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当前位置: 首页 > 文章 > 猪流行性腹泻病毒实时荧光RPA等温检测方法的建立 中国畜牧兽医 2017 (12) 3434-3439
Position: Home > Articles > Establishment of Real-time Recombinase Polymerase Amplification for Detection of Porcine Epidemic Diarrhoea Virus China Animal Husbandry & Veterinary Medicine 2017 (12) 3434-3439

猪流行性腹泻病毒实时荧光RPA等温检测方法的建立

作  者：
吕继洲;范燕茹;冯春燕;袁向芬;吴绍强
关键词：
猪流行性腹泻病毒;分子检测;重组酶聚合酶扩增(RPA);等温扩增
摘  要：
为建立一种简单、快速的猪流行性腹泻病毒(PEDV)分子检测方法,本研究基于PEDV病毒M基因保守序列,设计一系列扩增引物及其荧光探针,以包含PEDV病毒M基因片段的pUC57质粒为模板,同时以包含猪传染性胃肠炎病毒(TGEV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)及古典猪瘟病毒(CSFV)等病毒膜蛋白或核衣壳蛋白基因序列的质粒作为对照,建立了一种PEDV实时荧光重组酶聚合酶扩增(RPA)等温检测方法,测定了方法的特异性与敏感性。结果表明,该实时荧光RPA方法可在39℃恒温反应20min特异性扩增PEDV病毒M基因,与TGEV、PRRSV等对照病毒基因无交叉反应,其检测限为10拷贝/μL。应用该实时荧光RPA方法可有效检测出血浆及血浆蛋白粉中的PEDV核酸。本研究建立的实时荧光RPA检测方法简单、快速、灵敏度高,可为PEDV的检测防控提供一种新的、可靠的技术支持。
译  名：
Establishment of Real-time Recombinase Polymerase Amplification for Detection of Porcine Epidemic Diarrhoea Virus
作  者：
LV Ji-zhou;FAN Yan-ru;FENG Chun-yan;YUAN Xiang-fen;WU Shao-qiang;Institute of Animal Quarantine,Chinese Academy of Inspection and Quarantine;
单  位：
Institute of Animal Quarantine,Chinese Academy of Inspection and Quarantine
关键词：
porcine epidemic diarrhoea virus(PEDV);;molecular diagnosis;;recombinase polymerase amplification(RPA);;isothermal amplification
摘  要：
To develop aprecise and rapid diagnosis method for detecting porcine epidemic diarrhoea virus(PEDV),a series of recombinase polymerase amplification(RPA)primers and exoprobes were established based on the highly conserved M gene of PEDV.Then a Real-time RPA assay was developed to detect PEDV using pUC57 plasmid carrying M gene fragment of PEDV as template,and the membrane or nucleotide capsid proteins from TGEV,PRRSV,PCV2 and CSFV were utilized as control.Then the sensitivity and specificity of this Real-time RPA assay was evaluated.The results showed that the Real-time reaction could detect PEDV specifically at 39℃within 20 min with the detection limit of 10 copies/μL of plasmid DNA,and there was no crossreaction with other control viral pathogens.Besides,the established Real-time PRA method could successfully detecte the PEDV Mgene in the plasma and plasma protein power.The Real-time established in this study was simple,rapid and sensitive,which could be a novel and reliable method for diagnosing and control of PED.

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