PCR扩增技术联合CRISPR-Cas13a系统对MTB DNA检测方法的初步研究  

Preliminary study on detection method of MTB DNA by PCR amplification combined with CRISPR-Cas13a system

作　　者：于佳佳 张旭霞[1] 张雨晴[1] 任卫聪[1] 姚丛[1] 李传友[1] 刘毅[1] 唐神结[2] YU Jia-jia;ZHANG Xu-xia;ZHANG Yu-qing;REN Wei-cong;YAO Cong;LI Chuan-you;LIU Yi;TANG Shen-jie(Department of Bacteriology and Immunology,Beijing Key Laboratory on Drug-resistant Tuberculosis Research,Beijing Chest Hospital,Capital Medical University,Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China)

机构地区：[1]北京市结核病胸部肿瘤研究所,首都医科大学附属北京胸科医院,耐药结核病研究北京市重点实验室细菌免疫室,101149 [2]北京市结核病胸部肿瘤研究所,首都医科大学附属北京胸科医院,结核病多学科诊疗中心,101149

出　　处：《中国防痨杂志》2020年第12期1280-1288,共9页Chinese Journal of Antituberculosis

基　　金：首都卫生发展科研专项(2020-2-1042);“十三五”国家科技重大专项(2018ZX10301407-006);北京市教育委员会科技计划一般项目(KM202010025001)。

摘　　要：目的建立一种聚合酶链式反应(polymerase chain reaction,PCR)联合CRISPR(clustered regularly interspaced short palindromic repeats)-Cas13a识别靶基因核酸序列对结核分枝杆菌脱氧核糖核酸(Mycobacterium tuberculosis deoxyribonucleic acid,MTB DNA)进行检测的方法。方法将MTB保守序列IS6110片段插入pMDTM19-Tsimple Vector克隆载体,构建含待检测靶序列的模拟MTB质粒。同时针对待检测靶序列MTB保守序列IS6110设计可以检测MTB DNA的3条不同的特异性规律成簇间隔短回文重复序列RNA(CRISPR RNA,crRNA)探针(IS6110-1crRNA、IS6110-2crRNA、IS6110-3crRNA),引导CRISPR-Cas13a识别转录产物。将筛选出的特异性crRNA、不同待检测标本的PCR扩增转录产物、Cas13a、crRNA和Background RNA等按比例混合构建PCR-CRISPR反应体系。利用荧光定量PCR仪对含有MTB DNA不同稀释浓度的质粒模板、标准菌株H37Rv及6种非结核分枝杆菌进行检测。通过测定的相对荧光强度值分析检测的敏感度及特异度,最终建立基于MTB CRISPR-Cas13a系统的PCR-CRISPR检测方法。结果选择相对荧光强度值最大的IS6110-1crRNA[相对荧光强度值为197680.64(98364.94,304271.25)]作为后续MTB DNA检测的crRNA探针。PCR-CRISPR检测最低拷贝数为101拷贝/μl的质粒和100拷贝/μl的H37Rv扩增产物的相对荧光强度值[分别为38655.34(31975.51,45410.32)和17691.50(17612.36,17793.29)]明显高于阴性对照[29989.48(29435.72,30263.20)和13725.83(13652.43,13804.95)](Z=-6.713、-9.448;P值均<0.001),显示敏感度较好;阴性对照[37635.57(37168.74,38199.20)]和戈登分枝杆菌[39351.83(38903.70,39769.53)]、胞内分枝杆菌[39191.30(39018.51,39434.95)]、堪萨斯分枝杆菌[25172.20(24586.95,26046.45)]、脓肿分枝杆菌[37328.03(36959.01,37546.78)]、鸟分枝杆菌[37942.29(37455.63,38401.13)]、偶发分枝杆菌[29491.19(29148.63,30058.62)]等6种非结核分枝杆菌的相对荧光强度值均明显低于106拷贝/μl的MTB DNA质粒的相对荧光强度�Objective To establish a method for detection of Mycobacterium tuberculosis deoxyribonucleic acid(MTB DNA)by polymerase chain reaction(PCR)combined with CRISPR(clustered regularly interspaced short palindromic repeats)-Cas13 ato identify target gene nucleic acid sequence.Methods IS6110 fragment of MTB conserved sequence was inserted into pMDTM19-Tsimple Vector cloning vector to construct simulated MTB plasmid containing the target sequence to be detected.At the same time,according to the conserved sequence IS6110 of MTB,three different specific probes(IS6110-1 crRNA,IS6110-2 crRNA,IS6110-3 crRNA)of detecting MTB DNA with clustered regularly interspaced short palindromic repeats RNA(CRISPR RNA,crRNA)were designed,and used to guide CRISPR-Cas13 ato recognize transcripts.PCR-CRISPR reaction system was constructed by mixing selected specific crRNA,PCR amplified transcripts of different samples,Cas13 a,crRNA and Background RNA in proportion.Plasmid templates containing MTB DNA with different dilution concentrations,standard strain H37 Rv and six kinds of non-tuberculous mycobacteria were detected by the fluorescence quantitative PCR instrument.The sensitivity and specificity of detection were analyzed by the measured relative fluorescence intensity(Avalue),and finally the PCR-CRISPR detection method based on MTB CRISPR-Cas13 asystem was established.Results IS6110-1 crRNA with the strongest relative fluorescence intensity(A value:197680.64(98364.94,304271.25))was selected as crRNA probe for subsequent MTB DNA detection.PCR-CRISPR detection of low copy number of101 copies/μl plasmid and 100 copies/μl H37 Rv relative fluorescence intensity of amplification products(Avalues:38655.34(31975.51,45410.32)and 17691.50(17612.36,17793.29),respectively)was obviously higher than that of negative control(29989.48(29435.72,30263.20)and 13725.83(13652.43,13804.95);Z=-6.713,-9.448;Ps<0.001),with better sensitivity.Negative control(37635.57(37168.74,38199.20)),Mycobacterium gordonae(39351.83(38903.70,39769.53)),Mycobacterium intracel

关 键 词：结核 分枝杆菌 聚合酶链反应 DNA探针 微阵列分析 CRISPR-Cas13a 

分 类 号：R52[医药卫生—内科学] R440[医药卫生—临床医学]