Abstract:

A total of 546 patients with pulmonary tuberculosis who were hospitalized in Xi’an Chest Hospital between September 2017 to August 2019 were selected as subjects to evaluate the clinical application value of fluorescence PCR probe melting curve method (melting curve method) in detecting drug resistance of Mycobacterium tuberculosis (MTB). The sputum samples of the subjects with the amount larger than 2 ml were collected. After strain identification, 531 cases (strains) were finally included as the research objects. The same sputum sample of each subjects was performed drug resistance gene detection using BACTEC MGIT 960 liquid culture drug susceptibility test (960 liquid drug susceptibility test) and fluorescence PCR probe melting curve method. For subjects with inconsistent results of MGIT 960 drug sensitivity test and melting curve method for rifampicin and isoniazid resistance, the melting curve method was verified by gene chip method to detect drug resistance gene. Based on the results of MGIT 960 liquid drug susceptibility test as reference standard, the sensitivity, specificity, coincidence rate and Kappa value of the melting curve method in detecting streptomycin resistance were 86.67% (39/45), 99.38% (483/486), 98.31% (522/531) and 0.887; the sensitivity, specificity, coincidence rate and Kappa value of the melting curve method for detecting isoniazid resistance were 69.23% (54/78), 98.68% (447/453), 94.35% (501/531) and 0.751; the sensitivity, specificity, coincidence rate and Kappa value of the melting curve method for the detection of rifampicin resistance were 87.50% (42/48), 96.89% (468/483), 96.05% (510/531) and 0.778; the sensitivity, specificity, coincidence rate and Kappa value of the melting curve method for detecting ethambutol resistance were 50.00% (9/18), 98.83% (507/513), 97.18% (516/531) and 0.531. Among the 30 cases that were resistant by the melting curve method and sensitive by the MGIT 960 liquid drug sensitivity method, the melting curve method showed that the 6 cases resistant to isoniazid were katG 315 codon mutation; among the 15 cases resistant to rifampicin, the mutations at codons of ropB 507-512, ropB 521-528, and ropB 529-533 were 3 cases, 3 cases, 9 cases, respectively; the 3 cases resistant to streptomycin were rrs 513-517 codon mutation; the 6 cases resistant to ethambutol were embB 306 codon mutation. The results of gene chip detection of drug resistance genes revealed that among the 6 cases of resistance to isoniazid, there were 3 cases of katG 315 (AGC→ACC) mutation and 3 cases of katG 315 (AGC→AAC) mutation; and among the 15 cases of resistance to rifampicin, there were 3 cases of ropB 511 CTG→CCG mutation, 3 cases of ropB 526 CAC→TAC mutation and 9 cases of ropB 531 TCG→TGG mutation, which were consistent with the results of melting curve method. Fluorescence PCR probe melting curve method has good efficiency for the detection of streptomycin, isoniazid, rifampicin, ethambutol resistance mutation sites, and it can be used for rapid screening of clinical resistance to first-line anti-tuberculosis drugs.

Key words: Tuberculosis,multidrug-resistant, Polymerase chain reaction, Microbial susceptibility tests, DNA probes, Microarray analysis, Comparative study, Data interpretation,statistical