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猪生殖与呼吸综合征病毒的SYBR Green-Ⅰ
荧光定量PCR检测
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王小武1，符芳1，许红喜2，周艳君1，童光志1，李曦1*
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（1.中国农业科学院 哈尔滨兽医研究所，黑龙江 哈尔滨150001；
2.黑龙江八一农垦大学，黑龙江 大庆163319）
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摘要：
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根据猪生殖与呼吸综合征病毒(PRRSV) GP5蛋白基因核酸序列设计引物，经BLAST比较发现，该引物既可以扩增较早发现的PRRSV毒株，也可以扩增近期发现的在Nsp2基因上有较大变异的PRRSV毒株，以含有该引物扩增序列的重组质粒作为标准品建立了检测PRRSV的SYBR Green-Ⅰ荧光定量PCR方法。结果表明，该方法标准曲线的相关系数大于0.99，敏感性可以达到1×101拷贝/μL，约为普通PCR的100倍；用该方法检测人工感染PRRSV的猪全血，结果阳性检出率为100%。敏感性、特异性和稳定性试验表明，该方法具有很高的敏感性、特异性和稳定性，可以用来快速检测PRRSV。 |
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关键词：
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猪生殖与呼吸综合征病毒；SYBR Green-Ⅰ；荧光定量PCR |
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中图分类号：S 852.659.6：Q 503
文献标识码：
A 文章编号：1673-4696(2008)05-0401-06 |
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Establishment of SYBR Green-Ⅰ real-time fluorescent quantitative PCR
for detection of porcine reproductive and respiratory syndrome virus |
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WANG Xiao-wu1， FU Fang1，XU
Hong-xi2，ZHOU Yan-jun1，TONG Guang-zhi1，LI
Xi1 |
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(1.Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences，Harbin 150001,China;
2.Heilongjiang Eight-First Land Reclamation University,Daqing 163319，China) |
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Abstract: A pair of primers were designed based on the sequences of PRRSV GP5 protein gene.The amplicon was 502bp,which was used to develop SYBR Green-Ⅰ fluorescent quantitative PCR for detecting PRRSV.The results showed that r values exceeded 0.99 in the standard curve of the developed PCR met-hod.The detection limit of the PCR was 10copies/μL,which was 100 fold sensitive than that of the conventional PCR.The 54 sera from the pigs infected-experimentally with PRRSV were examined by the PCR,and the positive detection rate was 100%.This PCR assay was sensitive,specific and stable for rapid detection of PRRSV,with a positive detection rate of 100%. |
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Key words: porcine reproductive and respiratory syndrome virus(PRRSV);SYBR Green-Ⅰ;real-time quantitative PCR |
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