A novel quantitative real-time PCR with the GAPDH reference gene for peste des petits ruminants

Abstract

Peste des petits ruminants (PPR) is a serious acute, highly contagious disease caused by the peste des petits ruminants virus (PPRV). This study aims to establish a qRT-PCR assay with an internal amplification control for the rapid and accurate detection of PPRV. The primers and probes for PPRV N were based on the national standard of the diagnostic techniques for PPR of China, and a pair of primers and TaqMan probes for the internal reference gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was designed. Optimisation of the reaction conditions, specificity, sensitivity and reproducibility tests, and clinical sample detection were conducted. The results showed that the optimal primers and probe concentrations of PPRV were 0.4 μmol/l and 0.4 μmol/l, respectively, and were 0.4 μmol/l and 0.2 μmol/l for the reference gene GAPDH, respectively. The established method has no cross-reaction with other viruses. The minimum detection limit was 6.8 copies/µl for PPRV and 190 copies/µl for GAPDH. The coefficients of variation (CV%) of PPRV and GAPDH were both lower than 2%. The results suggest that the PPRV qRT-PCR method containing internal reference genes has strong specificity, high sensitivity, and good reproducibility. The addition of internal reference genes for the sample quality control improves the accuracy of the detection.

Keywords

• detection method
• fluorescence real-time quantitative pcr
• internal reference gene
• peste des petits ruminants virus