Abstract:

Objective Establishment of polymerase spiral reaction (PSR) assay for detection of Mycobacterium tuberculosis (MTB) and evaluate its effect. Methods The PSR primers targeting the specific genes IS 6110 of MTB were designed. The genomic DNA of MTB was extracted and added into RM2 × reaction solution containing Bst DNA polymerase. The reaction was carried out at 63℃ for 45 min and detected by fluorescence quantitative PCR. By optimizing the primer sequence, the best one was selected, and its specificity and minimum detection limit were evaluated. A total of 200 sputum samples were collected from Zhengzhou Sixth Peoples Hospital during May 2019 to August 2019, and three copies were collected from the same patient. Sputum smear and sputum culture, as well as PSR detection were performed on sputum samples. Sputum culture was used as the gold standard to evaluate the effectiveness of PSR in detecting tuberculosis patients. Results A set of PSR primers for detection of MTB was screened by optimizing the primer sequence, the limit detection of PSR application to MTB was 10 ³ CFU/ml. The detection specificity experiment showed that the method had no cross-reactivity with 15 strains of non-tuberculous mycobacteria. Among 200 pulmonary tuberculosis patients, 48 cases were positive in sputum smears, and the positive detection rate was 24.0% (48/200); 83 cases were positive in sputum culture test, and the positive detection rate was 41.5% (83/200); 87 cases were positive in PSR test, and the positive detection rate was 43.5% (87/200). Compared with the gold standard method, the results of 200 clinical samples test showed that the sensitivity, specificity and positive predictive value, negative predictive value were 96.4%(80/83),94.0%(110/117),92.0%(80/87),97.3%(110/113), respectively, which were consistency to sputum culture detection method (Kappa value was 0.898). Conclusion PSR detection is suitable for the rapid diagnosis of MTB.

Key words: Mycobacterium tuberculosis, Sputum, Polymerase spiral reaction, Diagnosis, Laboratory techniques and procedures