Abstract:
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.
journal_name
Clin Chemjournal_title
Clinical chemistryauthors
Chase CJ,Ulrich MP,Wasieloski LP Jr,Kondig JP,Garrison J,Lindler LE,Kulesh DAdoi
10.1373/clinchem.2005.051839subject
Has Abstractpub_date
2005-10-01 00:00:00pages
1778-85issue
10eissn
0009-9147issn
1530-8561pii
clinchem.2005.051839journal_volume
51pub_type
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journal_title:Clinical chemistry
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journal_title:Clinical chemistry
pub_type: 临床试验,杂志文章
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更新日期:1995-04-01 00:00:00
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更新日期:1997-12-01 00:00:00
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