炭疽杆菌双重可视化LAMP检测方法的建立
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摘要
目的建立同时检测炭疽杆菌cap A基因、PA基因的双重环介导等温扩增(Loop-mediated isothermal amplification,LAMP)方法,用于防范生物恐怖威胁。方法设计和合成分别针对炭疽杆菌cap A基因、PA基因的引物对,通过优化参数,建立可同时检测cap A基因、PA基因的双重LAMP方法,测试敏感性和特异性,并应用于模拟样本和实战检测。结果双重LAMP的检测cap A基因、PA基因的敏感性均可达到50模板拷贝每反应,并具有良好的特异性,在重大保障活动中得到检验。结论双重LAMP方法具有可以同时筛查、简单快速、灵敏度高等优点,在炭疽杆菌检测方面有良好应用前景。
Objective To develop a dupplex loop-mediated isothermal amplification( LAMP) assay for the detection of specific structural genes and virulence genes in bacillus anthracis. Methods Two sets of specific primers were designed and synthesized according to Cap A and PA genes of bacillus anthracis. The reaction parameters were optimized to develop the dupplex LAMP assay for the rapid detection of bacillus anthracis. This study also tested the sensitivity and specificity of the LAMP assay,and applied it to test simulated and actual samples. Results A double LAMP assay for rapid detection of bacillus anthracis PA gene( plasmid p X01) and cap A gene( p X02 plasmid) on visualization methods had been established. The detectable sensitivity of the duplex LAMP was 50 template copy per reaction,and it had good specificity,stability and reproducibility. Conclusion Considering LAMP's simplicity in operation and high sensitivity,there is potential use in clinical diagnosis and surveillance of bacillus anthracis.
Objective To develop a dupplex loop-mediated isothermal amplification( LAMP) assay for the detection of specific structural genes and virulence genes in bacillus anthracis. Methods Two sets of specific primers were designed and synthesized according to Cap A and PA genes of bacillus anthracis. The reaction parameters were optimized to develop the dupplex LAMP assay for the rapid detection of bacillus anthracis. This study also tested the sensitivity and specificity of the LAMP assay,and applied it to test simulated and actual samples. Results A double LAMP assay for rapid detection of bacillus anthracis PA gene( plasmid p X01) and cap A gene( p X02 plasmid) on visualization methods had been established. The detectable sensitivity of the duplex LAMP was 50 template copy per reaction,and it had good specificity,stability and reproducibility. Conclusion Considering LAMP's simplicity in operation and high sensitivity,there is potential use in clinical diagnosis and surveillance of bacillus anthracis.
引文
[1]中国疾病预防控制中心,全国炭疽监测方案(试行)(2005年7月26日),http://www.chinacdc.cn/n272442/n272530/n3479265/n3479308/31023.html.
[2]谈忠鸣,张忠献,董晨,等.一起皮肤炭疽疫情中炭疽杆菌分子特征分析[J].南京医科大学学报(自然科学版),2015,35(3):436-438.
[3]Zhang JH,Zhu J,Ren H,et al.Rapid visual detection of highly pathogenic streptococcus suis serotype 2 isolates by use of LoopMediated Isothermal Amplification[J].J Clin Microbiol,2013,51(10):3250-3256.
[4]Zhang JH,Feng YJ,Hu D,et al.Rapid and sensitive detection of H7N9 avian influenza virus using reverse transcription loop-mediated isothermal amplification[J].J Clin Microbiol,2013,51(11):3760-3764.
[5]吕恒,钟璟浩,张锦海,等.新型布尼亚病毒环介导等温扩增可视化检测方法的建立[J].中国病原生物学杂志,2016,11(3):225-228.
[6]Goto M,Honda E,Ogura A,et al.Colorimetric detection of loopmediated isothermal amplification reaction by using hydroxy naphthol blue[J].Biotechniques,2009,46(3):167-172.
[7]谭维国,陈文琦,吕恒,等.炭疽杆菌双重实时荧光PCR检测方法的建立[J].东南国防医药,2013,15(6):556-559.
[8]张锦海,王忠灿,王长军.用于检测炭疽杆菌的重组标准质粒、试剂盒及该质粒的构建方法[P].ZL201110114469.4.
[9]刘炬,徐俊杰,陈薇.炭疽芽孢杆菌建立感染的过程及其机制[J].军事医学,2014(8):651-654.
[10]吕恒,张锦海,顾海涛,等.肠出血性大肠杆菌O157:H7的快速可视化检测[J].东南国防医药,2013,15(4):321-324.
[11]Njiru ZK.Loop-Mediated isothermal amplification technology:towards point of care diagnostics[J].Plo S Negl Trop Dis,2012,6(6):692-694.
[12]Tomita N,Mori Y,Kanda H,et al.Loop-mediated isothermal amplification(LAMP)of gene sequences and simple visual detection of products[J].Nat Protoc 2008,3(5):877-882
[2]谈忠鸣,张忠献,董晨,等.一起皮肤炭疽疫情中炭疽杆菌分子特征分析[J].南京医科大学学报(自然科学版),2015,35(3):436-438.
[3]Zhang JH,Zhu J,Ren H,et al.Rapid visual detection of highly pathogenic streptococcus suis serotype 2 isolates by use of LoopMediated Isothermal Amplification[J].J Clin Microbiol,2013,51(10):3250-3256.
[4]Zhang JH,Feng YJ,Hu D,et al.Rapid and sensitive detection of H7N9 avian influenza virus using reverse transcription loop-mediated isothermal amplification[J].J Clin Microbiol,2013,51(11):3760-3764.
[5]吕恒,钟璟浩,张锦海,等.新型布尼亚病毒环介导等温扩增可视化检测方法的建立[J].中国病原生物学杂志,2016,11(3):225-228.
[6]Goto M,Honda E,Ogura A,et al.Colorimetric detection of loopmediated isothermal amplification reaction by using hydroxy naphthol blue[J].Biotechniques,2009,46(3):167-172.
[7]谭维国,陈文琦,吕恒,等.炭疽杆菌双重实时荧光PCR检测方法的建立[J].东南国防医药,2013,15(6):556-559.
[8]张锦海,王忠灿,王长军.用于检测炭疽杆菌的重组标准质粒、试剂盒及该质粒的构建方法[P].ZL201110114469.4.
[9]刘炬,徐俊杰,陈薇.炭疽芽孢杆菌建立感染的过程及其机制[J].军事医学,2014(8):651-654.
[10]吕恒,张锦海,顾海涛,等.肠出血性大肠杆菌O157:H7的快速可视化检测[J].东南国防医药,2013,15(4):321-324.
[11]Njiru ZK.Loop-Mediated isothermal amplification technology:towards point of care diagnostics[J].Plo S Negl Trop Dis,2012,6(6):692-694.
[12]Tomita N,Mori Y,Kanda H,et al.Loop-mediated isothermal amplification(LAMP)of gene sequences and simple visual detection of products[J].Nat Protoc 2008,3(5):877-882