| Abstract: | Objective To analyze the application value of DNA real-time fluorescence isothermal amplification method in the clinical detection of tuberculosis. Methods A total of 2421 patients with suspected pulmonary tuberculosis in Xi’an Chest Hospital from January to June 2019 were checked. Sputum samples collected from 356 patients were simultaneously subjected to BACTEC MGIT 960 liquid culture, real-time fluorescent quantitative nucleic acid detection of rifampicin resistance (GeneXpert MTB/RIF method), traditional real-time fluorescent quantitative nucleic acid detection of DNA (FQ-PCR method), detection of M.tuberculosis RNA by real-time fluorescence constant temperature amplification (SAT-RNA method) and isothermal amplification method. Taking the MGIT 960 test results as the gold standard, we calculated the sensitivity, specificity, positive predictive value, negative predictive value, coincidence rate, and Kappa value of the other four tests. The melting curve method was used to detect rifampin ropB gene in 121 nucleic acid samples positive for isothermal amplification method, to study the value of extracting nucleic acid with isothermal amplification method for drug resistance gene detection. Results Taking MGIT 960 test results as the gold standard, the sensitivity of isothermal amplification method (78.83%, 108/137) was lower than GeneXpert MTB/RIF (95.62%, 131/137), higher than FQ-PCR (74.45%, 102/137) and SAT-RNA (59.12%, 81/137), differences were statistically significant (χ 2=47.437, 43.654, 29.467; P values were all <0.001).The area under curve of the ROC curve for GeneXpert MTB/RIF was the largest (0.910), followed by the isothermal amplification method (0.860), FQ-PCR (0.854) and SAT-RNA (0.789). Conducting melting curve test on isothermal amplificated nucleic acid samples, when the dt value (detection time,1 dt=1 min) of the original nucleic acid and the 5 times diluted nucleic acid was 26-30, the detection rates of the ropB gene were 93.55% (29/31) and 90.32% (28/31) respectively; when the dt value was 15-25, the detection rates were 94.44% (34/36) and 100.00% (36/36) respectively. Conclusion Comparing with various detection technologies, the isothermal amplification method has clinical application value and is suitable for tuberculosis diagnosis in primary hospitals. |
