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

doi:10.3969/j.issn.1000-6621.2020.12.006

摘要/Abstract

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pMD^TM19-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值[89204.07(66253.60,108819.13)](Z值均=-9.448,P值均<0.001),显示特异度良好。结论首次成功建立并验证了针对MTB的PCR-CRISPR检测技术,具有敏感度及特异度高、稳定性好、成本低等特点,有望进一步用于临床样本的检测。

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

Abstract:

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)-Cas13a to identify target gene nucleic acid sequence. Methods IS6110 fragment of MTB conserved sequence was inserted into pMD ^TM19-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 (IS6110-1crRNA,IS6110-2crRNA,IS6110-3crRNA) of detecting MTB DNA with clustered regularly interspaced short palindromic repeats RNA (CRISPR RNA, crRNA) were designed, and used to guide CRISPR-Cas13a to recognize transcripts. PCR-CRISPR reaction system was constructed by mixing selected specific crRNA, PCR amplified transcripts of different samples, Cas13a, crRNA and Background RNA in proportion. Plasmid templates containing MTB DNA with different dilution concentrations, standard strain H37Rv 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 (A value), and finally the PCR-CRISPR detection method based on MTB CRISPR-Cas13a system was established. Results IS6110-1crRNA 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 of 10¹ copies/μl plasmid and 10⁰ copies/μl H37Rv relative fluorescence intensity of amplification products (A values: 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 intracellulare (39191.30 (39018.51,39434.95)), Mycobacterium kansasii (25172.20 (24586.95,26046.45)),Mycobacterium abscessus (37328.03 (36959.01,37546.78)),Mycobacterium avium (37942.29 (37455.63,38401.13)),Mycobacterium fortuitum (29491.19 (29148.63,30058.62)) were significantly lower than the 10⁶ copies/μl MTB DNA plasmid of relative fluorescence intensity (89204.07 (66253.60,108819.13))(Zs=-9.448,Ps<0.001), showing good specificity. Conclusion For the first time, we established and verified the PCR-CRISPR detection scheme and technology for MTB, which could be used to detect the presence of MTB in samples. The detection method is characterized by high sensitivity, high specificity, good stability and low cost, and is expected to be further used in the detection of clinical samples.

Key words: Mycobacterium tuberculosis, Polymerase chain reaction, DNA probes, Microarray analysis, CRISPR-Cas13a

于佳佳, 张旭霞, 张雨晴, 任卫聪, 姚丛, 李传友, 刘毅, 唐神结. PCR扩增技术联合CRISPR-Cas13a系统对MTB DNA检测方法的初步研究[J]. 中国防痨杂志, 2020, 42(12): 1280-1288. doi: 10.3969/j.issn.1000-6621.2020.12.006

YU Jia-jia, ZHANG Xu-xia, ZHANG Yu-qing, REN Wei-cong, YAO Cong, LI Chuan-you, LIU Yi, TANG Shen-jie. Preliminary study on detection method of MTB DNA by PCR amplification combined with CRISPR-Cas13a system[J]. Chinese Journal of Antituberculosis, 2020, 42(12): 1280-1288. doi: 10.3969/j.issn.1000-6621.2020.12.006

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名称	crDNA序列(5'→3')
IS6110-1crDNA	5' GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGTGGTCCGAAGCGGCGCTGGACGAGAT 3'
IS6110-2crDNA	5' GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACTAGGTGAGGTCTGCTACCCACAGCCGGT 3'
IS6110-3crDNA	5' GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACACCCTGCCCAGGTCGACACATAGGTGAG 3'
crDNA-F	5' TAATACGACTCACTATAGGGGATTTAGACTACCCCAA 3'
IS6110-1crDNA-R	5' ATCTCGTCCAGCGCCGCTT 3'
IS6110-2crDNA-R	5' ACCGGCTGTGGGTAGCAGAC 3'
IS6110-3crDNA-R	5' ACCCTGCCCAGGTCGACACA 3'

名称	用量	名称	用量
crDNA	X μl (1μg)	T7 RNA Polymerase Mix	2μl
NTP Buffer Mix	10μl	nuclease-free water	up to 30μl

名称	识别序列	crRNA序列(5'→3')
IS6110-1crRNA	ACCGGCTGTGGGTAGCAGACCT CACCTA	5' GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUA GGUGAGGUCUGCUACCCA CAGCCGGU 3'
IS6110-2crRNA	ATCTCGTCCAGCGCCGCTTCGG ACCACC	5' GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGG UGGUCCGAAGCGGCGCUGGACGAGAU 3'
IS6110-3crRNA	CTCACCTATGTGTCGACCTGGG CAGGGT	5' GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAC CCUGCCCAGGUCGACACA UAGGUGAG 3'