Real-time PCR assays targeting a unique chromosomal sequence of Yersinia pestis.
Standard culture and biochemical identification of the plague bacilli may be delayed because the organisms are relatively slow growers (48 h). Newer, rapid fluorescence-based immunosensors and immunostaining methods can detect the Y. pestis capsular antigen designated fraction 1 (F1), which forms during growth at temperatures above 33[degrees]C (3,4). A striking characteristic of these 2 Yersinia species is their sequence identity similarity, which is intriguing given their markedly different epidemiologic and clinical features. In this regard, it should be noted that Y. pestis has been proposed to be a recently emerged clone of Y. pseudotuberculosis (5). Within the Yersinia spp. makeup, the only significant differences appear in the plasmids, which are referred to by several alternative names in the literature (6). Three characteristic virulence plasmids are pCD1, which is found in both Y. pseudotuberculosis and Y. pestis, and pPCP1 and pMT1, which are unique to Y. pestis. Y. pestis strains isolated from plague patients usually contain all 3 virulence plasmids, but these may be lacking in atypical strains; therefore, molecular detection strategies usually include targets on each plasmid. Recent standard PCR methods (7-11) and real-time PCR assays (12-15) have primarily used specific virulence gene targets encoded on these 3 plasmids. However, in the opinion of Chain et al. (16), "the presence of these plasmids by themselves cannot account for the remarkable increase in virulence observed in Y. pestis". The 16S rRNA genes are not useful for the specific detection of Y. pestis because identical sequences are found in Y. pseudotuberculosis; however, the 1-nt difference in the 23S rRNA gene has been exploited for molecular detection (17). In our hands, the performance of assays based on a single-nucleotide difference has been remarkably dependent on assay chemistry, cycling conditions, and the real-time PCR instrument used. Small alterations can lead to the loss of sensitivity and/or specificity, particularly with TagMan[R]-based probes.
Although Leal and Almeida (7) did include a chromosomal target, irp2, in their standard PCR assay, a real-time differential assay based on a stable genomic target would be extremely desirable. Thus, a real-time PCR assay based on a unique chromosomal nucleotide sequence found in either Y. pestis or Y. pseudotuberculosis is needed to complement other available assays for the identification of each organism. Because virulence plasmids can potentially be transmitted between bacterial species, a chromosomal marker to confirm the identity of Y. pestis as the organism harboring the plasmids is important in a time of terrorist threats and bioengineering. The rapid rate of transmission and high mortality of Y. pestis infection make this organism a bioweapon candidate. If an aerosolized form were released, treatment would need to be administered within 24 h to prevent fatalities (18), making it necessary to verify the presence of the pathogen quickly and to easily distinguish it from Y. pseudotuberculosis. With the recent sequencing of 3 strains of Y. pestis (19-21) and the completion of the sequence for Y. pseudotuberculosis IP 32953 (16), we were able to pinpoint the exact location of a 25-bp sequence that exists in the Y. pseudotuberculosis yp48 gene but is deleted in the Y. pestis yp48 gene originally identified by Buchrieser et al. (22). The Y. pestis gene is similar to the Escherichia coli gene for MalK, which is an ATP-binding component of the maltose transport system. The deletion/ insertion site in the yp48 gene allowed us to design and test a chromosome-based real-time PCR assay for the differential identification of these 2 closely related organisms.
Materials and Methods
Genomic DNA samples from Y. pestis C092 and Y. pseudotuberculosis 6904 were amplified with the yp48-specific primers yp48-F751 (5'-GGC GAA TTG GTA GCA GGA AA-3') and yp48-R977 (5'-CCG TAC CAA CAT CGG ATC AAA-3'). The resulting yp48 PCR gene fragments were sequenced on an ABI Prism[TM] 3100 genetic analyzer [Applied Biosystems (ABI)]. The amplified DNA was used as template in BigDye[TM] Terminator Cycle Ready Reactions (ABI) according to the manufacturer's instructions. All sequence data were assembled and analyzed with DNA Star SegMan II software (DNASTAR Inc.).
PCR PRIMERS, TARGET SEQUENCES, AND FLUOROGENIC PROBES
yp48 assay. The real-time PCR assay primers and TagMan minor groove binder (MGB) probe (ABI), Simple-Probe[R] (Roche Applied Science), and MGB Eclipse probe (Epoch Biosciences) sequences for the yp48 target gene are listed in Table 1. The 25-bp insertion/ deletion between the Y. pestis and Y. pseudotuberculosis yp48 gene (GenBank accession nos. AL031866, NC 004088, NC 003143, NC_005810, and NC 006155) was used as a target sequence for the specific assays. The primer and TagMan MGB probe sequences were designed by use of Primer Express (Ver. 2.0) for Windows (ABI) with the Simple-Probes designed by use of the LightCycler Probe Design Software 2.0 (Ver. 1.0.R.36; Roche). All primers were synthesized by Invitrogen, using standard phosphoramidite chemistry followed by column desalting. The TagMan MGB probes were synthesized by PE Biosystems and contained 6-carboxyfluorescein (FAM) at the 5' end. A nonfluorescent quencher (NFQ) and the MGB were added to the 3' end. The Simple-Probes were synthesized by Roche Applied Science and contained a fluorescenn and the Simple-Probe chemistry (SPC) at the appropriate 5' or 3' end and a phosphate block at the end opposite the SPC-fluorescein label. The MGB Eclipse probes were synthesized by Epoch Bioscience. Their sequences were identical to those of the TagMan MGB probes, but the FAM was put at the 3' end and the MGB Eclipse Dark Quencher (MGBEDQ) on the 5' end. The melting temperature ([T.sub.m]) for the Simple-Probe was determined both through the software (theoretical) and empirically with the appropriate control DNA on the LightCycler, but the [T.sub.m] of the MGB Eclipse probe was determined only empirically.
5'-Nuclease PCR (TagMan MGB) assays. After using the Primer Express 2.0 software to design potential Y. pestis/ Y. pseudotuberculosis yp48-specific TagMan MGB assays, we optimized the assays according to a standard protocol instituted by the Diagnostic Systems Division at the US Army Medical Research Institute of Infectious Diseases (USAMRIID). Potential primer pairs were initially tested in the LightCycler with the fluorescent dye SYBR Green I (Roche Biochemicals). The optimum primer pair was selected on the basis of specificity (a single, appropriately sized amplicon) and efficiency of amplification [lowest threshold crossing point (Ct) value, defined as the realtime PCR cycle at which the LightCycler software determines the reaction to be positive]. The selected primer pair was then optimized by use of symmetric PCR primer concentrations (0.1-1.0 [micro]M) with the final concentration determined by lowest [C.sub.t] value and highest fluorescent signal. We then tested several potential TagMan MGB probes with the optimized primer pair by varying the probe and Mg[Cl.sub.2], concentrations. The final assay consisted of the primer/probe pair concentrations and reaction conditions that combined the lowest limit of detection (LOD), defined as the gene copy number that was detected by the assay at least 58/60 times; the lowest [C.sub.t] value; and the highest fluorescent signal-to-noise ratio. The LODs of the assays were determined from serial dilutions of genomic DNA purified from Y. pestis CO92 and Y. pseudotuberculosis 6904.
All TagMan MGB assays were carried out in 20-[micro]L volumes for the LightCycler 2.0, with each reaction made up in PCR buffer [50 mM Tris (pH 8.3), 25 ng/[micro]L bovine serum albumin, and 0.2 mM deoxynucleotide triphosphate mixture (Idaho Technology)]. Platinum Taq DNA polymerase (0.8 U; Invitrogen) was added to each reaction. The final Mg[Cl.sub.2], primer, and probe concentrations for each assay are listed in Table 1. Thermal cycling for the LightCycler was performed as follows: 1 cycle at 95[degrees]C for 2 min, followed by 45 cycles of 95[degrees]C for 1 s and 60[degrees]C for 20 s. A fluorescence reading was taken at the end of each 60[degrees]C step. Each reaction capillary tube was read in the 530 channel, and data were analyzed by the LightCycler Data Analysis software (Ver. 4.0): Amplification Analysis-Qualitative Analysis and/or Absolute Quantification depending on the nature of the data.
Simple-Probes. After using the LightCycler Probe Design Software 2.0 to design potential Y. pestis/Y. pseudotuberculosis yp48-specific Simple-Probe assays, we optimized the assays according to a Roche Application Report (23). The same primer pairs selected for the TagMan MGB assays (Table 1) were tested with potential Simple-Probes as follows: asymmetric PCR was performed with a final concentration of 0.1 [micro]M for the forward primer and 0.5 [micro]M for the reverse primer. The Simple-Probes were used at a final concentration of 0.1 [micro]M. The concentration of M[gCl.sub.2] was 3 mM. All Simple-Probe assays were carried out in 20-[micro]L volumes for the LightCycler 2.0, with each reaction made up in PCR buffer [50 mM Tris (pH 8.3), 25 ng/[micro]L bovine serum albumin, and 0.2 mM deoxynucleotide triphosphate mixture; Idaho Technology]. K1enTaq LA Polymerase Mix (0.8 U; BD Biosciences) was inactivated with an equal volume of TagStart Ab (BD Biosciences) and then added to each reaction. Thermal cycling for the LightCycler was performed as follows: 1 cycle at 95[degrees]C for 2 min, followed by 50 cycles of 95[degrees]C for 1 s, 55[degrees]C for 10 s, and 72[degrees]C for 10 s. The temperature ramp rate during these 2 steps was 20[degrees]C/s. A single fluorescence reading was taken at the end of each 55[degrees]C step. At the end of the 50 amplification cycles, a melting curve was generated as follows: 95[degrees]C for 1 s and 45[degrees]C for 20 s followed by an increase to 90[degrees]C at a ramp rate of 0.1[degrees]C/s. A continuous read was made in the 530 channel during the entire 0.1[degrees]C/s ramping, and data were analyzed by the LightCycler Data Analysis software (Ver. 4.0) Amplification Analysis: Qualitative Analysis and Melting Curve Analysis-Tm Calling.
MGB Eclipse probes. The yp48 MGB Eclipse probes were identical in sequence to the TagMan MGB probes except that the FAM dye was placed at the 3' end, whereas the MGBEDQ was placed at the 5' end. The MGB Eclipse probe assay primer and probe information are listed in Table 1. Cycling conditions and melting curve analysis were identical to the Simple-Probe assay conditions except that the final MgCl, concentration was 5 mM (exactly as for the TagMan MGB assays).
EXTENDED ASSAY EVALUATION
Inclusivity and exclusivity panel testing. All 8 assays were evaluated extensively, first against various strains of Y. pestis and Y. pseudotuberculosis genomic DNAs (see Table 1 of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vo151/issue10/). The panel consisted of DNAs from 16 Y. pestis strains, 7 Y. pseudotuberculosis strains, and 6 additional Yersinia spp. strains (10-100 pg/[micro]L) and human genomic DNA (see Table 1 of the online Data Supplement). All assays were also tested against various bacterial isolates and strains from the USAMRIID bacterial DNA cross-reactivity panel (70 DNAs at 100 pg each; see Table 1 of the online Data Supplement). Briefly, 5 [micro]L of each sample was added to the appropriate assay LightCycler Master Mix (15 [micro]L) and cycled as described above. All test runs included at least 1 positive control that contained 1.1 x [10.sup.3] copies (5 pg total) of purified Y. pestis C092 and Y. pseudotuberculosis 6904 DNAs and 2 no-template controls (NTCs), a reagent NTC and a sample NTC.
Calibration curve determination for the TagMan MGB assays. Y. pseudotuberculosis 6904 and Y. pestis CO92 genomic DNA samples were diluted and tested in triplicate in the LightCycler with the corresponding TagMan MGB assay at the following final concentrations in 20 [micro]L of Master Mix: 2.0: 4.8 ng (1000 000 gene copies), 2.4 ng (500 000 gene copies), 240 pg (50 000 gene copies), 24 pg (5000 gene copies), 2.4 pg (500 gene copies), 240 fg (50 gene copies), and 24 fg (5 gene copies). Calibration curves and the efficiency of the real-time PCR reactions were determined by the Absolute Quantification module of the LC 4.0 software.
LOCATION AND SEQUENCE OF THE yp48 GENE 25-BP INSERTION/ DELETION
The Yersinia spp. yp48 gene was chosen as our chromosomal target because of the discovery of a 25-bp deletion in Y. pestis 6/69M that makes it distinguishable from Y. pseudotuberculosis IP32637 (22). However, we first had to design yp48-specific sequencing primers to determine the exact position of the 25-bp insertion/ deletion because we could not locate it at the previously annotated position (22). Our yp48 sequencing results confirmed the position of the 25-bp insertion as being identical to that found in the recently published complete Y. pseudotuberculosis genomic sequence (16).
DEVELOPMENT OF REAL-TIME PCR ASSAYS
The final primer/TagMan MGB probe, Simple-Probe, and MGB Eclipse probe sequences and reaction conditions for each assay are shown in Table 1. Fig. 1 shows the location of the yp48 forward and reverse primers and outlines the probe region for the TagMan MGB, Simple-Probe, and MGB Eclipse assays. Also indicated are the position and sequence of the yp48 25-bp insertion/ deletion (bp 826-850). During the testing phase of the TagMan MGB assays, the Y. pseudotuberculosis-specific assay detected only the Y. pseudotuberculosis DNA (see Table 1 and Fig. 1A of the online Data Supplement), and the Y. pestis-specific assay always produced some small amount of cross-reactivity to Y. pseudotuberculosis DNA, indicated by a different detection curve (see Fig. 1B of the online Data Supplement). We tested 10 different TagMan MGB probes but were never able to eliminate the slight cross-reactivity of the Y. pestis-specific assay with Y. pseudotuberculosis DNA (data not shown). Importantly, the Y. pseudotuberculosis DNA curves (Fig. 1B of the online Data Supplement) were designated as positive by the LC 4.0 software Qualitative Detection module, and the small curves of Y. pestis DNA (Fig. 1A of the online Data Supplement) were designated as negative by the same module. The TagMan MGB assays also reproducibly detected 100 fg of the appropriate Yersinia spp. genomic DNA, which represented ~21 copies of each genome.
Because the TagMan MGB probe is destroyed during the reaction, we leveraged 2 alternative melting-probe technologies (Roche Simple-Probes and Epoch MGB Eclipse probes) to design assays that would clearly distinguish Y. pestis DNA from Y. pseudotuberculosis DNA. Using the same primers as in the TagMan MGB assay, we first designed Simple-Probes for both assays. The Y. pseudotuberculosis-specific Simple-Probe (yp48-SP828-53) detected all tested strains of Y. pseudotuberculosis DNA [[T.sub.m] = 67.84 (0.06)[degrees]C; Table 1 of the online Data Supplement], and the Y. pestis probe (yp48-SP812-05) detected all tested strains of both Y. pestis and Y. pseudotuberculosis DNA (Table 1 of the online Data Supplement). However, the [T.sub.m]s for the yp48-SP812-05 probe were significantly different for the 2 DNAs: 69.29 (0.03)[degrees]C for Y. pestis DNA compared with 62.35 (0.11)[degrees]C for the Y. pseudotuberculosis DNA ([DELTA]6.94[degrees]C; strains 6904 and CO92 in Fig. 2A). The Simple-Probe assays also reproducibly detected 100 fg of genomic DNA, which represented ~21 copies of Yersinia spp. yp48 gene (data not shown).
With the same primers as in the TagMan MGB assay, we designed MGB Eclipse probes for both assays. The Y. pseudotuberculosis-specific MGB Eclipse probe (yp48-p834S-ECL) detected all tested strains of Y. pseudotuberculosis DNA [[T.sub.m] = 58.73 (0.28)[degrees]C], and the Y. pestis probe (yp48-p815S-ECL) detected all tested strains of both Y. pestis and Y. pseudotuberculosis DNA (see Table 1 of the online Data Supplement). However, the [T.sub.m]s for the yp48-p815S-ECL probe were significantly different for the 2 DNAs: 70.49 (0.14)[degrees]C for Y. pestis DNA compared with 66.13 (0.17)[degrees]C for the Y. pseudotuberculosis DNA ([DELTA]4.36[degrees]C; strains 6904 and CO92 in Fig. 2B). The MGB Eclipse probe assay also reproducibly detected 100 fg of genomic DNA, which represented -21 copies of Yersinia spp. yp48 gene (data not shown).
USAMRIID DNA PANEL EVALUATIONS
Each assay was also tested against 2 USAMRIID DNA panels (see Table 1 of the online Data Supplement), a Yersinia spp. specialty panel and a DNA cross-reactivity panel. The results indicated that the Y. pseudotuberculosis-specific TagMan MGB assay detected only the genomic DNA from the Y. pseudotuberculosis strains in both panels and that the Y. pestis-specific assays detected Y. pestis DNA along with an unacceptable amount of Y. pseudotuberculosis DNA. The Y. pseudotuberculosis-specific Simple-Probe and MGB Eclipse probe assays detected only Y. pseudotuberculosis DNA. However, the Y. pestis-specific Simple-Probe and MGB Eclipse probe assays detected both Y. pestis and Y. pseudotuberculosis genomic DNA, but each was easily differentiated based on the [T.sub.m] profiles of the respective probes. In addition, as shown in Table 1 of the online Data Supplement, none of the 6 assays detected any other Yersinia DNA strains or non-Yersinia spp. genomic DNAs (to include human genomic DNA) in the USAMRIID cross-reactivity panel (70 DNAs in total). The obvious distinction between the 2 assays was that the difference in the [T.sub.m] for the Simple-Probes was almost 7[degrees]C and that for the MGB Eclipse probes was slightly more than 4[degrees]C; both were easily distinguishable by the LightCycler Melting Curve Analysis: Genotyping module of the LightCycler software 4.0.
CALIBRATION CURVE FOR THE TagMan MGB ASSAYS
The yp48 chromosomal TagMan MGB assays produced positive results with as few as 5 gene copies (3 of 3 for the Y. pseudotuberculosis-specific assay and 2 of 3 for the Y. pestis-specific assay; see Fig. 2, A and B, of the online Data Supplement). However, the LOD for both assays was 100 fg (21 gene copies), the lowest gene copy number for which all 60 samples tested simultaneously produced a positive call by the "Qualitative Detection" module of the LC 4.0 software. The "Absolute Quantification" module of the LC 4.0 software determined a PCR efficiency for the Y. pestis-specific assay of 1.894 and an efficiency of 1.908 for the Y. pseudotuberculosis-specific assay. For an assay to produce unambiguous and identical results, its amplification efficiency should be as close as possible to an efficiency value of 2. The values for our assays thus correspond favorably with this theoretically optimum efficiency value.
Plague is both an endemic disease and a biological warfare threat. Mortality from endemic plague throughout the world continues at a low rate despite the availability of effective antibiotics. The anthrax attacks during the fall of 2001 demonstrate the potential for disease organisms as bioterrorism agents, and mass production of Y. pestis or a genetically engineered variant is conceivable. It is crucial that fast, reliable but simple molecular diagnostic tests be developed.
[FIGURE 2 OMITTED]
The real-time PCR assays presented here take advantage of a recently discovered 25-bp insertion target sequence within the chromosomal yp48 gene of Y. pseudotuberculosis that is not present in Y. pestis (22). We initially identified the sequence and exact location of this 25-bp insertion by sequencing amplicons generated with primers designed to surround the region that contained the presumed deletion. Our results were confirmed by the recent publication of the entire Y. pseudotuberculosis genome. We chose to use TagMan MGB probe technology because of its hybridization properties (24). TagMan MGB probes are more stable and display increased mismatch discrimination, and their signal-to-noise ratio is improved by an NFQ in place of the fluorescent quencher dye 6-carboxytetramethylrhodamine (TAMRA) (25). In addition, the MGB stabilizes A/T-rich duplexes, increasing the probe [T.sub.m]. The MGB probes therefore simplified assay design for the short 25-bp insertion.
We used 2 melting curve-capable probe technologies (Simple-Probes and MGB Eclipse probes) to distinguish Y. pestis DNA from Y. pseudotuberculosis DNA. Simple-Probe technology was first described by Crockett and Wittwer (26) and is now used primarily for single-nucleotide polymorphism (SNP) analysis on the Roche LightTyper. Our method is the first, as far as we are aware, to use Simple-Probe technology in the LightCycler to identify 2 closely related strains of bacteria based on differential [T.sub.m]s. Simple-Probes are single probes designed to specifically hybridize to a target sequence that contains the SNP (in this case the insertion/ deletion site) of interest. Each probe contains a fluorescein molecule at either the 5' or 3' end with the opposite end blocked with a monophosphate molecule. Once hybridized, the SimpleProbe emits a greater fluorescent signal than when it is not hybridized. Because the probe is not destroyed during the PCR amplification reaction, a post-PCR melting curve can be generated. The [T.sub.m] of an individual probe is directly related to the number of mismatches present within the target sequence. The [T.sub.m] is highest when the probe and target sequence are 100% complementary. As mismatches occur between the Simple-Probe and target sequence, the [T.sub.m] is lowered.
The MGB Eclipse probe is very similar to the TagMan MGB probe (24, 27) in that they both contain a fluorescent dye (FAM in this case) at one end and an MGBNFQ at the other (28). The primary difference is that the MGB Eclipse probe has the FAM at the 3' end and the MGBEDQ at the 5' end, effectively blocking the probe from digestion during the PCR amplification cycle. MGB Eclipse probe assays were recently used successfully in a SNP [T.sub.m] analysis of challenging sequences on the ABI Prism[R] 7000 sequence detection system (29). Again, we believe our method is the first time MGB Eclipse probe technology has been used to identify 2 closely related strains of bacteria based on differential [T.sub.m]s in the LightCycler.
Initial reactions incorporating genomic DNAs available at USAMRIID increased our confidence in the overall specificity of each assay. A greater difference in the MGB Eclipse probe [T.sub.m] may be achieved by redesigning the probes with the software available on the Epoch Biosciences web site (http://www.epochbiosciences.com/ products/mgbe software.htm). Simple-Probe assays can also be analyzed in the Roche LightTyper if a LightCycler is unavailable. However, we have not tested the MGB Eclipse probe assay in the LightTyper, and our data showed that the Y. pestis and Y. pseudotuberculosis assays were highly specific and exceptionally sensitive.
Further testing with other strains of Y. pestis and Y. pseudotuberculosis from other sources must be performed to ensure that this 25-bp insertion/ deletion sequence is conserved among additional Yersinia species and strains. All Y. pestis and Y. pseudotuberculosis strains tested in this study were differentiated based on the disparate [T.sub.m]s of their respective probes, but the insertion/deletion region targeted in this study may not be conserved. Molecular diversity and genetic variation in the yp48 gene sequence could decrease the sensitivity and specificity of these particular assays. However, molecular identification of Y. pestis and its differentiation from the closely related Y. pseudotuberculosis is a multiphase process, requiring initial identification by assays specific for the virulence gene targets encoded on the 3 virulence plasmids of Y. pestis. Our assays add to the battery of Y. pestis-specific molecular diagnostic tests and allow for a real-time PCR method for the differentiation of Y. pestis from Y. pseudotuberculosis.
In conclusion, this study demonstrates the reliable and specific chromosomal target identification of Y. pestis DNA and its differentiation from its closest neighbor, Y. pseudotuberculosis, by a combination of TagMan MGB, Simple-Probe, and MGB Eclipse probe real-time PCR on the LightCycler. The assays did not cross-react with any genetic neighbors, and all strains of Y. pseudotuberculosis were distinguished from all strains of Y. pestis by easily measurable Simple-Probe and MGB Eclipse [T.sub.m]s. These real-time assays can be used to complement other molecular diagnostic assays for virulence plasmids by providing a background chromosomal target for organisms containing any of the plasmids.
We graciously thank Kathryn Kenyon for reviewing the manuscript. The research was sponsored by the Defense Technology Objective CB.56: Methodology to Facilitate Development of Biological Warfare (BW) Threat Agent Detection and Medical Diagnostic Systems, US Army Medical Research and Materiel Command. The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army or the Department of Defense.
(1.) Franz DR, Jahrling PB, Friedlander AM, McClain DJ, Hoover DL, Bryne WR, et al. Clinical recognition and management of patients exposed to biological warfare agents. JAMA 1997;278:399-411.
(2.) Inglesby TV, Dennis DT, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA 2000;283:2281-90.
(3.) Rowe CA, Scruggs SB, Feldstein MJ, Golden JP, Ligler FS. An array immunosensor for simultaneous detection of clinical analytes. Anal Chem 1999;71:433-9.
(4.) Wadkins RM, Golden JP, Pritsiolas LM, Ligler FS. Detection of multiple toxic agents using a planar array immunosensor. Biosens Bioelectron 1998;13:407-15.
(5.) Achtman M, Zurth K, Morelli G, Torrea G, Guiyoule A, Carniel E. Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A 1999; 96:14043-8.
(6.) Perry RD, Fetherston JD. Yersinia pestis--etiologic agent of plague. Clin Microbiol Rev 1997;10:35-66.
(7.) Leal NC, Almeida AM. Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR. Rev Inst Med Trop Sao Paulo 1999;41:339-42.
(8.) Neubauer H, Meyer H, Prior J, Aleksic S, Hensel A, Splettstosser W. A combination of different polymerase chain reaction (PCR) assays for the presumptive identification of Yersinia pestis. J Vet Med B Infect Dis Vet Public Health 2000;47:573-80.
(9.) Kulichenko AN, Norkina OV, Gintsburg AL, Popov I, Ddrozdov IG. [Improvement of a method for detecting of strains of the plague microbe using polymerase chain reaction]. Genetika 1994;30: 167-71.
(10.) Norkina OV, Kulichenko AN, Gintsburg AL, Tuchkov IV, Popov Y, Aksenov MU, et al. Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction. J Appl Bacteriol 1994; 76:240-5.
(11.) Melo AC, Almeida AM, Leal NC. Retrospective study of a plague outbreak by multiplex-PCR. Lett Appl Microbiol 2003;37:361-4.
(12.) Higgins JA, Ezzell J, Hinnebusch BJ, Shipley M, Henchal EA, Ibrahim MS. 5' Nuclease PCR assay to detect Yersinia pestis. J Clin Microbiol 1998;36:2284-8.
(13.) Tomaso H, Reisinger EC, AI DS, Frangoulidis D, Rakin A, Landt O, et al. Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes. FEMS Immunol Med Microbiol 2003;38:117-26.
(14.) Loiez C, Herwegh S, Wallet F, Armand S, Guinet F, Courcol RJ. Detection of Yersinia pestis in sputum by real-time PCR. J Clin Microbiol 2003;41:4873-5.
(15.) Iqbal SS, Chambers JP, Goode MT, Valdes JJ, Brubaker RR. Detection of Yersinia pestis by pesticin fluorogenic probe-coupled PCR. Mol Cell Probes 2000;14:109-14.
(16.) Chain PS, Carniel E, Larimer FW, Lamerdin J, Stoutland PO, Regala WM, et al. Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A 2004;101:13826-31.
(17.) Trebesius K, Harmsen D, Rakin A, Schmelz J, Heesemann J. Development of rRNA-targeted PCR and in situ hybridization with fluorescently labelled oligonucleotides for detection of Yersinia species. J Clin Microbiol 1998;36:2557-64.
(18.) McGovern T, Friedlander A. Plague. In: Sidell F, Takafuji E, Franz D, eds. Medical aspects of chemical and biological warfare: part 1--warfare, weaponry, and the casualty. Washington, DC: Office of The Surgeon General at TMM Publications, 1997:479-502.
(19.) Deng W, Burland V, Plunkett G III, Boutin A, Mayhew GF, Liss P, et al. Genome sequence of Yersinia pestis KIM. J Bacteriol 2002; 184:4601-11.
(20.) Parkhill J, Wren BW, Thomson NR, Titball RW, Holden MT, Prentice MB, et al. Genome sequence of Yersinia pestis, the causative agent of plague. Nature 2001;413:523-7.
(21.) Song Y, Tong Z, Wang J, Wang L, Guo Z, Han Y, et al. Complete genome sequence of Yersinia pestis strain 91001, an isolate avirulent to humans. DNA Res 2004;11:179-97.
(22.) Buchrieser C, Rusniok C, Frangeul L, Couve E, Billault A, Kunst F, et al. The 102-kilobase pgm locus of Yersinia pestis: sequence analysis and comparison of selected regions among different Yersinia pestis and Yersinia pseudotuberculosis strains. Infect Immun 1999;67:4851-61.
(23.) Roche. LightTyper Application Report: demonstration of Simple-Probes on the LightCycler. Indianapolis, IN: Roche Diagnostics, September 18, 2003.
(24.) Afonina IA, Reed MW, Lusby E, Shishkina IG, Belousov YS. Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence. Biotechniques 2002; 32:940-9.
(25.) Afonina I, Zivarts M, Kutyavin I, Lukhtanov E, Gamper H, Meyer RB. Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res 1997;25:2657-60.
(26.) Crockett AO, Wittwer CT. Fluorescein-labeled oligonucleotides for real-time PCR: using the inherent quenching of deoxyguanosine nucleotides. Anal Biochem 2001;290:89-97.
(27.) de Kok JB, Wiegerinck ET, Giesendorf BA, Swinkels DW. Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes). Hum Mutat 2002;19:554-9.
(28.) Afonina I, Belousov Y, Metcalf M, Mills A, Sanders S, Kutyavin I, et al. Single nucleotide polymorphism detection with MGB Eclipse assays. J Clin Ligand Assay 2005;25:268-75.
(29.) Belousov YS, Welch RA, Sanders S, Mills A, Kulchenko A, Dempcy R, et al. Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse Probe System in challenging sequence environment. Hum Genomics 2004;1:209-17.
CATHERINE J. CHASE,  MELANIE P. ULRICH,  LEONARD P. WASIELOSKI, JR.,  JOHN P. KONDIG,  JEFFREY GARRISON,  LUTHER E. LINDLER,  and DAVID A. KULESH  *
 Diagnostic Systems Division, The United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD.
 Battelle, Columbus, OH.
 National Biodefense Analysis and Countermeasures Center, Department of Homeland Security, Frederick, MD.
 Nonstandard abbreviations: MGB, minor groove binder; FAM, 6-carboxyfluorescein; NFQ, nonfluorescent quencher; SPC, Simple-Probe chemistry; EDQ, Eclipse dark quencher; [T.sub.m], probe melting temperature; USAMRIID, US Army Medical Research Institute of Infectious Diseases; Ct, threshold cycle number; LOD, lower limit of detection; NTC, no-template control; and SNP, single-nucleotide polymorphism.
* Address correspondence to this author at: The United States Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, Frederick, MD 21702-5011. Fax 301-619-2492; e-mail David.Kulesh@amedd. army.mil.
Received March 30, 2005; accepted July 8, 2005.
Previously published online at DOI: 10.1373/clinchem2005.051839
Table 1. Primer/probe sequences of the Y. pestis and Y. pseudotuberculosis yp48-specific assays. Amplicon Organism size, bp Primers/Probes Sequence, 5'-3' Y. pestis 88 yp48-F763 GCA GGA AAT GCG CAA TGC yp48-R850 GGG CGG ATC CCC ACT TTA yp48-p815S-MGB 6FAM-AGG TTC AGG TGA GCA CG-MGBNFQ yp48-SP812--05 Phosphate-TCG AGG (a) TTC AGG TGA GCA CGT TAA-SPC- Fluorescein yp48-p815S-ECL MGBEDQ-AGG TTC AGG TGA GCA CG-FAM Y. pseudotuberculosis 113 (b) yp48-F763 GCA GGA AAT GCG CAA TGC yp48-R850 GGG CGG ATC CCC ACT TTA yp48-p834S-MGB 6FAM-CGC CGC TCG TTC A-MGBNFQ yp48-SP828-53 Fluorescein-SPC-TAC CGC CGC CGC TC-Phosphate yp48-p834S-ECL MGBEDQ-CGC CGC TCG TTC A-FAM Final concentrations in TaqMan MGB/Simple-Probe/Eclipse Organism assays, [micro]M Mg[Cl.sub.2], mM Y. pestis 0.5/0.1/0.1 0.5/0.5/0.5 0.1 5 0.1 3 0.1 5 Y. pseudotuberculosis 0.5/0.1/0.1 0.5/0.5/0.5 0.1 5 0.1 3 0.1 5 (a) SP, Simple-Probe (self-quenching); ECL, Eclipse probe. (b) Although identical primers were used, the larger size is attributable to the 25-bp insertion in the Y. pseudotuberculosis yp48 gene. Fig. 1. Primer/Probe location on Y. pestis/Y. pseudotuberculosis (Y. pseudoTB) yp48 gene. Organism Strain GenBank Forward Accession # Primer (bp) Y. pestis KIM NC_004088 2682504-21 (a) CO92 NC_003143 2134277-94 91001 NC_005810 1654943-60 (a) Y. Pseudotuberculosis IP32953 NC_006155 2248787-804 Organism Reverse 25 bp insert Primer (bp) Y. pestis 2682434-51 Not Present 2134347-64 Not Present 1654873-90 Not Present Y. Pseudotuberculosis 2248882-99 2248850-74 (a) Complementary strand
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||Molecular Diagnostics and Genetics|
|Author:||Chase, Catherine J.; Ulrich, Melanie P.; Wasieloski, Leonard P., Jr.; Kondig, John P.; Garrison, Jef|
|Date:||Oct 1, 2005|
|Previous Article:||High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution.|
|Next Article:||Circulating placental RNA in maternal plasma is associated with a preponderance of 5' mRNA fragments: implications for noninvasive prenatal diagnosis...|