A new method for the detection of Mycobacterium tuberculosis based on the CRISPR/Cas system

Abstract

Object: Mycobacterium tuberculosis (MTB) is a bacterium that can cause zoonoses by aerosol transmission. Tuberculosis (TB) caused by MTB places a heavy burden on world public health security. The development of efficient, specific, convenient, and inexpensive MTB assays is important for the prevention and control of TB. Methods: Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) is a special DNA repeat family widely present in bacterial and archaeal genomes. In this study, we established a specific detection method for MTB using the CRISPR system, combined with recombinase mediated isothermal nucleic acid amplification (RAA) to improve the sensitivity of the detection system and achieve "two-level" amplification of the detection signal. The sensitivity and specificity of RAA combined with the CRISPR/Cas system were analyzed. Using BACTEC 960 culture as the gold standard for the detection of MTB, we established the TB-CRISPR technique by testing 504 samples from patients with suspected tuberculosis. Results: MTB H37Ra could be detected as low as 3.13 CFU/mL by the CRISPR-Cas12a system targeting IS6110. With BACTCE960 culture (120 positives and 384 negatives) as the gold standard, the sensitivity of the TB-CRISPR technique was 0.883 (0.809-0.932) and the specificity was 0.940 (0.910-0.961). According to the receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) reached 0.944 (0.914-0.975) within 95% CI. The positive likelihood ratio (PLR) was 14.747 (9.870-22.035) and the negative likelihood ratio (NLR) was 0.124 (0.076-0.203). The positive predictive value (PPV) was 0.822 (0.742-0.881) and the negative predictive value (NPV) was 0.963 (0.937-0.979). Conclusion: TB-CRISPR realizes the rapid screening and diagnosis of MTB. The whole detection time is less than 1.5h. It is easy to operate and does not need to rely on complex instruments. It is of great significance for the rapid detection of MTB and the clinical diagnosis of TB.