[1]王建华*,陈小金,赵 丹,等.非洲猪瘟病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒多重TaqMan荧光定量RT-PCR检测方法的建立[J].中国预防兽医学报,2017,(11):907-911.[doi:10.3969/j.issn.1008-0589.201611038]
 WANG Jian-hua*,CHEN Xiao-jin,ZHAO Dan,et al.Establishment of multiplex TaqMan-MGB real-time RT-PCR for detection of classical swine fever virus, african swine fever virus,and porcine reproductive and respiratory syndrome virus[J].Chinese journal of preventive veterinary medicine,2017,(11):907-911.[doi:10.3969/j.issn.1008-0589.201611038]
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非洲猪瘟病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒
多重TaqMan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:
2017年11
页码:
907-911
栏目:
诊断和检测技术
出版日期:
2017-11-15

文章信息/Info

Title:
Establishment of multiplex TaqMan-MGB real-time RT-PCR for detection of classical swine fever virus, african swine fever virus,and porcine reproductive and respiratory syndrome virus
文章编号:
1008-0589(2017)11-0907-05
作者:
 

王建华*陈小金赵 丹王玉玲张俊哲肖 妍董志珍赵祥平

 (天津出入境检验检疫局  动植物与食品检测中心,天津 300456)
Author(s):
 

WANG Jian-hua* CHEN Xiao-jin ZHAO Dan WANG Yu-ling ZHANG Jun-zhe XIAO Yan DONG Zhi-zhen ZHAO Xiang-ping

 (The Animals and Plants and Food Inspection Center of Tianjin Entry-Exit Inspection and Quarantine, Tianjin 300456, China)
关键词:
非洲猪瘟病毒猪瘟病毒猪繁殖与呼吸综合征病毒多重荧光定量RT-PCR
Keywords:
  ASFV  CSFV  HP-PRRSV  multiplex real-time RT-PCR
分类号:
S852.65
DOI:
10.3969/j.issn.1008-0589.201611038
文献标志码:
A
摘要:
为建立同时检测非洲猪瘟病毒(ASFV)、猪瘟病毒(CSFV)和高致病性猪繁殖与呼吸道综合征病毒(HP-PRRSV)的方法,本研究根据ASFV CP530R基因、CSFV 5’UTR基因和HP-PRRSV NSP2基因,分别设计了3对特异性引物和TaqMan水解探针,建立了同时检测这3种病毒的多重荧光定量RT-PCR方法,并对其反应条件进行优化。结果表明,该方法仅对ASFV、CSFV和HP-PRRSV呈现特异性扩增,不与伪狂犬病毒、猪细小病毒和猪圆环病毒2型的DNA以及猪流行腹泻病毒和猪流感病毒的cDNA发生交叉反应;该方法对ASFV、CSFV和HP-PRRSV的最低检出量分别为61拷贝/μL、11拷贝/μL和41拷贝/μL;组内和组间重复性试验的Ct值变异系数均小于2.5 %,具有良好的重现性。用该方法对276份临床样品进行ASFV、CSFV和HP-PRRSV的检测,所有样品的ASFV检测结果均为阴性,CSFV检测单阳性样品6份,HP-PRRSV检测单阳性样品22份,CSFV和HP-PRRSV检测双阳性样品4份。本研究建立的方法为临床样品中ASFV、 CSFV和HP-PRRSV的同时检测提供了一种快速、敏感和特异的技术手段。
Abstract:
To develop an assay for simultaneous differential detection of African swine fever virus(ASFV), classical swine fever virus (CSFV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), a multiplex real-time RT-PCR method was established with 3 pairs of specific primers targeting the sequence of ASFV CP530R gene, CSFV 5’UTR and HP-PRRSV NSP2 gene, along with 3 kinds of TaqMan probes labeled with different, distinguishable reporter dye for each virus. After optimizing the reaction conditions, the multiplex real-time RT-PCR assay was able to specifically detect ASFV, CSFV and HP-PRRSV simultaneously with the detection limit of 61 copies/μL for ASFV, 11copies/μL for CSFV and 41 copies/μL for HP-PRRSV, respectively, but no genomic DNA or cDNA from other virus were amplified such as pseudorabies virus. Moreover, the variation coefficient of intra- and inter-assays were both less than 2.5%. Furthermore, 276 samples tested by the multiplexed assay, all samples were negative for ASFV, whilst 6 were positive for CSFV, 22 positive for HP-PRRSV and 4 positive for both CSFV and HP-PRRSV. The method established in this study could provide a rapid, sensitive and specific technique for simultaneous detection of ASFV, CSFV and HP-PRRSV in clinical samples.

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(本文编辑:李    爽)

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更新日期/Last Update: 2017-12-13