Abstract: To establish a method for rapid detection of Peste des petits ruminants virus (PPRV). Two pairs of primers was designed according to H gene sequence of PPRV Nigeria75/1 that published by GenBank,the full-length cDNA of 1 830 bp H gene of PPCV was amplified by RT-PCR.The amplicon was subcloned to pMD-18 to construct a standard plasmid pMD-18-H for Real-time fluorescence PCR.After optimization of the conditions,the H gene of Peste des petits ruminants virus (PPRV) SYBR Green Ⅰ Real-time fluorescence qPCR detection method was established.The 25 μL optimal reaction system was composed of 2×SYBR qPCR Mix 12.5 L,PPRV cDNA 1 μL,F3 and F4 primer 0.2 μL each (0.2 mmol/L),DEPC H₂O 10.5 μL.The best reaction conditions was 94℃,2 min; followed by 40 cycles with 94℃ 5 s,57℃ 30 s.The standard curve displayed a good linear relationship between Ct value and initial amounts of tatal virus DNA at a range of 3.28×10⁴~3.28×10^-2 copies/μL.The correlation coefficient,amplification efficiency and slope were 0.994, 120.5% and -2.913,respectively.The established Real-time fluorescence quantitative PCR,which has high stability and strong specificity,is 10 000 times sensitive than normal PCR.The method is of great significance to the accurate diagnosis and quantitative detection of PPRV.

Key words: Peste des petits ruminants virus(PPRV), SYBR Green Ⅰ, Real-time fluorescence qPCR

CLC Number: 

• S852.65