摘要
目的基于环介导恒温扩增(LAMP)结合纳米生物传感条(LFB)技术,建立并评价黄疸出血群钩端螺旋体(钩体)的LAMP-LFB快速检测方法。方法以黄疸出血群钩体O抗原基因簇中的糖基转移酶基因(gtf)为检测靶标并设计特异性LAMP引物。通过对LAMP引物的特定标记(FIP-FAM和LF-Biotin)和优化试验,建立LAMP-LFB快速检测方法,并分析其灵敏度和特异性。应用建立的LAMP-LFB、普通PCR方法和显微凝集试验(MAT),分别对53株钩体分离菌株进行鉴定,比较分析鉴定结果并评价LAMP-LFB方法的实用性。结果灵敏度和特异性试验显示,LAMP-LFB方法可检出黄疸出血群赖型56601基因组DNA的最低浓度为100 fg/μL,检测特异性为100%,与其余血清群和非钩体菌株核酸无交叉反应。菌株鉴定结果显示,LAMP-LFB与MAT鉴定结果完全一致,符合率为100%,其敏感性高于PCR方法。此外,LAMP扩增产物经LFB验证,可直接通过观察检测线(TL)和质控线(CL)进行结果判定。结论基于gtf基因建立的LAMP-LFB检测技术方便、快捷且重复性好,可快速、灵敏、特异地鉴定黄疸出血群钩体菌株,能够作为有价值的黄疸出血群钩体菌株快速鉴别或诊断方法。
Objective To develop and evaluate a rapid detection assay for Leptospira interrogans serogroup icterohaemorrhagiae based on loop-mediated isothermal amplification(LAMP)combined with nanoparticle-based lateral flow biosensor(LFB).Methods The LAMP primers targeting the glycotransferase gene(gtf)in the O-antigen gene cluster of L.serogoup icterohaemorrhagiae were designed.After specific label of primers(FIP-FAM and LF-Biotin)and condition optimization,we evaluated the sensitivity,specificity and feasibility of gtf-LAMP-LFB assay.A total of 53 strains of Leptospira were identified by using LAMP-LFB assay,PCR and microscopic agglutination test(MAT),and the results were analyzed to evaluate the practicality of LAMP-LFB assay.Results Our data showed that the detection limit of the assay was 100 fg/μL for genomic DNA of reference strain of L.icterohaemorrhagiae(56601),and the specificity was 100%because there were no cross reactions with nucleic acids of other Leptospira serogroups and non-Leptospira strains.For the application examination,LAMP-LFB and MAT identification results were completely consistent,while the sensitivity of LAMP-LFB assay was higher than PCR.In addition,the color of CL and TL bands could be observed directly to determine the results by using LFB to detect LAMP amplicons.Conclusion The LAMP-LFB assay developed based on LAMP technique has high repeatability,sensitivity and specificity,which can be used for the rapid and accurate identification of the strains of L.interrogans serogroup icterohaemorrhagiae,and can be used as a potential screening and diagnosis tool for L.interrogans serogroup icterohaemorrhagiae.
作者
王毅
杨幸贵
刘英
黄俊飞
王铭
营夏
谭勤琴
胡勇
李世军
Wang Yi;Yang Xinggui;Liu Ying;Huang Junfei;Wang Ming;Ying Xia;Tan Qinqin;Hu Yong;Li Shijun(Experimental Research Center,Capital Institute of Pediatrics,Beijing 100020,China;School of Public Health,the Key Laboratory of Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guiyang 550025,Guizhou,China;Guizhou Center for Disease Control and Prevention,Guiyang 550004,Guizhou,China)
出处
《疾病监测》
CAS
CSCD
北大核心
2022年第5期646-651,共6页
Disease Surveillance
基金
国家自然科学基金(No.81760366,No.81960622)
贵州省高层次创新型人才培养项目[No.黔科合(2016)4021]。
关键词
环介导恒温扩增
纳米生物传感
钩端螺旋体
黄疸出血群
Loop-mediated isothermal amplification
Lateral flow biosensor
Leptospira
Serogoup icterohaemorrhagiae
