Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction

Abstract

A 501 bp caf1 gene fragment and a 443 bp of pla gene fragment carried by 100 kb (pFra) and 10 kb (pPst) species-specific extrachromosomal replicons, respectively, were used as targets to study the conditions under which DNA amplification by polymerase chain reaction (PCR) may be applied to detect and identify Yersinia pestis DNA in cell lysates of pure cultures and biological samples. The sensitivity limit of PCR with the crude cell lysates of Y. pestis EV was estimated as 10-50 cfu in reaction mixture. When target Y. pestis EV cells were mixed with fresh blood of white mice, which contained 0.4% potassium citrate, the PCR detection level varied from 400 to 100 cfu ml-1 of blood depending on the method used for preparing the sample. In our tests PCR was effective for the detection of yersinia in the blood of white laboratory mice experimentally infected with virulent Y. pestis KM638 strain. This method can be considered convenient for routine detection and identification of Y. pestis.

Yersinia pestis and Bubonic Plague

“…Extremely sensitive methods that utilize oligonucleotide targets to unique determinants are now used in many public health centers routinely to screen clinical specimens received from the field. Paramount in this effort was the use of polymerase chain reaction (PCR) for amplification of DNA sequences capable of interacting with probes prepared from pla, caf1 or both (Campbell et al, 1993;Hinnebusch and Schwan, 1993;Norkina et al, 1993;Tsukano et al, 1996). Another such method that does not rely on unique structural genes is the procedure of Trebesius et al (1998), which depends upon amplification of a 23S rRNA sequence specific to Y. pestis.…”

Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia 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".…”

Virulence of pPst+ and pPst- strains of Yersinia pestis for guinea-pigs

“…The plasmid profile data (not shown) were confirmed by PCR amplification of plasmidencoded gene (plasminogen activator, pla, and capsular antigen fraction 1, c@7) fragments as described previously [20].…”