:
Is anthropophilic dermatophytes. It is most common agents of
dermatophytes, primarily causing tinea pedis, onychmycosis, tinea
corporis and tinea capitis. This fungus was first described by
Malmsten in 1845(Blank, 1955). Two types of Trichophyton rubrum
12
Chapter One
Literature Review
may be distinguished down type and granular type. The downy type
has become the most widely distributed dermatophytes of human. It
frequently causes chronic infection of skin,nail and rarely scalp. The
granular type is frequent cause of tinea corporis in South East Asia
(Rebell and Taplin, 1974). The growth rate of T. rubrum in the lab can
be slow to rather quick. Their texture is waxy, smooth and even to
cottony. From the top, the color is white to bright yellowish beige or
red violet. Reverse is pale yellowish, brown, or reddish–brown.
Microscopically, the downy type is characterized by the production of
scanty to moderate numbers of slender cleavage microconidia and no
macroconidia. The granular type is characterized by the production of
moderate to abundant numbers of cavetto pyriform microconidia and
moderate to abundant numbers of thin–walled, cigar-shaped
macroconidia. The macroconidia may or may not have terminal
appendages. This organism may remain viable in the environment for
over six months, thus accounting for widespread infection.
Transmission occurs most often from person to person (Rippon,
1988).
1.7. Identification of dermatophytes:
There are two methods to identification of dermatophytes, the
first method which depended on the phenotype differences
(conventional method) and the second method which depended on the
molecular differences like PCR method.
13
Chapter One
Literature Review
1.7.1. Conventional method:
These methods were considered the classical method to
identificated dermatophytes. The conventional laboratory method
based on detection of phenotypic characteristic such as (microscopy
and in-vitro culture) and physiologic properties which played an
essential role in dermatophytes identification (Rippon, 1988).
Conventional method was divided into two ways, the first way
direct microscopic examination which based on detected septate of
hyphae and shape of conidia. The advantage of this way was rapid and
inexpensive but did not provide genus or species identification and
results were negative in 5% to 15% (Weitzman and Summerbell,
1995; Mohanty et al., 1999).
Microscopic examination was not species-specific, therefore
they were used the second way in-vitroculture for identification of
species but this way needed long time to give result about 3-4 weeks
and require a range of culture media such as Urea test medium,
Dermatophyte test medium, Sabourauds dextrose agar, Corn meal agar
which
used
to
stimulate
condition
or
pigment
production,
Phenotypic identification can be difficult because intra species
morphological polymorphism and phenotypic pleomorphism (Robert
and Pihet, 2008).
1.7.2. PolymeraseChain Reaction (PCR):
Polymerase chain reaction a biochemical technology in
molecular biology to amplify a single or few copies of a piece of DNA
14
Chapter One
Literature Review
across several orders of magnitude, generating thousand to millions of
copies of a particular DNA sequence. Developed in 1983 by
KaryMullis (Bartlett and Stirrling, 2003). PCR is common and often
indispensable technique used in medical and biological research labs
for a variety of applications (Saiki et al., 1985; Saiki et al., 1988).
These include DNA cloning for sequencing; DNA-based phylogeny or
functional analysis of genes; the diagnosis of hereditary disease; the
identification of genetic fingerprints (used in forensic sciences and
paternity testing); and the detection and diagnosis of infectious
diseases. In 1993, Mullis was awarded the Nobel Prize in chemistry
along with Michael Smith for his work on PCRthe method relies on
thermal cycling consisting of cycles of repeated heating and cooling of
the reaction for DNA melting and enzymatic replication of the DNA.
Primers (short DNA fragments) containing sequences complementary
to the target region along with a DNA polymerase (after which the
method is named) are key components to enable selective and
repeated amplification. As PCR progresses, the DNA generated is
itself used as a template for replication, setting in motion a chain
reaction in which the DNA template is exponentially amplifiedPCR
can be extensively modified to perform awide array of genetic
manipulations.
1.7.2.1. Requirement of PCR:
A basic PCR set up requires several components and
reagents(Sambrook and Russel, 2001) these components include:
15
Chapter One
1-
Literature Review
DNA template that contains the DNA region(target)to be
amplified.
2-
Two primers that are complementary to the 3,(three prime) ends
of each of the sense and anti-sense of the DNA target.
3-
Taq polymerase or anther DNA polymerase with a temperature
optimum at around 70ºC.
4-
Deoxynuclosidetriphosphate (dNTPs; nucleotide containing
triphosphate groups), the building-blocks from which the DNA
polymerase synthesizes a new DNA strand.
5-
Buffer solution, providing a suitable chemical environment for
optimum activity and stability of DNA polymerase.
6-
Metal Ion cofactor, magnesium chloride is an essential in PCR,
it act as cofactors for the DNA polymerase and catalyzes the extension
reaction of a primed template at 72ºC.
1.7.2.2.PCR stage:
There are three major steps in a PCR, which are repeated for 30 or 40
cycles. Each cycle of PCR includes steps for template denaturation
primer annealing and primer extension (Rychlik et al., 1990).
·
Initialization step: this step consists of heating the reaction to a
temperature of 94-96ºC, which is held for1-9minutes. It is only
required for DNA polymerases that require heat activation by hot-start
PCR (Sharkey et al., 1994).
·
Denaturation step: this step is the first regular cycling event and
consists of heating the reaction to 94-98ºC for 20-30seconds. It
16
Chapter One
Literature Review
causesDNA melting of the DNA template by disrupting the hydrogen
bonds between complementary bases, yielding single-trended DNA
molecules.
·
Annealing step: the reaction temperature is lowered to50-65ºC
for 20-40seconds allowing annealing of the primers to the singletrended DNA template. Typically the annealing temperature is about
3-5 degrees Celsius below the Tm of the primers used. Stable DNADNA hydrogen bonds are only formed when the primer sequence very
closely matches the templatesequence. The polymerase binds to the
primer–template hybrid and begins DNA formation.
·
Extension–elongation step: the temperature at this step depends
on the DNA polymerase used; Taqpolymerase has its optimum
activity temperature at 75-80ºC (Chien et al., 1967; Lawyer et al.,
1993). And commonly a temperature of 72ºC is used with this
enzyme. At this step the DNA polymerase synthesizes a new DNA
strand complementary to the DNA template strand by adding dNTPs
that are complementary to the template in 5’ to 3’direction,condensing
the 5’-phosphate group of the dNTPs with the3’-hydroxyl group at the
end of the nascent DNAstrand. The extension time depends both on
the DNA polymerase used and on the length of the DNA fragment to
be amplified.
·
Final extension: this single step is occasionally performed at a
temperature of 70-74ºC for 5-15 minutes after the last PCR cycle to
ensure that any remaining single-stranded DNA is fully extended.
17
Chapter One
·
Literature Review
Final hold: this step at 4-15ºC for an indefinite time may be
employed for short term storage of the reaction.
1.7.2.3 Types of PCR:
1.7.2.3.1. Hot start PCR:
The specificity and DNA yield of PCRs are often improved by the
"hot start" technique and analogous method. It reduces non-specific
amplification during the initialsetup stages of the PCR. It inhibits the
polymerase activity at ambient temperature, either by the binding of
an antibody. Oligonuclotides that amplify the conserved region of the
minicircle molecules of Leishmania were used in a hot- start PCR.
The technique successfully identified Leshmania kinetoplast DNA
present in patient (Pirmez et al., 1999).
1.7.2.3.2. Methylation-specific PCR:
It can rapidly assess the methylation status of virtually any group
of CpG sites within a CpG island, independent of the use of
methylation-sensitive restriction enzymes. It analysis uses bisulfatetreated DNA but avoids the need to sequences the area of interest.
Primer pairs are designed to be "methylation-specific"by including
sequences
complementing
only
unconverted
5-mrthylated,
or"unmethylation-specific", complementing thymine converted for
unmethylatedcytosienes. Methylation is determined by the ability of
the specific primer to achieve amplification (Goessl, 2000).
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Chapter One
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1.7.2.3.3. Reverse transcription PCR:
It is one of the most commonly used polymerase chain reaction
used in molecularbiology. It is a sensitive method for detection of
mRNA expression levels. It involves two steps: RNA is first reverse
transcribed into cDNA using a reverse transcriptase and then the
resulting cDNA is used as templates for subsequent PCR
amplification using primers specific for one or more genes.RT-PCR is
widely used in expression profiling, which detects the expression of
the gene (Athale et al., 2001).
1.7.2.3.4. Quantitative PCR:
Quantitative PCR is used widely to detect and quantify specific
DNA sequences in scientific fields that range from fundamental
biology to biotechnology and forensic sciences. It quantitatively
measure starting amount of DNA, cDNA or RNA. HIV infected
patients not responding to antiretroviral therapy is at risk of
cytomegalo virus (CMV) disease. Quantitative PCR had sensitivity
and specificity of 47% and 70%, respectively for detecting
cytomegalovirus (CMV) in HIV infected patients (Brantseater et al.,
2007).
1.7.2.3.5.Intersequence-specific PCR:
It amplifies the region between simple sequence repeats to
produce a unique fingerprint of amplified fragment lengths. It is an
efficient tool in phylogenetic classification of prokaryotic genomes in
general and diagnostic genotyping of microbial pathogens in
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Chapter One
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particular. Vibrio cholera pathogenic and nonpathogenic can be
identified by using Intersequence-specific PCR (Ravi Kumar et
al.,2007).
1.7.2.3.6. Touchdown PCR:
Touchdown PCR also called step-down PCR is another
modification of conventional PCR that may result in a reduction of
nonspecific amplification. It involves the use of an annealing
temperature that is higher than the target optimum in early PCR
cycles. The annealing temperature is decreased by one degree
centigrade every cycle or every second cycle until a specified or
"touchdown"annealing temperature is reached. The touchdown
temperature is then used for the remaining number of cycles. This
allows for the enrichment of correct product over any non-specific
product (Schiavoni et al., 2010).
1.7.2.3.7. Multiplex-PCR:
It uses multi primers sets within a single PCR mixture to
produce amplicons of varying sizes that are specific to different DNA
sequences. Multiplex-PCR was very useful in known mitochondrial
DNA mutation in Chinese patients with Lebers hereditary optic
neuropathy (LHON). This disease is a maternally transmitted disease
(Du et al., 2011).
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1.7.2.3.8. Nested PCR:
Increase the specificity of DNA amplification, by reducing
background due to non-specific amplification of DNA. Two sets
(instead of one pair) of primers used in two successive PCRs. In the
first reaction, one pair of primers is used to generate DNA products,
which besides the intended target, may still consist of non-specifically
amplified DNA fragments. The product(s) are then used in a second
PCR with a set of primers whose binding sites are completely or
partially different from and located 3’of each of primers used in the
first reaction. Nested PCR is often more successful in specifically
amplifying long DNA fragments then conventional PCR, but it
requires more detailed knowledge of the target sequences (Chow et
al., 2008).
1.7.2.3.9. Random Amplified Polymorphic DNA (RAPD-PCR):
RAPD is a DNA finger print technique based on polymerase
chain reaction (PCR) amplification of random fragments of genomic
DNA with single short primer of arbitrarily nucleotides sequence
usually 10-bases.In this process a single type of primer binds to the
genomic DNA at two different sites on opposite strands of the
template DNA at low annealing temperature 36ºC under low
stringency conditions. Primers are usually able to amplify fragments
from
3-10
genomic
sites
simultaneously
(Williams
et
al.,
1990).DNAamplificationreaction is repeated on a set of DNA samples
with several different primers, undercondition that results in several
21
Chapter One
Literature Review
amplified bands from each primer (Williams et al., 1993). Generate
RAPD fragments are commonly separate using agarose gel
electrophoresis and then visualize by staining with ethidium bromide
and observe under UV light (Hashemi et al., 2009). The number of
generated amplified fragments depends on the size of the genome and
the sequence and the length of the primer,the number of priming sites,
as well as the reaction condition, in which amplification with RAPD
primers is extremely sensitive to single-base change in the primertarget sitei.e. a single base change in the primer sequence or in the
genome may prevent amplification and cause complete change in the
pattern of amplified DNA segments (Williams et al.,1990;Williams
etal.,1993).
The amplified products usually consist of 5-10 discrete bands
and may reach to 21 with different molecular weights ranges between
200-4000bp (McPherson and MØller,2001). Hence, RAPD primer can
detect polymorphism that are usually noted by the presence or absence
of an amplification product from a single locus (Tingey et al., 1994)
resulting in different patterns of amplified DNA fragments with
different molecular size (McPherson and Moller,2001).
TheRAPD technique can simply and rapidly detect genetic
alteration in the entire genome without knowledge of specific
DNAsequence information and it works effectively with tiny amount
of DNA (Baeze et al., 2006). Therefore RAPD-PCR become a
particularly
popular
technique
and
thus
was
applied
in
geneticmapping, for plant and animal breeding applications, DNA
22
Chapter One
Literature Review
fingerprinting, study of population genetics, epidemiological studies
and typing of microorganisms and for identification of pathogenic
strain of bacteria and fungi (Abdel-Rahman, 2008).
Variation in the RAPD technique have been developed which
share the same principle as RAPD analysis but detecting methods use
different longer primers. These methods are called arbitrarily primed
polymerasechain reaction (AP-PCR) (Welsh and McClelland, 1990),
and DNA amplification fingerprint (DAF) (Caetano-Anolles et al.,
1991). AP-PCR methods used primers about 15 nucleotides long,
acryl amide gels for product separation and detection was achieved by
using autoradiography.On the other hand primers shorter than 10
nucleotides were used in DAF method and product were also
separated on acrylamide gels but were detected by using silver
staining methods(Wang et al., 1998).
More application of RAPD-PCR which using for identification
of plant, fungi, and bacteria (Younan, 2010 and Al-araji, 2003).
23
Chapter Two
Material and Method
2.1 Materials
2.1.1 Apparatus
Table (2-1): Illustrates the apparatus used in this study.
Apparatus Names
Company and Origin
Autoclave
Express -Japan
Deep freeze
Sanyo-Japan
Distiller
Controls-England
Electrophoresis unit
Consort-Belgium
Gel documentation system
Consort-Belgium
Hot plate magnetic stirrer
IKA-USA
Laminar air flow hood
Techne- UK
Magnetic stirrer
Scientific Industries-USA
Microcentrifuge
Eppendorf- Germany
pH-Meter
Bio Red – Italy
Power supply
Consort- German
Refrigerator
Ishtar-Iraq
Sensitive electronic Balance
Sortorius-Germany
Spectrophotometer
Shimadzu- Japan
Ultra violet transilluminator
Consort-Germany
Vortex
Stuart Scientific-UK
Water bath
Memmert – Germany
24
Chapter Two
Material and Method
2.1.2 Chemicals and Biological Materials
Table (2-2): Illustrates the Chemicals and Biological Materials used in this
Study.
Chemicals and Biological Materials
Company and origin
Absolute ethyl alcohol
BDH-England
Agarose
Promega-USA
Ammonium Acetate (CH3COONH4)
Thomas Bakar-India
Boric acid
Fluka – Switzerland
Bromophenol Blue
Sigma-USA
Chloroform
BDH-England
CTAB (cetyltrimethyl ammonium
bromide)
Riedel-de Haën
Ethanol Alcohol 70%
Hazard-UK
Ethidium bromide
Promega- USA
EDTA(ethylene diamintetra acetate)
BDH-England
Go Taq®Green master mix
Promega – USA
Glycerol
Fluka – Switzerland
HCl
BDH – England
Isoamyl Alcohol
Thomas Bakar-India
Isoprobanol
BDH-England
(1) Kb DNA Ladder (250-10000)bp
Promega – USA
Na2EDTA
Riedel-de Haën
Sodium chloride (NaCl2)
BDH-England
Sodium hydroxide (NaOH)
Fluka – Switzerland
Tris-Base
Thomas Bakar-India
TBE-buffer (10x)
Promega – USA
Peptone
Difco-USA
Phenocrystals
Fluka-Germany
Yeast extract
Fluka – Germany
Acetone
BDH-England
25
Chapter Two
Material and Method
2.1.3. Culture media
2.1.3.1. Prepared medium
2.1.3.1.1. Sabourauds Dextrose agar
Dextrose
Peptone
Agar
Distilled water
40 g
10 g
20 g
1000 ml
Twenty gm of agar was dissolved in 500ml distilled water
Dextrose and peptone were dissolved in another 500ml of distilled
water and warmed to 500C combined, distributed in tubes, bottles or
flask, then it was autoclaved at 1210C for 15 minutes at15psi
(Emmon’s et al .,1977) it was used for routine cultivation of
dermatophytes .
2.1.3.1.2.Sabourauds Dextrose agar with chloramphenicol
and cyclohexamide.
Dextrose
Peptone
Agar
Chloramphenicol
40 g
10 g
20 g
0.05 g
Cyclohexamide
Distilled water
0.5 g
1000 ml
Five hundred mg cyclohexamide in 10ml of acetone were added to
moltenmedium and sterilized for15minutes at 1210Cat 15/psi in
autoclave (Mackenzie et al., 1986). it was used for isolation of fungi
from clinical materials.
26
Chapter Two
Material and Method
2.1.3.1.3. Urea test medium
Peptone
1.0 g
Sodium chloride
5.0 g
Potassium dihydrogenorthophosphate(KH2po4)
2.0 g
Glucose
5.0 g
Agar
15.0 g
Distilled water
1000 ml
Forty gm of already prepared medium was dissolved in 1L and
warmed; 6ml of phenol red solution (0.2% in 50% alcohol) was added
and sterilized at 1210C at 15 psi for 15 minutes, then cooled to 500C.
100ml urea solution was added (20% aqueous solution sterilized by
filtration). It was distributed aseptically into 10 ml screw cap bottles
and slope. Most strain of Trichophyton mentagrophytes caused the
medium to change from yellow to red. Most strain of Trichophyton
rubrum did not change (Philpot,1967;Philpot, 1977).
2.1.3.2.Ready used medium
2.1.3.2.1.Potato Dextrose agar:
It was used for routine cultivation and identification of fungi
(Richardson and Warnock, 1993).
2.1.3.2.2. Sabouraud’s dextrose agar with penicillin and streptomycin.
It was used for isolation of fungi from clinical materials (Roberts
and Mackenzie, 1986).
27
Chapter Two
Material and Method
2.1.3.2.3. Yeast extract:
It was utilized during the perforation test to distinguish
Trichophyton mentagrophytes isolates from Trichophyton rubrum.
2.1.3.2.4.Corn meal agar:
This medium was used for pigment production by Trichophyton
rubrum strain, (Beneke and Rogers, 1970).
2.1.3.2.5. Rice Grain Medium:
It was used to distinguish between Microsprum audouinii from
Microsprum canis where the first failed to grow and the second grew well
(Mackenzie et al., 1986; Rippon, 1988).
2.1.3.2.6. Dermatophyte Test Medium (DTM):
It’s a selective and differential medium used for isolation of
dermatophytes (Rebell and Taplin, 1974).
These media were prepared according to manufactures instruments.
2.1.4. Reagents:
2.1.4.1. Potassiumhydroxide:
KOH crystals
10 g
Glycerol
10 g
Distilled water
80 ml
This reagent was used for direct detection of fungal elements in
clinical specimens by microscopically examination (Suhnen et al.,
1999).
28
Chapter Two
Material and Method
2.1.4.2.Lacto phenol cotton blue stain:
It was used for staining and microscopic identification of fungi
(Ellis, 1994).
Cotton blue
Phenol crystals
Glycerol
Lactic acid
Distilled water
0.05 g
20 g
40 ml
20 ml
20 ml
This stain is prepared over two days:
On the first day,the cotton blue was dissolved in D.W.then left
overnight to eliminate insoluble dye.
On the second day, thephenol crystals were added to lactic acid
in a glass beaker,placed on magnetic stirrer until the phenol was
dissolved.The glycerol was added.The cotton blue and D.W. solution
were filtered into the phenol/glycerol/lactic acid solution.It was mixed
and stored at room temperature.
2.1.5.Solution used in DNA extraction:
2.1.5.1. Extraction buffer (CTAB buffer):
It was prepared by dissolving 2gm of CTAB, 8.1816gm of Nacl,
0.7444gm of Na2EDTA, 1.2114gm of Tris-base in D.W,pH was
adjusted to 8.0, volume completed with D.W to 100ml, sterilized by
autoclaving for 15 min at 1210C at 15psi and stored at 40C (Maniatis
et al ., 1982).
29
Chapter Two
Material and Method
2.1.5.2. Chloroform: Isoamylalchol (24:1):
To prepare 100ml, 96ml of chloroform was added to 4ml of
isoamyl alcohol (Maniatis et al., 1982).
2.1.5.3. Washing buffer:
It was prepared by dissolving 0.140gm of ammonium acetate in
76ml ethanol, volume completed with D.W to 100ml (Sambrook et
al., 1989).
2.1.5.4. Tris-EDTA (TE)buffer:
It was prepared by dissolving 0.1211gm of Tris-base,0.0372gm
of Na2EDTA in D.W, pH was adjusted to 8.0,volumecompleted with
D.W to 100ml, sterilized by autoclaving and stored at 40C (Sambrook
et al ., 1989).
2.1.6. Solution used in Agarose gel electrophoresis:
2.1.6.1. Tris-borate (TBE)buffer:
To prepare 10x TBE solution, the components used as
following:108gm of Tris-base, 55gm of Boric acid, 40ml of 0.5µ
EDTA(pH=8.0) in an appropriate amount of D.W, pH was adjusted to
7.8 and volume completed to 1L with D.W. The solution was
sterilized by autoclave and stored at room temperature (Sambrook et
al., 1989).
2.1.6.2.Loading buffer:
It was prepared by dissolving 0.25gm bromphenol blue dye in
50ml D.W, 30ml glycerol was added, volume completed with D.W to
100ml (Sambrook, 1989).
30
Chapter Two
Material and Method
2.1.6.3.Ethidium bromide dye(10mg/ml):
It was prepared by dissolving 1gm of ethidium bromide in 100ml of
sterile D.Wand the bottle kept in a dark(Maniatiset al., 1982).
2.1.6.3.Molecular weight marker:
The DNA marker(1)kb ladder(250-10000)bp was prepared
according to the manufacture instructions.
2.2Methods
2.2.1.Collection of specimen:
Fifty samples including hair, nail, and skin were collected from
patients admitted to Dermatology Department of Al-Yarmuk Hospital
during the period of beginning of January 2011 till end of April 2011.
These samples were collected by using sterilized forceps, sterile
fine scissors, sterile blunt scalpel, so that, the scarping skin was
collected by sterilized the affected area with 70% alcohol and entire
edge scrape with a sterile blunt scalpel, while infected hair were
removed by a sterile forceps, but the infected nail was collect by used
sterile fine scissors. Then all the samples were diagnosed by direct
examination and laboratory culture.
2.2.2.Mycological Examination
The evidence of infection based on demonstration of fungal
elements (branching septate hyphae and spore (Arthroconidia) by the
direct microscopically examination of hair, scales, and nail from the
lesion by KOH mounting. Sufficient materials of specimen was taken
to permit repeated direct examination, since, a single preparation may
not provide enough evidence for the presence of an infecting fungus.
31
Chapter Two
Material and Method
2.2.2.1.Potassium hydroxide (KOH)mounts:
Skin scrapings were placed on surface of clean slide flooded
drops of 10%KOH heated gently for about 5-10 minutes, after that let
the slide to be cooled and put the cover slip and examined under the
microscope under 40x lens. Gentle warming of the preparation aids to
speed up the reaction.
2.2.2.2.Cultivation of dermatophytes:
The standard medium for isolation of dermatophytes from
clinical materials is Sabouraud's dextrose agar containing
cyclohexamide (0.5gm) to suppress the growth of saprophytic fungi
and chloramphenicol (0.05gm) to suppress the growth of fast growing
bacteria.In addition another modified sabourauds dextrose agar
containing procaine penicillin and streptomycin used for isolation of
fungi from clinical materials.Plates were inoculated with skin scraping
by aid of sterile scalped.Cultures were incubated at (280C). Cultures
were firstly examined after 7 days, then twice weekly for at least 3–
4weeks before being considered negative. The distinguishing features
of dermatophytes are usually produced within 10-20 days. Most
dermatophytes lose their distinctive cultural and microscopically
features when kept for a long time in culture. Identification of the
growth depends on the following (Rippon, 1988):
1- Colony observation (color, consistency and topography).
2- Colony reverses (color, significant pigment).
3- Microscopic morphology (microconidia and macroconidia: their
size, shape, arrangement, and hyphal structures).
In this study, identification was based on colonial and
microscopic feature, and selected media according to Emmon’s et al.,
(1977); Koneman et al., (1979) and Rippon (1988). Microscopic
32
Chapter Two
Material and Method
examination was made by examination of many preparations from
different areas of fungal growth mounted with lacto phenol cotton
blue stain to reveal spores which include large septate macroconidia
and small, single-celled microconidia.
2.2.3.Other tests
Physiological tests are sometimes performed especially in some
species which where seldom, if ever, produce spores, and have
colonies which resemble each other's so closely that they cannot be
identified on morphological basis alone (Al-Hamadani, 1997) these
tests include:
1. Sub culturing on corn meal agar medium for pigment production
by Trichophyton rubrum strain and to differentiate between it and
Trichophyton mentagrophytes.
2. Sub culturing on rice grain medium to differentiate
Microsporum canis which grows well than Microsporum audauinii
which fails to grow on this medium.
3. Growth at 370C of Trichophyton verrucosum.
4. Hair perforation test to determine the manner of hair digestion in
vitro which differentiates between T. rubrum and T.
mentagrophytes.
2.2.4. Hair perforation test:
The hair perforation test was used to distinguish T.
mentagrophytes isolates from T. rubrum by their ability to penetrate
human hair in vitro. This test can also be used as an ancillary
diagnostic feature for other species of dermatophytes as Microsporum
equinum from Microsporum cains (Mackenzie et al., 1986).
Autoclaved human hair (preferably from a child) was cut into short
pieces (1cm) and placed in a Petri-dish. Sterile distilled water (25ml)
33
Chapter Two
Material and Method
containing 2-3 drops of 10% yeast extract was added. The plates were
incubated at 28ₒC. individual hairs were removed at intervals up to 4
weeks and examined microscopically in lacto phenol cotton blue stain.
Isolates of T. mentagrophytes localized areas of spitting and marked
erosion. This useful test has the disadvantage that 3-4 weeks may be
required to give a definitive result (Richard et al., 1988).
2.2.5.DNA Extraction:
The DNA was extracted from mycelium according to the method
described by Delsal et al., (1989) as follows:
1. Approximately 1to 2gm of mycelia placed into a cold mortar.
2. Carefully, Liquid nitrogen was added to the mortar to freeze the
mycelia then grinding by the pestle agnist the mortar in a circular
motion. (This step repeated several times).
3. The powder was transferred to an eppendrof tube and 500µL of
extraction buffer (CTAB buffer) were added.
4. The eppendrof tubes were incubated at 680c in water bath for 60
minutes; mixed the tubes several times while there were incubated let
eppendrof tubes cool down for a couple minutes to relive the
pressure.
5. In the fume hood,10ml of chloroform/isoamyl-alcohol(24:1)was
added to each eppendrof, The samples were shaken 5-10 minutes at
room temperature then centrifuged at (4000rpm) for 15 minutes.
6. The upper phase was transferred into 1.5ml eppendrof tube.
7. The 0.6ml of total volume was added cold isopropanol (-200C)
and covered tightly before mixed by gently inverting tube several
times. Awhite, stringy precipitate consisting of DNA.
8. Spooled out nucleic acid (DNA) with glass hook (the hook is a
Pasteur pipette that has been bent at the end) from the wash buffer
and were dried at room temperature.
34
Chapter Two
Material and Method
9. Re-suspended a nucleic acid (DNA) pellet in 100µl of TE buffer
and incubated at 650c for 20-30 minutes or until the pellet was
dissolved and stored at -800C until using.
2.2.6.
Estimation
of
the
DNA
concentration
by
the
spectrophotomrter
Five micro liters of each sample were added to 495µl of D.W
and mixed well to determine the DNA concentration and its purity by
using the spectrophotometer.A spectrophotometer was used to
measure the optical density (O.D) at wave length of 260nm and
280nm. An O.D of 1 corresponds to approximately 50µg/ml for
double stranded DNA (Sambrook et al., 1989) the concentration of
DNA was calculated according to the formula:
DNA concentration (µg/ml) =O.D 260nm *50* dilution factor
The spectrophotometer was used also to estimate the DNA
purity ratio according to this formula:
DNA purity ratio = O.D 260 / O.D 280
This ratio was used to detect nucleic acid contamination in
protein preparation. DNA quality can be also assessed by simply
analyzing the DNA by agarose gel electrophoresis (Maniatis, 1982).
2.2.7.Agarose gel electrophoresis:
To separate DNA fragments, agarose gels in different
concentrations were used 0.8%for extracted DNA, 1.2%for visual
checking of RAPD product, 2.5%for visual checking of specific PCR
product. Gels were run horizontally in 0.5X TBE buffer. Samples of
DNA with loading buffer (loadingbuffer: DNA) 2/7(v/v) and loaded
35
Chapter Two
Material and Method
into the wells on the gel in checking step of total DNA, while in
checking steps of PCR products there were no need to mix with
loading dye because the green master mix reaction buffer is
proprietary buffer containing a compound that increases sample
density, and blue and yellow dyes,which function as loading dyes
when reaction products are analyzed by agarose gel electrophoresis.
Electrophoresis buffer was added to cover the gel and run for1-2 hours
at 5 v/cm Agarose gels were stained with ethidium bromide 0.5 µg/ml
for 20-30 minutes. DNA bands were visualized by UV
transilluminator at 365 nm wave length (Maniatis,1982). Agel
documentation system was used to document the observed bands.
2.2.8. Molecular analysis of genomic DNA of Trichophyton rubrum
by using RAPD PCR techniques.
2.2.8.1.RAPD PCR
Polymerase chain reaction was performed using the followings:
1- Random primer:
Fifteen random sequence decamer primers were used, synthesis
by (Alpha DNA-Canada) from different series (A, B, C, H, O, Pha,
Gs4 and E) in a lyophilized form and were dissolved in sterile distilled
water to give a final concentration of (10 picmol/µl)as recommended
by provider. The primers used and their sequences are listed in table
(2-3).
2- Go Taq Green master mix(2X)
Go Taq Green master mix is ready to use mixture that contains
Taq DNA polymerase, Mgcl2, pure deoxynuclotides (dNTPs), reaction
buffer and two dyes (blue and yellow) that allow monitoring of
progress during electrophoresis, with concentration (2x). Go Taq
Green master mix was provided by (promega–USA). Amplification
36
Chapter Two
Material and Method
was performed on ice in aseptic condition in laminar air flow using
0.2ml tight cap eppendorf tubes. In order to achieve homogeneity of
reagents and reduce risk of contamination a master mix for all samples
was prepared containing all components of the reaction except of
genomic DNA (template DNA), mixed gently with sterile distilled
water to achieve the appropriate volume. A negative control reaction
in each PCR experiment was set up containing all components of the
reaction without template DNA so that any contamination DNA
present in the reaction would be amplified and detected on agarose
gel.
2.2.8.1.1. The protocol of RAPD PCR:
PCR was performed with a protocol includes the following:
*PCR primer:the random PCR primer as indicated in table(2 -3)
*PCRmix:about 12.5µl of PCR ready mix(Go Taq Green master
mix)was added when the final reaction volume was 25µl to obtain a
final concentration (1x)as recommended by provider and sterile
distilled water was used to achieve a total volume of 25µl after added
each of primers and DNA template.
37
Chapter Two
Material and Method
Table(2-3): The names of the random primers used in the study
and their sequences:
NO. Primers name
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Sequence5-3
R03
A08
C011
C015
B01
B04
B014
AS02
Pha06
GS04
A013
H07
E02
R01
O16
ACGGTTCCAC
GTGACGTAGG
AAAGCTGCGG
GACGGATCAG
GTTTCGCTCC
GGACTGGAGT
TCCGCTCTGG
GTCCTCGTGT
CCCGTCAGCA
AAGAGCCCGT
CAGCACCCAC
CTGCATCGTG
GGTGCGGGAA
CACACCGTGT
TCGGCGGTTC
(Ahmadikhah, 2009; Rabhani et al., 2008)
Amplification reaction
Amplification of random fragments of genomic DNA was
performed with the following master amplification reaction.
RAPD-PCR master mix(final reaction volume=25µl)
Materials concentration
and manufacturer
D.W
Promegagreen mix(2x)
Primer(10pmol/µl)
Total reaction volume
Final concentration
Volume for (1) tube
1x
10 picmol/µl
9.5µl
12.5µl
1µl
23µl
Genomic DNA 2µl (50 ng / µl) + mix 23µl
38
Chapter Two
Material and Method
* PCR program
The amplification program was run as follow:(Rabbaniet al., 2008).
Temp:940C
Time:5min
Denaturation
Temp:940C
Time:1min
Annealing
Temp:360C
Time:1min
Extension
Temp:720C
Time:2min
Final extension
Temp:720C
Time:10min
Initial denaturation
No.of cycles =45 cycles
Approximately20µl of PCR amplified products were separated
by electrophoresis in 1.2% agarose gels (2 hr,5v/cm)0.5x Tris–borate
buffer).
Gels stained with ethidium bromide, PCR products were
visualized by UV transilluminator and then were imaged by gel
documentation system (Hashemi et al., 2009). The amplified products
usually consist of 1–10 discrete bands and may reach to15bands, the
size of RAPD–PCR products estimated by comparing with the marker
1 kb DNA ladder (250–10,000).
39
Chapterthree
Results and Discussion
3.1. Age and sex distribution of patients with dermatophytes
infection:
The results in table (3-1) showed that the incidence of
dermatophytes was higher in males than females, according to the sex.
While it was higher in patients under age tan years, according to the
age.
Table (3-1): Age and sex distribution of fifty patients with
dermatophytes infection.
Age (years)
Sex
≤ 10
11-20
21-30
31-40
41-50
Total
%
Males
12
7
10
2
3
34
68
Females
8
4
2
1
1
16
32
Total
20
11
12
3
4
50
100
Percentage%
40
22
24
6
8
100
In the present study, it was found that dermatophyte infection is
still a major problem among Iraqi population. It is commonly found
among families in certain localities where the standard of living and
hygiene are unhealthy and where is animal’s husbandry. It affects
children between three to twelve years with a mean of 6 years.
The incidence in, males 34 (68%) was almost twice the
incidence in females 16 (32%). These results are in agreement with
(Yehia, 1980; Ali, 1990; Abass, 1995). Ried et al (1968) motioned
40
Chapterthree
Results and Discussion
that erroneous health practices of boys including theuse of other
combs and caps and few times their hair washing than girls, have been
associated with dermatophytes infection Also, females have less
exposure to sporting facilities and institution when tinea is rife (Du.
Vivier, 1990).These reason examples why incidence in males was
almost twice the incidence in females.
3.2. Mycological Examination:
Out of the fifty cases of dermatophytes 30(60%) cases were
positive by both direct KOH mount smear and culture (Table
3.2),whereas 10 (20%) cases were culture negative after six weeks
incubation at room temperature although the direct KOH mount
examination was positive.
The remaining 10 (20%) cases gave apositive culture
resultwhereas the direct KOH mount smear examination was negative.
The total positive cases detected by the mycological method were 40
(80%) of total suspectedcases. Out of fifty cases 10 (20%) were
negative culture for dermatophytes. According to Singh et al (2003),
the possible reason for negative culture from microscopically positive
specimens was the increased use of topical antifungal agents for short
period of time on skin lesion of unknown etiology. The other possible
reason was the highly contaminated specimens over grown by the fast
growing saprophytic species which prevent the growth of
dermatophytes even on medium with cyclohexamide. The isolation
rate of the dermatophyte species was (80%). This result is similar to
the finding of Gumar and Guirges (1978) who reported (80.5%) and
Malhorta et al (1979) who reported (82%), Ali (1990) found a rate of
(65.7%) while Abass (1995) found a rate of (81.8%). These different
41
Chapterthree
Results and Discussion
findings of the isolation rate of dermatophytes attributed to the
different ways on which the workers depend on in selecting cases for
culture. In this study, all cases of dermatophytes were cultured
whether the direct KOH mount examinations were positive or
negative. With regard to correlation between positivity of the
microscopic examination and culture, the following findings were
reported in this study: positive direct KOH examination and positive
culture was (6o %), positive direct KOH examination and negative
culture was (20%) and negative KOH examination and positive
culture was (20%). Similar results were reported by Abass (1995) and
Mohmmed (2012) while Silverio et al (1989) reported different
results. It seems that the direct KOH examination is more practical
than culture for dermatophytes although both remain complementary.
The possible reason for negative culture from microscopically positive
specimens was the samples might not takefrom the active border of
the lesion where invading fungal elements are expected to be found.
Table(3.2): Relation between direct KOH mount smear and
Culture of fifty samples (hair and scale)
Examination cases
Percentage %
*
D+
*
C+
30
60
*
D+
*
C-
10
20
*
D-
*
C+
10
20
50
100
Total
*
Number
D+: positive direct KOH examination
*
D-: negative direct KOH examination
*
C+: positive culture
C-: negative culture
*
42
Chapterthree
Results and Discussion
3.3. Culture media:
Media contained in Petri-dishes were found better for isolation of
dermatophytes than media in screw-cap bottle, because plates offered
layer surface area for more clinical material to be incubated and plates
were easily scanned for developing dermatophyte colonies (Rippon,
1988). The best standard medium for isolation of dermatophytes from
clinical materials and producing the distinguishing feature of
dermatophytes was modified Sabouraud’s dextrose agar supplement
with (0.5mg/ml) cyclohexamide to suppress the growth of saprophytic
fungi and antibacterial antibiotic chloramphenical (0.05mg/ml). The
primary, dermatophytes test medium as selective and differential
medium for isolation of dermatophytes, gave less accurate diagnostic
test in this study, because in some times, some yeasts and saprobic
filamentous fungi also caused color change from yellow to red, this is
attributed to the alteration of the commercial sources of the
ingredients from that of the originator. On the other hand,
dermatophyte test medium did not produce distinguishing features of
dermatophyte species. Rice grain medium was used in this study to
distinguish Microsporum canis. All M. canis isolates grow on this
medium. Urea test medium was used to distinguish T. rubrum from T.
mentagrophyte which the first was urease negative and the second was
urease positive. Most dermatophytes isolates have varied in their
distinctive cultural and microscopic features when kept for long time
on culture media, so subcultures have been done to delay this
pleomorphism. Culture on slopes of maintenance media (Sabouraud’s
agar without glucose) had been done in present work to obtain
dermatophytes stock culture for few months in a refrigerator
(Mackenzie et al., 1986).
43
Chapterthree
Results and Discussion
Hair perforation test was used in this study to help in identification of
T. mentagrophyte isolates. All T. mentagrophyte isolates penetrated
hair invitro and produced marked localized areas of pitting and
marked ersion of hair shaft(figure3.1).
Figure (3.1): Hair perforation test in vitro by Trichophyton mentagrophyte
isolate. The isolate penetrated the hair and produced marked localized
areas of pitting and marked erosion of hair shaft (40x)
Out of fifty cases of dermatophytes only forty plates positive
culture after four weeks incubation at 37ºCthe other ten plates gave
negative culture result after maximal six weeks incubation(table
3.2).Out of these ten plates 4(40%) plates showed the growth of
Candida spp. 3(30%) plates showed the growth of Aspergillus spp.
And 3(30%) plates were sterile (i.e. no growth at all). Aspergillus
species were rapidly growing fungus as well. With five days of
incubation at room temperature, the colonies developed as gray-green
dry fluffy colonies. Microscopically, the characteristic fruiting body
44
Chapterthree
Results and Discussion
was the conidia borne in aradial arrangement upon the tip of
vesicle(figure3.2). Candida species rapidly growing yeast within 2-3
days, the colonies develops as creamy-white in color with waxy
surface. Microscopically, gram positive budding cell, oval budding
was seen (figure3.3).
Figure (3.2): Microscopically appearance of Aspergillus species
frutting structure (conidia) using lactophenol cotton
Figure (3-3): Microscopically appearance of Candidia species Using
lactophenol cotton blue stain (100x oil).
45
Chapterthree
Results and Discussion
The prances of the non-dermatophyte due to contamination
during the culture or there were secondary infection due to the local or
systemic factors.
3.4. Isolation and identification of the dermatophyte species:
Outof fifty patientscultures of hair and scales on SDA, 40(80%)
showed the presence of dermatophyte growth (table3.2), the remaining
specimens 10(20%) showed no growthin 3(6%) and growth of nondermatophytes in 7(14%). The non-dermatophytes species noticed in
this study were Aspergillus and Candida. Species identification of
dermatophyte isolates revealed that T. rubrum was the predominant
species identified in 17(42.5%) of the positive dermatophyte culture,
followed by T. mentagrophyte in 13(32.5%), M. canis in 10(25%).
On the basis of the test used for identification, three species were
recognized of both anthropophilic and zoophilic groups (table3.3).
The genus Trichophyton was responsible for the maximium cases of
the dermatophytosis (75%). Karla et al (1964) and Vasu, (1966), in
India found that among the cases studied by them (95%) and (95.15%)
respectively were infected by Trichophyton species. While Ali (1990)
and Abass (1995) in Iraq found that among the cases studied by them
(44.9%) and(61%) respectively were infected by Trichophyton
species.
46
Chapterthree
Results and Discussion
Table (3.3): Dermatophyte species isolated from forty cases of
positive culture in patients with dermatophytosis.
Dermatophyte species
Number
Percentage %
Trichophytonrubrum
17
42.5
Trichophytonmentagrophyte
13
32.5
Microsporumcanis
10
25
Total
40
100
3.5.Macroscopic and microscopic findings of the dermatophyte
Isolate:
3.5.1. Trichophyton rubrum:
Upon culturing on Sabouraud’s dextrose agar with
cyclohexamide and chloramphenicol, colonies of T. rubrum were
slow-growing, white, and cottony to velvety appeared on this medium,
and diameter reaching 2.5cm after 3-4 weeks of incubation at 37ºC
(figure 3.4).
This isolate had ability to produced pink pigment on the reverse
side when cultured on potato dextrose agar (figure 3.5).
Microscopically, this organism produced very small microconida
which oval and born along the sides of hyphae (figure3.6).
The overall incidence of this fungus was found to be (42.5%).
Review of literature revealed on increasing predominance of T.
rubrum in the United Kingdom (Blaschke-Hellmessen et al.,1975).
Moreover, it is the main agent responsible for ringworm infection in
India (Philpot, 1978), while T. rubrum is a major etiological agent of
dermatophytosis in Brazil making up about (80%) (Evans, 1998).
47
Chapterthree
Results and Discussion
Trichophyton rubrum is cosmopolitan but appears to have a more
restricted distribution in the past, having been transported widely as a
result of human migration (the anthropophiles travel with their human
hosts) (Rippon, 1985).
Figure (3.4): Colonial morphology of Trichophyton rubrum on Sabourauds dextrose agar
at 37C0 for 3-4 weeks incubation.
Figure (3.5): Colonial morphology of Trichophyton rubrum on potato
dextrose agar, (background color) at 37C for 3-4 weeks
incubation.
48
Chapterthree
Results and Discussion
Figure (3.6): Microscopic morphology of Trichophyton rubrum, Colony mounted with
lacto phenol cotton blue stain (40x).
Microscopically, macroconidia were rarely formed and they
were cylindrical with thin, smooth wall, while the microconidia were
spherical, abundant and born both in clusters along the hyphae (figure
3.8).
All isolates(13) were subcultureon corn meal agar to detect
pigment production on the reverse side of the colony, to allow
differentiation it from T. rubrum (the former produced yellow color
which different from the red color produced by later), and the
definitive differentiation between T. rubrum andT. Mentagrophyte
was done by performance of positive conical ersion of the hair shaft
after four weeks incubation at 37ºC (figure3.1). Table (3.3) shows that
T. mentagrophyte was the second common isolate (32.5%) in this
study. It was the main cause of dermatophytosis in Alberta (Padhye
and Sekhon, 1973) and was isolated from (41.4%) of infection. The
position of T. mentagrophyte as the second most common species
isolated from patients of ringworm infection has also been reported by
Gumar and Guirges (1978) in Bagdad (28.02%) and Karaoul et al
(1979) in Kuwait (14.1%).
49
Chapterthree
Results and Discussion
Figure (3.7): colonial form of Trichophyton mentgarophyte on sabourauds dextrose agar
at 37ₒC for two weeks incubation.
Figure (3.7): Microscopic morphologyof Trichophyton mentgarophyte, colony mounted
with lacto phenol cotton blue stain (40x).
50
Chapterthree
Results and Discussion
3.5.3. Microsporumcanis:
Colonies of M. canis have a distinctive rapid growth within 5-8
days, colonies were appeared glabrous with radial grooves, white to
buff in color (figure 3.9).Microscopically, the macroconidia were
many with spindle-shaped, thick walled and the external surface were
pitted. The microconidia were rarely formed except in old
culture(figure3-10). All the isolates(10) were subcultured on polished
rice grain agar to distinguish them from M. audouinii. M. canis grows
well on rice grain agar and produced intense yellow color on the
reverse side of the colony. M. canisis a zoophilic fungus. Most
infection in man is acquired from an animal rather than from man
(Emmon’s et al., 1977). It is the third etiological agent of
dermatophytosis isolated in (25%) of the cases in this study.
Microsporum canis was the main etiological agents of tinea capitis
reported by Shrquie and Al-Zubadi (1985) in Iraq, Al-Fouzan et
al.,(1993) in Kuwait.
Figure (3.9): colonial morphology of Microsporum canis on sabourauds dextrose agar at
37ₒC for 5-8 days incubation
51
Chapterthree
Results and Discussion
Figure (3.10): Microscopic morphology ofMicrosporum canis Colony mounted with
lactophenol cotton blue stain Showing the macroconidia(40x).
3.6. Clinical etiologic correlation of dermatophyte infection in (40)
cases:
The results revealed that there were three clinical types. The
predominant one is tinea capitis which represent 50%, followed by
tinea cruris which represent 32.5%, and tinea corporis which represent
17.5% (table 3.4). The commonest clinical presentation that was
observed in the present study was tinea capitis with a frequency of
50%. Previous reports in Iraq shows that tinea capitis was
predominant clinical type (Yehia, 1980; Rahim, 1966). The second
most predominant type of dermatophytoses was tinea cruris. Tinea
cruris was detected in 32.5% of total cases. Rothman (1985) stated
that ringworm of the groin is mainly a post pubertal disease of male
probably due to pubertal development of sex specific a porcine glands
in which its secretion plays a role in susceptibility to infection. On the
other hand, the scrotum and wearing of trousers facilitate localization
52
Chapterthree
Results and Discussion
of fungi in the groin of male and that female have less chance of
contagion (Herbra, 1964). The third most predominant type of
dermatophytose was tinea corporis with frequency of 17.5%. Previous
reports in Iraq showed that tinea capitis and tinea corporis were
probably the most prevalent clinical types existing in this country
(Yehia, 1980; Ali, 1990; Abass, 1995).
Table(3.4): Distribution of dermatophytes isolated from patients
according to their clinical types.
Type of
Trichophyton
Trichophyton
Microsporum
Tinea
rubrum
mentagrophyte
canis
Total
No.
%
No.
%
No.
%
No.
%
Tinea capitis
9
22.5
5
12.5
6
15
20
50
Tinea cruris
6
15
5
12.5
2
5
13
32.5
Tinea corporis
2
5
3
7.5
2
5
7
17.5
17
42.5
13
32.5
10
25
40
100
Total
53
Chapter three
Results and Discussion
3.7. DNA extraction:
The DNA was extracted efficiently by using CTAB method
depending upon Weigand et al., (1993) extraction protocols. Purity
and concentration of DNA were measured using the standard method
(Sambrook et al., 1989). The yield of the DNA extracted from the T.
rubrum isolates was in range of (750-1360) µg per gram with purity of
(1.3-1.6).
The CTAB method was suitable method for DNA extraction
from fungi and plants (Weising et al., 1998). There are several
problems during DNA extraction from fungi, these problems were
contamination, fungi have thick wall which contains chitin and
cellulose, and the DNA of fungi destroyed by DNase.
So that, to avoid all these problems, The fungi were cultured on
SDA medium which contain cyclohexamide and chloramphenicol and
after well growth the1-3g of mycelium taken and grind with liquid
nitrogen at low temperature of liquid nitrogen up to (-190ºC)
tostopped the activity of nucleases when liberated from the cell wall.
The presence of CTAB materials allows the DNA to react with it and
formed complex structure.So the CTAB material can dissolved the
complex (Weigand et al., 1993), in addition the EDTA material get
the Mg+2ions which important to DNase activity, so the presence of
EDTA material in extraction buffer prevent the DNase activity
(Wilson and Walker, 2004).The CTAB materialmust be removed by
adding chloroform which precipitatedtheCTAB but the DNA still in
liquid phase (Maniatis et al., 1982). Then DNA was precipitated by
isopropanol then wash by washing buffer which contain ammonium
acetate.Finally the DNA was dissolved in TE buffer and preserves in –
200C. Total genomic DNA was shown in figure (3-11).1 1
2
3
54
Chapter three
Results and Discussion
Figure(3-11): Agarose gel electrophoresis of total genomic DNA samples,
were fractionated by electrophoresis on a 0.8% agarose gel (1hr, 5v/cm,
0.5x Tris-borate buffer) and visualized under U.V.light after staining with
ethidium bromide.
Lanes of seventeen samples of Trichophyton rubrum
3.8. RAPD-PCR analysis:
The RAPD-PCR indicators characterized by sensitivity to any
change in the component of interaction. Therefore, these indicators
were difficult back reaction and get the same results, so the results
were changed with concentration component and circumstance
surrounding changed, the purpose to obtain good result it must be
perform several experiments to get the optimum condition for
reaction, there were several factors that can control through rereaction and get the same results:
1- Genomic DNA concentration
2- Appropriate part on program annular polymer thermocycler
3- The primer concentration
4- Mg+2 ion concentration
5- The accuracy of micropipette used in the test.
55
Chapter three
Results and Discussion
Therefore, several experiments were conducted to reach the
optimum condition for reaction through use the different concentration
of DNA and primer, the suitable concentration of DNA was ranged
between (25-50)ng/µl, the suitable concentration of primer was 10
picmol for the best results.
The experiments of RAPD-PCR for genomic DNA of T. rubrum
isolates were started after obtaining the optimum condition and the
following results have been obtained.
In this study, 15 primerswere used; only two primers did not
give result (R01and O16) because there primers do not find the
priming site in the genomic DNA of Trichophyton rubrum isolates
(Devos and Gale, 1992).
Primer B014:
The primer B014 was used to amplify the DNA of 17 isolates of
Trichophyton rubrum. The results showed that the primer B014
produced amplified bands totaling at 26. And ranging between (2351323) the rang of number of bands for isolates was (1-3) bands, the
highestnumber of bands was generated with sixth and eleventh
sample, the isolates 3 and 4 do not produce amplified band ,show in
(figure 3.12).
Primer B014 has been able to identify the fingerprinting of DNA
by marker bands (518,470, and 369 bp) for three isolates (6, 7, 8)
respectively.
56
Chapter three
Results and Discussion
Figure (3-12): Agarosegel electrophoresis of RAPD-PCR reaction for
random primer B014 for DNA samples of Trichophytonrubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T. rubrum.
M. 1kb ladder, NC: negative control
Table(3-5): The polymorphic, unique bands with their
molecular weight for primer B014.
No.
1
2
3
4
5
6
7
8
9
10
Band
Mwt in bp
1.323
1.000
950
570
531
518
470
369
275
235
1
2
5
6
7
8
9
10 11 12 13 14 15 16 17
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
1
0
0
0
1
0
0
0
0
1
0
0
1
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
Unique
1: present of band
0
1
0
0
1
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
polymorphic
0: absent of band
57
Chapter three
Results and Discussion
Primer B01:
Primer B01 appeared after reacted with the genomic DNA of T.
rubrum isolates polymorphism according to the number of bands for
each isolate and in the molecular weight of these bands. The total
number of bands were (58) bands (figure3.13), there were distributed
into (17) main bands, the number of polymorphic bands was (13) with
molecular weight between (393)bp and (2070)bp. The range of bands
for this primer was (2-8) bands the first sample produced only two
bands while the sixth sample produced the high number of bands (8).
Four unique bands were produced with this primer (table3.6), the
fourth and fifth band with molecular weight of about (680)bp and
(758)bp were differentiated sample seventh, while the first and eighth
bands with molecular weight of about (2.070)bp and (442)bp were
differentiated samples sixth.
Figure (3-13): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer B01 for DNA samples of Trichophytonrubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T. rubrum.
M. 1kb ladder, NC: negative control
58
Chapter three
Results and Discussion
Table(3-6): The polymorphic, unique bands with their molecular
weight for primer B01.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Band
Mwt in
bp
2.070
1.490
1.462
1.285
1.215
1.072
930
850
790
758
700
680
552
517
468
442
393
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
1
1
0
0
0
0
0
0
1
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
1
0
0
1
0
1
0
1
0
0
1
0
Unique
1: present of band
0
0
0
0
1
1
0
0
1
1
0
1
1
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
1
0
1
1
0
0
0
1
0
0
1
1
0
0
0
1
0
1
0
0
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
1
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
1
1
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
polymorphic
0: absent of band
Primer E02:
When primers E02 amplified genomic DNA of
Trichophytonrubrum isolates by using PCR technique, polymorphism
in the number of bands and in the molecular weight of these bands
were generated. (34) bands as total number of bands were produced
and were divided into (12) main bands, there were (6) polymorphic
bands with size ranged from (445)bp to (1018)bp, the range of number
of band for isolates was (1-4) bands (figure3.14), the highest number
of bands was generated with firstand seventh sample while the second
sample generated only one band.
59
Chapter three
Results and Discussion
Primer E02 produced six unique bands(table3.7), sample sixth
have two unique bands with molecular weight (1345)bp and (1227)bp,
sample seventh have two unique bands in the first and fourth band
with molecular weight (1124) bp and (592) bp, the second band with
molecular weight (886) bp distinguished the first sample and the third
band with molecular weight (418)bp distinguished the eleventh
sample.
Figure (3-14): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer E02 for DNA samples of Trichophytonrubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T. rubrum.
M. 1kb ladder, NC: negative control
60
Chapter three
Results and Discussion
Table (3-7): The polymorphic, unique bands with their molecular
weight for primer E02.
No.
1
2
3
4
5
6
7
8
9
10
11
12
Band
Mwtinbp
1.345
1.227
1.124
1.018
886
722
645
592
564
511
445
418
1
2
5
6
7
8
9
10 11 12 13 14 15 16 17
0
0
0
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
0
1
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
1
0
1
0
0
1
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
Unique
polymorphic
1: present of band
0: absent of ban
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
Primer C015:
PCR results of primer C015, that amplified genomic DNA of
Trichophytonrubrum isolates, shown (38) bands as total number of
bands. These total number of bands were distributed in (7) bands were
polymorphic, the size of these bands ranged from (315)bp to
(1151)bp. The range of bands between (1-7) bands, the seventh
sample produced the highest number of bands (7) as show in figure (315).
Primer C015 generated (8) unique bands, the three unique bands
with molecular weight (1765)bp, (954)bp, and (827)bp which
distinguished the seventh sample from other isolates as show in the
table (3-8).
61
Chapter three
Results and Discussion
Figure (3-15): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer C015 for DNA samples of Trichophytonrubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T.rubrum
M 1 2 3 M. 1kb ladder, NC: negative control
Table (3-8): The polymorphic, unique bands with molecular
weight for the primer C015.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Band Mwt in 1
bp
1.765
0
1.562
0
1.340
0
1.151
1
1.052
0
970
1
954
0
827
0
665
0
542
1
530
0
472
0
436
0
315
0
230
0
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
1
0
0
0
1
0
0
1
0
1
1
0
0
0
0
0
1
1
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
1
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
Unique
1: present of band
polymorphic
0: absent of band
62
Chapter three
Results and Discussion
Primer C011:
Primer C011 appeared after reacted with genomic DNA of
Trichophytonrubrum isolates polymorphism in the number of bands
for each isolate and in the molecular weight of these bands. The total
numbers of bands were (53). They were distributed into (16) main
bands, the number of polymorphic bands was (10) with molecular
weight of about (397)bp and (1425)bp, the range of the bands for this
primer was (2-5) bands show in figure (3.16).
Six unique bands were produced with this primer (table3.9), the
second and third bands with molecular weight of about (1630) bp and
(974)bpwere differentiated sample six.
Figure (3-16): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer C011 for DNA samples of Trichophytonrubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T.rubrum
M. 1kb ladder, NC: negative control
63
Chapter three
Results and Discussion
Table(3-9): The polymorphic, unique bands with molecular
weight for the primer C011.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Band Mwt
in bp
1.720
1.630
1.425
1.325
1.115
1.083
974
838
720
657
611
480
455
397
274
230
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1
0
1
0
0
0
0
1
0
1
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
1
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
Unique
1: present of band
0
1
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
1
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
1
0
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
1
1
0
0
1
0
0
0
0
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
polymorphic
0: absent of band
Primer B04:
When primers B04 amplified genomic DNA of Trichophyton
rubrum isolates by using PCR technique, polymorphism in number of
bands and in the molecular weight of these bands were generated. (31)
bands as atotal number of bands were produced and were divided into
(10) main bands, there were (7) polymorphism bands with size ranged
from (452)bp to (1411)bp, the range of number of bands for isolates
was (1-5) bands, the highest number of bands was generated with
sample tenth and eleventh shown in figure (3-17).
64
Chapter three
Results and Discussion
Primer B04 produced three unique bands(table3.10), the
seventeenth sample generated only one band which was unique band
with molecular weight (685)bp, the fourth bands with molecular
weight (142)bp distinguished sample sixteenth, but the third band with
molecular weight (212)bp distinguished sample fifth. So that, the
sample second, third, and fourth do not produced amplified product
with primer B04 because these sample do not have priming site which
can recognized by primer B04.
1
Figure (3-17): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer B04 for DNA samples of Trichophytonrubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T.rubrum
M. 1kb ladder, NC: negative control
65
Chapter three
Results and Discussion
Table (3-10): The polymorphic, unique bands with molecular
Weight for primer B04.
NO. Band Mwt 1
inbp
1
1.411
0
2
1.184
0
3
1.159
0
4
760
0
5
685
0
6
630
0
7
550
1
8
452
0
9
212
0
10
142
0
Unique
5
6
7
8
0
0
1
1
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
1: present of band
9
10 11 12 13 14 15 16 17
0 1 1
0 0 0
0 1 1
0 1 1
0 0 0
1 1 1
0 1 1
0 0 0
0 0 0
0 0 0
polymorphic
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
1
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
1
0
1
0
0
0
1
0
1
0
0
0
0
1
0
0
0
0
0
0: absent of band
Primer A013:
Primer A013 recognized to the priming site into genomic DNA
of Trichophytonrubrum isolates, this primer produced (33) bands as
total number of bands for all isolates, that were distributed into (13)
main bands. These (13) bands included, (6) polymorphic bands with
molecular weight between (250)bp and (1425)bp. The number of
bands for each isolate ranging (1-4) bands, the sample tenth generates
high number of bands, four bands, show in the table (3-11).
The primer A013 produced (7) unique bands, the sample seventh
have two unique bands in the first bands with molecular weight
(1211)bp, and the third band with molecular weight (473)bp. So that
the sample fifth produced only one band which unique band with
molecular weight (495)bp, the sample thirteenth produced only one
band which unique band with molecular weight (588)bp, the first band
of sample twelfth with molecular weight (1600)bp, the first band of
sample fifteenth with molecular weight (1792)bp, and the first band of
samples eighth with molecular weight (1.113)bp. In term unique
banding pattern, the sample seventh, fifth, eighth, twelfth and fifteenth
possessed special banding pattern.
66
Chapter three
Results and Discussion
Figure (3-18): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer A013for DNA samples of Trichophyton rubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T.rubrum
M. 1kb ladder, NC: negative control
Table (3-11): The polymorphic, unique bands with molecular
weight for primer A013.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
Band Mwt
in bp
1.792
1.600
1.425
1.211
1.180
1.113
810
660
588
495
473
430
250
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
Unique
1: present of band
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
1
0
0
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
1
0
1
0
0
0
0
1
0
0
0
0
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
polymorphic
0: absent of band
67
Chapter three
Results and Discussion
Primer AS02:
The genomic DNA of the T.rubrumisolates was amplified by
using primer AS02 and the results that appeared were (44) bands as
total number of bands for all isolates, that were distributed into (11)
main bands, of these (11) main bands, (8) bands were polymorphic
with molecular weight between (350)bp and (1400)bp.The number of
bands for each isolate ranging (1-4), the sample thirteenth and
sixteenth produced high number of band show in (figure3.19).
Primer AS02 appeared three unique bands, the one unique band
was the second band of sample fifth with molecular weight (866)bp
and the other unique band was the second band of the sample seventh
with molecular weight (577)bp, the second band of the sixteenth
sample with molecular weight (757)bp, in the term unique banding
pattern, the fifth, seventh, sixteenth sample have special banding
pattern shown in the table (3-12).
Figure (3-19): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer AS02 for DNA samples of Trichophyton rubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T.rubrum
M. 1kb ladder, NC: negative control
68
Chapter three
Results and Discussion
Table (3-12): The polymorphic, unique bands with molecular
weight for primer AS02.
No. Band Mwt
in bp
1
1.400
2
1.247
3
1.200
4
886
5
757
6
670
7
615
8
577
9
540
10
415
11
350
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
0
1
0
1
0
0
0
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
Unique
1: present of band
1
0
0
0
0
0
1
0
1
1
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
1
0
1
0
0
1
0
0
0
0
1
0
0
1
0
1
0
0
0
0
0
0
1
0
1
0
1
1
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
1
0
1
1
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
polymorphic
0: absent of band
Primer H07:
Genomic DNA of the Trichophyton rubrum isolates was
amplified by using primer H07 and the results included, the total
number of bands were (52) bands. A total number of bands were
distributed into (13) main bands. Out of (13) bands, (9) bands were
polymorphic, ranging in molecular weight of (415-1410)bp. Primer
H07 produced bands in range (1-8) bands, the sample tenth produced
high number of bands so the sample fifteenth produced only one band
shown in figure (3-20).
Primer H07 generated four unique bandstable(3-13), the fourth
band of sample thirteenth with molecular weight (825)bp, the fifth
band of sample twelfth with molecular weight (674)bp, the second
band of sample fourteenth with molecular weight (523)bp and the
third band of sample sixteenth with molecular weight (381)bp.
69
Chapter three
Results and Discussion
Figure (3-20): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer H07for DNA samples of Trichophyton rubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T.rubrum
M. 1kb ladder, NC: negative control
Table (3-13): The polymorphic, unique bands with molecular
weight for primer H07.
NO. Band M.Wt 1 2 3 4 5 6 7 8 9 10 11 12 13
in bp
1
1.410
0 0 0 0 0 0 0 0 0 1 1 1 0
2
1.100
1 0 0 0 0 0 0 1 1 1 1 1 1
3
970
0 0 0 0 1 0 0 0 0 1 1 1 1
4
850
0 1 1 0 0 1 0 1 1 1 1 1 1
5
825
0 0 0 0 0 0 0 0 0 0 0 0 1
6
674
0 0 0 0 0 0 0 0 0 0 0 1 0
7
633
1 0 0 0 1 0 0 0 0 1 0 0 0
8
600
0 1 0 1 0 0 1 0 0 1 1 0 0
9
543
0 0 0 1 1 1 1 0 0 1 0 0 0
10
523
0 0 0 0 0 0 0 0 0 0 0 0 0
11
465
0 0 0 0 1 1 0 0 0 0 0 0 0
12
415
0 1 0 0 0 0 0 0 1 1 0 0 0
13
381
0 0 0 0 0 0 0 0 0 0 0 0 0
Unique
polymorphic
1: present of band
0: absent of band
14 15 16 17
0
1
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
70
Chapter three
Results and Discussion
Primer R03:
Genomic DNA of the Trichophyton rubrum isolates was
amplified by using primer R03 and the results included, the total
product from react the primer R03 with the genomic DNA number of
bands were (45) bands. A total number of bands were distributed into
(13) main bands. Out of (13) bands, (12) bands were polymorphic,
ranging in molecular weight (151-1873)bp. Primer R03 produced
bands in range (1-5) bands, the sample twelfth produced highest
number of bands while the sample third, eighth and sixteenth
produced only one band.Primer R03 produced one unique bands,
which found in the third band of the sixth sample with molecular
weight (617)bp shown in the table (3-14).
Figure (3-21): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer R03 for DNA samples of Trichophyton rubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T.rubrum
M. 1kb ladder, NC: negative control
71
Chapter three
Results and Discussion
Table (3-14): The polymorphic, unique bands with molecular
weight for primer R03.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
Band Mwt in
bp
1.873
1.684
1.233
1.053
984
825
798
617
527
466
395
230
151
1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1
0
0
0
1
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
1
0
0
0
1
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
1
0
1
0
0
0
0
0
0
0
0
0
0
1
0
1
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
1
0
0
1
0
0
0
1
0
0
0
0
0
0
1
0
1
0
0
1
0
0
1
0
0
0
0
1
0
1
0
0
0
1
0
0
0
1
0
1
1
0
1
0
0
0
1
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
Unique
1: present of band
polymorphic
0: absent of band
Figure (3-22): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer GS04 for DNA samples of Trichophyton rubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T.rubrum
M. 1kb ladder, NC: negative control
72
Chapter three
Results and Discussion
Primer GS04:
Results of primer GS04interaction with genomic DNA of
T.rubrumisolatesshowed (62) bands on agarose gel as a total number
for all isolates. They were distributed into (24) main bands, of which
(15) bands were polymorphic bands, the range of molecular weight of
polymorphic bands were (235-1200)bp. The number of bands that
produced by this primer had ranged between (1-7) bands; the first
sample produced highest number of bands while the thirteenth sample
was the leastfigure(3-22).This primer produced (9) unique bands as
following, (3), (1), (1), (2), (1) and (1)unique bandswere found in the
first, third, sixth, ninth and tenthsample respectively.
Table (3-15): The polymorphic, unique bands with molecular
weight for primer GS04.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Band
Mwtinbp
2.174
1.806
1.200
1.123
1.010
956
920
868
858
843
742
734
700
679
630
570
557
537
494
450
420
336
310
235
1
2
3 4
5
6
7
8
9
10
11
12
13
14
15
16
17
1
1
0
1
0
0
1
0
0
0
0
1
1
0
1
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
1
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
1
0
0
1
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
Unique
1: present of band
polymorphic
0: absent of band
73
Chapter three
Results and Discussion
Primer Pha6:
Results of primer GS04interaction with genomic DNA of T.
rubrum isolates showed(68) bands, were distributed into (21) main
bands, the size of these bands ranged between (288-3372)bp, through
these bands only (14) bands were polymorphic, and seven bands were
unique bands, the unique bands distributed into five isolates of
T.rubrum, two in the sixth and seventh isolate at molecular weight
(1856)bp, (649)bp,(1335)bp and (1033)bp respectively, and one in the
first, third and twelfth isolate at molecular weight (971)bp, (661)bp
and (434)bp respectively. In the term of unique banding pattern, these
five isolates of T.rubrum have possessed special banding pattern with
this primer show in (Table3.16).
Figure (3-23): Agarose gel electrophoresis of RAPD-PCR reaction for
random primer Pha06 for DNA samples of Trichophyton rubrum (under
optimum condition). Bands were fractionated by electrophoresis on 1.2%
agarose gel (2hr, 5v/cm, 0.5xTris-borate buffer) and visualized by ethidium
bromide staining.
Lanes of seventeen samples of T.rubrum
M. 1kb ladder, NC: negative control
74
Chapter three
Results and Discussion
Table (3-16): The polymorphic, unique bands with molecular
weight for the primer Pha06.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Band
Mwtinbp
3.372
1.856
1.730
1.595
1.335
1.295
1.255
1.242
1.178
1.140
1.115
1.033
971
790
661
649
552
472
434
387
288
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 1 0 0 0 0
0 0 0 0 0 1
0 0 0 1 1 0
1 1 1 1 1 1
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 1 1
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 1
0 0 0 0 0 0
1 0 0 0 0 0
1 1 1 1 0 1
0 0 1 0 0 0
0 0 0 0 0 1
0 0 0 0 0 0
1 1 0 1 0 1
0 0 0 0 0 0
0 0 0 0 1 0
0 0 0 0 0 0
Unique
1: present of band
0
0
1
1
1
0
0
0
0
0
0
1
0
1
0
0
1
1
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0 0 0 0
0 0 0 0
0 1 0 0
0 1 1 1
0 0 0 0
1 1 0 0
0 0 0 1
0 0 0 0
1 1 1 0
0 0 0 0
0 0 0 1
0 0 0 0
0 0 0 0
0 1 1 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 1 0
1 1 0 1
0 0 1 0
polymorphic
0: absent of band
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
1
0
Primer A08:
This primer can produced amplified products with the genomic
DNA of the seventeen isolates of T.rubrum, and there results were
about (46) bands which distributed into (20) main bands with
molecular weight ranged between (180)bp and (1984)bp figure (3-24),
of these (20) bands only (15) bands were polymorphic and (5) unique
bands, these unique distributed in five isolates of T. rubrum as
following, one in the second, third, tenth, eleventh and seventeenth
isolate at molecular weight (180)bp, (1038)bp, (1528)bp, (566)bp, and
(535)bp respectively, Table (3-17).
75
Chapter three
Results and Discussion
Figure (3-24): Agarose gel electrophoresis of RAPD-PCR reaction for random
primer A08 for DNA samples of Trichophyton rubrum (under optimum
condition). Bands were fractionated by electrophoresis on 1.2% agarose gel (2hr,
5v/cm, 0.5xTris-borate buffer) and visualized by ethidium bromide staining.
Lanes of seventeen samples of T.rubrum
M. 1kb ladder, NC: negative control
Table(3-17): The polymorphic, unique bands with molecular weight for
primer A08.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Band Mwtbp
1.984
1.528
1.351
1.150
1.098
1.038
980
911
830
740
718
694
670
566
535
495
475
412
270
180
2
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
0
1
3
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
5
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
1
0
6
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
Unique
1: present of band
7
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
8
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9
1
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
10 11 12 13 14 15 16 17
0 0 0 0 0 0 0 0
1 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
1 0 0 0 0 0 0 0
1 1 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 1 0 0 1 0 0 0
0 1 0 0 0 0 0 0
0 0 1 0 1 0 1 0
0 0 1 1 0 0 0 0
1 1 0 0 0 0 0 0
1 0 0 0 0 0 0 0
0 0 0 0 0 1 1 1
0 1 0 0 0 0 0 0
0 0 0 0 0 0 0 1
0 0 1 0 0 1 0 0
0 0 0 1 0 0 0 0
0 0 0 1 0 1 0 0
0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0
polymorphic
0: absent of band
76
Chapter three
Results and Discussion
In this study, when the RAPD indicters were applied by using
PCR technique on the genomic DNA of the seventeen samples of
Trichophytonrubrum isolates which diagnosis by conventional
method, including morphology of conidia (shape and color) and
culture examination (Graseretal., 2007). Fifteen primers were used in
this study, two primers (R01 and O16) did not amplify the DNA, and
hence, they were eliminated from the analysis. Each of remaining (13)
primers which gave the result for the genetic variation between the
Trichophyton rubrum isolates. Some primers generated several bands,
while others generated only few.
The (13) random primers produced a total of (195) useful bands
across the seventeen samples isolates (table3.18), the (129) bands
were polymorphic (66%) and remaining bands were unique bands.
The primers (A08 and GS04) were produced the highest number of
polymorphic bands (15), while the primers (E02 and A013) were
produced the minimum number of polymorphic bands (6) this resultin
agreement with reported (Baezeetal., 2004) which give polymorphism
of T. rubrumisolates (70%).Polymorphism of each primer was
calculated as the percentage of polymorphic bands to the number of
total bands produced by the designated primer (Ali, 2003).
Polymorphism ranged between (46-92%), primer R03 produced the
highest percentage of polymorphism compared with primer A013.
Each primer generates a different number of bands, Pha06
generated maximum number of bands (68) while B014 amplified
minimum number of bands (26), the differentiation in the number of
77
Chapter three
Results and Discussion
bands amplified by different primers affected by several factors such
as primer structures, template quantity, and less number of priming
sites in the genomic DNA (Braschetal.,2010).
In the term of unique banding pattern, eight of Trichophyton
rubrum isolates were distinguished by different primers which
produced the most important unique bands, such as the second sample
was distinguished by using primer (A08(180bp), sample(14) was
distinguished
by
using
primer
(H07(523bp)),
sample(15)
was
distinguished
by
using
primer
(A013(1792bp),
sample(9)
was
distinguished by using primer (C015(1052bp and Gs04(1010)bp), sample
(8) was distinguished by using primer (A013(1113bp and C015(436)bp),
but the sample(17) was distinguished by two primers (A08(535bp and
B04(685bp),
sample(16)
was
distinguished
by
two
primers
(As02(757bpand B04(142)bp), and the last sample(11) was distinguished
by using primer(A08(566bp and E02(418)bp) show in the table(3-18).
According to these results, they were discriminated sixteen
isolates from seventeen samples of Trichophyton rubrum because the
sample(4) did not have special unique bands (Fernando et al ., 2001),
and for best result they were must exterminated several primers to
choose the primer which give the large number of variation between
the samples which isolates (Asemato et al., 1996).
The samples (1, 3, 4, 6, 8, 9, and 16) were obtained from hair of
patients but the others were obtained from skin of patients.
78
Chapter three
Results and Discussion
Table (3- 18): Trichophyton rubrum isolates and primers that
appeared unique bands and molecular weight of these
bands, and polymorphism percentage of isolates.
79
Conclusion and Recommendation
Conclusion
1. Trichophytonrubrum
was
predominant
species;
higher
percentage of infection was recorded in male than female.
2. Isolation rate of dermatophytosis was (80%), so that
tineacapitis was predominant clinical type (50%) followed by
tinea cruris (32.5%) and tinea corporis (17.5%).
3. The RAPD-PCR method was suitable for detect the genetic
variation between the Trichophyton rubrum isolates.
4. Unique band was obtained from T. rubrum isolates using
fingerprinting
80
Conclusion and Recommendation
Recommendation
1. Application of RAPD indicatorsto found the fingerprinting for other
isolates of Trichophytonrubrumand other fungi which causes the
infection to human for rapid detection and helping to find ways to
control limiting their spread.
2. The employment the RAPD indicators in diagnosis and classified
Trichophytonrubrum.
3. The utilization of other DNA indicators for the other studies in future,
for getting the large information about the genetic materials of
Trichophytonrubrum and differences between isolates of this fungus.
4. Trying to designation specific primer for differentiation and early
detection for Trichophytonrubrumand its isolates.
81
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‫ﺍﻟﺧﻼﺻﺔ‬
‫ﺗﻀﻤﻨﺖ ﺍﻟﺪﺭﺍﺳﺔ ﺟﻤﻊ ﺧﻤﺴﻮﻥ ﻧﻤﻮﺫﺝ )ﺍﻟﺠﻠﺪ ﻭﺍﻟﺸﻌﺮ ﻭﺍﻻﻅﺎﻓﺮ( ﻣﻦ ﺍﻻﺷﺨﺎﺹ‬
‫ﺍﻟﻤﺸﻜﻮﻙ ﺑﺎﺻﺎﺑﺘﻬﻢ ﺑﺎﻟﻔﻄﺮﻳﺎﺕ ﺍﻟﺠﻠﺪﻳﺔ ﻣﻦ ﺍﻟﻔﺘﺮﺓ ﻛﺎﻧﻮﻥ ﺍﻟﺜﺎﻧﻲ‪ ۲۰۱۱ /‬ﻭﻟﻐﺎﻳﺔ ﻧﻴﺴﺎﻥ‪۲۰۱۱/‬‬
‫ﻓﻲ ﻣﺴﺘﺸﻔﻰ ﺍﻟﻴﺮﻣﻮﻙ‪.‬‬
‫ﺧﻀﻌﺖ ﺟﻤﻴﻊ ﺍﻟﻨﻤﺎﺫﺝ ﺍﻟﻰ ﺍﻟﻔﺤﺺ ﺍﻟﻤﺒﺎﺷﺮ ﺑﺎﺳﺘﺨﺪﺍﻡ ﻣﺤﻠﻮﻝ ‪ KOH %۱۰‬ﻭﻓﻲ ﻧﻔﺲ‬
‫ﺍﻟﻮﻗﺖ ﺗﻢ ﺯﺭﻉ ﺍﻟﻨﻤﺎﺫﺝ ﻋﻠﻰ ﻭﺳﻂ ﺍﻛﺎﺭ ﺍﻟﺴﺎﺑﺮﻭﻳﺪ ﺍﻟﺪﻛﺴﺘﺮﻭﺯ‪ .‬ﺍﻅﻬﺮ ﺍﻟﻔﺤﺺ ﺍﻟﻤﺒﺎﺷﺮ‬
‫ﺑﻤﺤﻠﻮﻝ ‪ KOH%۱۰‬ﻧﺘﺎﺋﺞ ﻣﻮﺟﺒﺔ ﻓﻲ‪ ٤۰‬ﻧﻤﻮﺫﺝ ﻣﻦ ﻣﺠﻤﻮﻉ ‪ ،٥۰‬ﺑﻴﻨﻤﺎ ﻛﺎﻥ ﻧﻤﻮ ﺍﻟﻔﻄﺮﻳﺎﺕ‬
‫ﺍﻟﺠﻠﺪﻳﺔ ﻣﻮﺟﺒﺎ ﻓﻲ ‪ ٤۰‬ﺣﺎﻟﺔ ﻣﻦ ﻣﺠﻤﻮﻉ ‪ .٥۰‬ﺍﻥ ﺍﻟﻤﺴﺒﺒﺎﺕ ﺍﻟﺮﺋﻴﺴﻴﺔ ﻟﻼﺻﺎﺑﺎﺕ ﺍﻟﻔﻄﺮﻳﺔ‬
‫ﺍﻟﺠﻠﺪﻳﺔ ﺍﻟﺘﻲ ﻅﻬﺮﺕ ﻫﻲ‪ ۱۷Trichophyton rubrum :‬ﻋﺰﻟﺔ ‪،‬‬
‫‪Trichophyton‬‬
‫‪ ۱۳ mentagrophyte‬ﻋﺰﻟﺔ‪ ۱۰ Microsporu mcanis ،‬ﻋﺰﻟﺔ‪ .‬ﺷﺨﺼﺖ ﺍﻟﻔﻄﺮﻳﺎﺕ‬
‫ﺍﻟﺠﻠﺪﻳﺔ ﺍﻟﻤﺴﺒﺒﺔ ﻟﻼﻣﺮﺍﺽ ﺍﻟﺠﻠﺪﻳﺔ ﺑﺎﻻﻋﺘﻤﺎﺩ ﻋﻠﻰ ﺍﻟﺸﻜﻞ ﺍﻟﻤﻈﻬﺮﻱ ﻟﻨﻤﻮ ﺍﻟﻔﻄﺮﻱ ﻭ ﺍﻟﻔﺤﺺ‬
‫ﺍﻟﻤﺠﻬﺮﻱ ﻟﻬﺎ‪.‬‬
‫ﺗﻢ ﺍﺳﺘﺨﺪﺍﻡ ﺗﻘﻨﻴﺔ ﺍﻝ ‪ RAPD-PCR‬ﻟﺘﺤﺪﻳﺪ ﺍﻟﺒﺼﻤﺔ ﺍﻟﻮﺭﺍﺛﻴﺔ ﻟﺴﺒﻌﺔ ﻋﺸﺮﻋﺰﻟﺔ ﻣﻦ‬
‫ﺍﻟﻔﻄﺮ‪ .T. rubrum‬ﺗﻢ ﺍﺳﺘﺨﻼﺹ ﺍﻟﺪﻧﺎ ﺍﻟﻤﺠﻴﻨﻲ ﻣﻦ ﻛﻞ ﻋﺰﻟﺔ ﻭﻋﻨﺪ ﺗﺮﻛﻴﺰ ﻧﻬﺎﺋﻲ ﺗﺮﺍﻭﺡ‬
‫ﻣﻦ )‪ (750-1360‬ﻣﺎﻳﻜﺮﻭﻏﺮﺍﻡ‪ 1-2/‬ﻏﻢ ﻏﺰﻝ ﻓﻄﺮﻱ ﺟﺎﻑ ﻭﺑﻨﻘﺎﻭﺓ ﺗﺮﺍﻭﺣﺖ ﻣﻦ)‪.(1.3-1.6‬‬
‫ﺣﻴﺚ ﺗﻢ ﺍﻧﺘﻘﺎء ‪ ۱٥‬ﺑﺎﺩﺉ ﻋﺸﻮﺍﺋﻲ ﻟﺘﻄﺒﻴﻘﻬﺎ ﻋﻠﻰ ﺍﻟﺪﻧﺎ ﻋﺰﻻﺕ ﻓﻄﺮ ﻟﻜﺸﻒ ﻋﻦ ﺍﻟﺘﺒﺎﻧﻴﺎﺕ‬
‫ﺍﻟﻮﺭﺍﺛﻴﺔ ﻓﻴﻤﺎﺑﻴﻨﻬﺎ‪ .‬ﻭﻣﻦ ﺑﻴﻦ ﺗﻠﻚ ﺍﻟﺒﺎﺩﺋﺎﺕ ﺍﻟﻤﺴﺘﺨﺪﻣﺔ‪ ۱۳ ،‬ﻣﻨﻬﺎ ﺍﻧﺘﺠﺖ )‪ (195‬ﺣﺰﻣﺔ‬
‫‪۱۲٦ (68%)،‬ﺣﺰﻣﺔ ﻣﺘﺒﺎﻧﻴﺔ )‪ .(polymorphic bands‬ﺍﻧﺘﺞ ﺍﻟﺒﺎﺩﺋﻴﻦ‬
‫‪(A08 and‬‬
‫)‪GS04‬ﺍﻛﺒﺮﻋﺪﺩ ﻣﻦ ﺍﻟﺤﺰﻡ ﺍﻟﻤﺘﺒﺎﻧﻴﺔ ﻭ ﻛﺎﻧﺖ )‪ (15‬ﺣﺰﻣﺔ‪ ،‬ﺑﻴﻨﻤﺎ ﻛﺎﻥ ﺍﺻﻐﺮ ﻋﺪﺩ ﻣﻦ ﺍﻟﺤﺰﻡ‬
‫ﺍﻟﻤﺘﺒﺎﻧﻴﺔ ﻫﻮ )‪ (6‬ﺍﻟﺘﻲ ﺍﻧﺘﺠﺖ ﻣﻦ ﻗﺒﻞ ﺍﻟﺒﺎﺩﺋﻴﻦ)‪ .(E02 and A013‬ﺗﻢ ﺗﻘﺪﻳﺮ ﻗﻴﻤﺔ ﺍﻟﺘﺒﺎﻳﻦ‬
‫ﺍﻟﻮﺭﺍﺛﻲ ﻟﻜﻞ ﺑﺎﺩﺉ ﻭﻗﺪ ﺗﺮﺍﻭﺣﺖ ﻣﺎﺑﻴﻦ )‪ (46-85%‬ﺣﻴﺚ ﺍﻧﺘﺞ ﺍﻟﺒﺎﺩﺉ ‪ R03‬ﺍﻋﻠﻰ ﻧﺴﺒﺔ ﻣﺌﻮﻳﺔ‬
‫ﻣﻦ ﺍﻟﺘﺒﺎﻳﻦ ﺍﻟﻮﺭﺍﺛﻲ ﻣﻘﺎﺭﻧﺔ ﺑﺎﻟﺒﺎﺩﺉ ‪ A013‬ﺍﻟﺬﻱ ﺍﻧﺘﺞ ﺍﻗﻞ ﻧﺴﺒﺔ ﻣﺌﻮﻳﺔ‪.‬‬
‫ﺗﻢ ﺗﺤﺪﻳﺪ ﺍﻟﺒﺼﻤﺔ ﺍﻟﻮﺭﺍﺛﻴﺔ ﻝ)‪ (16‬ﻋﺰﻟﺔ ﻣﻦ ﻋﺰﻻﺕ ﻓﻄﺮ ‪ T.rubrum‬ﻭﺫﻟﻚ ﻣﻦ ﺧﻼﻝ‬
‫ﻅﻬﻮﺭ ﻋﺪﺩ ﻣﻦ ﺍﻟﺤﺰﻡ ﺍﻟﻔﺮﻳﺪﺓ )‪ (unique bands‬ﻟﻜﻞ ﻋﺰﻟﺔ ﺑﺤﻴﺚ ﺗﻤﻴﺰﻫﺎ ﻋﻦ ﺍﻟﻌﺰﻻﺕ‬
‫ﺍﻻﺧﺮﻯ‪ ،‬ﺣﻴﺚ ﻳﻤﻜﻦ ﺍﻋﺘﺒﺎﺭ ﻫﺬﺓ ﺍﻟﺤﺰﻡ ﻛﻤﺆﺷﺮﺍﺕ ﻭﺭﺍﺛﻴﺔ ﺧﺎﺻﺔ ﺏ)‪ (16‬ﻋﺰﻟﺔ ﻳﻤﻜﻦ‬
‫ﺍﻻﺳﺘﻔﺎﺩﺓ ﻣﻨﻬﺎ ﻣﺴﺘﻘﺒﻼ ﻓﻲ ﺗﺼﻤﻴﻢ ﺍﻟﺒﺎﺩﺋﺎﺕ ﺍﻟﻤﺸﺨﺼﺔ ﻟﺘﻠﻚ ﺍﻟﻌﺰﻻﺕ ﻣﺒﺎﺷﺮﺓ‪.‬‬