One-step SYBR green-based real-time RT-PCR assay for detection of foot-and-mouth disease virus circulating in India

Abstract

Rapid, sensitive, and reliable laboratory detection of foot-and-mouth disease virus (FMDV) infection is essential for containing and controlling virus infection in any geographical area. In this report a SYBR green-based 3D^pol-specific one-step real-time RT-PCR (rRT-PCR) assay was developed for the pan-serotype detection of FMDV in India. The detection limit of the SYBR green-based rRT-PCR was 10^-2 TCID₅₀/50 µl, which is 10 times more sensitive than the traditional agarose gel electrophoresis-based RT-multiplex PCR (RT-mPCR). The standard curve exhibited a linear range across 8-log₁₀ units of viral RNA dilution. The reproducibility and specificity of this assay were reasonably high suggesting that the 3D^pol-specific SYBR green rRT-PCR could detect FMDV genome specifically and with little run-to-run variation. The new 3D^pol-specific SYBR green rRT-PCR assay was evaluated alongside the established RT-mPCR using the archived FMDV isolates and clinical field samples from suspected FMD outbreaks. A perfect concordance was observed between the new rRT-PCR and the traditional RT-mPCR on viral RNA in the archived FMDV cell culture isolates tested. Furthermore, 73% of FMDV-suspected clinical samples were detected positive through the 3D^pol-specific SYBR green rRT-PCR, while the detection rate through the traditional RT-mPCR was 57%. Therefore, the SYBR green-based 3D^pol-specific one-step rRT-PCR could be considered as a valuable assay with higher diagnostic sensitivity to complement the routine assays that are being used for FMD virus diagnosis in India.

Keywords: Diagnosis; FMDV; Real-time PCR; SYBR green.