Use of internally controlled real-time genome amplification for detection of variola virus and other orthopoxviruses infecting humans

Abstract

Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3'-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube.

FIG. 1.

Alignment of orthopoxvirus and probe sequences within the crmB gene. BPXV, buffalopox virus; GBLV, taterapox virus. VIR 130 is specific probe for variola virus; ortopox 143 is generic probe. Accession numbers are shaded.

FIG. 2.

Detection of chimerical VARV DNA by TaqMan real-time PCR. Amplification plots (cycle number versus delta Rn) of serial 10-fold dilutions (10⁶ to 10 copies per tube) of chimerical VARV DNA tested in triplicate in the same experiment. NC is the negative control. Rn, the normalized reporter signal, is the fluorescence signal of the reporter dye divided by the fluorescence signal of the passive, internal reference dye. Delta Rn is determined by the formula Rn⁺ − Rn⁻, where Rn⁺ is the Rn value for a reaction involving all components, including the template, and Rn⁻ is the value for an unreacted sample.

FIG. 3.

Detection of VACV (Western Reserve strain) by TaqMan real-time PCR. The amplification plot was realized on 10-fold dilutions (10⁶ to 1 PFU per tube), assayed in triplicate, of extracted DNA from a VACV stock. See the Fig. 2 legend for Delta Rn definition.

FIG. 4.

Linearity of the TaqMan PCR assay. The standard curve is generated by plotting the observed Ct value, measured in triplicate, against (a) the log₁₀ of the input copy number (10⁶ to 10 copies per tube) of chimerical VARV DNA and (b) the log of serial dilutions (10⁶ to 1 PFU per tube) of VACV.