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Yersinia pestis, the etiological agent of pneumonic and bubonic plague, is a member of the Enterobacteriaceae family. Historically, it has been responsible for approximately 200 million deaths, including 3 pandemics.1 Y. pestis is generally an enzoonotic infection that circulates between more than 200 susceptible rodent species and their associated fleas.2 Humans can acquire plague through the bite of an infectious flea, contact with contaminated tissue, or direct inhalation of the bacterium via respiratory droplets.3
Currently, detection and presumptive identification of Y. pestis in flea vectors and animals is determined by direct fluorescent antibody assay or PCR. Confirmation of Y. pestis requires isolation of the bacterium either from direct plating of infected flea triturates or animal tissues or by inoculating laboratory mice with suspect infectious material and subsequent isolation of Y. pestis.4,5 Methods for quantification of the bacterium include performing 10-fold serial dilution plating followed by a 48- to 72-hr incubation period or a quantitative competitive standard PCR assay.6,7 Both methods are more time-consuming than real-time PCR, and plating methods require laboratory technicians to work with infectious material for extended periods of time.
Advances in molecular assays such as the polymerase chain reaction (PCR) have become important tools for more rapid and accurate detection of bacterial species. Numerous PCR assays have been developed for detecting Y. pestis including nested PCR, multiplex-PCR, real-time PCR, and a quantitative competitive PCR assay.8–19 Here we report a method of quantitative PCR utilizing a FAM-labeled TaqMan DNA oligonucleotide probe and unlabeled primers for amplifying a region of the chromosomal ferric uptake regulator (fur) gene for enumerating Y. pestis bacteria. Employment of this single-copy chromosomal target ensures detection of all Y. pestis strains regardless of their plasmid content. This 5'-nuclease assay can be performed on various thermocycler platforms and enables simultaneous large-scale screening utilizing 96 or 384 sample formats. A plasmid harboring the target region was constructed to quantify fur copy number, and the assay was validated using Y. pestis culture and laboratory-infected Oropsylla montana flea samples.
Y. pestis reference strains used in this study were UG0453-05 (biovar antiqua), KIM6 (biovar medievalis), CO96-3188 (biovar orientalis), and A1122 (biovar orientalis). Bacteria were inoculated from frozen stocks onto Congo red agar and incubated at 26°C for 48–72 hr. Colonies were then inoculated into heart infusion broth (HIB) and incubated at 28°C, with shaking at 160 rpm overnight for 12–14 hr. Overnight cultures were then diluted in fresh HIB and incubated at 28°C with shaking at 160 rpm until a final optical density reading of 1.0 (approximately 6 x 108 cfu/mL) was achieved, as determined by spectrophotometer at 620 nm (OD620).
The Y. pestis published fur gene sequence was used with Beacon Designer 4.0 software (Biosoft International, Palo Alto, CA) to identify sequences within this gene that were suitable for PCR primers and a FAM-labeled fluorescent TaqMan probe.20 The optimal forward primer, YpfurF (5'-TCT GGA AGT GTT GCA AAA TCC TG-3'), reverse primer, YpfurR (5'-AAG CCA ATC TCT TCA CCA ATA TCG -3'), and probe, YpfurP (5'-FAM-TGT CAC CAC GTC AGC GCG GAA GAT-BHQ1-3'), corresponding to nucleotides 66–88, 132–155, and 91–114, respectively, were selected from the fur coding region. Real-time PCR reactions contained the TaqMan Universal PCR Master Mix (Applied Bio-systems, Foster City, CA), YpfurF and YpfurR primers (final concentration 800 nM), YpfurP fluorescent probe (final concentration 200 nM), and 5 µL of DNA template. The samples were placed in a 96-well microtiter plate in a final volume of 50 µL and run under the following cycling conditions: 95°C for 10 minutes, 45 cycles of 95°C for 30 seconds, followed by 60°C for 1 minute. Fluorescent amplicons were then detected using a Stratagene Mx3005 qPCR thermocycler (Agilent Technologies, La Jolla, CA).
A plasmid standard curve was constructed by ligating the PCR product from Y. pestis, UG-0454, into the commercial plasmid vector, 2.1-TOPO plasmid vector (Invitrogen, Carlsbad, CA) following the manufacturer’s recommendations to produce plasmid ypUGfur. This plasmid was transformed into competent TOP10 Escherichia coli cells (Invitrogen), and transformants were selected on media containing 50 µg/mL kanamycin. Plasmid DNA was extracted and purified from 1 transformant using a Qiaprep Spin Miniprep Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, resuspended in elution buffer (Qiagen) and sequenced to determine the presence of the fur insert and its orientation. Plasmid DNA concentration was determined spectrophotometrically, and samples with an OD260 > 0.05 were used as plasmid DNA reference material. Reference plasmid DNA was then serially diluted to obtain 101 to 106 plasmid genome equivalents for standard curve analysis. Standard curves consistently demonstrated correlation coefficients (R2) of 0.94–0.99 and PCR efficiencies ranging from 95% to 100% when analyzed by MxPro aPCR Software, version 3.0 (Stratagene).
The reproducibility of the assay was determined by comparing colony counts from standard serial dilution plating results to qPCR results using the same culture (Figure 1
). Because plating enables only enumeration of viable bacteria, whereas qPCR detects all genome equivalents, a culture with the fewest number of dead bacteria present was needed. Therefore, viable bacterial samples were generated by harvesting Y. pestis UG005-0454 cells during exponential growth. The correlation between genome equivalents detected using qPCR and counts of colony forming units (cfu) obtained from 40 different aliquots of this culture were similar [qPCR = 1.1 (colony count) – (4.8 x 104); F = 4829, df = 1.0, P < 0.0001; Figure 1]. The 1.1 slope value of the linear regression indicates a near 1:1 relationship between the 2 methods.
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]. However, as noted in Figure 2, some deviation from this linear relationship was observed as colony counts decreased.
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). The specificity of primers YpfurF and YpfurR and probe YpfurP was determined using genomic DNA templates from 4 Y. pestis strains (UG05-0454, KIM6, CO96-3188, and A1122) representing 3 biovars (orientalis, medievalis, anti-gua) of this bacterium. The appropriate amplicon was generated in all 4 Y. pestis strains. and amplicon size was confirmed by agarose gel electrophoresis (data not shown). The capability of the assay to distinguish between Y. pestis and other Yersina spp., Bartonella spp., Rickettsia spp., and E. coli was then determined by PCR using DNA reference material from 28 species (data not shown). The primer/probe pair set showed cross-reactivity with 6 of 13 species of Yersinia tested, including Yersina rohdei, Yersina bercovieri, Yersina aldovae, Yersina kristensenii, Yersina frederiksenii, and Yersina entercolitica, organisms that have not been shown to be flea-borne pathogens.22 Of interest, cross-reactivity was not observed with Y. pseudotuberculosis or any of the non-Yersinia bacteria tested (data not shown). Although our quantitative assay is intended primarily for experimental studies and not diagnostic purposes, a 2-tiered assay could be established for field specimens using primers that are highly sensitive and Y. pestis-specific23,24 to first establish Y. pestis-positive samples, followed by the qPCR assay to rapidly quantify Y. pestis in collected samples.
In this report, we have described a real-time quantitative PCR assay for Y. pestis utilizing a TaqMan DNA oligonucleotide probe and a DNA plasmid standard curve. The chromosomal Y. pestis fur gene is used in this assay to quantitate genome equivalents and, by extension, the number of bacteria present in a sample by comparing the real-time cycle threshold (Ct) values of known recombinant plasmid ypUGfur DNA concentrations to that of unknown samples. For experimental studies, this assay enables more rapid enumeration of Y. pestis bacteria compared with conventional serial dilution plating and does not carry the risk of prolonged human exposure to infectious materials. Further studies are needed to determine the utility of this assay for determination of Y. pestis bacterial loads in field-collected mammalian and flea-derived tissues.
Received January 9, 2008. Accepted for publication March 25, 2008.
* Address correspondence to Nordin S. Zeidner, Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, 3150 Rampart Road, Fort Collins, CO 80521. E-mail: Naz2{at}cdc.gov 
Authors’ addresses: Elizabeth S. Gabitzsch, Rommelle Vera-Tudela, Rebecca J. Eisen, Scott W. Bearden, Kenneth L. Gage, and Nordin S. Zeidner, Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, CO, Tel: +1 (970) 221-6495, Fax: +1 (970) 225-4257, E-mail: Naz2{at}cdc.gov.
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  B. M. Carpenter, J. M. Whitmire, and D. S. Merrell This Is Not Your Mother's Repressor: the Complex Role of Fur in Pathogenesis Infect. Immun., July 1, 2009; 77(7): 2590 - 2601. [Full Text] [PDF]
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