Abstract
Peste des petits ruminants (PPR) isaserious acute, highly contagious disease caused bythe peste des petits ruminants virus (PPRV). This study aims toestablish aqRT-PCR assay with aninternal amplification control for the rapid and accurate detection ofPPRV. The primers and probes for PPRV Nwere based onthe national standard ofthe diagnostic techniques for PPR ofChina, and apair ofprimers and TaqMan probes for the internalreference gene ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was designed. Optimisation ofthe reaction conditions, specificity, sensitivity and reproducibility tests, and clinical sample detection were conducted. The results showed that the optimal primers and probe concentrations ofPPRV were 0.4μmol/l and 0.4μmol/l, respectively, and were 0.4μmol/l and 0.2μmol/l for the reference gene GAPDH, respectively. The established method has nocross-reaction with other viruses. The minimum detection limit was 6.8copies/μl for PPRV and 190copies/μl for GAPDH. The coefficients ofvariation (CV%) ofPPRV 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 ofinternal reference genes for the sample quality control improves the accuracy ofthe detection.
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