羊痘病毒实时荧光定量PCR检测方法的建立
发布时间：2018-09-02 08:44
【摘要】：本研究参考GenBank中山羊痘病毒基因组序列(AY077836)和绵羊痘病毒基因组序列(AY077832),针对A29L基因的核苷酸序列,设计1对特异性引物和两条TaqMan探针,同时制备重组质粒标准品。通过退火温度、引物浓度和探针浓度的筛选及优化,将标准阳性质粒10倍倍比稀释后,作实时荧光定量PCR标准曲线,建立了检测羊痘病毒的实时荧光定量PCR方法,.对所建立的实时荧光定量PCR方法的特异性、灵敏性和重复性进行了评价,并用此方法对临床样品进行了检测。结果显示：建立的实时荧光定量PCR的反应条件为退火温度58.4℃,引物浓度400 nmol/L,探针浓度200 nmol/L。该诊断方法与其它四种病毒不发生交叉反应,并且山羊痘病毒和绵羊痘病毒之间也不发生交叉反应。该方法的重复性试验变异系数均低于2%,并且双重实时荧光定量PCR最低浓度检测限分别为470fg和440fg。对标准阳性模板进行定量检测,生成的标准曲线相关系数分别为0.995和0.997。结果表明所建立的方法具有良好的特异性、灵敏性、稳定性,可以对GTPV和SPPV进行准确的定量检测。对19份临床样品进行检测,GTPV阳性有14份,SPPV阳性有4份,GTPV和SPPV混合感染有1份。
[Abstract]:According to the genomic sequence of GenBank Zhongshan sheep pox virus (AY077836) and sheep pox virus (AY077832), a pair of specific primers and two TaqMan probes were designed for the nucleotide sequence of A29L gene. Through the screening and optimization of annealing temperature, primer concentration and probe concentration, a real-time fluorescent quantitative PCR method for the detection of sheep pox virus was established by diluting the standard positive plasmid by 10 times specific dilution and using real-time fluorescence quantitative PCR curve. The specificity, sensitivity and reproducibility of the established real-time fluorescent quantitative PCR method were evaluated, and the clinical samples were detected by this method. The results showed that the reaction conditions of real-time fluorescent quantitative PCR were: annealing temperature 58.4 鈩，
本文编号：2218842
[Abstract]:According to the genomic sequence of GenBank Zhongshan sheep pox virus (AY077836) and sheep pox virus (AY077832), a pair of specific primers and two TaqMan probes were designed for the nucleotide sequence of A29L gene. Through the screening and optimization of annealing temperature, primer concentration and probe concentration, a real-time fluorescent quantitative PCR method for the detection of sheep pox virus was established by diluting the standard positive plasmid by 10 times specific dilution and using real-time fluorescence quantitative PCR curve. The specificity, sensitivity and reproducibility of the established real-time fluorescent quantitative PCR method were evaluated, and the clinical samples were detected by this method. The results showed that the reaction conditions of real-time fluorescent quantitative PCR were: annealing temperature 58.4 鈩，
本文编号：2218842
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