Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis

Abstract

Background: Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results. Methods: Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan ® minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe

Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

“…The Y. pestis TaqMan-MGB exhibited results similar to those described previously (8 ). At all concentrations of near-neighbor Y. pseudotuberculosis, false positives occurred approximately 3 cycles later than detection of an equivalent concentration of Y. pestis.…”

“…(b) Additionally, 7 bases flanking the 5Ј end of the insertion are repeated in the 3Ј end of the insertion, causing exact matches to shorter probes, an especially problematic feature for probes that depend on short lengths for increased specificity. TaqMan-MGB is an example of a probe that requires short lengths for improved differentiation, thus explaining why false positives were present even after 10 design iterations of TaqMan-MGB probes (8 ). Because Tentacle Probes use a strong hairpin for enhanced differentiation and do not require short probes, they are ideal candidates for specifically detecting Y. pestis.…”

“…In this study we applied Tentacle Probes to real-time PCR for the 1st time. These assays were selected because of ongoing difficulties with the selective detection of these genes in quantitative PCR using TaqMan-minor groove binder (MGB) probes (8 Tentacle Probes act through cooperative binding with both a detection and a capture probe designed around the principles of specific cell targeting (12)(13)(14)(15)(16). These probes have been shown to have increased binding kinetics compared to standard stem-loop fluorescent DNA probes, to have concentration-independent melting curves, and to increase specificity without sacrificing sensitivity (7 ).…”

Recent Advancement in the Development of Vaccines Against Y. pestis - A Potential Agent of Bioterrorism

“…PCR-gel electrophoresis based methods have been developed for detecting Y. pestis in fleas and other specimens (Hinnebusch and Schwan, 1993;Norkina et al, 1994, Tsukano et al, 1996. Real-time PCR assays in various formats have also been developed for detecting and identifying Y. pestis (Higgins et al, 1998;Iqbal et al, 2000;Lindler and Tall, 2001;Loiez et al, 2003;Tomaso et al, 2003;Chase et al, 2005;Woron et al, 2006). Real-time PCR based methods are more specific, and require less time and labor than conventional PCR assays.…”

“…Real-time PCR based methods are more specific, and require less time and labor than conventional PCR assays. Real-time PCR methods include SYBR Green (Saikaly et al, 2007), molecular beacons (Varma-Basil et al, 2004), TaqMan probes (Loiez et al, 2003;Chase et al, 2005) and minor groove binding (MGB) probes (Skottman et al, 2007), and target specific sequences on the chromosome and (or) plasmids. However, PCR based diagnosis is expensive compared to immunoassays, which may be useful for mass screening during epidemics.…”