[Improvement of a method for detecting of strains of the plague microbe using polymerase chain reaction]

Article · March 1994with4 Reads
Source: PubMed
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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    Article · Dec 2006
  • [Show abstract] [Hide abstract] ABSTRACT: Real-time PCR has been used previously to detect Yersinia pestis; this study applies this rapid, specific, and sensitive nucleic acid-based method to the detection and quantitation of Y. pestis specifically in food. Five sets of primers and corresponding TaqMan dual-labelled fluorogenic hybridization probes for Y. pestis were designed and optimized for specificity testing using genomic DNA from 71 bacterial strains. Four Y. pestis -specific primer and probe sets were developed, based on the virulence plasmid targets, and used to distinguish this bacterium from the various Yersinia and other bacterial species tested. An additional primer and probe set, based on a chromosomal gene target, distinguished Y. pestis and Yersinia pseudotuberculosis from the various Yersinia and other bacterial species tested. With optimized conditions, the quantitative detection limit of the probes for Y. pestis pure cultures ranged from 13 to 220 CFU. Standard curves were generated for the probes and used to determine the amplification efficiencies. The primers and probes demonstrated high amplification efficiencies, and their performance was evaluated using spiked milk and ground beef samples. The quantitative detection limit was 10(1) to 10(3) CFU/ml in milk and 10(2) to 10(5) CFU/g in ground beef without any preenrichment step. Testing the hybridization probes on food samples demonstrated the detection of Y. pestis in a foodborne application; this is the first such report, to our knowledge.
    Article · Jan 2010