TaqMan real-time RT‒PCR assay with an internal amplification control for rapid detection of Bluetongue virus

Abstract

Bluetongue (BT) is an arbovirus disease caused by Bluetongue virus (BTV), which has spread all over the world. In this study, a qRT-PCR assay with an internal amplification control was established for preventing false-negative results in Bluetongue virus (BTV) detection. The primers and probes for BTV NS3 were based on the qRT-PCR method for the detection of BTV in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (2022) of the World Organization for Animal Health (WOAH). The GAPDH gene was selected as the internal amplification control, and the primers and probes for the GAPDH gene were designed based on the analysis of conserved sequences in the genome sequences of cattle, sheep and goats. Optimization of the reaction conditions, specificity, sensitivity and repeatability tests were conducted, and a qRT-PCR method was established for the simultaneous detection of BTV and the reference gene GAPDH. The results showed that the optimal primers and probe concentrations of BTV-NS3 were 0.30 μmol/L and 0.25 μmol/L, and were 0.15 μmol/L and 0.20 μmol/L for the reference gene GAPDH. The established method has no cross-reaction with other viruses, which showed good specificity. The minimum detection of BTV- and GAPDH-positive plasmid standard copies was 6.685 copies/μL and 209 copies/μL, respectively. The intra-assay and interassay CVs of the Ct values measured by BTV were less than 1.2%, and the intra-assay and interassay CVs of the Ct values measured by GAPDH were less than 0.7%, which suggested a high degree of repeatability and stability for this assay. The established BTV qRT-PCR method was used to detect BTV in 88 sheep serum samples stored in the laboratory, revealing 4 BTV-positive samples with a positive rate of 4.55%, which was consistent with the result obtained by nested PCR for detecting BTV recommended by WOAH. The qRT-PCR method established in this study for simultaneously detecting BTV and the internal reference gene GAPDH has high sensitivity, strong specificity and high repeatability. The addition of an internal reference gene can effectively control the detection accuracy and enable false-negative results to be avoided. It can provide a reference for the standardization of qRT-PCR methods for Bluetongue disease.