Real-Time PCR System for Detection of Orthopoxviruses and Simultaneous Identification of Smallpox Virus

Abstract

A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus Orthopoxvirus. A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (T m s) determined for 46 strains of variola virus (T

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

“…The assay also exhibited high sensitivity (96%) and specificity (98%) that was comparable with assays based on liquid reagents. The detection limits of real-time PCR assays reported previously for orthopoxviruses ranged from 2 to 25 genome copies per PCR reaction (Espy et al, 2002;Ibrahim et al, 2003;Nitsche et al, 2004;Kulesh et al, 2004a,b;Olson et al, 2004;Li et al, 2006;Scaramozzino et al, 2007). In this study, the LOD of dried reagent based PCR assays was 25 and 50 copies for plasmid and genomic DNA, respectively.…”

“…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). These previous reports describing the use of real-time PCR for detecting orthopoxviruses are based on liquid-dispensed reagents that require freezing or refrigeration.…”

Real-Time PCR to Identify Variola Virus or Other Human Pathogenic Orthopox Viruses

“…Another approach, the 5Ј nuclease PCR or the TaqMan assay, first developed by Holland et al (27 ) and improved by Lee et al (28 ), allows for the simultaneous amplification and detection of nucleic acids in real time. Some assays targeting the 14-kD protein gene (29 ) or the rpo18 gene (30 ) and relying on the concept of melting temperature analyses correctly discriminate nonvariola orthopoxviruses from variola virus, but they can not be used on all real-time PCR platforms.…”

Detection of human orthopoxvirus infections and differentiation of smallpox virus with real‐time PCR

“…Conventional PCR together with restriction fragment length polymorphism assay [Ropp et al, 1995;Loparev et al, 2001] has been used as a tool to differentiate OPV species but the sensitivity was not optimal. Real-time PCR has been shown to be applicable as a specific and sensitive detection method [Espy et al, 2002;Nitsche et al, 2004;Olson et al, 2004]. Also microarray technology has been applied for detection and specific typing of OPVs [Laassri et al, 2003].…”