Exploratory development of PCR-fluorescent probes in rapid detection of mutations associated with extensively drug-resistant tuberculosis

Abstract

This study aims to evaluate the clinical value of PCR-fluorescent probes for detecting the mutation gene associated with extensively drug-resistant tuberculosis (XDR-TB). The molecular species identification of 900 sputum specimens was performed using polymerase chain reaction (PCR)-fluorescent probe. The mutations of the drug resistance genes rpoB, katG, inhA, embB, rpsL, rrs, and gyrA were detected. The conventional drug susceptibility testing (DST) and PCR-directed sequencing (PCR-DS) were carried out as control. DST demonstrated that there were 501 strains of rifampicin resistance, 451 strains of isoniazid resistance, 293 strains of quinolone resistance, 425 strains of streptomycin resistance, 235 strains of ethambutol resistance, and 204 strains of amikacin resistance. Furthermore, 427 (47.44%) or 146 (16.22%) strains were MDR-TB or XDR-TB, respectively. The mutations of the rpoB, katG, inhA, embB, rpsL, rrs, and gyrA genes were detected in 751 of 900 TB patients by PCR-fluorescent probe method, and the rate of drug resistance was 751/900 (83.44%). No mutant genes were detected in the other 149 patients. Compared with DST, the mutant rates of rpoB, katG/inhA, rpsL, rrs, embB, and gyrA of six drugs were higher than 88%; five of six drugs were higher than 90% except for SM (88.11%). The MDR and XDR mutant gene types were found in 398 (42.22%) and 137 (15.22%) samples. PCR-DS was also employed and confirmed the PCR-fluorescent probe method with the accordance rate of 100%. The PCR-fluorescent probe method is rapid and straightforward in detecting XDR-TB genotypes and is worthy of being applied in hospitals.

Keywords: Drug susceptibility testing; Extensive drug resistance; Mycobacterium tuberculosis; PCR-fluorescent probe.