RANDOM AMPLIFIED POLYMORPHIC DNA-POLYMERASE
CHAIN REACTION (RAPD-PCR) FINGERPRINTING OF
Escherichia coli O157:H7
NOR'AISHAH BINTI HASAN
INSTITUTE OF BIOLOGICAL SCIENCES
FACULTY OF SCIENCE
UNIVERSITI MALAYA
2008
RANDOM AMPLIFIED POLYMORPHIC DNA-POLYMERASE
CHAIN REACTION (RAPD-PCR) FINGERPRINTING OF
Escherichia coli O157:H7
NOR'AISHAH BINTI HASAN
A GRADUATION EXERCISE SUBMITTED TO THE
FACULTY OF SCIENCE
UNIVERSITI OF MALAYA
IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE
DEGREE OF MASTER OF BIOTECHNOLOGY
INSTITUTE OF BIOLOGICAL SCIENCES
FACULTY OF SCIENCE
UNIVERSITI MALAYA
2008
ABSTRACT
Twenty (n=20) among beef isolates of Escherichia coli O157:H7 were examined
for the detection of Shiga- toxin 1 and 2 (stx1 and stx2) genes and characterized using
Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR)
fingerprinting. All isolates were obtained from the laboratory of Food Science and
Biotechnology, University Putra Malaysia, Serdang, Selangor.
In the detection of stx1 and stx2 genes, 14 of isolates (14/20) were positive to stx1
and stx2.
Whereas, 5 isolates (5/20) were positive to stx1 and 1 isolate (1/20) was
negative by either of stx1 or stx2 genes.
Using RAPD-PCR analysis, two
oligonucleotides were chosen because they yielded clearly and reproducible band. There
were
OPAR8
(5’-TGGGGCTGTC-3’)
and
OPAR20
(5’-ACGGCAAGGA-3’).
Subsequently, all 20 isolates of E.coli O157:H7 were subtyped using OPAR8 and
OPAR20. Primer OPAR8 produced 8 RAPD-PCR fingerprinting namely P1 to P11.
Whereas, OPAR20 produced 16 RAPD-PCR fingerprinting of Q1-Q18. Combination of
two primers was analyzed using Unweighted Pair Group Method with Arithmetic mean
(UPGMA). Dendogram performed from cluster analysis showed that all the 20 isolates of
E.coli O157:H7 differentiated into 20 individual isolates which may suggest the high
level of local geographical genetic variation.
ABSTRAK
Dua puluh (n=20) isolat Escherichia coli O157:H7 yang diasingkan daripada
daging lembu telah dikaji untuk mengesan gen toksin Shiga 1 dan 2 (stx1 dan stx2) dan
pencirian dengan menggunakan cap jari Amplifikasi Polimorfik Asid Deoksiribonukleiktindakbalas rantaian polimerase (RAPD-PCR). Kesemua isolat telah di perolehi dari
Makmal Sains Makanan dan Bioteknologi, Universiti Putra, Serdang, Selangor.
Dalam pengesanan untuk gen stx1 dan stx2, 14 isolat (14/20) positif terhadap
stx1 dan stx2. Manakala, 5 isolat (5/20) positif terhadap stx1 dan 1 isolat (1/20) negatif
terhadap stx1 atau stx2 gen. Menggunakan analisis RAPD-PCR, 2 oligonukleotida telah
dipilih kerana menghasilkan jalur yang jelas dan keputusan berulang. Mereka adalah
OPAR8 (5’-TGGGGCTGTC-3’) dan OPAR20 (5’-ACGGCAAGGA-3’).
Oleh itu,
kesemua 20 isolat E.coli O157:H7 telah disubtaipkan dengan menggunakan OPAR8 dan
OPAR20. Primer OPAR8 menghasilkan 8 cap jari RAPD-PCR dinamakan P1 hingga
P11. Manakala, OPAR20 menghasilkan 16 cap jari RAPD-PCR dinamakan Q1 hingga
Q18. Gabungan dua primer dilakukan dengan menggunakan Kaedah Kumpulan Pasangan
Tanpa penimbang dengan Keertian Arithmatik (UPGMA).
Paparan dendrogram dari
analisis kelompok menunjukkan kesemua isolat E.coli O157:H7 boleh dibezakan kepada
20 isolat individu di mana mencadangkan paras tinggi genetik variasi di geografi
tempatan.
ACKNOWLEDGEMENT
Assalamualaikum.
Praise to Allah for the strength, wisdom and for the unconditional love that I got
that, I am able to pursue and complete my Degree of Master of Biotechnology. Without
that, I would never have the perseverance to make it until the end.
First of all, I would like to express my utmost appreciation to my supervisor, Dr
Noraida bt Ismail and to my co-supervisor, Dr. Sahilah bt Abdul Mutalib, for their
patience, kindness, guidance and useful advice given throughout this thesis project. Her
wisdom and encouragement has inspired us to work harder, to make this thesis a special,
successful and memorable one.
A million thanks to Prof. Dr. Yaakob Che Man, who is the Director of Halal
Products Research Institute, Universiti Putra Malaysia, for the facilities and helps in
making this, project a successful one
My thanks also go to my coursemates, Kak Suraya, Nadzimah, Faiza, Kak Lina
and Lani who worked together in the Food Biotechnology laboratory for the happy
moment that we shared in the lab. I also would like to express my deepest gratitude to
Amirul Asyraf bin Ismail for his support and guidance. Without your endless love,
support and encouragement, I could never have finished this thesis. Thank you for always
being there for me.
Finally, I would like to dedicate the thesis to my family; to my beloved mom, Ijal,
Aidah, Ajip, Ika, (my loving sisters and brothers) and my dearest relatives, my deepest
appreciation of love and thanks goes to all of you. I can never express my gratefulness
for everything that you have brought into my life. I love you all and this I can do for you.
DEDICACIÓN CON AMOR
-POR MI MADRE Y MI PADRE (1924-2004) POR CONFIANZA EN
MI Y MI FAMILIA. ESTE ES MI REGALITOS PARA TODOS.
TABLES OF CONTENTS
CHAPTER
PAGE
ABSTRACT
i
ABSTRAK
iii
ACKNOWLEDGEMENTS
v
LIST OF TABLES
viii
LIST OF FIGURES
ix
LIST OF ABBREVIATIONS & SYMBOLS
x
CHAPTER ONE
INTRODUCTION
1
CHAPTER TWO
LITERATURE REVIEW
2.1
Classification of Escherichia coli
4
2.2
Morphology & Colony Characteristics
8
2.3
Biochemical profile
10
2.4
Risk assessment acquiring Escherichia coli
11
2.5
Molecular Epidemiology & Bacterial Typing
13
2.6
Polymerase Chain Reaction (PCR) – Based Methods
14
2.6.1 Random Amplified Polymorphic DNA (RAPD–PCR)
14
2.6.2
Repetitive Element PCR Fingerprinting (REP–PCR)
15
2.6.3
Restricted Fragment Length Polymorphic (RFLP–PCR)
16
2.6.4
Amplified Restriction Fragment Polymorphic
(AFLP – PCR)
16
2.6.5
2.7
PCR Ribotyping
17
Pulsed Field Gel Electrophoresis ( PFGE )
CHAPTER THREE
17
MATERIALS & METHODS
3.1
Bacterial Strains
18
3.2
Preparation of Whole-cell DNA for PCR and RAPD
-PCR
19
3.3
Detection of Shiga toxin (stx1 and stx2)genes using multiplex PCR
19
3.4
RAPD-PCR Fingerprinting
20
3.5
Data Analysis
21
CHAPTER FOUR
RESULTS
4.1
Detection of Shiga toxin (stx1 and stx2)genes
23
4.2
RAPD-PCR Fingerprinting
24
4.3
Dendogram analysis using Unweighted Pair Group
29
Method with Arithmetic mean (UPGMA)
CHAPTER FIVE
DISCUSSION and CONCLUSION
34
CHAPTER SIX
REFERENCES
35
APPENDICES
A: GENERAL MEDIA AND SOLUTIONS
B: SOLUTION FOR PCR
LIST OF TABLES
Table
Page
2.1
Serotype of Escherichia coli associated with human diarrhea
8
2.2
Comparison between biochemical characteristics of Escherichia coli
O157:H7 and Escherichia coli
10
3.1
Number isolates of E.coli O157:H7
18
3.2
The 2 pairs of primer (Shiga Toxin) with their sequences
20
3.3
The ten oligonucleotides of RAPD-primers with their sequences
21
4.1
Genotypic diversity of Escherichia coli using RAPD-PCR
28
LIST OF FIGURES
Figure
Page
2.1
The six different pathogenic schemes of diarrheagenic Escherichia .coli. 7
Each of the six categories has unique features in their interaction with
eukaryotic cells.
4.1
Representative profile of multiplex detection with 2 pairs of different
primers (stx2F, stxR, stx1F and stx1R) of Escherichia coli O157:H7
isolates
4.2
Representative profile of arbitrarily primed-PCR patterns (P1-P4)
26
obtained with primer OPAR 8 of the Escherichia coli O157:H7
isolates.
24
4.3
Representative profile of arbitrarily primed-PCR patterns (P5-P11)
obtained with primer OPAR 8 of the Escherichia coli O157:H7
isolates
26
4.4
Representative profile of arbitrarily primed-PCR patterns (Q1-Q8)
27
obtained with primer OPAR 20 of the Escherichia coli O157:H7
isolates
4.5
Representative profile of arbitrarily primed-PCR patterns (Q9-Q18)
obtained with primer OPAR 20 of the Escherichia coli O157:H7
isolates
27
4.6
Dendogram generated from the arbitrarily primed-PCR patterns
with primer OPAR 8 and OPAR 20 of the Escherichia coli O157:H7
isolates from meat.
30
ABBREVAITIONS & SYMBOLS
Bp
Base pair
o
Degree celcius/Centigrade
C
DNA
Deoxyribonucleic acid
dNTPs
Dinucleotide(s) triphosphate
ds
Double stranded
et al.
Consensus and associates
g
Gram(s)
>
Greater than
H
Hour
Kb
Kilo base pair
µg
Microgram(s)
µl
Microliter(s)
µm
Micrometer(s)
M
Molar
min
Minute
mg
Milligram(s)
mM
Millimole(s)
ml
Milliliter(s)
%
Per cent
Pmol
Per mole
rmp
Rotation per minute
UV
Ultra violet
CHAPTER ONE
INTRODUCTION
1.1
INTRODUCTION
Enterohaemorrhagic Escherichia coli (EHEC) are collectively, one of the greatest
microbiological challenges to hit the food industry since the scourge of botulism some 80
years ago and are highly infectious in humans (International Life Science Report, 2001).
These organisms can cause illness including very serious illness such as haemorrhagic
colitis and haemolytic uraemic syndrome (HUS). About 5% of cases of haemorrhagic
colitis progress to HUS, in which the case fatality rate is approximately 10%
(Anonymous, 2000).
Outbreaks of infection, generally associated with beef, have been reported in
Australia, Canada, Japan, United States, in various European countries, and in southern
Africa (World Health Organization, 2007). In an outbreak in Japan in 1996, more than
6,300 school children were affected with three deaths (Fukushima et al., 1997). In the
United States in 1999, more than 1000 people (11 children with HUS and two deaths
recorded) were believed to have been infected through contaminated water (Charatan,
1999).
Five types of pathogenic E.coli are categorized according to their differences in
epidemiology, different interaction with intestinal mucosa, clinical syndromes of disease
caused and distinct O: H serotypes. There are Enterohaemorrhagic (EHEC),
Enterotoxigenic (ETEC), Enteropathogenic (EPEC), Enteroinvassive (EIEC), and
Enteroaggregative/Enteroadherent (EnaggEC) (Kauffman, 1947).
Food products associated with E. coli outbreaks include raw ground beef (Institute
of Medicine of the National Academies, 2002), raw seed sprouts or spinach, (Sabin,
2007), raw milk, unpasteurized juice, and foods contaminated by infected food workers
via fecal-oral route (Food Safety, 2006). Transmission of pathogenic E. coli often occurs
via fecal-oral transmission (Sabin, 2007). Dairy and beef cattle are primary reservoirs of
E. coli O157:H7 and they can carry it asymptomatically and shed it in their faeces (Bach
et al., 2002).
An outbreak caused by E.coli O157:H7 requires rapid isolation and identification
in order to identify the reservoir or the vector. Therefore, improved epidemiological
surveillance is essential and bacterial typing is of great value in epidemiologic
investigations. Various molecular typing have been developed; includes plasmid
profiling, PCR-ribotyping, IS 200 profiles, pulsed field gel electrophoresis (PFGE),
random amplified polymorphic DNA(RAPD) and repetitive element PCR fingerprinting
(Gillings and Holley, 1997; Rodrigue et al., 1995; Stubbs et al., 1994; Olsen et al., 1993;
Lagotolla et al., 1996).
It has been shown that E.coli O157:H7 had created a major problem in many
developing countries such as United States, Japan and Europe. In Malaysia, no report has
been obtained for E.coli O157:H7 outbreak. Nevertheless, from the data collected in
Malaysia, it shows that the incidence of E.coli O157:H7 isolated from animals and food
sources has increased noticeably in recent years especially from raw ground meat (Son et
al., 1997). The incidence of E.coli O157:H7 in meat samples in local market indicated the
exposures of these bacteria to public. It can be serve as a vehicle for the transmission of
the disease to man.
The aim of the project was to study the genetic diversity of E.coli O157:H7
species isolated from meat samples using molecular approaches of RAPD-PCR. In
this
study, in view of emerging important of E.coli O157:H7, the prevalence of E.coli
O157:H7 in food samples especially beef will be conducted to determine the presence of
Shiga-toxin 1 (stx1) and Shiga-toxin 2 (stx2) genes in E.coli O157:H7 and to subtype the
Escherichia coli 0157:H7 isolates using randomly RAPD-PCR. This would allow
discrimination below the species level. Besides that the data obtained will provide some
baseline information about the molecular epidemiology of E.coli O157:H7 in Malaysia.
CHAPTER TWO
LITERATURE
REVIEW
2.1 CLASIFICATION OF Escherichia coli
There are five distinct groups of E.coli cause gastrointestinal illness, which are
Enterotoxigenic (ETEC), Enteropathogenic, (EPEC), Enteroinvassive (EIEC),
Enterohaemorrhagic, (EHEC) and Enteroaggregative/Enteroadherent (EnaggEC) (Nataro
and Kaper 1998). However there is one group which is Diffuse Adherent E.coli (DAEC)
cannot be categorized due to the controversial of its pathogenicity (Tamura et al., 1996).
The clinical syndrome, made discovery of a new E.coli phenotype known as
EHEC (Karmali, 1989; Mead and Griffin, 1998). EHEC produce one or both of two
cytotoxin, VT1 and VT2. Gamage et al. (2003) reported that when both toxins are
produced by the same strains, VT1 predominates in cell lysates and VT2 is more active
toxin in supernates (Strocbine et al., 1986). Although this virulence factor (VT) found in
many different serotypes, only a few well characterized bioserotypes and clones (Dorn et
al., 1993) namely 0157:H7 and O26:H11 are the most important host for the verotoxin
phage(s). EHEC strains have become prominent in recent years as causes of hemorrhagic
enteritis and the hemolytic uremic syndrome (Coia, 1998)
Hemolytic Uremic Syndrome (HUS), first described in 1955, is a public human
disease caused by EHEC (International Life Science Report, 2001). E. coli O157:H7 is
responsible for over 90% of the cases of HUS that develop in North America. The
essence of the syndrome is described by its three central features: destruction of red blood
cells (hemolytic anemia), destruction of platelets (those blood cells responsible for
clotting, resulting in low platelet counts, or thrombocytopenia), and acute renal failure
(Karmali et al., 1989). Thrombotic thrombocytopenic purpura (TTP) is a clinical
syndrome defined by the presence of thrombocytopenia and microangiopathic hemolytic
anemia. TTP frequently leads to neurological and renal impairment in patients (World
Health Organization, 2005). Hemorrhagic colitis is an acute disease and a type of
gastroenteritis which produce a toxin that causes bloody diarrhea (Griffin and Tauxe,
1991).
ETEC strains cause diarrhea in infants and young children in tropical developing
countries, in travelers and contaminated food and water (Public Health, 2005). ETEC
produces two toxins, a heat-stable toxin (ST) and a heat-labile toxin (LT) (Fegan and
Desmarchelier, 2002). LT will influence the metabolism of prostaglandins and stimuli
neurotransmitters of the enteric nervous system (Beuble and Schuligoi, 2000; Mourad
and
Nassar,
2000).
Sculling
mouse,
radioimmunoassay
and
enzyme-linked
immunosorbent assay (ELISA) was the standard test to detect ST (Gianella and Vijg,
1981; Cryan, 1990). Recently, PCR assay has been developed, which are quite sensitive
and specific when used directly on clinical samples or on isolated bacterial colonies
(Lang et al., 2004; James and James 1998; Schultsz et al., 1994; Tornieporth et al.,
1997).
EIEC closely resemble Shigella (Kopecko, 1994) and causes diarrhea in humans
(DuPont et al., 1971). EIEC are non-motile and anaerogenic (Peter and Weagent, 2002).
The mechanism is uncertain, but it may attribute to a distinct plasmid encoded
enterotoxin (Robin et al., 1999). PCR assay was reported with primer derived from ial is
effective in multiplex PCR system to identify EIEC strains simultaneously with other
E.coli categories (James and James, 1998; Frankel et al., 1998).
EPEC causes enteric disease (Larry, 1985). Ability to produce A/E
(attaching/effacing) lesions and intimate adhere of bacteria to the intestinal epithelium is
the main mechanism of EPEC pathogenesis (Roger et al., 2004). There are two
approaches to detection of EPEC in the laboratory; phenotypic and genotypic. The
phenotypic approaches require the use of cell cultures and fluorescence microscopy.
Whereas the genotypic method requires the use of DNA hybridization of PCR, DNA
probes. Makino et al., (1994) suggested that PCR using eae- and bfp-specific primers and
HEp-2 adherence assay are useful to identify EPEC.
Konowalchuk and co-researchers 1977 reported VTEC produced cytotoxic for
vero cells and include representatives of both EPEC and EHEC (Pollard et al., 1990).
Verotoxin (VT) and Shiga-like toxin (SLT) are synonymous (Carter et al., 1987). Toxin
neutralization assay is sensitive and specific technique (immunochromatography and
latex agglutination) (Cermelli et al., 2002) suffer from limited sensitivity and a lack of
specificity (Watanabe et al., 1996). Simple and rapid detection of characteristic (toxin
production and type serotype) is possible in a single dipsticks test device, directly from a
food enrichment culture (Aldus et al., 2003).
Source: Nataro and Kaper (1998)
Figure 2.1: The six different pathogenic schemes of diarrheagenic E. coli. Each of the six
categories has unique features in their interaction with eukaryotic cells
2.2 MORPHOLOGY AND COLONY CHARACTERISTICS
E.coli was discovered in 1885 by Theodor Escherich, a German pediatrician and
bacteriologist (Feng and Weagent, 2002). A specific combination of O and H antigens
defines the serotype of an isolates but a complete O (somatic): H (flagellar): K (capsular)
serotype gives better identification for clonal relationships between strains (Orskov and
Orskov, 1992; Whittam et al., 1993).
Kauffman (1947) described that the O: H system (Table 2.1) was established in
order to differentiate E.coli on the basics of liposaccaharide O, flagellar H and
polysaccharide K antigen.
Table 2.1: Serotypes of Escherichia coli associated with human diarrhea
Enterotoxigenic
Enteropathogenic
Enteroinvassive
Enterohaemorrhagic
(ETEC)
(EPEC)
(EIEC)
(EHEC)
06
026
028ac
026
08
055
029
0111
015
086
0124
0157
020
0111
0136
025
0119
01432
027
0125
0144
063
0126
0152
078
0127
0164
080
0128ab
0167
Source: Levine, 1987
E. coli O157:H7 is a Gram negative bacillus straight rod bacterium. It is
distinguished by their inability to ferment sorbitol rapidly and by their lack of production
of glucuronidase (Hayes et al., 1995, Doyle and Schoeni, 1984). The size of E.coli
O157:H7 is 1.1-1.5 by 2.0-6.0 μm (living) and 0.4-0.7 by 1.0-3.0μm (dried and stained)
(Orskov, 1974). E.coli O157:H7 is motile by peritrichous flagella. However, there are a
few strains, which are non-motile. It is anaerobe and facultative anaerobe bacteria. The
temperature range for E.coli O157:H7 growth is 10-400C (optimum 370C). It can be
grown on ordinary media. Colonies are moist, smooth, large, thick or partially translucent
discs. Some strains may occur in mucoid form. Many pathogenic strains are hemolytic on
blood agar. It ferments lactose, glucose, mannitol, maltose and many other sugars.
E. coli O157:H7 has its ability to produce one or more Shiga toxins (ST) and it is
indistinguishable from Shiga toxin produced by Shigella dysenteriae (Anantharnarayan
and Jayaram, 1996). Shiga toxin 2 is a more divergent molecule, with only 56% amino
acid homology with Shiga toxin 1. Most E. coli O157:H7 strains produce Shiga toxin 2;
the percentage that also produce Shiga toxin 1 ranges from less than 25% in a series from
Europe to greater than 80% in a series from North America (Slutsker et al., 1997 , Law
and Kelly, 1995 , Mead and Griffin, 1998) . Other factors thought to contribute to the
virulence of E. coli O157:H7 include a virulence plasmid (pO157) and the locus of
enterocyte effacement (LEE). The LEE contains genes for an adhesion molecule (intimin)
2.3 BIOCHEMICAL PROPERTIES FOR Escherichia coli O157:H7
Table 2.2: Comparison between biochemical characteristics of E.coli O157:H7 and
Escherichia coli
Biochemical characteristic
Ornithine decarboxylase
Arginine dihydrolase
Lysine decarboxylase
Urease
L-Arabitol
Galacturonate
5-Ketogluconate
Phenol red
-Glucosidase
Mannitol
Adonitol
Palatinose
-Glucuronidase
Indole
N-Acetyl- -glucosaminidase
-Galactosidase
Glucose
Saccharose
L-Arabinose
D-Arabitol
-Glucosidase
-Galactosidase
Trehalose
Rhamnose
Inositol
Cellobiose
Sorbitol
-Maltosidase
L-Aspartic acid arylamidase
Escherichia coli
+
+
+
+
+
ND
ND
+
ND
+
+
ND
ND
+
ND
+
+
+
+
+
+
+
+
+
+
+
ND
ND
Escherichia coli O157:H7
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Source: Farmer et al., 2002)
+
ND
: positive reaction
: negative reaction
: not determined (different reaction given by different strains of a serotype)
2.4 RISK ASSESSMENT OF ACQURING Escherichia coli O157:H7
Risk assessment studies have been conducted to determine the actual risk of
disease and the parameters contribute to the susceptibility of populations to the disease.
Morgan et al. (1993) suggested that low levels of E. coli O157:H7 can play a role in
acquiring immuno-protection and building disease resistance. More research is needed to
quantify the risk in relation to public health, and to determine the approaches to
government regulation.
The outbreak of E.coli O157:H7 have been reported in many developed countries
such as United States, Japan and European (Beuchat 1996b). In Malaysia, the true burden
of infectious diarrhoea disease is largely unknown. In year 2004, around 6,000 cases of
food poisoning were reported (Ministry of Health, 2000). An attempt was made more
than a decade ago to estimate the burden of diarrhoeal disease in children in Malaysia
using a community-based study (Lim, 2007). In this study, based on the 2000 Malaysian
census, which showed a total population of 23.27 million with 33.3% under the age of 15
years, there would be nearly two million episodes of diarrhoea annually among
Malaysian children. However there have been no outbreaks of E.coli O157:H7 reported
in Malaysia (Health Ministry, 1999) but data collected showed an increasing incidence
among Malaysian (Bach et al., 2002). Son et al. (1998) reported that E. coli O157:H7
strains possessing important virulence traits were shown to be distributed at a
considerable frequency in the beef retailed in Malaysia.
In the last two decades, many new pathogens have been shown to be associated
with diarrhoeal diseases. They include Escherichia coli O157:H7 and other
diarrhoeagenic E. coli. Many of these agents are readily transmitted through food and
water (Walterspeil et al., 2003). Because of the manner processed food is now mass
produced and distributed, a single contaminated food source can cause an outbreak
involving many thousands of cases spread over a wide geographical area (Science Daily,
2007).
Laboratory tests to ascertain the cause of the diarrhoeal episode is not often
performed especially in general practice in Malaysia due to cost factors. The lack of a
specific diagnosis can hinder the institution of appropriate therapeutic and preventive
measures. There is a need for improved surveillance systems including syndromic and
laboratory surveillance to enable us to detect outbreaks in a timely manner in order to
take the necessary preventive measures (Son et al., 1997). Due to improvement in socio
economic status of the population and good access to medical facilities, severe
dehydration resulting from diarrhea has become relatively uncommon in Malaysia.
2.5
MOLECULAR EPIDEMIOLOGY AND BACTERIAL TYPING
Molecular epidemiology is based on characterization of bacterial isolates by
phenotypic assays such as biochemical testing, antimicrobial susceptibility testing and
serotyping (Versalovic et al., 1993). Several approaches had been tried to differentiate
different bacterial strains with biochemical, immunological and molecular methods
(Jordens et al., 1995). Biochemical methods are simple but time consuming and less
sensitive where as immunological method are not reveal full variability of bacterial
(Anand, 2001).
Bacterial typing systems for discriminating bacteria below the species level
subtyping from a single species have been based on phenotype, such as serotype, biotype,
phage typing, or antibiogram (susceptibility to one or more antibiotics)(Eisen et al.,
1995). There are two major categories; phenotypic and genotypic. Phenotyping includes
serotyping, bacteriocins, bacteriophages typing, biotyping, and antibiotic resistance
pattern and protein profiles. Whereas, genotyping have the potential of providing more
consistent, reproducible data and are applicable to other species and genera involve direct
DNA-based analysis of chromosome, plasmid and insertion sequences/ transposon (IS).
The popular approaches for characterizing individual strains are found in the practical
application of molecular biology such as Polymerase Chain Reaction, nucleic acid
fingerprinting and DNA sequence analysis (Relman and Persing, 1996).
2.6.
POLYMERASE CHAIN REACTION (PCR) – BASED METHODS
The polymerase chain reaction (PCR) is a biochemistry and molecular biology
technique for isolating and exponentially amplifying a fragment or sequence of interest of
DNA in a short time (Joseph et al., 2001). PCR offer speed and simple protocol (Wernars
and Huevelman, 1993). PCR has an enormous impact and variations of PCR-based
techniques as describe below:
2.6.1
RANDOM AMPLIFIED POLYMORPHIC
ARBITRARILY PRIMED PCR (AP-PCR)
DNA
(RAPD-PCR)
or
RAPD-PCR is a technique of utilizing arbitrary oligonucleotides to prime DNA
synthesis at low annealing temperature to divulge genomic diversity. RAPD is great
power and general applicability because does not require any specific knowledge of DNA
sequence of the target organism (Versalovic et al., 2002).
RAPD reaction mixture creates several arbitrary nucleotide sequence, short
primers, and then proceeds with the PCR using to amplify. During the annealing process,
the primer which sequence are not directed to any known genetic locus attaches to the
target DNA at random sites with a complementary sequence to permit initiation of
polymerization. If such sites are located, kilo bases of each other on opposite DNA strand
and in the proper orientation, then amplification of the intervening fragment will occur
(William et al., 1990).
RAPD has been applied to microorganisms such as Camphylobacter jejuni (Owen
and Hernandez, 1993), Listeria monocytogenes (Mazurier and Wernars, 1992) and
Pseudomonas fragi (Tanaka et al., 1993). In Escherichia coli, many reports have been
published regarding RAPD as a typing method such as in E. coli O157:H7 (Son et al.,
1998)
2.6.2
REPETITIVE ELEMENT PCR FINGERPRINTING (REP – PCR)
In rep-PCR technique, primers are designed complementary to bacterial
interspersed repetitive sequences (Versalovic et al., 2002). It is enable a rational approach
to primer design and a limited set of primers can be used with virtually any bacteria. The
primers are longer in length (18 to 22 mers) and, therefore, higher annealing temperatures
that enable greater stringency variation of the PCR can be used. Rep-PCR has multiple
applications in molecular epidemiology; including, Staphylococcus, Xanthomonas and
Pseudomonas (Gillings and Holey, 1997). This technique also widely applied to monitol
enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) (Dalla et al., 1998).
2.6.3 PCR–RESTRICTED FRAGMENT LENGTH POLYMORPHISM
(PCR RFLP)
This method involves amplifying a known sequence, cleaving the amplicon with
the suitable restriction endonucleases and comparing the restricted fragments of amplified
DNA from different strains. PCR-RFLP analysis has been applied most widely to genes
encoding toxins or other structural/functional molecules from Camphylobacter (Owen et
al., 1993; Burnens et al., 1995) and Vibrio (Faruque et al., 1994; Suthienkul et al., 1996).
In E.coli, report has been published regarding RFLP as a typing method (Buysse et al.,
1995).
2.6.4 PCR-AMPLIFIED FRAGMENT LENGHT POLYMORPHISM
(AFLP – PCR)
AFLP-PCR is a highly sensitive, higher reproducible and resolution method,
described by Vos and Zabeau in 1993. In AFLP analysis, bacterial genomic DNA is
digested with restriction enzymes, ligated to adapters and a subset of DNA fragments are
amplified using primers containing 16 adapter defined sequences with one additional
arbitrary nucleotide (Lin et al., 1996).
AFLP has become widely used for the identification of genetic variation in strains
or closely related species of bacteria such as Vibrio cholerae (Jiang et al., 2000),
Xanthomonas spp.(Rademaker, et al., 2003) and E. coli (Guan et al., 2002, Arnold et al.,
1999)
2.6.5 PCR RIBOTYPING
PCR ribotyping is based on the amplification of the spacer sequences between the
genes coding for 16S and 23S rRNA (Kostman et al., 1992). The rRNA loci are present
in 2 to 11 copies on the chromosomes of the most bacterial species. Whereas, a high
degree of sequence homology exist for rRNA genes, the intergenic spacer regions show
intensive sequence and length variation which can be used to characterize bacteria at the
genus (Jensen et al., 1993), species (Dolzani et al., 2004; Jensen et al., 1993) and
subspecies level (Dolzani et al., 2004; Kostman et al., 1992). This method has been
successfully applied to Salmonella spp. by Jensen et al., (1993) and Lagatolla et al.,
(1996). This technique also had been used by E. coli (Mehwish et al., 2007).
2.7 PULSED FIELD GEL ELECTROPHORESIS ( PFGE )
Pulsed Field Gel Electrophoresis (commonly abbreviated as PFGE) described by
Chu et al. (1986) is a method for separating large DNA molecules. The ability of PFGE
to separate large DNA fragments as large as 1.2 megabases (Zhang et al., 2000), provides
the opportunity to study complete microbial genomes. The use of PFGE has been applied
to other analysis such as taxonomy and bacterial genome mapping (Rompling et al.,
1992). Subtyping has made it easier to discriminate among strains of Listeria
monocytgenes, Salmonella weltevreden (Sahilah et al., 2001) and thus to link
environmental or food isolates with clinical infections. PFGE has been reported in E. coli
O157:H7 by (Son et al., 2001).
CHAPTER THREE
MATERIALS
&
METHODS
3.1
Bacterial strains
The Escherichia coli O157:H7 isolates were obtained from the laboratory of Halal
Product Research Institute, Infoport Centre, Universiti Putra Malaysia (UPM), 43400,
Serdang, Selangor Darul Ehsan.
E.coli
Strains no.
O157:H7
1
EC1
2
EC2
3
EC3
4
EC4
5
EC5
6
EC6
7
EC7
8
EC8
9
EC9
10
EC10
11
EC11
12
EC12
13
EC13
14
EC14
15
EC15
16
EC16
17
EC17
18
EC18
19
EC19
20
EC20
Table 3.1: Number of isolates of E. coli O157:H7
3.2
Preparation of whole-cell DNA for PCR and RAPD-PCR fingerprinting
DNA extraction was done by using boiling method. The cells were grown in
1.5 ml of Lauria-Bertani (LB)(Tryptone, 4.0 g/L, Yeast Extract, 5.0g/L, Sodium chloride,
10.0 g/L) at 35 °C for 20 h were harvested and centrifuged at 12,000 rpm for 1 min. The
supernatant was discarded. The pellet was then washed with 1.0 ml sterile distilled water
and vortex. Then, it was boiled at 97 oC for 10 min and immediately was frozen at -20 oC
for 10 min. The tube was centrifuged at 10,000 rpm for 3 min. The supernatant was used
as a template.
3.3
Multiplex PCR for detection of Shiga-toxin 1 and 2 (stx1and stx2) genes
The multiplex detection gene and PCR condition were optimized using
recommendations reported previously by Fode-Vaughan et al. (2003) using 20 strains and
2 pairs of primers were used which stx2F(5’-TTCTTCGGTATCCTATTCCC-3’), stx2R
(5’-ATGCATCTCTGGTCATTGTA-3’) , stx1F(5’-CAGTTAATGTGGTGGCGAAG3’), and stx1R (5’-CTGTCACAGTAACAACCGT-3’) designed by Olsvik and
Strockbine (1993). The detection assay was performed in a 25 μl volume containing
5.0 μl of 5× PCR buffer (100 mmol l−1 Tris–HCl, 35 mmol l−1 MgCl 2 , 750 mmol l−1
KCl, pH 8.8), 1.0 μl of 10 mmol l−1 dNTPs (Promega, Madison, USA) 1.0 μl of
10 pmol μl−1 primer stx2F, stx2R, stx1F and stx1R, 0.2 μl of 1.0 units of Taq DNA
polymerase (Promega, Madison, USA), 12.30 μl of sterile ultrapure deionized water and
2.0 μl of 100 ng DNA template. A negative-DNA control was performed by adding 1 μl
of sterile ultrapure deionized water. Amplification was performed in personal Eppendorf
thermal-cycler (Eppendorf, Germany) with a temperature program consisting of the
initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for
2 min, annealing for 1 min at 35 °C and polymerization at 72 °C for 2 min. Final
elongation was at 72 °C for 10 min.
3.4
Random Amplified Polymorphic DNA-polymerase chain reaction
(RAPD-PCR) fingerprinting
The discriminatory ability and stability of RAPD-PCR fingerprinting were tested
in a preliminary study against a panel of 4 different bacterial strains of E. coli O157:H7
with 10-mer random primers (Promega, USA). Primer OPAR 8 and OPAR 20 showed
the greatest stability and discriminatory ability among the E. coli O157:H7 isolates, and
was therefore used in this study. The RAPD-PCR fingerprinting assay was performed in
a 25 μl volume containing 2.5 μl of ×
10 PCR buffer (100
mmol l−1 Tris–HCl,
35 mmol l−1 MgCl 2 , 750 mmol l−1 KCl, pH 8.8), 0.5 μl of 10 mmol l−1 dNTPs (Promega,
Madison, USA) 1.0 μl of 10 pmol μl−1 primer OPAR 8 & OPAR 20, 0.3 μl of 1.5 units of
Taq DNA polymerase (Promega, Madison, USA), 18.95 μl of sterile ultrapure deionized
water and 1 μl of 100 ng DNA template. A negative-DNA control was performed by
adding 1 μl of sterile ultrapure deionized water. Amplification was performed in personal
Eppendorf thermal-cycler (Eppendorf, Germany) with a temperature program consisting
of the initial denaturation at 94 °C for 5 min followed by 45 cycles of denaturation at
94 °C for 1 min, annealing for 1 min at 35 °C and polymerization at 72 °C for 2 min.
Final elongation was at 72 °C for 7 min. The amplification products were analyzed by
electrophoresis in a 1.0% agarose in 0.5X TBE (0.1 M Tris, 0.1 M Boric acid, 0.1 mM
EDTA) at 90 V for 40 minutes. Gels were stained with ethidium bromide. The amplified
fragments were visualized with UV transilluminator (Syngene, USA). The 100bp DNA
ladder (Promega, USA) was used as a DNA size marker.
3.5
Data analysis
The banding patterns of individuals’ strains were scored based on the presence or
absence of the bands. The banding patterns scored were analyzed using the RAPDistance
Package Software (version 1.04) program. The scoring was made in the form of binary
code with the score ‘1’ indicating presence of band and ‘0’ the absence of band. The data
obtained were recorded and entered in the software CorelDRAW Graphic Suite X3 where
a dendogram was produced for further analysis. Clustering was based on the unweighted
pair of group average method (UPGMA) and was performed with the RAPDistance
software.
CHAPTER FOUR
RESULTS
4.1
Detection of Shiga-toxin 1 and 2 (stx1and stx2) genes
In this study, 2 pairs of primer (stx2F, stx2R stx1F and stx1R) were used for
detection and confirmation of E. coli O157:H7 as described by Fode-Vaughan et al.
(2003). Two pair of primers were used for multiplex PCR analysis of twenty (n=20)
isolates to detect stx gene. Most clinical signs of disease arise as a consequence of the
production of Shiga toxin stx1, stx2 or combinations of these toxins (Griffin et al., 1991).
The average sizes for the two primers are 180 bp and 255 bp, respectively (Paton and
Paton (2002); Genhua et al., 2002).
Among the twenty one samples, 14 isolates (14/20) were positive to stx1 and stx2,
indicated by formation of 2 bands in a range of molecular weight of 180 bp – 255 bp.
Whereas, 5 isolates (5/20); (EC2, EC7, EC12, EC14 and EC17) were positive to stx1,
indicated by formation of only one band in a arrange of molecular weight of 180 bp while
single isolate (1/20); (EC8) was lacked of both stx1 and stx2 which was indicated by no
formation of band (Figure 4.1).
M 1
2
3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
bp
1,517
500
100
Figure 4.1: The electrophoresis patterns of multiplex detection of Escherichia coli
O157:H7 isolates electrophoresed on 1.0 % agarose gel. M, Molecular weight sizes
(base pairs, bp) are indicated by numbers on the left; lane 1-20: EC1, EC2, EC3, EC4,
EC5, EC6, EC7, EC8, EC9, EC10, EC11, EC12, EC13, EC14, EC15, EC16, EC17,
EC18, EC19, EC20. Lane 21: Positive control (EC 12).
4.2
RAPD-PCR fingerprinting
A total of 10-mer of different oligonucleotide primers was used for RAPD-PCR
analysis of a subset of 4 isolates to detect polymorphism within Escherichia coli
O157:H7. The two primers produced a clear pattern and were used to analyze the whole
set of 20 E.coli O157:H7strains.
Twenty (n=20) strains of Escherichia coli O157:H7 were used for RAPD-PCR
analysis with OPAR8 and OPAR20. RAPD-PCR fingerprinting of E.coli O157:H7
obtained with primer OPAR8 represented by the Figure 4.2 and 4.3. The possible number
of RAPD-PCR fingerprinting was estimated on the basis of changes in one or more clear
bands or band sizes. Eleven (n=11) RAPD-PCR fingerprinting (P1-P11) were apparent
from primer OPAR8. The number of RAPD bands produced for a given primer ranged
from 1 to 7, with molecular sizes ranging from 0.1 to more than 2.0 kb with some of the
bands appeared weak. However, no band produced with isolates EC1 and EC8 (Figure
4.3) with primer OPAR8.
The RAPD-PCR fingerprinting of E.coli O157:H7 strains obtained from primer
OPAR20 is shown in Figure 4 and 5. Eighteen (n=18) RAPD-PCR fingerprinting (Q1Q18) were obtained from primer OPAR20. The number of RAPD bands produced for a
given primer ranged from 3 to 17, with molecular sizes ranging from 0.1 to more than 2.0
kb. Combination of both primers allowed the all E. coli O157:H7 differentiated into 20
genome types (Table 4.1).
M
1
2
3
4
5
6
7
8
9
10
11
bp
1,517
500
100
Fingerprintings:
ND P1 P1
P2 P3
P3 P1 ND P4
P2
Figure 4.2: RAPD-PCR fingerprinting (P1-P4) of Escherichia coli O157:H7
isolates obtained with primer OPAR8 electrophoresed on 1.0% agarose gel. Lane
M: 100 bp DNA ladder (molecular weight in base pair, bp); lane 1-10: EC1, EC2,
EC3, EC4, EC5, EC6, EC7, EC8, EC9, EC10; Lane 11: Negative control
M
1
2
3
4
5
6
7
8
9
10
11
bp
1,517
500
100
Fingerprintings:
P5
P6 P7 P8
P6 P9
P7
P10 P11
P8
Figure 4.3: RAPD-PCR fingerprinting (P5-P11) of Escherichia coli O157:H7 isolates
obtained with primer OPAR8 electrophoresed on 1.0% agarose gel. Lane M: 100bp
DNA ladder (molecular weight in base pair, bp); lane 1-10: EC11, EC12, EC13,
EC14, EC15, EC16, EC17, EC18, EC19, EC20; Lane 11: Negative control.
M
1
2
3
4
5
6
7
8
Q1
Q2
Q3
Q4
Q4
Q5
Q6
Q7
9
10
Q1
Q8
11
bp
1,517
500
100
Fingerprinting:
Figure 4.4: RAPD-PCR fingerprinting (Q1-Q8) of Escherichia coli O157:H7 isolates
obtained with primer OPAR20 electrophoresed on 1.0% agarose gel. Lane M: 100 bp
DNA ladder (molecular weight in base pair, bp); lane 1-10: EC1, EC2, EC3, EC4,
EC5, EC6, EC7, EC8, EC9, EC10; Lane 11: Negative control.
M
1
2
3
4
5
6
7
8
9
10
11
bp
1,517
500
100
Fingerprinting:
Q9
Q10 Q11 Q12
Q13 Q14 Q15 Q16 Q17 Q18
Figure 4.5: RAPD-PCR fingerprinting (Q9-Q18) of Escherichia coli O157:H7
isolates obtained with primer OPAR20 electrophoresed on 1.0% agarose gel. Lane M:
100bp DNA ladder (molecular weight in base pair, bp); lane 1-10: EC11, EC12,
EC13, EC14, EC15, EC16, EC17, EC18, EC19, EC20; Lane 11: Negative control
Table 4.1: Genotypic diversity of Escherichia coli using random amplified polymorphic
DNA-PCR (RAPD-PCR).
Strains no.
RAPD-PCR profiles for primer
Genome types
OPAR8
OPAR20
EC1
ND
Q1
1
EC2
P1
Q2
2
EC3
P1
Q3
3
EC4
P2
Q4
4
EC5
P3
Q4
5
EC6
P3
Q5
6
EC7
P1
Q6
7
EC8
ND
Q7
8
EC9
P4
Q1
9
EC10
P2
Q8
10
EC11
P5
Q9
11
EC12
P6
Q10
12
EC13
P7
Q11
13
EC14
P8
Q12
14
EC15
P6
Q13
15
EC16
P9
Q14
16
EC17
P7
Q15
17
EC18
P10
Q16
18
EC19
P11
Q117
19
EC20
P8
Q18
20
ND-Not detected
4.3
Combination of two primer using Unweighted Pair Group Method
with Arithmetic mean (UPGMA) analysis
Combination of two primers, OPAR8 and OPAR20 was analysed using
Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis. Figure
4.6 showed the combination of RAPD-PCR fingerprinting of E.coli O157:H7
obtained from primer OPAR8 and OPAR20, respectively. Dendogram performed
twenty E.coli O157:H7 strains into 2 major clusters. Cluster A contained 2 sub cluster
which are sub cluster I and sub cluster II. Sub cluster I has 6 strains of E. coli
O157:H7 which are EC18, EC14, EC5, EC9, EC11 and EC8. Sub cluster II contained
7 strains which are EC12, EC15, EC16, EC19, EC7, EC4 and EC10. Cluster B
divided into one sub cluster which is sub cluster III. It contained 7 strains which are
EC1, EC2, EC6, EC20, EC17, EC13 and EC3.
.1 8 7
.0 8 4
EC18
. 11 2
.0 6 9
.0 3 3
.1 0 6
EC14
EC5
.2 2 9
.0 3 5
EC9
.248
P 27c
P2 2 c(1 )
P 2 4 b (1 )
P 2 7 b (2 )
EC11
P 3 o b (1 )
.3 3
.0 4 2
EC8
P 2 b b (2 )
EC12
.1 9 8
.0 3 2
.1 5 6
.0 4 3
EC15 P 2 4 c ( 1 )
EC16 P 2 7 c 2 )
.218
.0 9
.327
.0 3 2
.2 8 1
EC7
.0 3
.3 4
EC1
.3 8
.3 6 5
.0 3 9
.0 1 3
EC6
EC20
.1 2 3
EC17
. 11 8
EC13
.0 9 3
.3 8 3
EC4
P 2 6 a (1 )
EC1
P 2 3 a (3 )
EC2
.3 9 6
.3
P 2 4 c(3 )
P 2ab
.062
.0 4 1
EC19
P 2 4 b (3 )
.1 0 8
.2 3 7
P 3 o b (2 )
P 24a
P2 4 b (2 )
P 3 o c (3 )
P 2 5 c(2 )
P 3 o b (3 )
EC3
P 2 4 a (1 )
Figure 4.6: Dendogram generated from the random amplified polymorphic DNAPCR (RAPD-PCR) fingerprinting among 20 beef isolates of the Escherichia coli
O157:H7 with primer OPAR 8 and OPAR20.
CHAPTER FIVE
DISCUSSION
&
CONCLUSION
5.1
DISCUSSION and CONCLUSION
In this study, the detection of stx1 and stx2 genes have been shown among 20 beef
isolates of Escherichia coli O157:H7 were examined. Samples were collected from 3
different wet markets (Location A: EC1-4, Location B: EC5-13 and Location C: EC14EC20) in Selangor, Malaysia. Wet markets were chosen due to the unhygienic situation
(Shiklomanov, 2000)
Fourteen (n=14) strains were positive to stx1 and stx2, 5 strains were positive to
stx1 and a single strain was negative by either of stx1 or stx2 genes while a single isolate
was negative either stx1 or stx2. All enterohemorrhagic Escherichia coli (EHEC) strains
cause serious disease in humans and possess at least one Shiga-like toxin (stx1 or stx2)
gene (Jothikumar and Griffiths, 2002). The detection of Shiga-like toxins is very useful
for the identification of EHEC and Non-EHEC strains were negative for both stx1 and
stx2.
The stx1 and stx 2 primers gave negative results from other bacteria tested,
including Listeria monocytogenes, Listeria grayii, Listeria ivanovii, Salmonella enterica
serovar Typhimurium var. Copenhagen PT 10 SA, S. enterica serovar Enteritidis,
Shigella sonnei, Yersinia enterocolitica, and Proteus vulgaris (Jothikumar and Griffiths,
2002). In this work, EC8 showed negative result to stx1 and stx2 primers which clearly
indicated the EC8 did not belong to the EHEC E. coli. The primers used are a powerful
primer to amplify stx1 and stx2 sequences in pathogenic EHEC E. coli and able to
distinguish among nonpathogenic E. coli isolates.
All E. coli isolates were examined for random amplified polymorphic DNA-PCR
(RAPD-PCR). Arbitrarily primed-PCR fingerprinting has been shown in recent years to
be useful for classifying a number of bacterial species (William et al., 1990). In addition,
this method can be used as a diagnostic tool in tracing the source of infections associated
with the consumption of beef meat because results can be obtained in less than 24h after
sampling. DNA products produced in RAPD-PCR fingerprinting analysis depend on the
primer used, with different primers producing different banding patterns. Two 10-mer
arbitrary primers (OPAR8 and OPAR20) were used to generate RAPD-PCR fingerprints.
The selections of those primers were based on good yield bands observed on the agarose
gel. Several isolates failed to produce any bands with the two primers used. This can be
interpreted as the loss of specific sites for primers binding in the chromosomal DNAs of
these isolates since these DNAs gave appropriate bands when they examined using the
primers in reciprocal (Table 4.1).
The OPAR20 primer was more powerful in discriminatory of all E. coli O157:H7
tested which generated 18 fingerprints compared to OPAR8 which was only produced 11
fingerprints.
The RAPD-PCR analysis using OPAR8 and OPAR20 in combination
allowed all strains of E. coli O157:H7 differentiated into 20 genome types. Cluster
analysis is used to establish the degree of the relatedness among strains, information that
may useful in epidemiological studies (Soto et al., 1991). Consistent with Unweighted
Pair Group Method with Arithmetic mean (UPGMA) analysis, dendogram performed
from cluster analysis showed that all the 20 isolates of E.coli O157:H7 differentiated into
20 individual isolates which may suggest the high level of local geographical genetic
variation.
CHAPTER SIX
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APPENDICES
APPENDIX A: GENERAL MEDIA AND SOLUTIONS
Maintenance of Bacterial Strains
All bacterial isolates were maintained on TSA slant in bijou bottle and kept at 4 oC. All
the stock cultures were subculture for every three months.
Luria-Bertani (LB Broth)
Tryptone
Yeast Extract
Sodium chloride
Distilled water
4.0 g
2.0 g
4.0 g
400 ml
Sterilize by autoclaving at 121oC for 15 min.
Sequences for RAPD-primers.
Arbitrary primer (RAPD-primer)
G+C contents (%)
Sequence (5’ to 3’)
OPAR8
70
TGGGGCTGTC
OPAR10
50
CCATTTACGC
OPAR20
60
ACGGCAAGGA
OPAC 01
60
TCCCAGCAGT
OPAC 02
60
GTCGTCGTCT
OPAC 04
70
ACGGGACCTG
OPAC 05
60
GTTAGTGCGG
OPAC 07
70
GTGGCCGATG
OPAC 09
60
AGAGCGTACC
OPAC 11
70
CCTGGGTCAG
OPAC 12
70
GGCGAGTGTG
APPENDIX B: SOLUTION FOR PCR
Taq polymerase and other PCR chemicals
(store at -20 oC)
Gel Electrophoresis
10x Tris-Borate-EDTA (TBE) Buffer
Tris-Base
108.0 g
Boric acid
55.0 g
EDTA
9.3 g
Distilled water
1000 ml
For routine electrophoresis, 10X TBE were diluted to 0.5 x TBE, with the pH
8.3.Solution can be kept at room temperature.
1.0% Agarose (for RAPD)
Agarose
1.0 g
0.5Xtbe Buffer
100ml
Heat until the medium boils. Do not overheat
Ethidium Bromide solution
Ethidium bromide1.0 g
Distilled water 100 ml