CRISPR-Based Diagnostic Method For Chlamydia Trachomatis in Patients With Non-Gonococcal Urethritis

Non-gonococcal urethritis (NGU) is a type of urethritis that is transmitted by sexual contact. About 20%–50% of NGU cases are caused by Chlamydia trachomatis . Most NGU cases are asymptomatic, but failure to intervene promptly can lead to severe sequelae of the reproductive system and other complications. Here, we aimed to develop a technique to routinely screen for C. trachomatis infections to prevent such adverse consequences. The proposed approach involves the use of clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) that is complementary to the cryptic plasmid fragments of C. trachomatis ( i.e. , the target sequence). In the presence of the target sequence, the RNase activity of the Cas13a protein is activated, and it cleaves a quenched RNA fluorescent probe such that fluorescence will be emitted. The proposed molecular diagnostic method based on the CRISPR-Cas13a technology leverages the high DNA-amplification efficiency of recombinase polymerase amplification (RPA) and the high specificity of crRNA. The sensitivity of the C. trachomatis -dsDNA detection based on CRISPR-Cas13a was 10 fM. A one-pot method based on CRISPR-Cas13a detection and qPCR was also developed. The results obtained by the two methods were closely correlated: χ 2 = 81.798 based on a Chi-squared test (P < 0.001), κ = 0.975 based on a Cohen’s kappa test (P < 0.001), and the area under the receiver operating characteristic curve area was 0.991 (95% confidence interval: 0.970–1.000 P < 0.001) with a sensitivity of 0.982 (95% confidence interval: 0.890–0.999) and specificity of 1.000 (95% confidence interval: 0.863–1.000). Further, the method can be carried out at room temperature and yields results within 1 hour. The developed technique does not require expensive instruments and, thus, can meet the needs of community hospitals and other institutions for screening. Future research to develop lyophilized reagents could enable the application of this technique to point-of-care testing.