[1]李 军,蔡良语,陈 兵,等.猪传染性胃肠炎病毒、猪流行性腹泻病毒和猪轮状病毒A多重荧光定量RT-PCR检测方法的建立[J].中国预防兽医学报,2018,(09):801-805.[doi:0.3969/j.issn.1008-0589.201805029]
 LI Jun,CAI Liang-yu,CHEN Bing,et al.Development of a multiplex real-time reverse transcriptase PCR assay for simultaneous detection of TGEV, PEDV and PRoVA[J].Chinese journal of preventive veterinary medicine,2018,(09):801-805.[doi:0.3969/j.issn.1008-0589.201805029]
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猪传染性胃肠炎病毒、猪流行性腹泻病毒和猪轮状病毒A多重荧光定量
RT-PCR检测方法的建立
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年09
页码:
801-805
栏目:
诊断和检测技术
出版日期:
2018-09-17

文章信息/Info

Title:
Development of a multiplex real-time reverse transcriptase PCR assay for simultaneous detection of TGEV, PEDV and PRoVA
文章编号:
1008-0589(2018)9-0801-05
作者:
 

李 军1蔡良语2陈 兵3刘建利3孙 洁3陈书琨3林庆燕3陶 虹3吴绍强4卢体康3陈金顶1秦智锋3*

 (1. 华南农业大学,广东 广州510642;2. 武汉轻工大学,湖北 武汉430023;3. 深圳出入境检验检疫局动植物
检验检疫技术中心,广东 深圳518045;4. 中国检验检疫科学研究院,北京 100176)
Author(s):
 

LI Jun1 CAI Liang-yu2 CHEN Bing3 LIU Jian-li3 SUN Jie3 CHEN Shu-kun3 LIN Qing-yan3 TAO Hong3 WU Shao-qiang4 LU Ti-kang3 CHEN Jin-ding1 QIN Zhi-feng3*

 (1. South China Agricultural University, Guangzhou 510642, China; 2. Wuhan Polytechnic University, Wuhan 430023, China;
3. Shenzhen Entry-Exit Inspection & Quarantine Bureau, Shenzhen 518045, China;
4. Chinese Academy of Inspection and Quarantine, Beijing 100176, China)
关键词:
猪传染性胃肠炎病毒猪流行性腹泻病毒猪轮状病毒A荧光定量RT-PCR检测多重检测
Keywords:
transmissible gastroenteritis  porcine epidemic diarrhea virus  porcine rotavirus A  real time RT-PCR multiplex test 
分类号:
S852.65
DOI:
0.3969/j.issn.1008-0589.201805029
文献标志码:
A
摘要:
为对引起腹泻症状的猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)和猪轮状病毒A(PRoVA) 3种病毒同时快速地鉴别检测,本研究对GenBank中登录的TGEV的S基因、PEDV的S基因、PRoVA的NSP基因进行序列分析,分别设计了针对3种病毒的特异性引物和用3种荧光基团标记的核酸探针,通过条件优化建立了针对这3种病毒的多重荧光定量RT-PCR方法。结果显示,该方法能够特异性检测PEDV、TGEV和PRoVA,而与其它病原无交叉反应;对TGEV、PEDV和PRoVA的最低检测量分别为1.12 拷贝/μL、39 拷贝/μL和25 拷贝/μL;组内和组间变异系数均小于5 %;该方法与商品化的试剂盒检测结果符合率达100 %。该方法快速、敏感,检测结果准确可靠,为猪病毒性腹泻疾病的流行病学调查和分子生物学快速诊断奠定了基础。
  
Abstract:
In order to simultaneous detection of the transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine rotavirus A (PRoVA) which have the similar clinical signs, a multiplex real-time reverse transcriptase PCR (mrRT-PCR) assay was developed with three sets of probes labeled with three different fluorophore and primers designed according to the S gene of TGEV, S gene of PEDV and NSP gene of PRoVA to rapidly distinguish TGEV, PEDV and PRoVA (TGEV/PEDV/PRoVAmrRT-PCR). The TGEV/PEDV/PRoVA mrRT-PCR assay was specific, and had no false positive and no false negative detection results. The sensitivity of the TGEV/PEDV/PRoVA mrRT-PCR assay was  1.12copies/μL for TGEV,  39copies/μL for PEDV, and 25copies/μL for PRoVA, respectively.  The repeatability of the assay was acceptable with CV≤5% and the consistency was 100% with commercial kits. The developed TGEV/PEDV/PRoVA mrRT-PCR assay was a promising method for monitor and surveillance of swine viral diarrhea diseases.

参考文献/References:

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(本文编辑:李 爽)

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更新日期/Last Update: 2018-09-28