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猪繁殖与呼吸综合征病毒PCR-DHPLC检测新技术的建立及应用
引用本文:杨春华,祝建新,钟毅,孙思扬,沈艳,陈庆富,王川庆.猪繁殖与呼吸综合征病毒PCR-DHPLC检测新技术的建立及应用[J].中国兽医学报,2012,32(2):167-171.
作者姓名:杨春华  祝建新  钟毅  孙思扬  沈艳  陈庆富  王川庆
作者单位:1. 河南农业大学牧医工程学院,河南郑州450002/江西出入境检验检疫局检验检疫综合技术中心,江西南昌330002
2. 江西出入境检验检疫局检验检疫综合技术中心,江西南昌,330002
3. 河南农业大学牧医工程学院,河南郑州,450002
基金项目:国家质检局科技计划项目(2009IK029)
摘    要:应用PCR结合变性高效液相色谱(DHPLC)技术检测猪繁殖与呼吸综合征病毒(PRRSV),根据PRRSV GP5基因的序列特点设计特异性引物,PCR扩增产物经DHPLC技术进行快速检测。以猪细小病毒、猪圆环病毒Ⅱ型、猪流感病毒、猪瘟病毒和猪伪狂犬病毒进行特异性试验,无交叉反应,具有较好的特异性和重复性;对阳性标准品的检测结果表明,所建立的PCR-DHPLC法灵敏度可达1.0×101拷贝/μL。对16份疑似病料分别应用本试验所建立的PCR-DHPLC法与SYBR GreenⅠ实时荧光PCR法、PCR-凝胶电泳法、病毒培养法进行检测,发现有15份荧光定量PCR阳性,15份PCR-DHPLC阳性,12份PCR-凝胶电泳阳性。结果表明,建立的PCR-DHPLC法具有特异、敏感、快速、重复性好等优点,可用于临床PRRSV感染的早期诊断以及分子流行病学调查。

关 键 词:猪繁殖与呼吸综合征病毒  GP5基因  变性高效液相色谱  实时荧光PCR

Development and application of PCR-DHPLC assays for detection of the porcine reproductive and respiratory syndrome virus
YANG Chun-hua,ZHU Jian-xin,ZHONG Yi,SUN Si-yang,SHEN Yan,CHEN Qing-fu,WANG Chuan-qing.Development and application of PCR-DHPLC assays for detection of the porcine reproductive and respiratory syndrome virus[J].Chinese Journal of Veterinary Science,2012,32(2):167-171.
Authors:YANG Chun-hua  ZHU Jian-xin  ZHONG Yi  SUN Si-yang  SHEN Yan  CHEN Qing-fu  WANG Chuan-qing
Institution:1(1.College of Animal Science and Veterinary Medicine,Henan Argricultural University,Zhengzhou 450002,China;2.Jiangxi Entry-Exit Inspection and Quarantine Bureau,Nanchang 330002,China)
Abstract:To identify the porcine reproductive and respiratory syndrome(PRRS),a PCR-DHPLC assay was performed in this study.Primers specific for the partial region of the PRRSV GP5 gene were selected to conduct the PCR-DHPLC assays.The specific testing was performed with PRRS and PPV,PCV-Ⅱ,SIV,CSFVand PRV,without cross reation,with good specification and nice repeatability.Sensitivity analysis showed that the developed PCR-DHPLC could detect 1.0×101 copy/μL.Then the established method was used to detect the clinical samples,15 positives could be observed by PCR-DHPLC for 16 suspicious positive samples,it is consistent with SYBR GreenⅠreal-time PCR test and 12 positive by normal RT-PCR.The method could be used in clinical diagnosis and epidemiological investigation.
Keywords:PRRSV  GP5 gene  DHPLC  real-time PCR
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