Comparison of 13 single-round and nested PCR assays
targeting IS900, IS, f57 and locus 255 for detection of
subsp.
Petra Möbius, Helmut Hotzel, Astrid Raßbach, Heike Köhler
To cite this version:
Petra Möbius, Helmut Hotzel, Astrid Raßbach, Heike Köhler. Comparison of 13 single-round
and nested PCR assays targeting IS900, IS, f57 and locus 255 for detection of subsp.. Veterinary
Microbiology, Elsevier, 2009, 126 (4), pp.324. .
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Accepted Manuscript
Title: Comparison of 13 single-round and nested PCR assays
targeting IS900, ISMav2, f57 and locus 255 for detection of
Mycobacterium avium subsp. paratuberculosis
Authors: Petra Möbius, Helmut Hotzel, Astrid Raßbach,
Heike Köhler
PII:
DOI:
Reference:
S0378-1135(07)00360-4
doi:10.1016/j.vetmic.2007.07.016
VETMIC 3766
To appear in:
VETMIC
Received date:
Revised date:
Accepted date:
23-3-2007
19-7-2007
20-7-2007
Please cite this article as: Möbius, P., Hotzel, H., Raßbach, A., Köhler, H., Comparison of
13 single-round and nested PCR assays targeting IS900, ISMav2, f57 and locus 255 for
detection of Mycobacterium avium subsp. paratuberculosis, Veterinary Microbiology
(2007), doi:10.1016/j.vetmic.2007.07.016
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Manuscript
1
Comparison of 13 single-round and nested PCR assays targeting IS900,
2
ISMav2, f57 and locus 255 for detection of Mycobacterium avium subsp.
3
paratuberculosis
4
Petra Möbius1*, Helmut Hotzel2, Astrid Raßbach2, and Heike Köhler1
6
Institute of Molecular Pathogenesis1 and Institute for Bacterial Infections and Zoonoses2,
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Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Naumburger Str.
8
96a, 07743 Jena, Germany
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*Corresponding author. Mailing address: Friedrich-Loeffler-Institute, Federal Research
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Institute for Animal Health, Naumburger Str. 96a, D-07743 Jena, Germany. Phone: + 49-
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3641-804280; Fax: + 49 3641 804228. E-mail: [email protected]
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Abstract
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For molecular biological detection of Mycobacterium avium subsp. paratuberculosis (MAP),
3
PCR methods with primers targeting different regions specific for MAP are used worldwide.
4
However, some uncertainties exist concerning the specificity of certain target regions and the
5
sensitivity. To identify the methods which are best suited for diagnostics, eight single-round
6
and five nested PCR systems including twelve different primer pairs based on IS900 (9x),
7
ISMav2 (1x), f57 (1x), and locus 255 (1x) sequences were compared regarding their analytical
8
sensitivity and specificity under similar PCR conditions. Reference strains and field isolates
9
of 17 Mycobacterium species and subspecies and of 16 different non-mycobacterial bovine
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pathogens and commensals were included in this study.
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Single-round PCR resulted in a detection limit of 100 fg to 1 pg, and nested PCR in 10 fg or
12
below. Depending on the specific primer sequences targeting IS900, false positive results
13
occurred with one of the five single-round and two of the four nested PCR systems. This also
14
applied to the single-round PCR based on ISMav2 and the nested PCR based on f57. A high
15
number of non-specific products was primarily detected for the single-round PCR assay based
16
on ISMav2, but also for a single-round PCR targeting the IS900 and the locus 255.
17
In conclusion, stringent selection of IS900-specific primers ensures that IS900 remains a
18
favourite target sequence for amplification of MAP specific loci. The studied PCR systems
19
based on f57, and locus 255 can also be recommended. Revision of ISMav2 primers is
20
necessary. Single-round PCR systems are very reliable. Nested PCR assays were occasionally
21
disturbed by contaminations, thus bearing a risk for routine diagnostics.
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Key words: bovine paratuberculosis, PCR, sequence dependent specificity, cross-reactions,
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by-products, analytical sensitivity
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Page 2 of 25
1. Introduction
2
At present, faecal culture on solid media is still the most sensitive and specific method for
3
direct diagnosis of paratuberculosis in animals, and thus the “gold standard” (Stabel, 1997).
4
However, the method is too laborious and time consuming to be applicable in large-scale
5
diagnostic programmes. Therefore, direct detection of Mycobacterium avium subsp.
6
paratuberculosis (MAP) in faecal samples by PCR seems to be a practicable alternative.
7
Different PCR methods including real-time and nested PCR are already in use for the
8
detection of MAP, although valid data regarding the specificity and sensitivity of these assays
9
are missing.
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Several primers for single-round and nested PCR assays based on the first discovered MAP
11
specific repetitive insertion segment IS900 have been published (Green et al., 1989; Moss et
12
al., 1991; Sanderson et al., 1992; Collins et al., 1993; Vary et al., 1990; Millar et al., 1996;
13
Englund et al., 1999; Marsh and Whittington, 2001; Bull et al., 2003; Vansnick et al., 2004;
14
Ikonomopoulos et al., 2004). Their specificity was evaluated using different numbers of
15
Mycobacterium species and other common bacteria (Collins et al., 1989; Vary et al., 1990;
16
Englund et al., 1999; Ikonomopoulos et al., 2004; Vansnick et al., 2004, Tasara et al., 2005).
17
The occurrence of IS900-like sequences in a non-MAP isolate (Cousins et al., 1999; Englund
18
et al., 2002) and false positive results obtained by IS900 PCR for some Mycobacterium avium
19
complex (MAC) strains and non-MAP isolates (Cousins et al., 1999; Motiwala et al., 2004;
20
Tasara et al., 2005) caused uncertainties about the specificity of PCR systems targeting IS900
21
for scientific purposes and routine diagnosis. In the meantime, additional gene loci specific
22
for MAP have been identified and suggested for use in diagnostics: ISMav2 (Strommenger et
23
al., 2001), f57 (Poupart et al., 1993), ISMap02 (Stabel et al., 2005), and 21 other MAP
24
specific coding sequences (Bannantine et al., 2002).
25
The aim of the current study was to find out, which of the proposed primer pairs published
26
and which MAP specific target region are most suitable for MAP detection. 13 single-round
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and nested PCR systems based on the target regions IS900, ISMav2, f57, and locus 255 were
2
involved. The specificity and analytical sensitivity of the primers was analysed under
3
comparable conditions.
4
2. Material and methods
6
2.1. Bacterial strains and DNA probes
7
Reference strains and field isolates of 17 mycobacterial species and subspecies, and of 16
8
other bacterial pathogens or commensals which can be isolated from cattle were included in
9
this study and are listed in Table 1. The MAP field isolates recovered from faecal and organ
10
samples were maintained on Loewenstein-Jensen based paratuberculosis medium containing
11
mycobactin as well as colistinsulfate, amphotericin, piperacillin and trimethoprim (PACT,
12
Bioservice Waldenburg, Waldenburg, Germany). Non-MAP mycobacterial field isolates
13
recovered from organ samples were maintained on Middlebrook 7H11 agar supplemented
14
with oleic acid, albumin, dextrose and catalase (OADC). Other bacterial species, not
15
belonging to Mycobacterium, were obtained from the German Collection of Microorganisms
16
and Cell Cultures (DSMZ) and were cultivated on special culture media recommended by the
17
DSMZ. Salmonella reference strains were cultivated on nutrient broth (Oxoid), Escherichia
18
coli field isolates on Luria-Bertani-Agar, and Coxiella burnetii field isolates on Buffalo Green
19
Monkey cells in Nephros LP or UltraCulture medium (BioWhittaker).
20
The identity of the bacterial species used was examined by different methods. The species of
21
different mycobacterial strains was confirmed by PCR amplification of hsp65 and restriction
22
enzyme analysis (Telenti et al., 1993). Field isolates of atypical mycobacteria were identified
23
by sequencing of 16S ribosomal RNA genes. Additionally, isolates of the subspecies
24
Mycobacterium avium (M. a.) paratuberculosis, M. a. avium and M. a. hominissuis were
25
differentiated by PCR detection of the species-specific target regions IS900 (Englund et al.,
26
1999), IS1245 (Guerrero et al., 1995), and IS901 (Kunze et al., 1992). Bacterial reference
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strains, not belonging to mycobacteria and purchased from DSMZ were identified by
2
morphological characteristics. The identity of other species was confirmed by species-specific
3
PCR.
4
Genomic DNA from mycobacteria was prepared by the cetyl-trimethyl-ammonium-bromide
5
(CTAB) method described by van Soolingen et al. (1991), which included a combination of
6
chloroform/isoamylalcohol and isopropanol for extraction and precipitation of the DNA. For
7
DNA extraction of non-mycobacteria strains the High Pure PCR Template Preparation Kit
8
was used according to the manufacturer’s instructions (Roche Diagnostics, Mannheim,
9
Germany). Nucleic acid concentration was measured by spectrophotometry (DU640
10
Photometer, Beckman Coulter GmbH, Krefeld, Germany) at 260 and 280 nm. Dilutions of
11
DNA were freshly prepared prior to PCR.
12
2.2. PCR Systems
13
Eight single-round and five nested PCR systems including twelve different primer pairs (see
14
table 2) were tested. PCRs were carried out in a final volume of 20 µl, containing 1 µl of
15
template DNA, 0.5 µM of each primer (final concentration), and 10 µl Taq DNA Polymerase
16
Master Mix (QIAGEN, Hilden, Germany) which results in a final concentration of 0.5 units
17
Taq DNA Polymerase, 1.5 mM MgCl2 and 200 µM of each dNTP, and distilled water
18
(QIAGEN). A positive control (1 ng DNA from Map reference strain) and a negative control
19
(1 µl sterile water) were included in each PCR run. PCR was performed using initial
20
denaturation (96°C, 1 min), followed by 35 cycles of denaturation (96°C, 15 s), primer
21
annealing depending on the specific PCR system (between 56°C and 68°C, for 15 s until 1
22
min), primer elongation (72°C, 1 min), and additionally a final extension (72°C, 5 min), all on
23
a Mastercycler (Eppendorf AG Hamburg, Germany). For nested and seminested PCR, 1 µl of
24
amplicons from the first PCR served as template for the second run of 17 cycles. For the
25
single PCR assay of Doran et al. (1994), a touch-down PCR was carried out, described by
26
Winterhoff (2000). PCR targeting locus 255 was established with primers described by
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Bannantine et al. (2002) using 35 cycles of denaturation (96°C, 15 s), primer annealing (64°C,
2
45 s), and synthesis (72°C, 1 min). Amplicons were detected by visualization under UV light
3
on 1.5 % agarose gels (13- by 13-cm) after staining with ethidium bromide. DNA isolation,
4
preparation of master mix, addition of template DNA, electrophoresis and nested PCR were
5
all carried out in separate rooms. All PCR reactions were repeated at least twice.
6
2.3. DNA sequencing
7
Hypervariable regions of 16S rRNA of atypical mycobacteria were amplified using primers
8
according to Kirschner and Böttger (1998). Sequencing was done with primers 285 and 259
9
after removal of PCR amplicons from the agarose gel and purification using QIAquick Gel
10
Extraction Kit (QIAGEN). Cycle sequencing was carried out with the BIGDYETM Terminator
11
1.1. Cycle Sequencing Kit (Applied Biosystems). All manipulations were done according to
12
the manufacturers’ instructions.
13
Additionally, PCR products obtained from MAP reference strains and different primer
14
systems were verified by DNA sequencing. Sequence data were aligned with the IS900
15
sequence (accession number X16293).
16
2.4. Analytical sensitivity
17
Detection limits of the PCR assays were assessed by application of decimal dilutions of
18
purified DNA extracted from cultures of the MAP reference strains ATCC 19698 and DSM
19
44135, and the field isolate 03A1961. For this purpose, DNA solutions were serially diluted
20
with sterile water to obtain end concentrations of 100 ng to 0.1 fg / per µl.
21
2.5. Specificity
22
To determine the specificity of the different PCR systems, DNA concentrations of 0.01, 1, 10
23
and 100 ng were used.
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Definitions. False positive products as a result of cross reaction with non MAP-DNA, which
25
have the same size as the specific amplicons, and non-specific products (also called by-
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products), which differed in size from the specific amplicons in the agarose gel, were
2
distinguished.
3
3. Results
5
Eight single-round and five nested PCR systems targeting the different gene loci IS900,
6
ISMav2, f57 and locus 255 were analysed for their specificity for MAP and their analytical
7
sensitivity. Using MAP DNA as template in these examined PCR assays, amplicons exhibited
8
the expected length, according to the published sequences. Additionally, sequence analysis
9
confirmed the identity with the expected target (data not shown). Regardless of different
10
concentrations of MAP DNA, an inhibition of the PCR process was never observed, and non-
11
specific products were never generated.
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For assessment of the analytical sensitivity, the concentration of MAP DNA was quantified.
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Depending on the primers and on the kind of PCR, the detection limit ranged from 1 pg to
15
less than 1 fg (Table 2). For single PCR systems, a detection limit between 1 pg and 100 fg
16
was determined. These detection limits correspond to data for single PCR systems published
17
by Englund et al. (1999, 2001), Schneider (2003), and Stabel et al. (2005). On average the
18
nested PCR assays showed a 10 to 100 times higher analytical sensitivity, a detection limit of
19
10 fg was ascertained. These coefficients are in agreement with the results of Ikonomopoulos
20
et al. (2004). The use of nested PCR described by Englund et al. (1999) or by Vansnick et al.
21
(2004) permitted determination of 1 fg of MAP DNA. Different estimations were published
22
for the relationship between MAP genome and DNA quantity. Sanderson et al. (1992)
23
considered one genome copy as equivalent to 5 fg of DNA, Schneider (2003) one genome
24
copy as equivalent to 1 fg of DNA. Based on this calculation, the current detection limits of
25
single-round PCR ranged from 20 to 100 or 200 to 1000 genomic copies, respectively. For
26
nested PCR, these limits averaged between 2 to 10 and 0.2 to 1 genome copies. In Table 2, the
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results are indicated after conversion of DNA quantities into genomic copies according to
2
Schneider et al. (2003). The detection limits of 10 and 1 fg MAP DNA, equal 2 to 10 and 0.2
3
to 1 genome copies, agree with the results of Moss et al. (1991), Collins et al. (1993), Riggio
4
et al. (1997), Englund et al. (1999; 2001), and Schneider (2003).
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The specificity of the primer pairs differed, as shown in Tables 3 and 4. Concerning the target
7
region IS900, four of the five PCR primer systems tested for single-round PCR (Bauerfeind et
8
al., 1996; Doran et al., 1994; Englund et al., 1999; Schneider, 2003) showed an excellent
9
specificity, no false positive products were detected. Only few by-products were found, but
10
only when a high concentration of non-MAP DNA (100 ng per PCR) was used. The PCR
11
assay using primers published by Vary et al. (1990) is an exception, because it resulted in
12
false positive products for DNA of M. fortuitum, M. intracellulare and Salmonella
13
Typhimurium. Additionally, a large number of by-products was detected with DNA of
14
different mycobacteria species and other bacteria. For the target region ISMav2, the used
15
primer system (Stratmann et al., 2004) also resulted in false positive amplification products
16
with DNA of M. fortuitum, M. smegmatis and two other non-mycobacterial species.
17
Remarkably, also a large number of non-specific products was seen, partially already starting
18
at a DNA concentration of 0.01 ng of non-MAP organisms (Figure 1). Using the proposed
19
primers for the target region f57 published by Vansnick et al. (2004), no false positive
20
products or by-products were found with any of the tested non-MAP strains, confirming the
21
high specificity of this target region. The primer system targeting the MAP specific locus 255
22
(Bannantine et al., 2002) showed no false positive results, but some by-products (starting
23
from 100 ng).
24
Generally, nested PCR showed a sufficient specificity. For three of the five tested systems,
25
false positive products were detected with one or two non-MAP bacteria, for another selection
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of three nested PCR systems only a few by-products were found. But carry-over and some
2
cross-contaminations became evident using nested PCR (data not shown).
3
4. Discussion
5
4.1. Specificity of primers
6
IS900 belongs to a family of related insertion elements found in the order Actinomycetales
7
(Bull et al., 2003). This group also includes IS901 (specific for M. a. avium), IS902 (specific
8
for M. a. silvaticum), IS1110 (detected in some M. avium complex isolates), and IS1613
9
(derived from M. a. avium), which share between 60-80% sequence identities with IS900
10
(Cousins et al., 1999). These sequence identities resulted in an only small number of by-
11
products with strains of M. avium subspecies other than MAP in four of the five studied single
12
PCR assays targeting the IS900 in the current study, but no false positive amplifications were
13
detected. Generally, all tested primer pairs based on IS900 showed a satisfying specificity
14
with one exception. The primers IS900/150 and IS900/921 published by Vary et al. (1990)
15
were not very specific in MAP detection, which was also described by Cousins et al. (1999).
16
PCR systems producing false positive amplifications were also found in the literature after
17
using primer pair p90+/p91+ (Millar et al., 1996) for single round PCR assay and in
18
combination with other primers for nested PCR assay (Cousins et al., 1999; Motiwala et al.,
19
2004; Tasara et al. 2005). Primer pairs IS900/150, IS900/921, and p90+ / p91+ (Vary et al.,
20
1992; Millar et al., 1996) cannot be recommended as a diagnostic tool for MAP detection.
21
The most specific single-round PCR assay of the current study targeting the IS900 used
22
primer sequences published by Bauerfeind et al. (1996) and a very stringent annealing
23
temperature of 65°C, which was possibly responsible for the lower sensitivity of this assay in
24
comparison to others. Although these highly specific primers are located in the 3’-region,
25
PCR targeting the 5’-region of IS900 has been considered to be more specific for MAP
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detection than the 3’-region which was explained by homologies to other IS elements (Green
2
et al., 1989; Millar et al., 1995).
3
An increased discussion about the specificity of the target region IS900 started, when
4
Englund et al. (2002) isolated a mycobacteria strain from a healthy dairy cow harbouring one
5
copy of a sequence with 94% identity to IS900 in the genome (M. sp. strain 2333). This
6
IS900-like element resulted in amplicons with several very specific PCR primer systems
7
based on IS900. Only a sequence analysis or the additional use of another MAP specific target
8
region for PCR could confirm the real identity of such isolates (Englund et al., 2002). In more
9
than 2000 isolates of M. a. avium, M. a. silvaticum, M. a. hominissuis and 213 field isolates
10
and strains of 19 other mycobacteria, Bartos et al. (2005) did not find any IS900-like element.
11
Rare single false positive results must and can be accepted in routine diagnosis when using
12
PCR assays based on the IS900 element.
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Gene locus f57 as another specific target region has no homology with known sequences and
15
was not found in any other Mycobacterium species including M. avium subspecies (Poupart et
16
al., 1993). The current study confirmed the high specificity of primers targeting the f57 locus,
17
tested also by Vansnick et al. (2004) and Tasara et al. (2005) on a large panel of
18
mycobacterial and other bacterial strains, and by Englund et al. (2002) on the above
19
mentioned M. sp. strain 2333. Except for some by-products, the specificity of primers
20
targeting the locus 255 (Bannatine et al., 2002) is acceptable.
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In contrast, the specificity of the primer system targeting the ISMav2 (Stratmann et al., 2002),
23
remains inadequate. Besides the false positive amplification products with non MAC bacteria,
24
the very high number of by-products caused by using these primers which was comparable to
25
the above mentioned PCR assay of Vary et al. (1992) was remarkable. In the cited literature,
26
details about non-specific products could not be found. Sometimes differences of fragment
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length between non-specific products and specific amplicons were very small so that results
2
on short running gels could be misinterpreted as false positive amplifications. Nevertheless,
3
by-products can cause misleading results using other PCR verification methods based on
4
unspecific probes or real-time systems with SYBR Green.
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4.2. Analytical sensitivity
7
Most of the primer pairs published for MAP are based on the repetitive element IS900. Since
8
IS900 occurs with 14 to 20 copies in the MAP genome, it was presumed that PCR with this
9
target region would be more sensitive than PCR based on other targets occurring in lower
10
copy numbers (Green et al., 1989; Collins et al., 1989). In the present study, the analytical
11
sensitivity of five different single PCR systems all targeting IS900 varied between 0.1 and 1.0
12
pg DNA. There were no differences to single PCR assays targeting ISMav2 (Stratmann et al.,
13
2003) which occurs in three copies (Strommenger et al., 2001), f57 with one copy (Vansnick
14
et al., 2004) and locus 255 also with one copy (Bannantine et al., 2002) in the genome.
15
Likewise, in our study, the nested PCR using primers of Vansnick et al. (2004) targeting the
16
f57 locus showed the same sensitivity of 1 fg DNA/PCR as the one based on IS900-specific
17
primers from Englund et al. (1999). This sensitivity confirms the results of Vansnick et al.
18
(2004) who had detected a limit of 1 CFU for nested PCR assays targeting both loci. The
19
different binding abilities of primers and PCR conditions seem to have a higher influence on
20
analytical sensitivity than the number of copies of the specific target region in the MAP
21
genome.
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5. Conclusion
24
In conclusion, stringent selection of IS900-specific primers ensures that IS900 remains a
25
favourite target sequence for amplification of MAP specific loci. A PCR system targeting two
26
different MAP specific regions would have a still higher specificity. The following six single11
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round PCR-systems can be recommended: IS900 based PCRs of Schneider (2003), Englund
2
et al. (1999), Bauerfeind et al. (1996), and Doran et al. (1994), the f57 based PCR assay
3
(Vansnick et al., 2004), and the locus 255 based PCR assay (Bannantine et al., 2002). At least
4
100 fg (equivalent to 100 MAP genomes) of DNA are necessary for a successful
5
identification of MAP by single-round PCR.
6
However, despite their advantages, such as a hundredfold enhancement of sensitivity, nested
7
PCR assays bear a high risk of contamination and crossing over and, therefore, cannot be
8
recommended as a reliable method for routine diagnosis of MAP.
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6. Acknowledgements
11
The authors would like to thank Kerstin Steger for her excellent technical assistance. We
12
thank Ulrich Methner from the Federal Research Institute for Animal Health, Jena, Germany
13
for providing the Salmonella reference strains, Lutz Geue and Klaus Henning from the
14
Federal Research Institute for Animal Health, Wusterhausen, for the field isolates of
15
Escherichia coli and Coxiella burnetii.
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6. References
18
Bannantine, J.P., Baechler, E., Zhang, Q., Li, L., Kapur, V., 2002. Genome scale comparison
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of Mycobacterium avium subsp. paratuberculosis with Mycobacterium avium subsp.
20
avium reveals potential diagnostic sequences. J. Clin. Microbiol. 40, 1303-1310.
21
Bartos, M., Hlozek, P., Svastova, P., Dvorska, L., Bull, T., Matlova, L., Parmova, I., Kuhn, I.,
22
Stubbs, J., Morakova, M., Kintr, J., Beran, V., Melicharek, I., Ocepec, M., Pavlik, I.,
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2006. Identification of members of Mycobacterium avium species by Accu-Probes,
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serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with
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internal standards. J. Microbiol. Methods 64, 333-345.
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Bauerfeind, R., Benazzi, S., Weiss, R., Schliesser, T., Williems, H., Baljer, G., 1996.
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Molecular characterization of Mycobacterium paratuberculosis isolates from sheep,
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goats and cattle by hybridization with a DNA probe to insertion element IS900. J.
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Clin. Microbiol. 34, 1617-1621.
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Benazzi, S., Bauerfeind, R., Weiss, R., 1995. Nachweis und Differenzierung von
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mit dem DNS-Fingerprinting. Tagungsbericht des 21. Kongresses der DVG, Bad
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Nauheim, Bd.2, S.170-178. (in Deutsch)
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Bull, T.J., McMinn, E.J., Sidi-Boumedine, K., Skull, A., Durkin, D., Neil, P., Rhodes, R.,
Pickup, G., Hermon-Taylor, J., 2003: Detection and verification of Mycobacterium
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avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from
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individuals with and without Crohn's disease. J. Clin. Microbiol. 41, 2915-2923.
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Collins, D.M., Gabric, D.M., De Lisle, G.W., 1989. Identification of a repetitive DNA
sequence specific to Mycobacterium paratuberculosis. FEMS Microbiol. Lett. 51,
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175-178.
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Collins, D.M., Stephens, D.M., De Lisle, G.W., 1993. Comparison of polymerase chain
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bovine faeces. Vet. Microbiol. 36, 289-299.
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Mycobacteria distinct from Mycobacterium avium subsp. paratuberculosis isolated
20
from the faeces of ruminants possess IS900-like sequences detectable by IS900
21
polymerase chain reaction: implications for diagnosis. Mol. Cell. Probes 13, 431-442.
22
Doran, T.J., Davies, J.K.; Radford, A.J.; Hodgson, A.L., 1994. Putative functional domain
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within ORF2 on the Mycobacterium insertion sequences IS900 and IS902. Immunol.
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Ebert, M.N., Kraft, S.A., Seyboldt, C., 2004. Entwicklung einer DNA-Extraktionsmethode
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2
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3
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and nested PCR based on the IS900 gene.Vet. Microbiol. 81, 257-271.
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Englund, S., Bolske G., Johansson K.E., 2002. An IS900-like sequence found in a
Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis. FEMS
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Microbiol. Lett. 209, 167-171.
Green, E.P., Tizard, M.L., Moss, M.T., Thompson, J., Winterbourne, D.J., McFadden, J.J.,
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an
11
Hermon-Taylor, J., 1989. Sequence and characteristics of IS900, an insertion element
15
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Guerrero, C., Bernosconi, C., Burki, D., Bodmer, T., Telenti, A., 1995. A novel insertion
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relatedness. J. Clin. Microbiol. 33, 304-307.
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Ikonomopoulos, J., Gazouli, M., Pavlik, I., Bartos, M., Zacharatos, P., Xylouri, E.,
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Microbiol. Methods 56, 315-321.
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Kirschner, P., Böttger, E.C., 1998. Species identification of Mycobacteria using rDNA
25
sequencing. In: Parish, T., Stoker, N.G. (Eds.), Methods in Molecular Biology, Vol.
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Kunze, Z.M., Portaels, F., McFadden, J.J., 1992. Biologically distinct subtypes of
2
Mycobacterium avium differ in possession of insertion sequence IS901. J. Clin.
3
Microbiol. 30, 2366-2372.
4
Marsh, I.B., Whittington, R.J., 2001. Progress towards a rapid polymerase chain reaction
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Millar, D.S., Withey, S.J., Tizard, M.L.V., Ford, J.G., Hermon-Taylor, J., 1995. Solid-phase
hybridization capture of low-abundance target DNA sequences: application to the
9
polymerase chain reaction detection of Mycobacterium paratuberculosis and
11
Mycobacterium avium subsp. silvaticum. Anal. Biochem. 226, 325-330.
Millar, D., Ford, J., Sanderson, J., Withey, S., Tizard, M., Doran, T., Hermon-Taylor, J.,
an
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us
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1996. IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of
13
whole pasteurized cows' milk in England and Wales. Appl. Environ. Microbiol. 62,
14
3446-3452.
15
dM
12
Moss, M.T., Green, E.P., Tizard, M.L., Malik, Z.P., Hermon-Taylor, L., 1991. Specific
detection of Mycobacterium paratuberculosis by DNA hybridisation with a fragment
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of the insertion element IS900. Gut 32, 395-398.
Motiwala, A.S., Amonsin A., Strother, M., Manning, E.J.B., Kapur, V., Sreevatsan, S., 2004.
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Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates
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21
Poupart, P., Coene, M., Van Heuverswyn, H., Cocito, C., 1993. Preparation of a specific RNA
22
probe for detection of Mycobacterium paratuberculosis and diagnosis of Johne's
23
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Riggio, M.P., Gibson, J., Lennon, A., Wray, D., MacDonald, D.G., 1997. Search for
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Mycobacterium paratuberculosis DNA in orofacial granulomatosis and oral Crohn’s
26
disease tissue by polymerase chain reaction. Gut 41, 646-650.
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2
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paratuberculosis DNA in Crohn's disease tissue. Gut. 33, 890-896.
3
Schneider, R., 2003. Ph.D. thesis. University of Munich, Germany.
4
Stabel, J.R., 1997. An improved method for cultivation of Mycobacterium paratuberculosis
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Stabel, J.R., Bannantine, J.P., 2005. Development of a nested PCR method targeting
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9
paratuberculosis in fecal samples. J. Clin. Microbiol 43:4744-4750.
10
us
8
Stratmann, J., Strommenger, B., Stevenson, K., Gerlach, G.F., 2002. Development of a
peptide-mediated capture PCR for detection of Mycobacterium avium subsp.
12
paratuberculosis in milk. J. Clin. Microbiol. 40, 4244-4250.
Strommenger, B., Stevenson, K., Gerlach, G.F., 2001. Isolation and diagnostic potential of
dM
13
an
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ISMav2, a novel insertion sequence-like element from Mycobacterium avium
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subspecies paratuberculosis. FEMS Microbiol. Lett. 196, 31-37.
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Tasara, T., Hoetzle, L.E., Stephan, R., 2005. Development and evaluation of a
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Mycobacterium avium subspecies paratuberculosis (MAP) specific multiplex PCR
18
assay. Int. J. Food Microbiol.104, 279-287.
19
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Telenti, A., Marchesi, F., Balz, F., Bally, F., Böttger, E.C., Bodmer, T., 1993. Rapid
20
identification of mycobacteria to the species level by polymerase chain reaction and
21
restriction enzyme analysis. J. Clin. Microbiol. 31, 175-178.
22
Van Soolingen, D., Hermans, P. W. M., de Haas, P. E., Soll, D. R., van Embden, J. D. A.,
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1991. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis
24
complex strains: evaluation of an insertion sequence-dependent DNA polymorphism
25
as a tool in the epidemiology of tuberculosis. J. Clin. Microbiol. 29, 2578-2586.
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Vansnick, E., De Rijk, P., Vercammen, F., Geysen, D., Rigouts, L., Portaels, F., 2004. Newly
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Page 16 of 25
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developed primers for the detection of Mycobacterium avium subspecies
2
paratuberculosis. Vet. Microbiol. 100, 197-204.
3
Vary, P.H., Andersen, P.R., Green, E., Hermon-Taylor, J., McFadden J.J., 1990. Use of
highly specific DNA probes and the polymerase chain reaction to detect
5
Mycobacterium paratuberculosis in Johne’s disease. J. Clin. Microbiol. 28, 933-937.
pte
dM
an
us
cri
p
Winterhoff, C., 2000. Ph.D. Thesis. University of Hanover, Germany.
Ac
ce
6
t
4
17
Page 17 of 25
Figure 1. Agarose gel electrophoresis of PCR products using different mycobacterial DNA (1
2
ng) and primers for ISMav2 (Stratmann et al., 2004) in the amplification process. The lanes
3
contain 1 M. a. subsp. avium ATCC 25291; 2 M. a. subsp. silvaticum DSM 44175; 3 M.
4
intracellulare 04A3497; 4 M. smegmatis ATCC 19420; 5 M. phlei ATCC 19249; 6 M. phlei
5
FI 04A0803; 7 M. diernhoferi ATCC 19340; 8 M. fortuitum ATCC 6841; M molecular
6
weight marker
7
nonchromogenicum 04A0874; 11 M. palustre DSM 44572; 12 M. malmoense ATCC 29571;
8
13 M. a. subsp. paratuberculosis DSM 44135; 14 M. a. subsp. paratuberculosis 03A1961 15
9
negative control. The size of the specific PCR product is indicated by the arrowhead to the
ATCC 19530; 10 M.
us
cri
p
9 M. nonchromogenicum
pte
dM
an
right.
Ac
ce
10
VIII (Roche);
t
1
19
Page 18 of 25
Ac
ce
pte
dM
an
us
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p
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Figure 1
Page 19 of 25
Table 1
Table 1. Type strains and isolates used to assess the specificity of PCR
Species and subspecies
Type Host species No.
Designation
M. avium subsp. silvaticum
M. bovis
M. diernhoferi
M. fortuitum
M. intracellulare
4
FI
FI
TS
Pig
Human
Wood pigeon
1
2
1
TS
TS
TS
TS
FI
TS
TS
FI
TS
TS
TS
FI
TS
TS
FI
TS
a
Environment
Human
a
Environment
Human
Environment
Cattle
Human
Environment
Phage
Cattle
Human
a
Cattle
Mycobacteriaceae
bacterium Ellin 5409
Bacteroides fragilis
Bifidobacterium thermophilum
Bifidibacterium pseudolongum
subsp. globosum
Campylobacter jejuni
Coxiella burnetii
Escherichia coli
Lactobacillus ruminis
Mannheimia haemolytica
Pasteurella multocida
Proteus mirabilis
Salmonella Dublinb
Salmonella Typhimuriumb
Staphylococcus aureus
subsp. aureus
Streptococcus agalactiae
a
ATCC 19698, DSM 44135
03A1961, 02A1037, 02A0238
04A0386, 03A1311, 03A2230
ATCC 25291
00A0270, 03A2754, 03A2746
03A0041, 00A0730
03A3159, 03A2530, 04A0613,
04A1277
00A0854, 00A0799, 03A2893,
03A3072
02A0730
00A0520, 01A1084
DSM 44175
t
Cattle
pte
M. obuense
M. palustre
M. phlei
M. scrofulaceum
M. smegmatis
M. terrae
M. tuberculosis
FI
dM
M. malmoense
M. nonchromogenicum
2
3
3
1
3
2
4
cri
p
M. avium
subsp. hominissuis
Cattle
Cattle
Sheep
Cattle
Cattle
Pig
Poultry
us
M. avium
subsp. avium
TS
FI
FI
TS
FI
FI
FI
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
ATCC 19210
ATCC 19340
ATCC 6841
ATCC 15985
04A3497
ATCC 29571
ATCC 19530
04A0874c
03A0262
DSM 44572
ATCC 19249
04A0803c
ATCC 19981
ATCC 19420
04A3183c
ATCC 27294
an
M. avium
subsp. paratuberculosis
Ac
ce
1
FI
Cattle
1
05A0154c
TS
TS
TS
Human
Cattle
1
1
1
ATCC 25285
ATCC 25866
ATCC 25865
TS
FI
FI
TS
TS
TS
TS
TS
TS
TS
Cattle
Sheep
Cattle
Cattle
Cattle
Cattle
Cattle
1
1
1
1
1
1
1
1
1
1
ATCC 33560
14A
CW0017
ATCC 27780
DSM 10531
DSM 5281
ATCC 29906
SD81
STM421
ATCC 25178
TS
Cattle
1
DSM 6784
a
a
a
a
Page 20 of 25
Streptococcus parauberis
TS
Human
1
total
DSM 6631
56
a
without details of host species, b see Hartmann et al. (1973), c identified by sequence analysis; TS = type
3
or reference strain of species or subspecies, FI = field isolate from Germany, cultivated at the FLI, Jena,
4
ATCC = designation of type strains by the American Type Culture Collection, Rockville, USA, DSM =
5
designation of type and reference strains of the German Collection of Microorganisms and Cell Cultures,
6
Braunschweig, Germany
Ac
ce
pte
dM
an
us
cri
p
t
2
Page 21 of 25
Table 2. Primers used in the current study and detection limit of single-round, nested and semi-nested PCR
Target
Sensitivitya
Product size
Reference
Primer
Sequence (5’ – 3’)
Bauerfeind et al., 1996
MP3c)
MP4c)
CTG GCT ACC AAA CTC CCG A
GAA CTC AGC GCC CAG GAT
Doran et al., 1994
MK5
MK6
Englund et al., 1999
pg
genome numberb
314
1.0
1000
TTC TTG AAG GGT GTT CGG GGC C
GCG ATG ATC GCA GCG TCT TTG G
560
1.0
1000
S204
S749
TGA TCT GGA CAA TGA CGG TTA CGG A
CGC GGC ACG GCT CTT GTT
563
0.1
100
Schneider, 2003
Ptb1fwd
Ptb1rev
GTC GGC GTG GTC GTC TGC TGG GTT GAT
GCG CGG CAC GGC TCT TGT TGT AGT C
587
0.1
100
Vary et al., 1990
IS900/150
IS900/921
CCG CTA ATT GAG AGA TGC GAT TGG
AAT CAA CTC CAG CAG CGC GGC CTC G
229
0.1
100
ISMav2
Stratmann et al., 2002
ISMav1
ISMav2
GTA TCA GGC CGT GAT GGC GG
CCG CAC CAG CGC TCG ATA CA
312
0.1
100
F57
Vansnick et al., 2004
F57
R57
CCT GTC TAA TTC GAT CAC GGA CTA GA
TCA GCT ATT GGT GTA CCG AAT GT
432
0.1
100
Locus 255
Bannantine et al., 2002
255F
255R
CAG TCA CCC CGC GGC CGG TA
TCT ACT GAC CCG CAG ATC GAA
402
0.1
100
Benazzi et al., 1995
MP1
MP2
CGC CTT CGA CTA CAA CAA GA
GTG CGT TTT CGG TCG TAG TA
579
0.01
10
0.01
10
0.01
10
Nested IS900
MP3/ MP4c)
Bull et al., 2003
Ebert et al., 2004
314
nu
TJ1
TJ2
GCT GAT CGC CTT GCT CAT
CGG GAG TTT GGT AGC CAG TA
356
TJ3
TJ4
CAG CGG CTG CTT TAT ATT CC
GGC ACG GCT CTT GTT GTA GT
294
S204
Mp4
TGA TCT GGA CAA TGA CGG TTA CGG A
GAA CTC AGC GCC CAG GAT
890
S204/ S749
Englund et al., 1999
Ma
Single IS900
sc
(bp)
ted
PCR
Ac
ce
p
1
rip
t
Table 2
563
S204/S749
1
Page 22 of 25
F57
Vansnick et al., 2004
F57/R57
210
0.001
1
0.001
1
432
TGG TGT ACC GAA TGT TGT TGT CAC
424
sc
F57Rnd)
R57
Limit of detection; bcalculation of genome number see Schneider, 2003; c primers for single PCR and inner primers for nested PCR; dsemi-nested PCR;
bold typed primers are inner primers for nested PCR
ted
Ma
nu
a
Ac
ce
p
2
3
GCC GCG CTG CTG GAG TTG A
AGC GTC TTT GGC GTC GGT CTT G
rip
t
S 347
S 535
2
Page 23 of 25
Table 3
avium
hominissuis
Mycobacteria
Bacteria
–
–
–
–
M. fortuitum (100 ng)
M. intracellulare (100 ng)
–
–
–
–
Salmonella Typhimurium
(100 ng)
–
–
–
–
–
–
–
–
–
–
ISMav2
Stratmann et al., 2002
–
–
M. fortuitum ( 1 ng)
M. smegmatis (100 ng)
M. bacteria Ellin 5409 ( 1 ng)
Bifidobacterium thermophilum
(10 ng)
F57
Vansnick et al., 2004
–
–
–
–
Locus 255
Bannantine et al., 2002
–
–
–
–
Benazzi et al., 1995
–
–
–
–
Bull et al., 2003
–
–
–
Ebert et al., 2004
–
–
Englund et al., 1999
–
–
M. obuense (100 ng)
M. palustre (1 ng)
–
Bifidobacterium pseudolongum
subsp. globosum (100 ng)
–
Vansnick et al., 2004
–
–
M. obuense (100 ng)
nu
sc
Bauerfeind et al., 1996
Doran et al., 1994
Englund et al., 1999
Schneider, 2003
Vary et al., 1990
F57
3
other species of
IS900
Nested IS900
2
M. avium
Reference
Ma
Single
Target
ted
PCR
rip
t
Table 3. False positive products with DNA of different bacterial species (see Table 1). Detection limits are given in brackets.
Ac
ce
p
1
–
–
Page 24 of 25
Table 4. By-products of PCR assays using DNA of different bacterial species.
Mycobacteria
Bacteria
–
+c
–
+c
+++ c
–
–
+c
–
+c
+++ b
+++ c
–
–
–
+++ c
+c
–
–
–
–
+c
+c
+c
–
–
+c
+b
–
–
–
–
–
+b
–
–
–
ISMav2
Stratmann et al., 2002
+++ a
+++ b
F57
Vansnick et al., 2004
–
–
Locus 255
Bannantine et al., 2002
–
Benazzi et al., 1995
Bull et al., 2003
Ebert et al., 2004a
Englund et al., 1999
–
–
–
–
Vansnick et al., 2004
–
nu
–
+b
+c
+c
+++ c
sc
hominissuis
Bauerfeind et al., 1996
Doran et al., 1994
Englund et al., 1999
Schneider, 2003
Vary et al., 1990
F57
Other species of
avium
IS900
Nested IS900
2
3
M. avium subsp.
Reference
Ma
Single
Target
ted
PCR
(–; +; +++) indicate that PCR assay produced no by-products (–), some by-products (+), or many by-products (+++);
small letters indicate minimum DNA quantity which produces visible PCR by-products on agarose gels: a 0.01 ng, b 1 ng, c 100 ng.
Ac
ce
p
1
rip
t
Table 4
Page 25 of 25