联合环介导等温扩增技术和横向流动试纸条检测金黄色葡萄球菌
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摘要
目的应用环介导等温扩增技术(LAMP)和横向流动试纸条检测技术(LFD)联合检测金黄色葡萄球菌。方法从GenBank数据库下载多条金黄色葡萄球菌nuc基因序列,在其保守区域设计LAMP引物,建立LAMP-LFD恒温扩增快速检测技术;优化反应温度和体系后通过检测多种病原菌验证检测特异度和灵敏度,并比较LAMP-LFD和荧光PCR法的检测效果。结果 LAMP-LFD在最佳反应温度60℃条件下检测金黄色葡萄球菌具有高特异度,构建的阳性质粒DNA灵敏度达到1.0×101copies/μL。LAMP-LFD和荧光PCR法检测12份临床标本的阳性率均为75.00%;LAMP-LFD检测时间为35 min,而荧光PCR法约为1 h。结论 LAMP-LFD有灵敏度高、检测时间短等特点,可应用于金黄色葡萄球菌快速检测。
引文
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[3]梁玉林,刘秀,周振森,等.基于反转录一环介导等温扩增技术检测金黄色葡萄球菌[J].食品与发酵工业,2018,44(1):217-223.
[4]NOTOMI T,OKAYAMA H,MASUBUCHI H,et a1.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):E63.
[5]XU C,FENG Y,CHEN Y,et al.Rapid detection of measles virus using reverse transcription loop-mediated isothermal amplification coupled with a disposable lateral flow device[J].Diagnostic Microbiology and Infectious Disease,2016,85(2):168-173.
[6]YAMAMOTO M,ALSHAHNI M M,TAMURA T,et al.Rapid detection of Candida auris based on Loop-mediated Isothermal Amplification(LAMP)[J].J Clin Microbiol,2018,56(9):e00591-18.
[7]LALLE M,POSSENTI A,DUBEY J,et al.Loop-Mediated Isothermal Amplification-Lateral-Flow Dipstick(LAMP-LFD)to detect Toxoplasma gondii oocyst in ready-to-eat salad[J].Food Microbiology,2018(70):137-142.
[8]张兵,徐昌平,程东庆,等.嗜肺军团菌核酸等温扩增快速检测技术研究[J].中国卫生检验杂志,2018,28(7):782-784,788.
[9]YU G,SHEN Y,ZHONG F,et al.Diagnostic accuracy of the loop-mediated isothermal amplification assay for extrapulmonary tuberculosis:a meta-analysis[J].PLoS One,2018,13(6):e0199290.
[10]WANG H,CONG F,ZENG F,et al.Development of a real time reverse transcription loop-mediated isothermal amplification method(RT-LAMP)for detection of a novel swine acute diarrhea syndrome Coronavirus(SADS-CoV)[J].J Virol Methods,2018(260):45-48
[11]LIU W,ZHOU Y,FAN Y,et al.Development of cross-priming amplification coupled with vertical flow visualization for rapid detection of infectious spleen and kidney necrosis virus(ISKNV)in mandarin fish,Siniperca chuatsi[J].J Virol Methods,2018(253):38-42.
[12]FENG T,LI S,WANG S,et al.Cross priming amplification with nucleic acid test strip analysis of mutton in meat mixtures[J].Food Chemistry,2018(245):641-645.