The potential of 5′ nuclease PCR for detecting a single-base polymorphism inOrthopoxvirus

Smallpox and pan -Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

“…LightCycler and the Cepheid (Sunnyvale, Calif.) Smart Cycler (5,(8)(9)(10)33). Several papers have dealt with the general application of real-time TaqMan PCR technology for identifying biological agents (11,13,19), as well as specifically for orthopox viruses (8,16,17,24,31). The TaqMan chemistry has recently undergone a significant improvement by the addition of a minor groove binding protein (MGB) (1,2,20) and a nonfluorescent quencher (NFQ) to the 5Ј end of the probe molecule.…”

Real-Time PCR Assay To Detect Smallpox Virus

“…One of the most promising approaches for rapid and sensitive diagnosis is the real-time 5Ј nuclease PCR assay, also known as the TaqMan assay. Previously, we reported on the selection of the gene for the hemagglutinin, a gene that distinguishes the genus Orthopoxvirus from other poxvirus genera, as a suitable sequence target for genus-and species-specific PCR tests (1,2,10,11) and the potential utility of 5Ј nuclease assays for the diagnosis of orthopoxvirus infections (10,11). Here, we report on the development of a real-time TaqMan assay based on the hemagglutinin gene for the sensitive and specific detection of variola virus, the causative agent of smallpox, on portable realtime detection devices: the Smart Cycler instrument (Cepheid, Sunnyvale, Calif.) and the LightCycler instrument (Roche Molecular Systems, Indianapolis, Ind.…”

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents

“…Because of its sensitivity, rapidity, and ease, real-time PCR is increasingly becoming the method of choice for preliminary detection of Orthopoxvirus infection, with isolation and growth in a high-level containment laboratory utilized for confirmation. Previously, the development of real-time PCR for detection of Orthopoxvirus infections was reported (Ibrahim et al, 1997(Ibrahim et al, , 1998(Ibrahim et al, , 2003Aitichou et al, 2005). Other reports have demonstrated the utility of real-time PCR in detecting variola and other Orthopoxvirus species (Espy et al, 2002;Kulesh et al, 2004a,b;Li et al, 2006;Nitsche et al, 2004;Olson et al, 2004;Scaramozzino et al, 2007).…”