Development and application of a one‐step real‐time RT‐PCR using a minor‐groove‐binding probe for the detection of a novel bunyavirus in clinical specimens

Abstract A highly sensitive one‐step real‐time RT‐PCR method using a minor‐groove‐binding (MGB) probe was developed for detection and quantitation of severe febrile with thrombocytopenia syndrome virus (SFTSV). The assay could discriminate SFTSV infection from other related viral diseases in human with a minimum detection limit of 10 viral RNA copies/µl and was 1,000 times more sensitive than the conventional PCR. Strong linear correlations (r 2 > 0.99) between the C t values and viral RNA standards over a linear range were obtained. The coefficients of variation of intra‐ and inter‐assay reproducibility were both less than 2%. The RT‐PCR was also shown to be highly specific, as no positive signals were detected for other related viruses. Evaluation of this assay with serum samples from laboratory confirmed cases and healthy donors showed 100% clinical diagnostic sensitivity and over 99% specificity. Clinical application with samples from 287 patients admitted to the hospital with suspected SFTSV infection showed that 15% were infected by SFTSV. This assay was rapid, requiring just over 2 hr, including the nucleic acid extraction step. J. Med. Virol. 85:370–377, 2013. © 2012 Wiley Periodicals, Inc.