山羊痘病毒TaqMan实时荧光定量PCR检测方法的建立及应用

根据GenBank中山羊痘病毒(AY077835)gp063基因DNA序列,应用Beacon Designer 7.0软件,设计合成1对引物和1条TaqMan探针,同时制备含有靶DNA序列的pEASY-T1重组质粒标准品。通过引物、探针浓度的筛选及反应条件的优化,建立了山羊痘病毒TaqMan法实时荧光定量PCR。该方法组内和组间重复试验变异系数均低于2%,最低浓度检测极限值为1×100copies/μL,上机检测时间少于40 min。运用该方法对不同时期流行于云南局部的山羊痘临床组织病料进行定量检测,生成的标准曲线斜率(Slope)为-3.36,截距(Intercept)为41.08,相关系数(R2)为0.999 989,阳性对照及送检样品抽提DNA中病毒含量依次为:P(TK)2.70×105copies/μL,华坪(HP)毒株1.45×106copies/μL,景谷(JG)毒株5.12×105copies/μL,保山(BS)毒株2.96×105copies/μL,宣威(XW)毒株1.86×105copies/μL,永胜(YS)毒株1.36×103copies/μL。结果表明:所建立方法具有灵敏、特异、安全、快速的特点,适合于山羊痘的早期检测。

Development and Application of TaqMan Real-time PCR for the Quantitative Detection of Goatpox Virus

In order to develop a fluorescence quantitative detection method for goat pox virus （ GTPV）, one pair of primers and a probe were designed for TaqMan real-time PCR based on gp063 gene sequence of goat pox virus genome（ AY077835 ） available from NCBI GenBank. Meanwhile, pEASY - T1 recombinant plasmid that contains the target DNA sequence was prepared for quantitative detection of GTPV. With optimization of reaction conditions and concentration of primers and probe, a TaqMan real-time PCR assay for goat pox virus was developed. The coefficient of intra- and inter-variation of the assay were both less than 2% . The sensitiv- ity of the assay was 1×100 eopies/μL and the detection time was less than 40 rain. The developed assay was applied to quantitatively detect GTPV strains from clinical samples, which ever prevailed in some areas of Yunnan province in different periods. The slope, intercept and R2 of the standard curve was - 3.46, 41.08 and 0.999 989 respectively. The copies of GTPV virus in extracted DNA samples of TK （positive control）, HP, JG, BS, XW and YS was 2.70×105 copies/μL, 1.45×106 copies/μL, 5. 12×105 copies/μL, 2.96×105 copies/μL, 1.86×105 copies/μL and 1.36×103 copies/μL respectively. The results showed that the TaqMan real-time fluorescence quantitative PCR assay is sensitive, specific, fast and safe, thus is suited for early detection of goat pox.