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 heavily burdens world public health security. Developing efficient, specific, convenient, and inexpensive MTB assays are essential for preventing and controlling TB.

Methods: In this study, we established a specific detection method for MTB using the Clustered Regularly Interspersed Short Palindromic Repeats (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 detecting MTB, we established the TB-CRISPR technique by testing 504 samples from patients with suspected TB.

Results: MTB H37Ra could be seen as low as 3.13 CFU/mL by the CRISPR-Cas12a system targeting IS6110. With BACTEC960 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 plays an essential role in the molecular diagnosis of TB. The whole detection time is less than 1.5 h. It is easy to operate and does not need complex instruments. It is of great significance for the rapid detection of MTB and the clinical diagnosis of TB.

Keywords: CRISPR; Diagnosis; Molecular testing; Mycobacterium tuberculosis; RAA.

Fig. 1

Scheme of TB-CRISPR

Fig. 2

Comparison of different gRNA detection capabilities (A) fluorescence curve; Blue:IS6110-gRNA1; Red: IS6110-gRNA2; Green: IS1081-gRNA1; Purple: IS1081-gRNA2; Gray: negative control; (B) Fluorescence value of four gRNAs NC: negative control; ***: P < 0.001

Fig. 3

Specificity test based on CRISPR system (A) CRISPR detection curves for the specificity of seven different strains; (B) column chart of CRISPR detection of specificity of seven different strains NC: negative control; ***: P < 0.001

Fig. 4

Sensitivity test based on CRISPR system (A) Real-time fluorescence intensity alteration of H37 Ra assay for different concentrations of targets; (B) The calibration plots of fluorescence intensity versus the logarithm of the target concentration. NC: negative control

Fig. 5

ROC curve 504 ROC Curve Analysis for Relative Fluorescence Values of Clinical Samples

Fig. 6

Relative fluorescence values for clinical samples. Samples included 217 sputa, 194 bronchoalveolar lavage fluid (BALF), 54 hydrothorax, 39 other (17 tissues, 10 urine, 4 puncture fluid, 3 cerebrospinal fluid, 2 ascites, 2 pus, and 1 lung puncture tissue)

Fig. 7

Specificity and sensitivity by clinical sample type