[ Objective] To develop a rapid and visualized detection method of classical swine fever virus （CSFV） using reverse transcriptase loopmediated isothermal amplification （RT-LAMP）. [ Method ] A total of six special ...[ Objective] To develop a rapid and visualized detection method of classical swine fever virus （CSFV） using reverse transcriptase loopmediated isothermal amplification （RT-LAMP）. [ Method ] A total of six special primers were designed based on the conserved sequences of CSFV gene. After optimizing, the reaction of RT-LAMP was carded out at 63℃ for 45 rain. The RT-LAMP products were analyzed by agarose gel electro- phoresis. The sensitivity, specificity and repeatability were verified, respectively. [ Result] The RT-LAMP method could be used for detecting CSFV rather than six generic viruses. The sensitivity of RT-LAMP was 100 times higher than that of RT-PCR. The detection of 27 clinical samples by RT- LAMP and RT-PCR showed that RT-LAMP is more reliable and convenient. [ Conclusion] The RT-LAMP method is sensitive and reliable for the detection of CSFV.展开更多
A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was perfor...A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.展开更多
为了建立一种便捷、灵敏的犬流感病毒(CIV)检测技术,本研究根据CIV相对保守的M片段设计3对特异性引物,建立CIV的RT-LAMP检测方法,对反应体系中的MgSO4、Betaine、Bst DNA PoLymerase、dNTP、引物浓度等分别进行了优化,并进行敏感性和特...为了建立一种便捷、灵敏的犬流感病毒(CIV)检测技术,本研究根据CIV相对保守的M片段设计3对特异性引物,建立CIV的RT-LAMP检测方法,对反应体系中的MgSO4、Betaine、Bst DNA PoLymerase、dNTP、引物浓度等分别进行了优化,并进行敏感性和特异性试验。结果表明:所建立的反应体系在恒温水浴锅中作用45min即可得到其特有的阶梯状条带,而且对犬细小病毒、犬瘟病毒和犬副流感病毒的扩增结果均为阴性。该方法对CIV RNA的最小检测限为0.1pg,灵敏性高于一步RT-PCR方法。RT-LAMP和普通RT-PCR方法检测临床样品的符合率为93.02%。该方法为犬流感病毒的临床检测提供了一种简便、实用的方法,为基层检疫提供了方便。展开更多
报道了一种可以快速、灵敏地检测七带石斑鱼神经坏死病毒(NNV)的逆转录环介导等温扩增方法(RT-LAMP)。该方法参考赤点石斑鱼NNV病毒的主衣壳蛋白(MCP)基因的保守序列,设计1对引物克隆出七带石斑鱼NNV的基因序列,利用Primer Explorer V4...报道了一种可以快速、灵敏地检测七带石斑鱼神经坏死病毒(NNV)的逆转录环介导等温扩增方法(RT-LAMP)。该方法参考赤点石斑鱼NNV病毒的主衣壳蛋白(MCP)基因的保守序列,设计1对引物克隆出七带石斑鱼NNV的基因序列,利用Primer Explorer V4软件设计了3对针对所克隆NNV基因序列的特异性引物,建立了RT-LAMP的反应体系,并分别对反应系统的引物浓度、反应温度和时间进行了优化,形成了七带石斑鱼NNV病毒RT-LAMP检测技术。实践应用结果表明,在63℃的最佳反应温度下,采用RT-LAMP检测技术经过45 min就能完成一次检测,准确率达到100%。本研究建立的RT-LAMP检测NNV技术设备要求简单,为七带石斑鱼等石斑鱼无NNV受精卵和仔稚鱼的生产现场检测和筛选提供了一种方便、灵敏的检测方法。展开更多
基金supported by Independent Innovation Specific Projects of Shandong Province (2008ZHZX1A1103)
文摘[ Objective] To develop a rapid and visualized detection method of classical swine fever virus （CSFV） using reverse transcriptase loopmediated isothermal amplification （RT-LAMP）. [ Method ] A total of six special primers were designed based on the conserved sequences of CSFV gene. After optimizing, the reaction of RT-LAMP was carded out at 63℃ for 45 rain. The RT-LAMP products were analyzed by agarose gel electro- phoresis. The sensitivity, specificity and repeatability were verified, respectively. [ Result] The RT-LAMP method could be used for detecting CSFV rather than six generic viruses. The sensitivity of RT-LAMP was 100 times higher than that of RT-PCR. The detection of 27 clinical samples by RT- LAMP and RT-PCR showed that RT-LAMP is more reliable and convenient. [ Conclusion] The RT-LAMP method is sensitive and reliable for the detection of CSFV.
文摘A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.
文摘为了建立一种便捷、灵敏的犬流感病毒(CIV)检测技术,本研究根据CIV相对保守的M片段设计3对特异性引物,建立CIV的RT-LAMP检测方法,对反应体系中的MgSO4、Betaine、Bst DNA PoLymerase、dNTP、引物浓度等分别进行了优化,并进行敏感性和特异性试验。结果表明:所建立的反应体系在恒温水浴锅中作用45min即可得到其特有的阶梯状条带,而且对犬细小病毒、犬瘟病毒和犬副流感病毒的扩增结果均为阴性。该方法对CIV RNA的最小检测限为0.1pg,灵敏性高于一步RT-PCR方法。RT-LAMP和普通RT-PCR方法检测临床样品的符合率为93.02%。该方法为犬流感病毒的临床检测提供了一种简便、实用的方法,为基层检疫提供了方便。
文摘报道了一种可以快速、灵敏地检测七带石斑鱼神经坏死病毒(NNV)的逆转录环介导等温扩增方法(RT-LAMP)。该方法参考赤点石斑鱼NNV病毒的主衣壳蛋白(MCP)基因的保守序列,设计1对引物克隆出七带石斑鱼NNV的基因序列,利用Primer Explorer V4软件设计了3对针对所克隆NNV基因序列的特异性引物,建立了RT-LAMP的反应体系,并分别对反应系统的引物浓度、反应温度和时间进行了优化,形成了七带石斑鱼NNV病毒RT-LAMP检测技术。实践应用结果表明,在63℃的最佳反应温度下,采用RT-LAMP检测技术经过45 min就能完成一次检测,准确率达到100%。本研究建立的RT-LAMP检测NNV技术设备要求简单,为七带石斑鱼等石斑鱼无NNV受精卵和仔稚鱼的生产现场检测和筛选提供了一种方便、灵敏的检测方法。
