| 基于TaqMan探针的猪细小病毒实时荧光定量PCR方法的建立 |
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| 引用本文： | 陈红英,李新生,崔保安,杨明凡,金钺,刘金朋.基于TaqMan探针的猪细小病毒实时荧光定量PCR方法的建立[J].湖南农业大学学报(自然科学版),2008,34(6). |
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| 作者姓名： | 陈红英 李新生 崔保安 杨明凡 金钺 刘金朋 |
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| 作者单位： | 1. 河南农业大学,牧医工程学院,河南,郑州,450002;河南省动物性食品安全重点实验室,河南,郑州,450002
2. 河南农业大学,牧医工程学院,河南,郑州,450002 |
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| 基金项目： | 河南省杰出人才创新基金项目(0621002100) |
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| 摘 要： | 根据GenBank公布的猪细小病毒VP2基因序列，利用Premierexpress软件设计并合成了1对引物和1条相应的TaqMan探针，从猪细小病毒感染的PK．15细胞中提取DNA，经PCR扩增VP2基因，纯化的PCR产物克隆入pGEM-TEasy载体中，构建重组质粒．转化大肠杆菌JM109，经PCR及测序鉴定后得到阳性重组质粒，作为荧光定量PCR标准品模板建立标准曲线．在25此扩增反应体系中，对探针浓度、引物浓度、镁离子浓度和退火温度进行优化，建立了最佳的荧光定量PCR反应体系和扩增程序．该方法能够特异、定量检测猪细小病毒，灵敏度达1．12TCID50／mL．
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| 关 键 词： | 猪细小病毒 检测 实时荧光定量PCR |
Development of rea1-time fluorescent quantitative PCR assay based on TaqMan probe for detection of porcine parvovirus |
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| CHEN Hong-ying,LI Xin-sheng,CUI Bao-an,YANG Ming-fan,JIN Yue,LIU Jin-peng.Development of rea1-time fluorescent quantitative PCR assay based on TaqMan probe for detection of porcine parvovirus[J].Journal of Hunan Agricultural University,2008,34(6). |
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| Authors: | CHEN Hong-ying LI Xin-sheng CUI Bao-an YANG Ming-fan JIN Yue LIU Jin-peng |
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| Abstract: | The structural protein 2(VP2) nucleotide sequences of porcine parvovirus(PPV) were retrieved from the GenBank and aligned using the software program DNAstar.One pair of primers and a TaqMan probe were designed using the Primer express(version 2.0) software based on a highly conserved sequence within the VP2 region of the PPV genome.VP2 gene was amplified by PCR from DNA extracted from the PK-15 porcine kidney cells infected by 7909 isolates of PPV,and then was cloned and sequenced.And the positive recombina... |
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| Keywords: | porcine parvovirus detection rea1-time fluorescent quantitative PCR |
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