基于环介导等温扩增技术建立的假丝酵母菌快速检测方法

Establishment of loop-mediated isothermal amplification forrapid detection of candida spp

摘要:

目的  基于环介导等温扩增技术（LAMP）建立一种假丝酵母菌的快速检测方法。方法  依据假丝酵母属18 S rDNA基因保守序列，采用Primer Explorer V4软件设计引物，建立LAMP扩增方法和反应体系，对体系的4种关键参数和3项性能指标进行优化和探讨，并用临床样本进行方法验证。结果  6种常见假丝酵母菌LAMP法检测均为阳性；最适扩增条件为：温度63～65℃、Bst酶活性4 IU、Mg2+浓度4 mol/L、扩增时间1 h；7种对照菌种及人全血DNA样本LAMP扩增结果均为阴性；检测限为102～107 CFU/ml。经52例临床样本验证，该法敏感性为100%，特异性为95%。结论  该研究建立的LAMP假丝酵母菌检测方法具有实用、简便、快速、特异和敏感的特点，可为临床提供一种新的假丝酵母菌诊断方法。

Abstract:

Objective To establish a rapid detection of candida spp on the basis of loop-mediated isothermal method. Methods According to the gene bank, 18 S rDNA gene sequence was provided. Primer Explorer V4 online design software design for the Candida genus-specific primers was used, and LAMP expansion method and the reaction system was set up. Four key parameters for the system and three performance were optimized and discussed, and then verified by clinical samples. Results Six kinds of candida were all positive, and the optimum temperature was 63℃ - 65℃, the activity of Bst was 4 u, concentration of Mg2+ was 4 mM, time was 1 h. All 7 kinds of clinical reference strains and Human whole blood DNA samples amplification results were negative. The detection limit was 107-102 CFU/ml. A total of 52 cases of clinical samples proved that the method sensitivity was 100%, and the specificity was 95%. Conclusions The method of the genus candida established by LAMP technology is significantly practical, simple, rapid, specific and sensitive.