环介导恒温核酸扩增技术快速检测志贺菌研究  

Rapid and sensitive detection of Shigella by Loop-Mediated Isothermal Amplification

作　　者：梅玲玲[1] 朱敏[1] 占利[1] 龚璞[1] 张俊彦[1] 

机构地区：[1]浙江省疾病预防控制中心,杭州310051

出　　处：《中国卫生检验杂志》2011年第2期389-391,共3页Chinese Journal of Health Laboratory Technology

基　　金：浙江省公共卫生检验检测重点学科群(XKQ-009-003)

摘　　要：目的:建立一种快速检测志贺菌的环介导恒温核酸扩增(LAMP)技术,为志贺菌食源性疾病监测、诊断提供技术支撑。方法:在对志贺菌基因序列进行分析、对比基础上,根据志贺菌ipaH基因序列保守区域设计6条特异性引物;通过对LAMP技术反应条件优化,实现对志贺菌的快速检测;用不同来源志贺菌及沙门菌、大肠杆菌、副溶血性弧菌、柠檬酸杆菌、金黄色葡萄球菌、乙型溶血性链球菌等菌株验证方法的敏感性和特异性。结果:当25μl反应体系中含1.6μmol/L内引物(FIP、BIP)、0.2μmol/L外引物(F3、B3)、0.2μmol/L环引物(LF、LB)、1 mmol/L dNTP、6 mmol/L Mg-SO4,0.8 mol/L甜菜碱、1×反应缓冲液、DNA模版,8 U Bst DNA聚合酶,扩增条件为60℃,60 min时,LAMP技术对志贺菌最低检测浓度为200 CFU/ml,经8 h增菌,最低检测浓度可达到20 CFU/ml。对32株不同来源的志贺菌检测结果,所有反应管内肉眼观察有混浊,为阳性,而13种139株其它非志贺菌株反应结果均呈透明为阴性。对50件食品检测结果与常规培养方法及实时荧光PCR方法相比无显著性差异(P>0.05,χ2=0.27)。结论:本研究建立的志贺菌LAMP检测方法具有特异性强、灵敏度高、方便快捷、成本低等特点,适合在基层、小型实验室以及无条件购买荧光定量PCR仪的病原微生物检验检测机构推广使用。Objective：To develop a technique for rapid detection of Shigella using a novel DNA amplification procedure designated Loop-mediated isothermal amplification（LAMP）.To provide technical support for foodborne disease surveillance and diagnose.Methods：Six Shigella-specific primers were designed from the published ipaH gene sequences of Shigella by using the LAMP primer supported software program.The specificity and sensitivity of LAMP were examined using Salmonella,Escherichia coli,Vibrio Parahemolyticus,Staphylococcus aureus and other species.Results： It showed that Shigella-specific primers amplified Shigella only and that the lowest detection limit of the LAMP reaction was 200 CFU/ml.If there was an 8 hours enrichment before the LAMP,the owest detection limit of the LAMP reaction was 20 CFU/ml.50 food samples were examined by the LAMP while they were examined by real-time quantitative Polymerase Chain Reaction.The result showed that there was no significant difference between these two methods（P0.05,χ2=0.27）.Conclusion：This LAMP-based assay is simple,rapid,sensitive and specific.No specialized equipment is needed which makes it is more economical and practical.This assay is expected to become a valuable tool for rapid diagnosis in Shigella infection especially in primary laboratory.

关 键 词：志贺菌 环介导恒温核酸扩增(LAMP)技术 IPAH基因 

分 类 号：R378[医药卫生—病原生物学]