Rapid detection ofYersinia pestiswith multiplex real-time PCR assays using fluorescent hybridisation probes

Abstract

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.

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".…”

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

“…The sensitivities remained the same for the type A.I and novicida assays, with an order of magnitude decrease in sensitivity for the type A.II and type B assays. Another multiplex RT-PCR assay for F. tularensis demonstrated sensitivities as low as 0.5 genome equivalents (Tomaso et al, 2003). This increased sensitivity may be explained by two factors: 1) the assay targeted the 16S rDNA, of which multiple copies are present in the genome; and 2) the multiplex assays only included two assays, thus reducing the chance of competitive PCR problems.…”

“…This increased sensitivity may be explained by two factors: 1) the assay targeted the 16S rDNA, of which multiple copies are present in the genome; and 2) the multiplex assays only included two assays, thus reducing the chance of competitive PCR problems. When Tomaso et al (2003) included three assays in their multiplex assay, no amplification curves were observed.…”

Specific detection and differentiation of Ochrobactrum anthropi, Ochrobactrum intermedium and Brucella spp. by a multi-primer PCR that targets the recA gene

“…Cycling was started with a 10 min denaturation step at 95 uC, followed by 45 cycles of 10 s denaturation at 95 uC, 10 s annealing at 55 uC and 12 s extension at 72 uC. As an internal amplification control, we included a bacteriophage l PCR system as described previously (Tomaso et al, 2003). No-template controls containing 2 ml molecular-grade water instead of DNA and positive controls containing Brucella DNA of the reference strains B. melitensis 16M T (biovar 1), B. abortus 1119-3 (biovar 1) and/or B. suis 1330 (biovar 1) were included in each run to detect any amplicon contamination or amplification failure.…”