TABLE 17.

Detection of othopoxvirus DNA by real-time PCR

ReferenceCultureReal-time PCRComments
No. of specimensNo. positive (%)CellsNo. of specimensNo. positive (%)Test platform/gene target
Espy et al. 2002 (114)Reference strains. American Type Culture Collection: vaccinia virus (vr-117); cowpox virus (vr-302); monkeypox virus (vr 267). Centers for Disease Control: vaccinia virus; variola virus (smallpox); plasmid insert of a 300-bp segment of the hemagglutinin geneLightCycler PCR/hemagglutinin geneVariola virus (Tm, 62.45°C) could be differentiated from vaccinia (Tm, 56.72°C) and monkeypox (Tm, 56.24°C) viruses.
Sofi Ibrahim et al. 2003 (457)Centers for Disease Control: variola virus, (smallpox): 48 strains of camelpox, cowpox, ectromelia, gerbilpox, monkeypox, rabbitpox, raccoonpox, skunkpox, vaccinia, herpes simplex virus, and varicella-zoster virusLightCycler, SmartCycler/hemagglutinin geneBoth PCR platforms were considered acceptable for the detection of variola virus DNA.
322 coded samples; 48 different stains (above)SmartCycler: 2 samples with false-positive results among 116 samples not containing variola virus (specificity, 98.3%): LightCycler: 5 samples with false-positive results among 116 samples not containing variola virus (specificity 95.7%)
Kelly et al. 2004 (217)ABI Prism, Laboratory Response Network assays for vaccinia virus and for varicella-zoster virus.Publication reports the detection of vaccinia virus-specific DNA in lesions of patients previously immunized against smallpox.
Kulesh et al. 2004 (241)16 coded samples: 8 variola virus (smallpox); 5 non-variola virus, orthopox virus isolates; 2 varicella-zoster virus; 1 herpes simplex virusLightCycler, SmartCycler/hemagglutinin gene. SmartCycler detected 92.3% of the orthopoxvirus DNAs and between 95% and 93.8% of the variola virus DNAs. LightCycler detected 96.2% of the orthopox DNAs and 93.8% of the variola virus DNAs.Both LightCycler and SmartCycler had 100% sensitivity with both platforms with samples above the limit of detection (≥12 gene copies).
Nitsche et al. 2004 (345)LightCycler/genes: A13L, rpo18, VETFA complete set of four hybridization probe-based real-time PCR assays provide for the specific detection of orthopoxvirus DNA by melting point analysis.
Olson et al. 2004 (352)LightCycler/fusion protein genePCR primers were designed to detected all Eurasian-African species of orthopoxvirus. A single nucleotide mismatch in the target sequence allowed differentiation of variola virus from other orthopoxviruses by melting-curve analysis.