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

The LUX(TM) [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(TM) approach, a variation of the LUX(TM) 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 con-elation 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. (C) 2005 Elsevier Ltd. All rights reserved.