Abstract:

Objective To establish the method of modified propidium monoazide (PMAxx)-quantitative PCR (qPCR) for identifying live and heat-inactivated Mycobacterium tuberculosis (MTB), and evaluate the value of PMAxx-qPCR assay to detect activity of antituberculosis drugs in vitro. Methods The pre-treatment conditions of PMAxx including light-exposure time, dark-incubation time, PMAxx concentration and DNA amplification size were optimized with live and heat-inactivated MTB (H37Rv). Through plotting the dose-effect curve and establishing the standard curve of the quantification cycle (Cq) value and bacterial load, the antibacterial activity of isoniazid (INH) and rifampin (RFP) in vitro were calculated by this assay, the detection sensitivity was evaluated, and comparing it to Microplate Alamar Blue assay (MABA) and colony forming unit (CFU) method, the colony count was analyzed by independent sample t-test, and the difference was statistically significant (P<0.05). Results When the bacterial load was set as 10 ⁷ CFU/ml, the dead and living bacteria could be identified effectively (ΔCq_dead-live=6.772±0.453) under the condition of the concentration of PMAxx with 10 μmol/L, dark incubation for 10 min and light-exposure for 15 min. The difference of the dead and living bacteria could be confirmed more effectively by using the >200 bp target DNA fragments. PMAxx-qPCR method obtained good MIC results comparing to MABA method in three days (INH: 0.049-0.076 μg/ml vs. 0.032-0.064 μg/ml, t=0.782, P=0.491; RFP: 0.102-0.145 μg/ml vs. 0.051-0.079 μg/ml, t=2.828,P=0.066). The Cq value of the quantitative standard curve showed a good linear relationship with the CFU (lgCFU/ml) (R ²=0.9863), and the limit number of detection was 10 ²CFU/ml. Meanwhile, the antibacterial counts of 16×, 8×, 4×, 2× and 1×MIC concentrations of INH by the PMAxx-qPCR method and CFU method were (4.376±0.344) and (4.325±0.318) CFU/ml, (4.232±0.106) and (3.936±0.194) CFU/ml, (4.122±0.277) and (3.874±0.105) CFU/ml, (3.950±0.113) and (3.675±0.250) CFU/ml, (3.770±0.228) and (3.618±0.257) CFU/ml, respectively. The antibacterial counts of 16×, 8×, 4×, 2× and 1×MIC concentrations of RFP by the PMAxx-qPCR method and CFU method were (4.577±0.216) and (4.675±0.250) CFU/ml, (4.445±0.054) and (4.374±0.675) CFU/ml, (3.627±0.173) and (3.154±0.076) CFU/ml, (1.946±0.359) and (2.159±0.083) CFU/ml, (1.552±0.423) and (0.960±0.202) CFU/ml respectively, all differences were not statistically significant ((t=0.165, P=0.880; t=1.894, P=0.199; t=1.186, P=0.357; t=1.419, P=0.292; t=0.626, P=0.595) and (t=0.469,P=0.671; t=0.199,P=0.855; t=3.535,P=0.071; t=0.784,P=0.490; t=1.777,P=0.174)). Conclusion The established PMAxx-qPCR method in this study is stable. It can be used to detect the activity of the first-line antituberculous drugs including INH and RFP with high sensitivity, rapid and accuracy.

Key words: Propidium monoazide (PMA), Antitubercular agents, Polymerase chain reaction, Microbial sensitivity tests, Comparative study