Dual-Probe Real-Time PCR Assay for Detection of Variola or Other Orthopoxviruses with Dried Reagents
ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FORT DETRICK MD VIROLOGY DIV
Pagination or Media Count:
A real-time, multiplexed PCR assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM 6-carboxyfluorescein-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET 6-carboxytetramethylrhodamine-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96 for variola and 98 for orthopoxviruses, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely-related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.
- Medicine and Medical Research