[Objective] This study aimed to detect the reliability of LAMP method for detecting Listeria monocytogenes in dairy food. [Method] Based on the sequence of hlyA gene encoding listeriolysin O in Listeria monocytogenes,...[Objective] This study aimed to detect the reliability of LAMP method for detecting Listeria monocytogenes in dairy food. [Method] Based on the sequence of hlyA gene encoding listeriolysin O in Listeria monocytogenes, the LAMP method was established for detecting Listeria monocytogenes in dairy food. [Result] The es- tablished LAMP rapid detection method has .high specificity and sensitivity, which are equivalent to those of real-time fluorescent quantitative PCR. The detection re- sults of Listeria monocytogenes in dairy food by established LAMP method were completely consistent with those by bacterial isolation method. [Conclusion] The de- tection results of Listeria monocytogenes by LAMP method can be directly identified by naked eye, so the established LAMP method was suitable for the detection of Listeria monocytogenes in dairy food in emergency situations.展开更多
根据GenBank发表的鼻气管鸟疫杆菌的16SRNA(登录号:JF810495.1)序列,利用Primer Explorer V4在线软件设计了1套环介导等温核酸扩增技术(LAMP)引物,并对反应体系和反应条件进行优化,同时评价该方法的灵敏性和特异性。结果显示,建立的LAM...根据GenBank发表的鼻气管鸟疫杆菌的16SRNA(登录号:JF810495.1)序列,利用Primer Explorer V4在线软件设计了1套环介导等温核酸扩增技术(LAMP)引物,并对反应体系和反应条件进行优化,同时评价该方法的灵敏性和特异性。结果显示,建立的LAMP检测方法能够在63℃1h内实现对目的片段的大量扩增,检测结果可直接用肉眼判断。特异性试验结果显示,该方法只能检测到鼻气管鸟疫杆菌,与其他细菌如鸡白痢沙门菌、大肠杆菌、金黄色葡萄球菌、多杀性巴氏杆菌、副鸡嗜血杆菌的核酸无交叉反应,特异性强。敏感性试验表明,该方法可检测到1×101拷贝/μL的质粒标准品,比普通PCR高104倍。利用该方法对分离的阳性菌株进行检测,检出率为83.33%。结果表明,建立的鼻气管鸟疫杆菌LAMP检测方法,具有快速、简便、特异性强、灵敏度高的特点,可用于鼻气管鸟疫杆菌的临床检测。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
基金Supported by Natural Science Foundation of Anhui Province(1508085QC64)~~
文摘[Objective] This study aimed to detect the reliability of LAMP method for detecting Listeria monocytogenes in dairy food. [Method] Based on the sequence of hlyA gene encoding listeriolysin O in Listeria monocytogenes, the LAMP method was established for detecting Listeria monocytogenes in dairy food. [Result] The es- tablished LAMP rapid detection method has .high specificity and sensitivity, which are equivalent to those of real-time fluorescent quantitative PCR. The detection re- sults of Listeria monocytogenes in dairy food by established LAMP method were completely consistent with those by bacterial isolation method. [Conclusion] The de- tection results of Listeria monocytogenes by LAMP method can be directly identified by naked eye, so the established LAMP method was suitable for the detection of Listeria monocytogenes in dairy food in emergency situations.
文摘根据GenBank发表的鼻气管鸟疫杆菌的16SRNA(登录号:JF810495.1)序列,利用Primer Explorer V4在线软件设计了1套环介导等温核酸扩增技术(LAMP)引物,并对反应体系和反应条件进行优化,同时评价该方法的灵敏性和特异性。结果显示,建立的LAMP检测方法能够在63℃1h内实现对目的片段的大量扩增,检测结果可直接用肉眼判断。特异性试验结果显示,该方法只能检测到鼻气管鸟疫杆菌,与其他细菌如鸡白痢沙门菌、大肠杆菌、金黄色葡萄球菌、多杀性巴氏杆菌、副鸡嗜血杆菌的核酸无交叉反应,特异性强。敏感性试验表明,该方法可检测到1×101拷贝/μL的质粒标准品,比普通PCR高104倍。利用该方法对分离的阳性菌株进行检测,检出率为83.33%。结果表明,建立的鼻气管鸟疫杆菌LAMP检测方法,具有快速、简便、特异性强、灵敏度高的特点,可用于鼻气管鸟疫杆菌的临床检测。
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。
