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