Two-color multiplex assay for the identification of orthopox viruses with real-time LUX- PCR.

The LUX™ [Light Upon eXtension] is a real-time detection system that can be used for the detection and quantification of pathogens nucleic acids. In this study we used a universal LUX™ approach, a variation of the LUX™ detection system, for identifying Orthopoxvirus nucleic acids in real time. This approach enables the design of sequence-specific primer sets in high identity genome sequences. The assay described here is designed to allow simultaneous detection of Variola and other orthopox viruses in a multiplex format, with a limit of detection in the range of 50–100 copies of the Orthopoxvirus genome. Regression analysis showed that the assay was linear over seven orders of magnitude, with 0.97 correlation coefficient. The sensitivity and specificity of the assay, as determined from a panel of 100 samples that contained nucleic acids from a variety of bacteria and viral species, were rated at 98%. Thus, the assay offers a sensitive and specific tool for simultaneous identification and quantification of Variola and other orthopox viruses, and the approach allows more flexible sequence-specific primers design for pox viruses as well as other microbial pathogens.