J Med Microbiol 57 (2008), 324-331; DOI: 10.1099/jmm.0.47485-0
© 2008 Society for General Microbiology
ISSN 1473-5644
A quadruplex real-time PCR assay for the detection of Yersinia pestis and its plasmids
Alvin Stewart,
Benjamin Satterfield,
Marissa Cohen,
Kim O'Neill and
Richard Robison
851 WIDB, Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA
Correspondence
Richard Robison
richard_robison{at}byu.edu
Received 29 June 2007
Accepted 3 December 2007
Yersinia pestis, the aetiological agent of the plague, causes sporadic disease in endemic areas of the world and is classified as a National Institute of Allergy and Infectious Diseases Category A Priority Pathogen because of its potential to be used as a bioweapon. Health departments, hospitals and government agencies need the ability to rapidly identify and characterize cultured isolates of this bacterium. Assays have been developed to perform this function; however, they are limited in their ability to distinguish Y. pestis from Yersinia pseudotuberculosis. This report describes the creation of a real-time PCR assay using Taqman probes that exclusively identifies Y. pestis using a unique target sequence of the yihN gene on the chromosome. As with other Y. pestis PCR assays, three major genes located on each of the three virulence plasmids were included: lcrV on pCD1, caf1 on pMT1 and pla on pPCP1. The quadruplex assay was validated on a collection of 192 Y. pestis isolates and 52 near-neighbour isolates. It was discovered that only 72 % of natural plague isolates from the states of New Mexico and Utah harboured all three virulence plasmids. This quadruplex assay proved to be 100 % successful in differentiating Y. pestis from all near neighbours tested and was able to reveal which of the three virulence plasmids a particular isolate possessed.
Abbreviations: NCBI, National Center for Biotechnology Information.
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INTRODUCTION
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Yersinia pestis, the causative agent of the plague, is a Gram-negative, non-spore-forming bacterium belonging to the family Enterobacteriaceae. Listed as a National Institute of Allergy and Infectious Diseases Category A Priority Pathogen, Y. pestis is endemic to small rodent populations in the south-western USA, parts of South America, southern Africa and south-eastern Asia. Brubaker et al. (1965) have reported that fewer than ten bacteria are required to infect a mouse via the intravenous route. Clinical manifestations of the disease in humans include acute lymphadenopathy (bubo), septicaemia, pneumonia and ultimately death (Sebbane et al., 2006). Along with Y. pestis, the genus Yersinia includes two other human pathogens: Yersinia pseudotuberculosis and Yersinia enterocolitica (Perry & Fetherston, 1997). All three species share a common plasmid termed pCD1, which encodes a type III secretion system (Hu et al., 1998). The divergence of Y. pestis from an ancestral strain of Y. pseudotuberculosis was believed to have occurred 1500–20 000 years ago, hence the high genomic similarity found between these two species (Achtman et al., 1999; Brubaker, 2004; Hu et al., 1998; Moore & Brubaker, 1975; Parkhill et al., 2001). Two other virulence plasmids exist only in Y. pestis and are known as pMT1 and pPCP1 (Ferber & Brubaker, 1981; Hu et al., 1998; Portnoy & Falkow, 1981).
The extreme virulence of Y. pestis makes detection and identification of this organism a priority for local, state and national health departments as well as laboratories that deal with pathogenic bacteria. Methods of detecting Y. pestis that do not use PCR include standard microbiological techniques (Norkina et al., 1994), radioisotope labelling (McDonough et al., 1988) and immunofluorescent staining (Devdariani et al., 1993; Drozdov et al., 1995; Feodorova & Devdariani, 2000). Due to the length of time needed to culture the bacterium and the relative insensitivities of other assays, several PCR tests have been developed. PCR-based assays have given researchers the ability to detect very small amounts of plague DNA (McAvin et al., 2003). Early assays employed standard PCR (Engelthaler et al., 1999; Hinnebusch & Schwan, 1993; Khushiramani et al., 2006; Leal & Almeida, 1999; Norkina et al., 1994) and nested PCR (Campbell et al., 1993; Leal et al., 1996). Disadvantages of standard PCR assays included the additional time required to analyse the products by gel electrophoresis and the accompanying contamination of the laboratory environment by these products that frequently occurs. Real-time PCR has given researchers the ability to visualize an ongoing PCR by using specific fluorescing probes in a time-saving format that does not require the use of separating gels for product detection and analysis. Real-time plague detection assays using 5'-hydrolysis probes were developed by Higgins et al. (1998), Iqbal et al. (2000), McAvin et al. (2003) and Woron et al. (2006), whilst Loiez et al. (2003) used a minor-groove-binder probe.
Numerous real-time and standard PCR assays have been developed in multiplex formats that allow the detection of multiple targets within a single reaction tube (Leal & Almeida, 1999; Tomaso et al., 2003; Tsukano et al., 1996; Woron et al., 2006). However, two main problems have persisted concerning Y. pestis detection: first, detection of the Y. pestis chromosome usually involves the amplification of genomic sequences common to Y. pseudotuberculosis or Y. enterocolitica, which does not give a clear indication of which organism is present without the combined analysis of Y. pestis-specific plasmid assays (Campbell et al., 1993; Iqbal et al., 2000; Neubauer et al., 2000; Tomaso et al., 2003; Tsukano et al., 1996; Woron et al., 2006); and secondly, most published assays have been validated with a very limited number of Y. pestis isolates (
36), which may be insufficient to prove adequately the efficacy of the assays (Higgins et al., 1998; Hinnebusch & Schwan, 1993; Iqbal et al., 2000; Khushiramani et al., 2006; Leal & Almeida, 1999; Leal et al., 1996; Loiez et al., 2003; Neubauer et al., 2000; Norkina et al., 1994; Tomaso et al., 2003; Tsukano et al., 1996; Woron et al., 2006).
The objective of this study was to develop a quadruplexed real-time PCR assay that could quickly and accurately identify Y. pestis isolates and characterize their associated virulence plasmid content in a single-tube format. This assay used Taqman probes to detect a unique Y. pestis chromosomal sequence and specific virulence genes on each of the three main plasmids: pCD1, pMT1 and pPCP1.
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METHODS
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Bacterial strains and culture conditions.
The following isolates were used in this study: 180 naturally occurring Y. pestis isolates, the well-characterized Y. pestis CO92, seven known Y. pestis mutants missing the pCD1 plasmid, four known Y. pestis mutants lacking the pgm locus, 20 Y. pseudotuberculosis, 28 Y. enterocolitica, two Yersinia kristensenii, one Yersinia frederiksenii, one Yersinia intermedia and a collection of seven members of the Enterobacteriaceae family belonging to other genera. Y. pestis isolates were obtained from the New Mexico State Department of Health (NM, USA), the Utah State Department of Health (UT, USA), Dr Bob Brubaker (Michigan State University, MI, USA) and Dugway Proving Grounds (Toole, UT, USA). Y. pestis isolates were grown on Columbia agar plates at 28 °C under 5 % CO2. Y. pseudotuberculosis isolates were grown on Columbia agar plates at 33 °C under 5 % CO2. The two Y. kristensenii isolates were grown on Columbia agar plates at 23 °C. All other species were grown on Columbia agar plates at 35 °C. Gram stains were made of each isolate to confirm Gram morphology and purity. The identity of each Y. pestis isolate was also confirmed by Congo red incorporation and MIDI fatty acid methyl ester analysis. No biovar determinations were made for the natural isolates from either New Mexico or Utah. However, all three biovars were represented in the known strains.
Preparation of DNA.
Total genomic DNA was extracted from each isolate by incubating a cell suspension in Tris/EDTA buffer [10 mM Tris/HCl (pH 8.0), 1 mM EDTA] containing 1.8 µg lysozyme µl–1 for 1 h at 37 °C, followed by an automated DNA extraction performed with a MagNA Pure LC system and the MagNA Pure LC DNA Isolation kit III as recommended by the manufacturer (Roche Diagnostics). DNA concentration was measured using an ND-1000 spectrophotometer (Nanodrop Technologies). For optimization purposes, DNA stock solutions were diluted to a concentration of approximately 10 ng µl–1.
Primer and probe design.
Real-time PCR assays using 5'-hydrolysis Taqman probes were designed for the unique chromosome gene yihN and three genes on different plasmids: lcrV on pCD1, caf1 on pMT1 and pla on pPCP1 (Table 1
). The sequences of these genes were obtained from completed genomic sequences for Y. pestis CO92 (GenBank accession no. AL590842) and the plasmids pCD1, pMT1 and pPCP1 (GenBank accession nos AL117189, AL117211 and AL109969, respectively). The BLASTN program at the National Center for Biotechnology Information (NCBI) confirmed that all of the PCR target regions had 100 % similarity to two sequenced isolates of Y. pestis, KIM and Medievalis strain 91001. The targeted genes were compared using BLAST against Y. pseudotuberculosis (GenBank accession no. BX936398) and Y. enterocolitica (Sanger Institute) sequences to confirm specificity of the sequences. The primer sequences for the chromosome, pMT1 and pPCP1 targets had no significant similarities to either the Y. enterocolitica or Y. pseudotuberculosis sequences. According to the NCBI BLAST search, Y. enterocolitica and Y. pseudotuberculosis showed high similarity to the targeted sequence for pCD1. No other significant similarities were found to any other bacteria in the genus Yersinia or in the family Enterobacteriaceae. The primers and probes were designed using software available from Integrated DNA Technologies and were purchased from the same company.
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Table 1. Quadruplex primer and probe sequence data
Chrom, Chromosome; F, forward primer; R, reverse primer; Pro, probe; BHQ1, Black Hole Quencher 1; BHQ2, Black Hole Quencher 2; IabRQ, Iowa Black Quencher RQ.
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PCR cycling conditions.
Real-time PCR assays were performed on a Cepheid SmartCycler II using a SmartMix HM real-time PCR master mix (Cepheid). Primers and probes for each individual real-time assay were optimized at final concentrations of 500 and 200 nM, respectively. The sample volume was 25 µl per assay. Individual assay conditions used the following concentrations: 500 nM forward primer, 500 nM reverse primer, 200 nM probe, 50 ng target DNA and HPLC-purified H2O to 25 µl. Quadruplex reaction conditions were optimized as follows: 500 nM forward primer (chromosome, pCD1, pMT1 and pPCP1), 500 nM reverse primer (chromosome, pCD1, pMT1 and pPCP1), 400 nM probe (chromosome, pCD1 and pMT1), 200 nM probe (pPCP1), 50 ng target DNA and HPLC-purified H2O to 25 µl. Thermal cycling conditions were 2 min at 95 °C, followed by 30 cycles of 12 s at 95 °C, 25 s at 58 °C and 25 s at 72 °C. SmartCycler program conditions were the same as the program defaults. A positive signal was determined by the crossing of a fluorescence threshold of 25 before cycle 30. If an abnormal curve was produced, the background substitution was removed and the curve was analysed manually to determine whether amplification had occurred.
PCR assay conditions for validation of plasmid absence.
To confirm the results of the quadruplex assay with regard to the absence of one or more plasmids, four additional PCR assays were optimized using different plasmid target areas. Published primers were used for the pst gene on pPCP1 (Iqbal et al., 2000) and for yopT on pCD1 and ymt on pMT1 (Tomaso et al., 2003). Additionally, published primers for virF on the pCD1-like plasmid pYV1 harboured by Y. enterocolitica were used (Thoerner et al., 2003). Sequence data for all four primer sets can be found in Table 2. All four assays were optimized to be run on a SmartCycler II. The reaction mixture for pst consisted of a final concentration of 500 nM forward and reverse primers, 12.5 µl AmpliTaq Gold (Applied Biosystems), 50 ng target DNA and HPLC-purified H2O to 25 µl. Thermal cycling conditions were 7 min 30 s at 95 °C, followed by 40 cycles of 10 s at 95 °C, 20 s at 54 °C and 20 s at 72 °C. The reaction mixture for yopT consisted of a final concentration of 400 nM forward and reverse primers, 12.5 µl AmpliTaq Gold, 50 ng target DNA and HPLC-purified H2O to 25 µl. Thermal cycling conditions were 7 min 30 s at 95 °C, followed by 40 cycles of 10 s at 95 °C, 20 s at 51 °C and 20 s at 72 °C. The reaction mixture for ymt consisted of a final concentration of 400 nM forward and reverse primers, 12.5 µl AmpliTaq Gold, 50 ng target DNA and HPLC-purified H2O to 25 µl. Thermal cycling conditions were 7 min 30 s at 95 °C, followed by 40 cycles of 10 s at 95 °C, 20 s at 50 °C and 20 s at 72 °C. PCR products were run on a 1.5 % agarose gel containing ethidium bromide (0.2 µg ml–1) at 100 V for 60 min. DNA bands were visualized using a Bio-Rad FluorS imager. The reaction mixture for virF consisted of a final concentration of 500 nM forward and reverse primers, 1.25 µl 25x SYBR Green, 25 ng target DNA, one Hot Start Mix RTG bead (GE Healthcare) and HPLC-purified H2O to 25 µl. Thermal cycling conditions were 95 °C for 2 min, followed by 35 cycles of 20 s at 95 °C, 50 s at 63 °C and 50 s at 72 °C. SYBR Green fluorescence was detected using a Cepheid SmartCycler II. Positive reactions were determined by crossing a threshold level of 25.
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RESULTS AND DISCUSSION
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Chromosomal identification of Y. pestis
One of the main goals of this project was to selectively differentiate Y. pestis isolates from other bacteria, especially closely related species such as Y. pseudotuberculosis and Y. enterocolitica. This real-time PCR assay was able to identify successfully the yihN gene for all Y. pestis isolates tested (Table 3). None of the other species tested produced a positive signal. One of the largest obstacles for specifically identifying a single species of bacteria occurs when two near neighbours share significant genomic similarity, as is the case for Y. pestis and Y. pseudotuberculosis. In the past, researchers have singled out Y. pestis chromosomal sequences such as the 16S rRNA gene (Neubauer et al., 2000; Tomaso et al., 2003), the inv gene (Tsukano et al., 1996) and the entF3 gene (Woron et al., 2006). However, the main drawback to all four of these assays is the amplification of genomic DNA from Y. pseudotuberculosis. Leal & Almeida (1999) and Leal-Balbino et al. (2004) used the irp2 gene found in the pathogenicity island on Y. pestis. Unfortunately, repeated culturing of Y. pestis has been shown to promote the loss of irp2, thereby rendering this assay useless for positive identification of Y. pestis (de Almeida et al., 1993). Unlike previous standard PCR or real-time PCR assays, the assay described in this report was specific for the Y. pestis chromosome and did not amplify DNA sequences from the genomes of Y. pseudotuberculosis, Y. enterocolitica or any other member of the Enterobacteriaceae tested (Table 3). In a singleplex format, the sensitivity of the chromosome assay was 150 pg Y. pestis DNA (Fig. 1).
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Fig. 1. Sensitivities of the individual target assays. Serial tenfold dilutions of Y. pestis DNA were tested as follows for the presence of the chromosome (a) and plasmids pPCP1 (b), pCD1 (c) and pMT1 (d): 60 ng, 6 ng, 0.6 ng, 60 pg, 6 pg, 0.6 pg and 60 fg.
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Plasmid detection
The identification of the plasmid virulence genes pla and caf1 produced results similar to the chromosomal target, both showing 98 % specificity. Surprisingly, the primer and probe set for the lcrV gene that showed significant similarity in NCBI BLAST searches for all three pathogenic Yersinia species produced positive results for Y. pestis, negative results for all of the Y. pseudotuberculosis isolates and positive results for only 10 of the 28 Y. enterocolitica isolates tested. None of the three plasmid targets were detected in the DNA of any other bacterial species tested (Table 3
).
The development of real-time PCR assays for the detection and characterization of Y. pestis isolates has wide applications for both public health organizations and research groups. Not only is it possible to detect the presence of this organism quickly, but important information on its virulence capabilities can also be obtained from a single assay. Most PCR-based tests have focused on the plasmids that carry virulence genes, as plasmids are often present in multiple copies in each cell, making PCR amplification more reliable. More than ten assays have targeted the pPCP1 plasmid as part of their detection (Engelthaler et al., 1999; Higgins et al., 1998; Hinnebusch & Schwan, 1993; Iqbal et al., 2000; Khushiramani et al., 2006; Leal & Almeida, 1999; Leal et al., 1996; Leal-Balbino et al., 2004; Loiez et al., 2003; Neubauer et al., 2000; Norkina et al., 1994; Tomaso et al., 2003; Tsukano et al., 1996; Woron et al., 2006), as this plasmid carries some of the classical Y. pestis virulence genes including pesticin, coagulase and the plasminogen activator (Sodeinde & Goguen, 1988). The pPCP1 assay developed in this study detected very small amounts of pPCP1 in a sample, to a lower limit of 1.5 pg (Fig. 1).
The second most targeted virulence plasmid is pMT1, which contains the caf1 gene (encoding the fraction 1 antigen) and the gene for the murine toxin, ymt. Leal & Almeida (1999), Leal et al. (1996), Leal-Balbino et al. (2004), Norkina et al. (1994), Tomaso et al. (2003), Tsukano et al. (1996) and Woron et al. (2006) included the detection of either caf1 or ymt in their assays. The individual assay developed in this project was able to detect pMT1 plasmid at a DNA concentration of 150 pg (Fig. 1).
The pCD1 plasmid, which contains a type III secretion system, has been the least targeted of the virulence plasmids (Khushiramani et al., 2006; Leal & Almeida, 1999; Leal-Balbino et al., 2004; Tomaso et al., 2003; Tsukano et al., 1996; Woron et al., 2006), most probably because this plasmid has a homologue, pYV1, in Y. pseudotuberculosis and Y. enterocolitica (Hu et al., 1998; Thomson et al., 2006). Only about one-third of the Y. enterocolitica isolates tested produced positive results for this marker. Surprisingly, none of the Y. pseudotuberculosis isolates tested positive, even though the sequences showed 98 % similarity on BLAST searches. It is likely that the lack of pCD1/pYV1 signals for both the Y. pseudotuberculosis and Y. enterocolitica isolates was not due to the specificity of the assay, but rather the absence of the plasmid in these isolates. Li et al. (1998) have shown that plasmids in Yersinia species are commonly lost through repeated laboratory culture. All of the Y. pseudotuberculosis isolates tested were obtained from ATCC. In addition, the Y. enterocolitica isolates that tested positive were well-characterized laboratory strains that undoubtedly have been passed multiple times. The Y. pseudotuberculosis and Y. enterocolitica isolates that tested negative for pCD1/pYV1 in the quadruplex assay were retested using two assays that identify pYV1, one targeting virF (Thoerner et al., 2003) and one targeting yopT (Tomaso et al., 2003). The results obtained from these two additional assays confirmed that pCD1/pYV1 was absent in these negative isolates, supporting the theory that these isolates have lost this plasmid. Our Y. pestis pCD1 assay showed a sensitivity of 150 pg DNA when run in a singleplex format (Fig. 1).
Detection sensitivity
Serial tenfold dilutions of genomic Y. pestis DNA were used in singleplex reactions to estimate the detection limit of each assay. The chromosome assay demonstrated a detection limit of about 150 pg DNA. The sensitivities for the two plasmid assays for pCD1 and pMT1 were similar at about 150 pg DNA. However, the sensitivity of the pPCP1 assay was highest, detecting as little as 1.5 pg DNA (Fig. 1). From research conducted by Parkhill et al. (2001) on genomic analysis of Y. pestis, the pPCP1 assay has greater sensitivity as a result of the nearly 200 copies of pPCP1 typically found within each bacterium.
Quadruplex capability
Optimization of primer and probe sets allowed the four individual assays to be combined successfully into a single-tube, quadruplex reaction (Fig. 2). The sensitivities of target detection decreased to approximately 1.5 ng stock DNA when using the quadruplex format. Multiplex PCR assays for Y. pestis are not uncommon. Tomaso et al. (2003) developed various duplex assays for the chromosome and plasmids, Leal & Almeida (1999) and Tsukano et al. (1996) developed a quadruplex assay using standard PCR and Woron et al. (2006) developed a quadruplex real-time assay that included similar targets to this assay. However, the assay of Woron et al. (2006) lacked Y. pestis specificity, as both Y. pestis and Y. pseudotuberculosis are positive for both the chromosome and pCD1 targets. The assay described here confirms that a single-tube, quadruplex real-time assay can detect the chromosome and pCD1, pMT1 and pPCP1 plasmids (Fig. 2) in Y. pestis using as little as 1.5 ng target DNA. Y. pseudotuberculosis and other members of the family Enterobacteriaceae did not show amplification of any of the markers with the exception of ten Y. enterocolitica isolates that showed amplification of the pCD1 marker (Fig. 2). No other assay described previously has demonstrated this level of specificity for a chromosomal marker. Due to the reduced sensitivity inherent in multiplex reactions, this quadruplex assay is most useful in the confirmation and characterization of culture isolates of Y. pestis. The use of this assay on clinical specimens containing potential PCR inhibitors has not been tested.
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Fig. 2. Individual assays were combined to form a quadruplex assay. Negative results are shown as lines falling below the threshold of 25. A Y. pestis positive control is shown as grey in the graphs. Detection of FAM (a), Cy3 (b), TexR (c) and Cy5 (d) was combined to yield the combined quadruplex assay (e) for Y. pestis using the isolate India against nine near neighbours (five Y. enterocolitica, three Y. pseudotuberculosis and one Y. kristensenii) and a negative control.
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Characterization of plasmid content in natural isolates from New Mexico and Utah
This quadruplex real-time PCR assay was used to evaluate a large collection of Y. pestis isolates from New Mexico and Utah to determine their plasmid content. Approximately 25 % of the isolates lacked the pCD1 plasmid (Table 3). The other two plasmids appeared to be much more stable, with only about 2 % of the isolates lacking pMT1 and only 2 % lacking pPCP1. These data showed that only 72 % of the natural isolates demonstrated a classical three-plasmid signature. This is similar to the results of Filippov et al. (1990), who reported that 71 % of 242 Y. pestis isolates showed the presence of all three plasmids (pCD1, pMT1 and pPCP1). These data confirm the inherent instability of the pCD1 plasmid in Y. pestis. Various hosts and growth temperatures, as well as unknown factors, may contribute to the loss of plasmids in Y. pestis (Iqbal et al., 2000).
Confirmation of plasmid content
As with all PCR-based assays, mutations in the target sequences can result in amplification failures, even though the specific genes may be present. To confirm that negative results from the quadruplex assay were in fact due to plasmid loss and not mutation, alternative targets were employed using four previously published assays. These assays were performed to ascertain the absence of the plasmids pCD1, pMT1 and pPCP1 in isolates that tested negative with the quadruplex assay. Two primer sets (yopT on pCD1 and ymt on pMT1) from the work of Tomaso et al. (2003) and one primer set (pst on pPCP1) from Iqbal et al. (2000) were used for the Y. pestis isolates that tested negative. In addition, one primer set for virF (Thoerner et al., 2003) on the pCD1-like plasmid pYV1 harboured by Y. enterocolitica was used on all Y. enterocolitica isolates. Results of these assays verified the absence of the plasmids in the isolates that tested negative in the quadruplex assay. All single PCR tests performed gave identical results to those obtained from the quadruplex assay (Fig. 3).
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Fig. 3. Verification of the absence of plasmids. PCR assays for yopT on pCD1 (a), ymt on pMT1 (b) and pst on pPCP1 (c) were used to verify the absence of plasmids in isolates whose plasmid composition was known: Y. pestis 9900200 (pCD1– pMT1+ pPCP1+), Y. pestis Nairobi (pCD1– pMT1– pPCP1+), Y. pestis 1901b (pCD1+ pMT1+ pPCP1–), Y. pestis 1584b (pCD1+ pMT1– pPCP1+) and Y. pestis 1998a (pCD1+ pMT1+ pPCP1+). The 100 bp ladder was from Fermentas.
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Conclusions
This study describes the first quadruplexed real-time PCR assay that solely identifies Y. pestis based on a novel chromosomal sequence encoding the yihN gene and specifically identifies the plasmid content of tested isolates. This rapid, single-tube assay selectively identified Y. pestis and its three main virulence plasmids (pCD1, pMT1 and pPCP1) with no amplification observed for Y. pseudotuberculosis, its closest near neighbour. This assay was validated using 181 natural isolates and seven known plasmid-deletion mutants of Y. pestis to confirm its functionality. Natural Y. pestis isolates can sometimes drop one or more plasmids, with pCD1 being the one most commonly eliminated. Only 72 % of all natural isolates evaluated in this study were found to harbour all three virulence plasmids.
The specificity and ease of use of this assay make it a useful tool for examining the external factors that lead to plasmid loss in Y. pestis. As 45 of the tested isolates lacked pCD1, three lacked pPCP1, two lacked pMT1 and one lacked both pCD1 and pMT1, it is clear that the organism can persist without the full complement of virulence plasmids. Growth in various organisms such as fleas, rodents and other presently unknown hosts may provide different evolutionary pressures with respect to maintaining all three plasmids. Furthermore, external environmental conditions such as weather patterns may also prove to be a factor in these pressures (Parmenter et al., 1999). The assay described in this report should greatly facilitate these types of studies.
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ACKNOWLEDGEMENTS
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We express appreciation to the New Mexico and Utah State Departments of Health for supplying the majority of the Y. pestis isolates evaluated in this study, Dr Bob Brubaker at Michigan State University for supplying the Y. pestis plasmid mutants, Joe Hinnebusch at Rocky Mountain Laboratories (NIH) and Virginia Miller at Washington University School of Medicine for kindly providing several Y. enterocolitica isolates, and Dugway Proving Grounds for providing additional Y. pestis isolates. We express gratitude to Janet Fowler for her help and information relative to Yersinia plasmids and plasmid mutant information. This work was supported by grants from the National Bioforensics Activity Center of the Department of Homeland Security and the BYU mentoring environment programme.
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INTRODUCTION
METHODS
