利用环介导等温扩增技术检测新型隐球菌  

Loop-mediated isothermal amplification for rapid detection of Cryptococcus neoformans

作　　者：李东冬[1] 王凯飞[1] 陈荣[1] 沈定霞[1] 

机构地区：[1]解放军总医院微生物科,北京100853

出　　处：《中华实验和临床感染病杂志（电子版）》2014年第1期74-76,共3页Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)

摘　　要：目的：建立一种快速检测新型隐球菌的方法。方法根据新型隐球菌的18 S rRNA基因序列设计内外引物各一对，在不同温度下对新型隐球菌进行环介导等温扩增，以探明环介导等温扩增法检测新型隐球菌最适宜的温度。对新型隐球菌、其他几种常见引起脑膜炎的病原菌以及部分种类的念珠菌进行扩增，以研究环介导等温扩增技术检测隐球菌的特异性。将新鲜培养的新型隐球菌稀释后加入非隐球菌感染患者的脑脊液中，模拟脑膜炎感染后的脑脊液环境，以研究环介导等温扩增技术检测新型隐球菌的敏感性。结果环介导等温扩增法检测新型隐球菌在60℃～65℃条件下均能实现良好扩增。环介导等温扩增法对其他病原菌的检测结果均为阴性，仅新型隐球菌的检测结果为阳性，特异性达到100%。用环介导等温扩增法从脑脊液中检测新型隐球菌的最低检出限为102 CFU/ml。用环介导等温扩增技术检测新型隐球菌性脑膜炎，能节省培养及传统鉴定的时间，从DNA的提取至反应结束在2～3h内即可完成，大大减少了诊断所需时间。结论环介导等温扩增是一种灵敏度高，特异性强，耗时短，且操作简便的检测新型隐球菌感染的方法。Objective To establish a method of loop-mediated isothermal ampliifcation （LAMP） assay for rapid detecting Cryptococcus neoformans （C. neoformans）. Methods Two pairs of primers were designed according to 18 S rRNA gene sequence of C. neoformans. Performing LAMP at different temperatures in order to ascertain the optimum temperature of LAMP method for detection of C. neoformans, other common pathogens of meningitis and some species of candida were all ampliifed through LAMP, to investigate the specificity of detecting C. neoformans by LAMP assay. Fresh cultured of C. neoformans was diluted into different concentrations with non-cryptococcal infection cerebrospinal fluid, to build C. neoformans meningitis cerebrospinal fluid simulation environments. Sensitivity was evaluated through detecting C. neoformans of different concentrations in cerebrospinal lfuid. Result LAMP assay could achieve good ampliifcation between 60℃and 65℃. In Speciifcity test, only C. neoformans showed ampliifcation, no speciifc reactions were found in other meningitis pathogens nor candidas, LAMP showed a speciifcity of 100%. The minimum detection limit of LAMP for C. neoformans from cerebrospinal lfuid is 102 CFU/ml. Detecting C. neoformans with LAMP assay could save time for culture and conventional identiifcation. From DNA extraction to the end of the reaction could be completed within 2-3 hours, greatly reducing the time needed for diagnosis. Conclusion LAMP could be a very simple, rapid method for detecting C. neoformans with high speciifcity and sensitivity.

关 键 词：环介导等温扩增 新型隐球菌 脑膜炎 

分 类 号：R446.5[医药卫生—诊断学]