Differentiation of Variola major and Variola minor Variants by MGB-Eclipse Probe Melt Curves and Genotyping Analysis

Smallpox, caused by the Variola major virus, is considered to be one of the most lethal of all potential biological weapons and has far-reaching consequences. Real-time polymerase chain reactions PCR assays are available as a reliable diagnostic tool to detect members of the genus Orthopoxvirus. In addition real-time PCR assays specific for variola virus have been developed that distinguish it from other orthopoxviruses. However, a positive identification of variola spp. does not classify the virus as the one that causes smallpox V. major or as the variant V. minor that causes a much less severe form of the disease. This study reports the development of a real-time PCR minor groove binder MGB -Eclipse probe assay utilizing a unique sequence within the variola B9RB10R gene complex that reliably differentiates V. major from V. minor by specific probe melting temperatures Tms and genotyping analysis. The probe Tms for V. major and V. minor were 62.71 - 0.05 and 53.97 - 0.44 deg C, respectively P 0.001. We also used the identical sequence to develop a TaqMan -MGB assay that specifically detected V. minor but not V. major variants by qualitative analysis.