LAMP和PCR法检测痢疾志贺菌的特异性和灵敏性比较
Specificity and sensitivity of LAMP method detecting Shigella dysenteriae and its comparison with PCR
目的 建立环介导等温扩增技术( Loop-mediated isothermal amplification , LAMP)快速检测痢疾志贺菌，并对其特异性、灵敏度与传统PCR进行比较。 方法 以痢疾志贺菌侵袭性质粒抗原H基因(ipaH)为靶序列，设计6条特异性引物(内引物、外引物和环引物各2条)，优化LAMP反应体系及反应条件，检测LAMP反应的灵敏度、 特异性及模拟样品，同步与PCR进行比较。 结果 确定LAMP检测痢疾志贺菌的反应体系与条件为：Mg2+浓度5mmol/L，dNTPs浓度0.3mmol/L，甜菜碱浓度0.6mol/L，外引物、内引物与环引物的浓度比 1:4:4，Bst DNA聚合酶8U，扩增温度63℃ 反应60min。LAMP法和PCR均能特异性地扩增出志贺菌靶DNA，而其他非志贺菌均未扩增出特有条带。该法检测细菌纯培养物的灵敏度为5.3×101cfu/ml，检测模拟食品的灵敏度为6.8×101cfu/ml；PCR法检测的灵敏度分别为 5.3×102 cfu/ml 和6.8×102 cfu/ml。结论 利用LAMP技术，可快速、灵敏、简便检测痢疾志贺菌，与PCR方法比较，特异性强、操作简便、检测成本低，耗时短，有望发展成为快速检测痢疾志贺菌的有效手段。
Objective The aim was to detect Shigella dysenteriae rapidly by using the loop-mediated isothermal amplification(LAMP) technology．And to compare the specificity and sensitivity of the LAMP to that of conventional PCR . Methods The sequences of ipaH of Shigella was used as target sequences to design the six primers. The reaction conditions of LAMP were established and optimized. Results The most optimal reaction conditions of LAMP contained 5mM MgCl2, 0.3 mM dNTPS, 0.6 mM Betain and 8 U of Bst DNA polymerase.The mixture was incubated at 63 C for 60 min.The LAMP assay detection limit was detecting 53 cfu/ml of pure Shigella dysenteriae and 68 cfu/ml in raw meat. The traditional PCR assay detection limit was 530 cfu/ml and 680 cfu/ml respectively. Conclusion LAMP technology for detection Shigella was rapid and specificity．It was a more sensitive method for shigella detection than the PCR method . This study is a simple tool for the detection of shigella dysenteriae in food and build a technology platform.
吴娴波、曾桂芬、范宏英、龙北国
微生物学生物科学研究方法、生物科学研究技术医学研究方法
微生物学环介导等温扩增技术（LAMP）PCR痢疾志贺菌ipaH基因
Loop-mediated isothermal amplification (LAMP)PCRShigella dysenteriaeipaH gene
吴娴波,曾桂芬,范宏英,龙北国.LAMP和PCR法检测痢疾志贺菌的特异性和灵敏性比较[EB/OL].(2012-04-13)[2025-08-11].http://www.paper.edu.cn/releasepaper/content/201204-196.点此复制
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