A quadruplex real-time PCR assay for the detection of Yersinia pestis and its plasmids

Abstract

Yersinia pestis, the aetiological agent of the plague, causes sporadic disease in endemic areas of the world and is classified as a National Institute of Allergy and Infectious Diseases Category A Priority Pathogen because of its potential to be used as a bioweapon. Health departments, hospitals and government agencies need the ability to rapidly identify and characterize cultured isolates of this bacterium. Assays have been developed to perform this function; however, they are limited in their ability to distinguish Y. pestis from Yersinia pseudotuberculosis. This report describes the creation of a real-time PCR assay using Taqman probes that exclusively identifies Y. pestis using a unique target sequence of the yihN gene on the chromosome. As with other Y. pestis PCR assays, three major genes located on each of the three virulence plasmids were included: lcrV on pCD1, caf1 on pMT1 and pla on pPCP1. The quadruplex assay was validated on a collection of 192 Y. pestis isolates and 52 near-neighbour isolates. It was discovered that only 72 % of natural plague isolates from the states of New Mexico and Utah harboured all three virulence plasmids. This quadruplex assay proved to be 100 % successful in differentiating Y. pestis from all near neighbours tested and was able to reveal which of the three virulence plasmids a particular isolate possessed.

Diagnostic Bioluminescent Phage for Detection of Yersinia pestis

“…The sensitivity limits of detection are between 6 ϫ 10 3 and 7 ϫ 10 3 CFU/ml, which is similar to the detection sensitivity described in this report (33). However, both of these sensitivity limits pale in comparison with that of real-time PCR, which can detect between 0.01 and 1 pg of extracted DNA; assuming a 100% extraction efficiency, this translates to approximately 1 CFU (24,32). Nevertheless, the reporter phage technology has a number of desirable traits compared to PCR-based assays.…”

A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies

“…In a quadruplex RT-PCR assay for Yersinia pestis, Stewart et al (2008) saw a tenfold decrease in the sensitivity when their assay was moved from singleplex to quadruplex: from 150 pg to 1.5 ng. The results for our triplex assay are similar to these results, showing a tenfold decrease in sensitivity for two of the assays when used in a multiplex format: from 114 to 1136 organisms.…”

Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

“…In order to specifically differentiate the pathogen from its closely related species, we have targeted chromosomal gene sequences specific for each pathogen. For B. anthracis, we have chosen a unique genomic locus, dhp61.183 [8] and for Y. pestis, a chromosomal gene target, YihN [9]. B. melitensis specific sequence was designed from BMEI1162 gene [10].…”