[Improvement of a method for detecting of strains of the plague microbe using polymerase chain reaction].
ABSTRACT Three pairs of oligonucleotide primers, complementary to nucleotide sequences of Yersinia pestis plasmids (pPst, 9.5 kb; pCad, 70 kb; pFra, 95 kb), were used in polymerase chain reaction for high-sensitive specific detection of plague pathogen. Primer pairs P1,P2,C1,C2, and F1,F2 were used to amplify fragments of pla gene (plasmid pPst), yop1 gene (plasmid pCad of plague microbe), and caf1 gene (plasmid pFra), respectively. The method developed enables specific detection of strains from all natural loci in Russia and contiguous states as well as strains of oceanic origin. Sensitivity of method is 50-100 CFU/ml. Primer sequences enable to amplify gene fragments, located in three own plasmides of plague microbe, in the same reaction mixture. The method offers identification of plague microbe and determination of its virulence and epidemic significance.
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ABSTRACT: 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. 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. 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. 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.Clinical Chemistry 11/2005; 51(10):1778-85. · 7.15 Impact Factor
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ABSTRACT: An approach for isolation of an autoagglutination factor (AF) from Hms(-) cells of the plague agent has been developed. Purified AF has been obtained and characterized in physicochemical properties. The AF is found to be a complex of a 17.5-kD protein with a low molecular weight peptide component, which binds iron ions and shows siderophore activity. This low molecular weight component is responsible for hydrophobic properties and immunochemical activity of the AF, as well as for its ability to interact with the plague diagnosticum L-413c bacteriophage.Biochemistry (Moscow) 12/2006; 71(11):1192-9. · 1.15 Impact Factor