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
A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive an...A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains.As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR),the RT-LAMP method only used a turbidimeter,exhibited a detection limit corresponding to a 10-4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV-containing cells,and no cross-reactivity was observed with other related viruses including porcine circovirus type 2,swine influenza virus,porcine rotavirus and classical swine fever virus.From forty-two field samples,33 samples in the RT-LAMP assay was detected positive,whereas three of which were not detected by RT-PCR.Furthermore,in 33 strains of PRRSV,an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates,especially in developing countries.展开更多
[ 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
建立一种便捷、灵敏的检测方法,即逆转录环介导等温核酸扩增技术(RT-LAMP)用于H5N1亚型禽流感病毒基因检测。该技术使用特异对应于靶序列中8个基因区段的6条特异引物,在等温条件下进行核酸扩增反应。对51份实验感染动物及病毒培养标本的...建立一种便捷、灵敏的检测方法,即逆转录环介导等温核酸扩增技术(RT-LAMP)用于H5N1亚型禽流感病毒基因检测。该技术使用特异对应于靶序列中8个基因区段的6条特异引物,在等温条件下进行核酸扩增反应。对51份实验感染动物及病毒培养标本的H5N1亚型禽流感病毒的HA、NA基因区进行了RT-LAMP检测,并以SYBR Green I为反应指示剂进行了逆转录环介导等温核酸扩增技术,对该反应进行实时监控,经对扩增产物做内切酶验证和测序分析,证明RT-LAMP技术的特异性;同时,用10倍系列稀释的RNA样品对该检测方法的灵敏度进行了测试。结果显示:利用RT-LAMP技术成功检测到H5N1禽流感病毒的HA、NA基因区,且RT-LAMP与Real-time PCR结果呈现很好的一致性。此方法的灵敏度可达到能检测10个拷贝RNA分子水平。因此,RT-LAMP技术应用于H5N1亚型禽流感病毒的快速检测是一种可行的方法。展开更多
本试验根据GenBank中登录的牛副流感病毒3型(BPIV-3)基因序列,利用在线软件Primer Explorer V4Software和Primer Premier 5.0,针对BPIV-3 NP基因序列的保守区设计并筛选了一套环介导逆转录等温核酸扩增(RT-LAMP)引物,建立BPIV-3特异性...本试验根据GenBank中登录的牛副流感病毒3型(BPIV-3)基因序列,利用在线软件Primer Explorer V4Software和Primer Premier 5.0,针对BPIV-3 NP基因序列的保守区设计并筛选了一套环介导逆转录等温核酸扩增(RT-LAMP)引物,建立BPIV-3特异性检测的RT-LAMP方法。在Bst DNA聚合酶作用下,63℃恒温反应1h即可完成扩增过程,扩增产物通过浑浊度比较、凝胶电泳和肉眼可视化进行判定。结果表明,该方法比RT-PCR敏感度更高,最低检出量可达0.069fg/μL。该方法可用于牛副流感病毒3型的实验室检测和临床初步诊断。展开更多
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A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains.As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR),the RT-LAMP method only used a turbidimeter,exhibited a detection limit corresponding to a 10-4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV-containing cells,and no cross-reactivity was observed with other related viruses including porcine circovirus type 2,swine influenza virus,porcine rotavirus and classical swine fever virus.From forty-two field samples,33 samples in the RT-LAMP assay was detected positive,whereas three of which were not detected by RT-PCR.Furthermore,in 33 strains of PRRSV,an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates,especially in developing countries.
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.
文摘建立一种便捷、灵敏的检测方法,即逆转录环介导等温核酸扩增技术(RT-LAMP)用于H5N1亚型禽流感病毒基因检测。该技术使用特异对应于靶序列中8个基因区段的6条特异引物,在等温条件下进行核酸扩增反应。对51份实验感染动物及病毒培养标本的H5N1亚型禽流感病毒的HA、NA基因区进行了RT-LAMP检测,并以SYBR Green I为反应指示剂进行了逆转录环介导等温核酸扩增技术,对该反应进行实时监控,经对扩增产物做内切酶验证和测序分析,证明RT-LAMP技术的特异性;同时,用10倍系列稀释的RNA样品对该检测方法的灵敏度进行了测试。结果显示:利用RT-LAMP技术成功检测到H5N1禽流感病毒的HA、NA基因区,且RT-LAMP与Real-time PCR结果呈现很好的一致性。此方法的灵敏度可达到能检测10个拷贝RNA分子水平。因此,RT-LAMP技术应用于H5N1亚型禽流感病毒的快速检测是一种可行的方法。
文摘本试验根据GenBank中登录的牛副流感病毒3型(BPIV-3)基因序列,利用在线软件Primer Explorer V4Software和Primer Premier 5.0,针对BPIV-3 NP基因序列的保守区设计并筛选了一套环介导逆转录等温核酸扩增(RT-LAMP)引物,建立BPIV-3特异性检测的RT-LAMP方法。在Bst DNA聚合酶作用下,63℃恒温反应1h即可完成扩增过程,扩增产物通过浑浊度比较、凝胶电泳和肉眼可视化进行判定。结果表明,该方法比RT-PCR敏感度更高,最低检出量可达0.069fg/μL。该方法可用于牛副流感病毒3型的实验室检测和临床初步诊断。
