[1]王贝贝,吴艳阳,高冬生,等. 禽偏肺病毒通用型EvaGreen 荧光定量RT-PCR检测方法的建立[J].中国预防兽医学报,2018,(10):908-912.[doi:10.3969/j.issn.1008-0589.201802014]
 WANG Bei-bei,WU Yan-yang,GAO Dong-sheng,et al. Development of a universal EvaGreen-based reverse transcription quantitative PCR method for detection of avian metapneumovirus[J].Chinese journal of preventive veterinary medicine,2018,(10):908-912.[doi:10.3969/j.issn.1008-0589.201802014]
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禽偏肺病毒通用型EvaGreen 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10
页码:
908-912
栏目:
诊断和检测技术
出版日期:
2018-11-15

文章信息/Info

Title:
 

Development of a universal EvaGreen-based reverse transcription quantitative PCR method for detection of avian metapneumovirus

文章编号:
1008-0589(2018)10-0908-05
作者:
 

王贝贝吴艳阳高冬生刘延珂万文妍杨宁霞王新卫*赵 军*

 

(河南农业大学 禽病研究所,河南 郑州 450002)

Author(s):
 

WANG Bei-bei WU Yan-yang GAO Dong-sheng LIU Yan-ke WAN Wen-yan

YANG Ning-xia WANG Xin-wei* ZHAO Jun*

 

(College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China)

关键词:
禽偏肺病毒通用型EvaGreenRT-PCR
Keywords:
 

avian metapneumovirus universal EvaGreen RT-PCR

分类号:
S852.65
DOI:
10.3969/j.issn.1008-0589.201802014
文献标志码:
A
摘要:
 

为建立各亚型禽偏肺病毒(aMPV)的快速检测方法,本研究对aMPV 4个亚型的核苷酸序列比对分析,针对aMPV N基因的保守区域设计了一对特异性引物,通过条件优化,建立了检测aMPV各亚型的通用型EvaGreen荧光定量 RT-PCR方法。结果显示,该方法在模板为103拷贝/μL~108拷贝/μL范围内具有良好的线性关系;检测下限为103拷贝/μL,敏感性为常规RT-PCR的10倍。与其它常见禽类病毒无交叉反应;组内重复试验和组间重复试验的变异系数分别为0.89 %~1.34 %和2.01 %~3.29 %,具有较好的重复性。利用所建立的方法对河南省部分地区鸡场送检病鸡样品进行检测,结果显示被检地区鸡场的aMPV的平均阳性率为46.66 %,与普通RT-PCR的符合率为100 %。本研究建立的EvaGreen 荧光定量RT-PCR方法可以用于aMPV的检测,为aMPV感染的流行病学调查提供了可行方法。

  

Abstract:
 

In order to establish a rapid assay for detect all subtypes of avian metapneumovirus (aMPV), a universal EvaGreen-based reverse transcription quantitative PCR (RT-qPCR) assay was developed with the primers targeting the conserved region of N genes of aMPV subtypes. The standard curve for each subtype of aMPV showed a linear relationship between cycle threshold (Ct) and template concentration ranging from 103copies/μL to 108copies/μL. The detection limit of the developed EvaGreen RT-qPCR was 103copies/μL, which was ten-fold more sensitive than that of conventional RT-PCR. The developed EvaGreen RT-qPCR had no cross reactivity with other common avian pathogens and had high reproducibility with coefficient variation of 0.89% to 1.34% and 2.01% to 3.29% in intra- and inter-assay, respectively. Moreover, the developed EvaGreen RT-qPCR was used to aMPV surveillance in some poultry farms in Henan province, and the average positive rate was 46.6%. The coincidence rate of the developed EvaGreen RT-qPCR with conventional RT-PCR was 94.7%. The developed universal EvaGreen qRT-PCR could be used for detecting all subtypes of aMPV, which provided a technical support for aMPV epidemiological investigation.

参考文献/References:

 

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(本文编辑:彭永刚)

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更新日期/Last Update: 2018-11-28