鸭坦布苏病毒快速实时荧光RT-LAMP方法的建立及初步应用
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S855.3

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国家现代农业产业技术(水禽)体系资助项目(CARS-42);云南省廖明专家工作站项目(202105AF150077);云南省重大科技专项(202102AE090029)


Establishment and Preliminary Application of a Rapid Real-time Fluorescent RT-LAMP for Duck Tembusu Virus
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    摘要:

    为建立检测鸭坦布苏病毒(DTMUV)的荧光逆转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)检测方法,根据DTMUV NS5基因保守序列设计1套特异性引物,对反应时间和温度进行优化,并评价该方法的特异性、敏感性、重复性和临床适用性。结果显示:建立的荧光RT-LAMP最优反应条件为65 ℃ 25 min;只特异性扩增DTMUV,而不扩增其他几种禽类病毒;最低检测限为4×102 copies/μL,与qRT-PCR方法敏感性一致,是普通RT-PCR的100倍;批内和批间重复性高;应用该方法检测DTMUV不同剂量人工感染鸭肝脏样品,对接毒剂量为103.7 TCID50和102.7 TCID50组的阳性检出率均为100%,对接毒量101.7 TCID50组的阳性检出率为90%(9/10)。结果表明,本研究建立的荧光RT-LAMP检测方法特异性强、重复性好、敏感性高,可成为快速诊断DTMUV的方法之一。

    Abstract:

    In order to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay for detection of duck Tembusu virus(DTMUV),one set of special primers were designed based on the highly conservative sequence of DTMUV NS5 gene,the reaction time and temperature were optimized,followed by evaluation on specificity,sensitivity,repeatability and clinical applicability. The results showed that the optimal conditions for the established method were 65 ℃ for 25 minutes,only DTMUV could be specifically amplified,and no reaction with other avian viruses was observed;its minimum detection limit was 4×102 copies/μL,which was consistent with qRT-PCR and 100 times higher than that of conventional RT-PCR;with high intra and inter-assay repeatability,the method was applied to detect the liver samples from artificially infected ducks at different doses,the positive detection rates of the samples vaccinated with the challenge doses of 103.7 TCID50 and 102.7 TCID50 were 100%,that of those with 101.7 TCID50 was 90%(9/10). In conclusion,the established method was with high specificity,repeatability and sensitivity,and could be used for rapid diagnosis of DTMUV.

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王巧萍,粟柯衡,元正菊,常志顺,张薇,张以芳,信爱国.鸭坦布苏病毒快速实时荧光RT-LAMP方法的建立及初步应用[J].《中国动物检疫》编辑部,2022,39(5):103-109.

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  • 在线发布日期: 2022-05-07
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