使用环介导等温扩增技术检测炭疽芽孢杆菌及其定量检测的研究

    易海华1，张阳1，房超2，孙涛1，徐政1，徐波1，刘继永3

    1.徐州出入境检验检疫局卫生检疫科，江苏 徐州221006；2.徐州出入境检验检疫局技术中心；3.南京医科大学

    摘要：目的建立一种适合公共卫生机构现场使用的环介导等温扩增（LAMP）技术检测炭疽芽孢杆菌的方法。方法根据LAMP方法的原理，建立LAMP以及荧光定量LAMP检测方法，对方法的特异性、灵敏度、重复性及模板浓度与反应时间之间的线性关系进行评估。结果LAMP检测可在1h内完成，检出限为103拷贝/反应，灵敏度比经典PCR高10倍，与其他杆菌群细菌无交叉反应，平均试验间变异系数小于5％。反应时间与模板浓度有良好的线性关系（R2= 0.9891）。结论 该方法快速、灵敏、特异、操作简单、设备要求低的特点，适合基层医疗卫生机构和现场查验机构的广泛应用。

    关键词：环介导等温扩增；炭疽芽孢杆菌；荧光定量LAMP；快速检测

    中图分类号：R331　文献标识码：B

    Study on detection of Bacillus anthracis andits quantitate based on

    Fluorescent Quantitative Loop-mediatedIsothermal Amplification

    YI Hai-hua*, ZHANG Yang, FANG Chao, SUN Tao,XU Zheng, XU Bo, LIU Ji-yong

    *Xuzhou Entry-exit Inspection and QuarantineBureau, Xuzhou, Jiangsu 221000, China

    Abstract:   Objective  To develop a method of Fluorescent Quantitative and QualitativeLoop-mediated Isothermal Amplification (LAMP) to Bacillusanthracis  rapidly ,specifically, sensitively and simplysuited for used in the primary public health agency.  Methods   According to conserved nucleotide of Bacillusanthracis and principle of LAMP, a set of LAMP primers was designedand a LAMP and a real-time LAMP reaction system was set up. Thespecificity, sensitivity and repeatability of the method wasevaluated. In addition, the linearity of the real-time LAMP betweeninitial template copies Log value and reaction time wasevaluated.  Results   The optimal assay showed thatit was no cross-reaction with the other bacteria belong to Bacilluscereus group , and the threshold values of detection for Bacillusanthracis was 103 copies per reaction ,10 times more sensitive thanPCR , and The Coefficient of Variance between tests was less than5%。The kinetics curves showed a good linearity between initialtemplate copies Log value and reaction time, which correlationcoefficient was 0.9891.  Conclusion   These findingsdemonstrate that the fluorescent quantitative and qualitative LAMPhas the potential clinical application for detection anddifferentiation of Bacillus anthracis in the primary hospital,inspection and quarantine institution for its sensitive, specificand simple feature.

    Key words:   Loop-MediatedIsthermal Amplification; Bacillus anthracis; Fluorescentquantitative and qualitative LAMP; Rapid detection

    《中国国境卫生检疫杂志》2013年第1期