Development of a Real-time Quantitative PCR Assay to Enumerate ...

Am. J. Trop. Med. Hyg., 79(1), 2008, pp. 99–101 Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

Short Report: Development of a Real-time Quantitative PCR Assay to Enumerate Yersinia pestis in Fleas 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, Colorado

Abstract. A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis. The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene (fur) to design primers and a fluorescent (FAM-labeled) TaqMan probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating. Y. pestis reference strains used in this study were UG045305 (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 × 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 Biosystems, Foster City, CA), YpfurF and YpfurR primers (final concentration 800 nM), YpfurP fluorescent probe (final concentration 200 nM), and 5 ␮L of DNA template. The samples were placed in a 96-well microtiter plate in a final volume of 50 ␮L 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 ␮g/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

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 singlecopy 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.

* 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: [email protected]

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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 × 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. To evaluate the sensitivity of the qPCR assay when analyzing infected fleas, laboratory-infected O. montana flea triturate samples, for which plated colony counts had been previously determined, were analyzed. Briefly, fleas were orally infected, triturated in 100 ␮L of HIB plus 10% glycerol, serially diluted in PBS, and plated in duplicate on sheep blood agar plates. Cultures were incubated at 25–28°C for 48 hr, and colony counts were determined as described previously.21 DNA was extracted from 25 ␮L of the triturate using a QIagen DNeasy tissue kit and resuspended in elution buffer (Qiagen) prior to quantification by qPCR. The number of viable bacteria in 38 flea triturate samples found by conventional plating and the number of genomic equivalents in those same samples as determined by qPCR were highly correlated [qPCR ⳱ 1.1 (colony count) – (5.1 × 105); F ⳱ 36.85, df ⳱ 1.0, P < 0.0001; Figure 2]. However, as noted in Figure 2, some deviation from this linear relationship was observed as colony counts decreased. Sensitivity of the qPCR assay, using serially diluted plasmid DNA and plate-enumerated genomic DNA, reproducibly demonstrated fluorescence levels above background for 1 and 5 copies, respectively. Moreover, detection of as few as 5 cfu from cultured organisms was routinely observed (data not

FIGURE 1. Scatter plot of the relationship between colony count and qPCR results of cultured Y. pestis.

FIGURE 2. Scatter plot of the relationship between colony count and qPCR results of Oropsylla montana fleas artificially infected with Y. pestis.

shown). Assay sensitivity was also checked using DNA extracted from plate-quantified infected flea triturate, which consistently detected as few as 300 cfu. This decrease in sensitivity in flea samples could be attributed to PCR inhibitors in the samples. Studies by Engelthaler and Gage demonstrated that fleas collected from burrows carried a mean bacterial load of 105.6 Y. pestis per flea (range ⳱ 103.3 to 106.9) while those collected from rodent carcasses harbored a mean bacterial load of 104.8 (range ⳱ 102 to 106.7) per flea.19 Although additional field testing is needed, it appears that these bacterial loads are well within the ability of this assay to detect and quantify Y. pestis bacilli in wild-caught fleas (Figure 2). 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, antigua) 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

REAL-TIME QUANTITATIVE PCR FOR Y. PESTIS IN FLEAS

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 fleaderived tissues.

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14. Received January 9, 2008. Accepted for publication March 25, 2008. 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: [email protected].

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