Fluorogenic RT-PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus

A single tube fluorogenic RT-PCR-based 'TaqMan' assay was developed for detection and classification of bovine viral diarrhea virus (BVDV). TaqMan-PCR was optimized to quantify BVD virus using the ABI PRISM 7700 sequence detection system and dual-labeled fluorogenic probes. Two different gene specific labeled fluorogenic probes for the 5' untranslated region (5' UTR) were used to differentiate between BVD types I and II. Sensitivity of the single tube TaqMan assay was compared with two-tube TaqMan assay and standard RT-PCR using 10-fold dilutions of RNA. Single tube TaqMan assay was 10-100-fold more sensitive than the two-tube TaqMan assay and the standardized single tube RT-PCR. Specificity of the assay was evaluated by testing different BVD virus strains and other bovine viruses. A total of 106 BVD positive and negative pooled or single serum samples, field isolates and reference strains were tested. Quantitation of cRNA from types I and II BVD virus was accomplished by a standard curve plotting cycle threshold values (C(T)) versus copy number. Single tube TaqMan-PCR assay was sensitive, specific and rapid for detection, quantitation and classification of BVD virus.