小反刍兽疫病毒实时荧光定量PCR检测方法的建立

Establishment of Real Time Fluorescence RT-PCR Detection Method for Peste des petits ruminants virus

为了建立一种快速检测小反刍兽疫病毒的方法。根据Gen Bank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 bpH基因,构建标准质粒p MD-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 GreenⅠ实时荧光定量PCR检测方法。最优反应体系25μL:2×SYBR qPCR Mix 12. 5μL,模板DNA 1μL,特异引物各0. 2μL(0. 2μmol/L),DEPC H2O 10. 5μL;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. 28×10-2copies/μL 7个浓度梯度呈良好线性关系y=-2. 913x+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。

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 H 2 O 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 3.28×10^4 ~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 10000 times sensitive than normal PCR.The method is of great significance to the accurate diagnosis and quantitative detection of PPRV.