摘要
目的优化双重分子信标的实验体系以快速检测结核杆菌及其耐药菌株。方法选择不同镁离子浓度、退火温度、引物浓度分别进行荧光定量PCR,最终得出最佳反应条件。结果为保证扩增效率且无非特异性扩增,最终选择最佳条件是:Mg^(2+)浓度3.0mmol/L;退火温度60℃;引物浓度0.3mmol/L。结论确立了双重分子信标实验的最优条件,从而保证了分子信标荧光定量PCR检测结核杆菌具有操作简便、快速、灵敏度高(最低可检测1CFU/mL)、特异性强(只能检测包括耐药菌株的结核杆菌复合群)、重复性好(变异系数<5%)等优势,为双重分子信标检测结核杆菌提供了必要条件。
Objective To optimize the experimental system of dual molecular beacon to rapidly detect Mycobacterium tuberculosis and its resistant strains.Methods Fluorescence quantitative PCR was carried out by selecting different magnesium ion concentration,annealing temperature and primer concentration respectively.Finally,the optimum reaction conditions were obtained.Results In order to ensure the efficiency of amplification and no non-specific amplification,the final selection of the best conditions were as follows,the concentration of Mg^2＋ was 3.0 mmol/L,annealing temperature was 60 ℃,and the concentration of primers was 0.3 mmol/L.Conclusion The optimal condition of dual molecular beacon experiment was established,which ensured that the detection of Mycobacterium tuberculosis by molecular beacon quantitative PCR had the advantages,such as simple operation,rapid speed,high sensitivity（the minimum detection limit was 1 CFU/mL）and specificity（only Mycobacterium tuberculosis complex including drug-resistant strains could be detected）,good reproducibility（coefficient of variation was 〈5%）and other advantages.The study provides the necessary conditions for the dual molecular beacon detection of Mycobacterium tuberculosis.
作者
余传星
张焕
黄明翔
赵自云
朱玲
YU Chuanxing;ZHANG Huan;HUANG Mingxiang;ZHAO Ziyun;ZHU Ling(Medicine of Second People＇s Hospital of Fujian Province ,Fuzhou ,Fujian 350003 ,China;Immunology Department and Center of Neuroscience, Fujian Medical University, Fuzhou, Fujian 350004, China;Department of Clinical Laboratory Pulmonary Hospital of Fujian Province ,Fuzhou ,Fujian 350008 ,China ,Fuzhou)
出处
《国际检验医学杂志》
CAS
2018年第9期1029-1033,共5页
International Journal of Laboratory Medicine
基金
福建省社会发展引导性重点项目(2014Y0058)
国家卫生计生委共建科学研究基金--第四轮福建省卫生教育联合公关计划项目(WKJ2016-2-32)
关键词
结核杆菌
微菌落
分子信标
体系优化
Mycobacterium tuberculosis
microcolony
molecular beacon
system optimization
