猪急性腹泻综合征病毒RT-PCR检测方法的建立及应用
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广东省生猪产业体系疾病岗位资金资助项目


Establishment and Application of a RT-PCR Assay for Detection of Porcine Acute Diarrhea Syndrome Coronavirus
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    摘要:

    为建立一种鉴定猪急性腹泻综合征病毒(SADS-CoV)的RT-PCR方法,根据NCBI登录的SADS-CoV N基因保守区域序列,设计了3对引物(P1、P2和P3),通过对SADS-CoV及其他6种病毒进行检测,筛选出最合适的引物,然后对退火温度、引物浓度、dNTPs浓度、rTaq DNA聚合酶浓度和循环次数等反应条件进行了优化。结果显示,P3引物对SADS-CoV特异性最好,与其他病毒无交叉反应。敏感性试验显示,该方法最低检测限可达9.55×102 copies/μL;重复性试验显示该方法重复性良好。运用建立的RT-PCR方法,对广东省的21份临床样品进行检测,结果检出SADS-CoV阳性样品4份,阳性样品的测序结果与其RT-PCR完全一致。结果表明,本研究成功建立了一种鉴定SADS-CoV的RT-PCR方法,且该方法具有检测快速、成本廉价、特异性强、敏感性高和重复性好的优点,可以用于SADS-CoV的临床诊断。

    Abstract:

    In order to establish a RT-PCR assay for detection of porcine acute diarrhea syndrome virus(SADS-CoV),three pairs of primers(P1,P2 and P3)were designed based on N gene sequence of SADS-CoV registered in NCBI,and the most appropriate primer was selected through the detection of SADS-CoV and other six kinds of viruses,followed by the optimization of reaction conditions including annealing temperature,primer concentration,dNTPs concentration,rTaq DNA polymerase concentration,cycle index,etc. The results showed that P3 primer was with the best specificity against SADS-CoV,and failed to react with other viruses. It was shown that,by sensitivity test,the lowest detection limit was 9.55×102 copies/μL;the established assay was with good repeatability as tested. 21 clinical samples from Guangdong were detected by the RT-PCR assay,4 positive samples were found,which was consistent with the sequencing results. Therefore,a RT-PCR assay was successfully established for detection of SADS-CoV,which was characterized by rapid detection,low cost,strong specificity,high sensitivity and good repeatability,and could be used for identification of SADS-CoV in practice.

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司广斌,陈志飞,梁文清,陈一波,贺东生.猪急性腹泻综合征病毒RT-PCR检测方法的建立及应用[J].《中国动物检疫》编辑部,2020,37(11):76-81.

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  • 在线发布日期: 2020-11-02
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