摘要： 为了建立一种快速检测小反刍兽疫病毒的方法。根据GenBank中公布的PPRV Nigeria75/1株H基因序列设计2对引物，PCR扩增出1 830 bp H基因，构建标准质粒pMD-18-H。以标准质粒优化反应条件，建立了小反刍兽疫病毒H基因SYBR Green Ⅰ实时荧光定量PCR检测方法。最优反应体系25 μL：2×SYBR qPCR Mix 12.5 μL，模板DNA 1 μL，特异引物各0.2 μL（0.2 μmol/L），DEPC H₂O 10.5 μL；最佳反应条件：94℃ 2 min；94℃ 5 s，57℃ 30 s，40个循环，Ct值与标准质粒模板浓度在3.28×10⁴~3.28×10^-2 copies/μL 7个浓度梯度呈良好线性关系y=-2.913 x+36.904，斜率为-2.913，扩增效率120.5%，相关系数R²=0.994。建立的实时荧光定量PCR检测方法比普通PCR灵敏度高10 000倍，稳定性好，特异性强，对PPRV的准确诊断和定量检测具有重要意义。

关键词: 小反刍兽疫病毒(PPRV), SYBR Green Ⅰ, 实时荧光定量PCR

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

中图分类号: 

• S852.65