CAJ | 학술논문

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。
목적：건립일충쾌속검측신형은구균적방법。방법근거신형은구균적18 S rRNA기인서렬설계내외인물각일대，재불동온도하대신형은구균진행배개도등온확증，이탐명배개도등온확증법검측신형은구균최괄의적온도。대신형은구균、기타궤충상견인기뇌막염적병원균이급부분충류적념주균진행확증，이연구배개도등온확증기술검측은구균적특이성。장신선배양적신형은구균희석후가입비은구균감염환자적뇌척액중，모의뇌막염감염후적뇌척액배경，이연구배개도등온확증기술검측신형은구균적민감성。결과배개도등온확증법검측신형은구균재60℃～65℃조건하균능실현량호확증。배개도등온확증법대기타병원균적검측결과균위음성，부신형은구균적검측결과위양성，특이성체도100%。용배개도등온확증법종뇌척액중검측신형은구균적최저검출한위102 CFU/ml。용배개도등온확증기술검측신형은구균성뇌막염，능절성배양급전통감정적시간，종DNA적제취지반응결속재2～3h내즉가완성，대대감소료진단소수시간。결론배개도등온확증시일충령민도고，특이성강，모시단，차조작간편적검측신형은구균감염적방법。
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.