Development of a 1-step TaqMan real-time PCR method for detection of the Bovine Group A Rotavirus

Abstract

Background: The purpose of this study was to develop a 1-step real-time quantitative fluorescence polymerase chain reaction (QF-PCR) method for detecting Bovine Group A Rotavirus (BRVA). The primers and probe were designed targeting the VP6 gene of BRVA. The standard substance was obtained through in vitro transcription. The primers, probe concentration, and annealing temperatures were optimized to determine the optimal system and conditions for the reaction. The specificity, sensitivity, and repeatability of the method were assessed and compared with a reported real-time QF-PCR method for clinical samples.

Results: The results indicated that the detection method can achieve a sensitivity of 3.47 copies/μL and exhibit good specificity by exclusively detecting BRVA without cross-reactivity to other common pathogens in cattle and sheep. The standard curve exhibited a robust linear correlation, and the amplification efficiency was calculated to be 105%. The intra-group and inter-group coefficients of variation were less than 2%. A total of 96 clinical samples were tested and compared with the real-time QF-PCR method that was reported. The coincidence rate was 90.63% (87/96). Furthermore, the clinical samples revealed that the prevalence of BRV in cattle from Fujian Province was 85.42% (82/96).

Conclusion: This study has successfully developed a 1-step real-time QF-PCR method for BRVA, which offers an efficient and sensitive technical support for the rapid diagnosis and epidemiological investigation of BRVA.

Keywords: Bovine rotavirus; Detection method; Real-time PCR; TaqMan.