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Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis
Published on: June 17, 2019
Real-time PCR assays targeting a unique chromosomal sequence of Yersinia pestis
Catherine J Chase1, Melanie P Ulrich, Leonard P Wasieloski
1Diagnostic Systems Division, The United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA.
Background:
Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results.
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 Eclipse 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 degrees C Tm difference and the MGB Eclipse probe a slightly more than 4 degrees 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.
Insights
New real-time PCR assays targeting chromosomal genes accurately detect Yersinia pestis (plague) and differentiate it from Yersinia pseudotuberculosis, improving bioweapon detection reliability.
Area of Science:
- Microbiology
- Molecular Biology
- Biosecurity
Background:
- Yersinia pestis is the causative agent of plague and a significant bioweapon concern.
- Existing real-time PCR detection methods for Y. pestis rely on plasmid targets, risking false positives.
- Accurate and reliable detection is crucial for biodefense and public health.
Purpose of the Study:
- To develop novel real-time PCR assays for Yersinia pestis detection using chromosomal targets.
- To differentiate Y. pestis from closely related species, particularly Yersinia pseudotuberculosis.
- To enhance the specificity and reliability of Y. pestis identification.
Main Methods:
- Designed and tested two real-time TaqMan minor groove binder (MGB) assays targeting the Y. pestis yp48 chromosomal gene.
- Developed additional assays using Simple-Probe and MGB Eclipse probe technologies targeting a specific insertion site in Y. pseudotuberculosis.
- Utilized post-PCR melting temperature (Tm) analysis for species differentiation.
Main Results:
- The yp48-based assay differentiated Y. pestis from other Yersinia species but showed low-level detection of Y. pseudotuberculosis.
- Probes targeting a 25-bp insertion site specifically detected Y. pseudotuberculosis.
- Melting temperature analysis clearly distinguished Y. pestis from Y. pseudotuberculosis, with Tm differences of up to 7°C.
Conclusions:
- Developed chromosomal real-time PCR assays that accurately discriminate Y. pestis from all tested Yersinia species, including Y. pseudotuberculosis.
- These assays provide reliable confirmation of Y. pestis presence using chromosomal targets.
- The method offers a robust tool for distinguishing Y. pestis from its close relatives, enhancing biosecurity detection capabilities.
