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Molecular Diagnostics and Genetics |
1 Diagnostic Systems Division, The United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD.
2 Battelle, Columbus, OH.
3 National Biodefense Analysis and Countermeasures Center, Department of Homeland Security, Frederick, MD.
aAddress 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{at}amedd.army.mil.
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Abstract |
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Methods: Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan® minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe® and MGB EclipseTM probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis.
Results: The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 °C Tm difference and the MGB Eclipse probe a slightly more than 4 °C difference.
Conclusions: Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 °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)(8)(9)(10)(11) and real-time PCR assays (12)(13)(14)(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 TaqMan®-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)(20)(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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pcr primers, target sequences, and fluorogenic probes
yp48 assay.
The real-time PCR assay primers and TaqMan minor groove binder (MGB)
1
probe (ABI), Simple-Probe® (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 TaqMan 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 TaqMan 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 fluorescein 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 TaqMan 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 (Tm) for the Simple-Probe was determined both through the software (theoretical) and empirically with the appropriate control DNA on the LightCycler, but the Tm of the MGB Eclipse probe was determined only empirically.
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5'-Nuclease PCR (TaqMan MGB) assays.
After using the Primer Express 2.0 software to design potential Y. pestis/ Y. pseudotuberculosis yp48-specific TaqMan 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 real-time 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 µM) with the final concentration determined by lowest Ct value and highest fluorescent signal. We then tested several potential TaqMan MGB probes with the optimized primer pair by varying the probe and MgCl2 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 Ct 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 TaqMan MGB assays were carried out in 20-µL volumes for the LightCycler 2.0, with each reaction made up in PCR buffer [50 mM Tris (pH 8.3), 25 ng/µL 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 MgCl2, 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 °C for 2 min, followed by 45 cycles of 95 °C for 1 s and 60 °C for 20 s. A fluorescence reading was taken at the end of each 60 °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 TaqMan MGB assays (Table 1
) were tested with potential Simple-Probes as follows: asymmetric PCR was performed with a final concentration of 0.1 µM for the forward primer and 0.5 µM for the reverse primer. The Simple-Probes were used at a final concentration of 0.1 µM. The concentration of MgCl2 was 3 mM. All Simple-Probe assays were carried out in 20-µL volumes for the LightCycler 2.0, with each reaction made up in PCR buffer [50 mM Tris (pH 8.3), 25 ng/µL bovine serum albumin, and 0.2 mM deoxynucleotide triphosphate mixture; Idaho Technology]. KlenTaq LA Polymerase Mix (0.8 U; BD Biosciences) was inactivated with an equal volume of TaqStart Ab (BD Biosciences) and then added to each reaction. Thermal cycling for the LightCycler was performed as follows: 1 cycle at 95 °C for 2 min, followed by 50 cycles of 95 °C for 1 s, 55 °C for 10 s, and 72 °C for 10 s. The temperature ramp rate during these 2 steps was 20 °C/s. A single fluorescence reading was taken at the end of each 55 °C step. At the end of the 50 amplification cycles, a melting curve was generated as follows: 95 °C for 1 s and 45 °C for 20 s followed by an increase to 90 °C at a ramp rate of 0.1 °C/s. A continuous read was made in the 530 channel during the entire 0.1 °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 TaqMan 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 MgCl2 concentration was 5 mM (exactly as for the TaqMan 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/vol51/issue10/). The panel consisted of DNAs from 16 Y. pestis strains, 7 Y. pseudotuberculosis strains, and 6 additional Yersinia spp. strains (10–100 pg/µL) 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 µL of each sample was added to the appropriate assay LightCycler Master Mix (15 µL) and cycled as described above. All test runs included at least 1 positive control that contained 1.1 x 103 copies (5 pg total) of purified Y. pestis CO92 and Y. pseudotuberculosis 6904 DNAs and 2 no-template controls (NTCs), a reagent NTC and a sample NTC.
Calibration curve determination for the TaqMan MGB assays.
Y. pseudotuberculosis 6904 and Y. pestis CO92 genomic DNA samples were diluted and tested in triplicate in the LightCycler with the corresponding TaqMan MGB assay at the following final concentrations in 20 µL of Master Mix: 2.0: 4.8 ng (1 000 000 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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development of real-time pcr assays
The final primer/TaqMan 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 TaqMan 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 TaqMan 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 TaqMan 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 TaqMan MGB assays also reproducibly detected 100 fg of the appropriate Yersinia spp. genomic DNA, which represented 
21 copies of each genome.
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Because the TaqMan 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 TaqMan 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 [Tm = 67.84 (0.06) °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 Tms for the yp48-SP812-05 probe were significantly different for the 2 DNAs: 69.29 (0.03) °C for Y. pestis DNA compared with 62.35 (0.11) °C for the Y. pseudotuberculosis DNA (
6.94 °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).
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With the same primers as in the TaqMan 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 [Tm = 58.73 (0.28) °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 Tms for the yp48-p815S-ECL probe were significantly different for the 2 DNAs: 70.49 (0.14) °C for Y. pestis DNA compared with 66.13 (0.17) °C for the Y. pseudotuberculosis DNA (4.36 °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 TaqMan 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 Tm 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 Tm for the Simple-Probes was almost 7 °C and that for the MGB Eclipse probes was slightly more than 4 °C; both were easily distinguishable by the LightCycler Melting Curve Analysis: Genotyping module of the LightCycler software 4.0.
calibration curve for the TAQMAN MGB assays
The yp48 chromosomal TaqMan 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 TaqMan MGB probe technology because of its hybridization properties (24). TaqMan 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 Tm. 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 Tms. 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 Simple-Probe 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 Tm of an individual probe is directly related to the number of mismatches present within the target sequence. The Tm is highest when the probe and target sequence are 100% complementary. As mismatches occur between the Simple-Probe and target sequence, the Tm is lowered.
The MGB Eclipse probe is very similar to the TaqMan 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 Tm analysis of challenging sequences on the ABI Prism® 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 Tms 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 Tm 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 Tms 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 TaqMan 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 Tms. 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.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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E. S. Gabitzsch, R. Vera-Tudela, R. J. Eisen, S. W. Bearden, K. L. Gage, and N. S. Zeidner Development of a Real-time Quantitative PCR Assay to Enumerate Yersinia pestis in Fleas Am J Trop Med Hyg, July 1, 2008; 79(1): 99 - 101. [Abstract] [Full Text] [PDF]
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M. G. Herrmann, J. D. Durtschi, L. K. Bromley, C. T. Wittwer, and K. V. Voelkerding Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dyes Clin. Chem., March 1, 2006; 52(3): 494 - 503. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
