尼帕病毒实时荧光RT-PCR检测方法的建立
|
摘要
目的建立快速检测尼帕病毒的实时荧光RT-PCR方法。方法分析尼帕病毒N基因片段核苷酸序列的保守性,设计实时荧光RT-PCR引物和探针,优化引物和探针浓度,利用尼帕病毒DNA质粒作为阳性对照,确定最佳反应体系,并进行灵敏性、重复性、特异性检测。结果该方法对尼帕病毒DNA的最适线性检测范围为102~108拷贝/μl,检测下限为102拷贝/μl。对2个不同浓度(102、104拷贝/μl)的尼帕病毒DNA进行4次重复检测,具有良好的重复性。与登革病毒、基孔肯雅病毒、黄热病毒、乙脑病毒和汉坦病毒等无交叉反应。结论建立的尼帕病毒实时荧光RT-PCR法能准确、快速地检测尼帕病毒。
Objective To estsblish a fast and sensitive method for the detection of Nipah virus(NIV)by using Real-time RT-PCR.Methods The N segment sequences of NIV strains were analyzed.A pair of primers and probe were designed in the conserved region and used for Real-time RT-PCR.The optimal reaction system,sensitivity and specificity were determined by using NIV DNA positive control.Results The optimal linear detection range of NIV-N-DNA was 102-108copies/μl,and the detection limit was 102copies/μl.Four replicates of two different concentrationsof NIV-N-DNA were tested with good repeatability,and had no cross reaction with Dengue virus,Chikungunya virus,Yellow fever virus,Japanese encephalitis virus,Hataan virus.ConclusionThe Real-time RT-PCR method with accurate,rapid for NIV detection had been established,which could be used for the rapid detection of NIV.
Objective To estsblish a fast and sensitive method for the detection of Nipah virus(NIV)by using Real-time RT-PCR.Methods The N segment sequences of NIV strains were analyzed.A pair of primers and probe were designed in the conserved region and used for Real-time RT-PCR.The optimal reaction system,sensitivity and specificity were determined by using NIV DNA positive control.Results The optimal linear detection range of NIV-N-DNA was 102-108copies/μl,and the detection limit was 102copies/μl.Four replicates of two different concentrationsof NIV-N-DNA were tested with good repeatability,and had no cross reaction with Dengue virus,Chikungunya virus,Yellow fever virus,Japanese encephalitis virus,Hataan virus.ConclusionThe Real-time RT-PCR method with accurate,rapid for NIV detection had been established,which could be used for the rapid detection of NIV.
引文
[1]李小波,黄吉城.当前传入我国风险较大的几种新发传染病[J].中国人兽共患病学报,2018,34(2):182-187.
[2]刘丽娟,孙肖红.尼帕病毒和尼帕病毒病[J].中国国境卫生检疫杂志,2018,41(6):455-457.
[3] Chadha MS,Comer JA,Lowe L,et al. Nipah virus-associated encephalitis outbreak,Siliguri,India[J].Emerg Infect Dis,2006,12(2):235-240.
[4] Kulkarni DD,Tosh C,Venkatesh G,et al. Nipah virus infection:current scenario[J].Ingian J Virol,2013,24(3):398-408.
[5]孟玲,常昭瑞,王哲,等. 2018年6月中国大陆需关注的突发公共卫生事件风险评估[J].疾病监测,2018,33(6):447-451.
[6]吴玙彤,谢丽丽.尼帕病毒的诊断技术研究进展[J].猪群保健,2015,2(11):106-107.
[7]王建华,柴明骏,赵祥平,等.一种基于M基因的尼帕病毒TaqMan-MGB荧光RT-PCR检测方法的建立[J].中国兽医科学,2015,45(12):1277-1282.
[2]刘丽娟,孙肖红.尼帕病毒和尼帕病毒病[J].中国国境卫生检疫杂志,2018,41(6):455-457.
[3] Chadha MS,Comer JA,Lowe L,et al. Nipah virus-associated encephalitis outbreak,Siliguri,India[J].Emerg Infect Dis,2006,12(2):235-240.
[4] Kulkarni DD,Tosh C,Venkatesh G,et al. Nipah virus infection:current scenario[J].Ingian J Virol,2013,24(3):398-408.
[5]孟玲,常昭瑞,王哲,等. 2018年6月中国大陆需关注的突发公共卫生事件风险评估[J].疾病监测,2018,33(6):447-451.
[6]吴玙彤,谢丽丽.尼帕病毒的诊断技术研究进展[J].猪群保健,2015,2(11):106-107.
[7]王建华,柴明骏,赵祥平,等.一种基于M基因的尼帕病毒TaqMan-MGB荧光RT-PCR检测方法的建立[J].中国兽医科学,2015,45(12):1277-1282.