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

Abstract

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.

Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays

“…The assay is also efficient since use of test plates specifically designed for the iCycler iQ ™ allowed assay of up to 384 samples/plate. The Weinstock, 2001 andMahlum et al, 2002), foot and mouth disease virus (Callahan et al, 2002), porcine endogenous retrovirus (Argaw et al, 2002), and rotavirus (Schwarz et al, 2002). Real-time RT-PCR assays were shown to be most efficient and sensitive for assay of clinical specimens infected with BVDV, when compared to gel-based RT-PCR assays, virus isolation tests, immunoperoxidase monolayer assays, and immunohistochemistry assays.…”

Disseminated Bovine Viral Diarrhea Virus in a Persistently Infected Alpaca (Vicugna Pacos) Cria

“…Real-time reverse transcription PCR (real-time RT-PCR) to detect and subtype BVDV in tissues was performed according to a duplex TaqMan real-time PCR procedure as previously described. 3 After extraction of total RNA with TRIzol c , BVDV-1 was detected in kidney, liver, spleen, lung, and thymus. Bovine viral diarrhea virus was isolated from the same tissues in BVDV-free bovine turbinate cells.…”

Johne’s disease, Mycoplasma and BVD in Utah-Bulk Tank Milk Testing and Comparison to Previous Regional Prevalence and Individual Herd Results over Time

“…Milk samples were screened for BVD virus using a VetMAX™-Gold BVDV Detection Kit (Applied Biosystems, Carlsbad, CA, USA) realtime reverse transcription PCR kit as per manufacturer's instructions as reported previously [36]. Before detection samples were extracted using the MagMAX™ Total Nucleic Acid Isolation Kit (Applied Biosystems, Carlsbad, CA, USA).…”