Detection of Yersinia pestis in Sputum by Real-Time PCR

Abstract

A 5 nuclease PCR assay for detection of the Yersinia pestis plasminogen activator (pla) gene in human respiratory specimens with simulated Y. pestis infection was developed. An internal positive control was added to the reaction mixture in order to detect the presence of PCR inhibitors that are often found in biological samples. The assay was 100% specific for Y. pestis. In the absence of inhibitors, a sensitivity of 10 2 CFU/ml of respiratory fluid was obtained. When inhibitors were present, detection of Y. pestis DNA required a longer sample treatment time and an initial concentration of bacteria of at least 10 4 CFU/ml. The test's total turnaround time was less than 5 h. The assay described here is well suited to the rapid diagnosis of pneumonic plague, the form of plague most likely to result from a bioterrorist attack.Protecting the population against an act of bioterrorism is a major concern for many governments. Yersinia pestis, the agent of plague, can be viewed as a potential bioweapon due to its ability to cause high rates of morbidity and mortality in humans. The intentional dissemination of plague by terrorists would most likely occur by aerosolization, causing fulminant pneumonia in exposed individuals. Since pneumonic plague is almost always fatal when untreated, clinical microbiology laboratories play a pivotal role in the early detection of this type of infectious agent, thus allowing rapid implementation of effective preventive measures. Availability of a rapid diagnostic procedure using molecular techniques (such as real-time PCR) is essential in the establishment of coordinated laboratory response systems. Several PCR assays have been developed (1, 3-7, 10, 13): the plasmidic plasminogen activator (pla) gene (located on the Y. pestis-specific pPst/pCP1 plasmid [5,11]) was found to be the most sensitive target, since its copy number can be as high as 186 per bacterium (8). In the present study, we describe a real-time PCR protocol for detection of the Y. pestis pla gene. The assay is based on 5Ј exonuclease assay technology coupled with automated DNA extraction and uses a minor groove binder-conjugated small DNA probe for DNA detection via hybridization-triggered fluorescence: subsequent multiplex development can be thus envisaged. Inclusion of an internal positive control (IPC) in each batch assay enabled the detection of endogenous PCR inhibitors.The primers and the fluorogenic probe for the pla gene (GenBank accession no. M27820) were designed with Primer Express software, version 2.0 (Applied Biosystems, Foster City, Calif.) and were obtained from Applied Biosystems (Warrington, United Kingdom). The nucleotide sequences of the forward and reverse primers were 5Ј-GAAAGGAGTGCGG GTAATAGGTT-3Ј (positions 816 to 838) and 5Ј-AACCAGC GCTTTTCTA-3Ј (positions 869 to 884), respectively. The sequence of the minor groove binder probe was 6-carboxyfluores cein-5Ј-GACTTGCAGGCC-3Ј (positions 840 to 851). PCR amplifications were performed in 25-l reaction volumes including 1ϫ TaqMan Universal Master Mix (A...

TheYersinia pestis caf1M1A1Fimbrial Capsule Operon Promotes Transmission by Flea Bite in a Mouse Model of Bubonic Plague

“…In this context, many efforts have been made to develop rapid diagnostic procedures. The caf1 and pla genes, as well as their products, are the most frequent targets used to detect Y. pestis in many different kinds of samples (11,17,28,33,43,62,65). Thus, even though diagnostic procedures based on caf1 and pla have been shown to have great value in the field (11), our current work, and our previous results showing that pla is not essential to establish an infection after flea bite (54), suggests that supplementary diagnostic procedures should be developed to detect atypical Y. pestis and that better surveillance of atypical Y. pestis strains in plague foci is needed, as was stressed more than 20 years ago (69).…”

Evidence of Yersinia pestis DNA in rodents in plague outbreak foci in Mbulu and Karatu Districts, northern Tanzania

“…This gene which is located on plasmid pPCP1, is incorporated into most Y. pestis PCR assays, and in several studies it has been used as the prime or sole marker (Riehmet al, 2011;Adjemian et al, 2008). Our reasons for including pla in our assay are its occurrence in multiple copies, its absence from closely related Yersinia species, and its role in Y. pestis virulence (Loïez et al, 2003;Tomaso et al, 2003).…”

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".…”