实时荧光环介导等温扩增检测毛癣菌属方法的建立  

Establishment of a real-time fluorescence loop-mediated isothermal amplification method for the detection of Trichophyton

作　　者：邓穗燕[1] 郭旭光[1] 何文茵[1] 易江华[2] 夏勇[1] Deng Suiyan;Guo Xuguang;He Wenyin;Yi Jianghua;Xia Yong(Department of Clinical Laboratory,The Third Affiliated Hospital of Guangzhou Medical University,Guangzhou 510150,China;Department of Dermatology,The Third Affiliated Hospital of Guangzhou Medical University,Guangzhou 510150,China)

机构地区：[1]广州医科大学附属第三医院检验科,广州510150 [2]广州医科大学附属第三医院皮肤科,广州510150

出　　处：《中华临床实验室管理电子杂志》2020年第4期211-216,共6页Chinese Journal of Clinical Laboratory Management(Electronic Edition）

基　　金：国家自然科学基金青年项目(81700004);广州市卫生健康科技一般引导项目(20201A011091)。

摘　　要：目的建立实时荧光环介导等温扩增(Real-time Loop-mediated Isothermal Amplification,LAMP)技术检测皮肤癣的方法,实现皮肤癣菌病的快速实验室诊断。方法通过在NCBI官网查找下载皮肤癣菌中毛癣菌属、小孢子菌属和表皮癣菌属临床分离率较高的菌种rRNA序列(18S rRNA,ITS1,5.8S rRNA,ITS2和28S rRNA),运用DNASTAR Lasergene软件进行分析比对,寻找出毛癣菌属内高度保守的靶序列,且该段靶序列要与其他属存在差异,运用Primer Explorer V5设计LAMP引物。提取须癣毛癣菌NBRC6202基因组DNA作为模板筛选引物以及建立LAMP检测毛癣菌属的反应体系,以红色毛癣菌ATCC28188基因组DNA作为模板测试其敏感性和稳定性,以毛癣菌属以外的其他真菌、细菌基因组DNA验证LAMP反应体系的特异性。结果筛选的4套引物里,primer2的扩增效果较好,可在22 min检测出须癣毛癣菌,且可以特异地检测出红色毛癣菌、指间毛癣菌、疣状毛癣菌、许兰毛癣菌、紫色毛癣菌。本研究建立的毛癣菌属RT-LAMP检测方法的最低检测限为1 pg/μL;在10000 pg/μL、100 pg/μL和1 pg/μL三个浓度的分别10次重复检测实验中平均CT值和CV%分别为9.24±0.30,3.29%、10.57±0.54,5.12%、15.95±0.52,3.24%。结论实验建立的毛癣菌属LAMP检测方法在特异性、敏感性和稳定性三方面均表现出优越的性能,能快速准确地检测出毛癣菌属常见菌种,具有良好的临床应用前景。Objective To establish an efficient,sensitive and accurate method for the detection of dermatophytosis by using the real-time fluorescence technique based on loop-mediated isothermal amplification,so as to realize the rapid laboratory diagnosis of dermatophytosis.Methods By searching and downloading the rRNA sequences of the strains with high clinical isolation rates of Trichophyton,Microsporum and Epidermophyfon from the NCBI website,DNASTAR Lasergene software was used for analysis and comparison to find the highly conserved target sequences of Trichophyton,which were different from those of other genera.LAMP primers that included a pair of internal primers,external primers and ring primers were designed using Primer Explorer V5.The NBRC6202 genomic DNA of Trichophyton mentagrophytes was extracted as a template for screening primers and a LAMP reaction system was established for the detection of Trichophyton.The sensitivity and stability of this reaction system were tested by using the ATCC28188 genomic DNA of T.rubrum.The specificity of LAMP reaction system was verified by the genomic DNA of other fungi and bacteria other than Trichophyton.Results Among the 4 sets of primers screened,primer2 had a good amplification effect and was able to detect T.mentagrophytes within 22 min.Moreover,T.rubrum,T.interdigitale,T.verrucosum,T.schoenleinii and T.violaceum could be specifically detected.The minimum detection limit of LAMP detection system established in this study was 1 pg/μL.The average CT value and CV%were 9.24±0.30,3.29%,10.57±0.54,5.12%,15.95±0.52,3.24%at 10000 pg/μL,100 pg/μL,and 1 pg/μL,respectively.The peaking time was stable.Conclusion The LAMP method of Trichophyton showed superior performance in specificity,sensitivity and stability,which can quickly and accurately detect common strains of Trichophyton,and had a good clinical application prospect.

关 键 词：实时荧光环介导等温扩增 皮肤癣菌 毛癣菌属 快速鉴定 

分 类 号：R756[医药卫生—皮肤病学与性病学]