[Study on the method for rapid detection of Mycobacterium tuberculosis by phage amplified biologically assay]

To set up a method for rapid detection of Mycobacterium tuberculosis (MTB) by phage amplified biologically (PhaB) assay and to investigate the optimal test condition.

Various test conditions were compared in order to observe the influence on detective results after MTB was infected by Mycobacteriophage. The test condition established was used for detection of sputum samples, and the results were compared with BIOTEC Lab test.

The bacterial concentration of MTB in 200 - 500/ml was detected by PhaB assay at 1 x 10(9) plaque forming unite (PFU)/ml of Mycobacteriophage, 37 degrees C for 60 min. The optimal concentration of virucidal for inactivation of Mycobacteriophage was 100 mmol/ml for 5 min at room temperature. The bacteriolytic plaque was clear at the concentration of 1 x 10(8)/ml indicator cells. Bacterium inactivated by heat can not be infected by Mycobacteriophage. Positive result was observed for control strains of H(37)Rv, H(37)Ra and M. bovis while negative result was obtained for 7 strains of non-Mycobacterium and 16 control strains of non-Tuberculosis Mycobacteria (NTM). The 4 strains of NTM (M. fortuitum, M. intrcellulare, M. aurum, M. phlei) showed positive reaction at higher concentrations (> 1 x 10(5)/ml). The repetition test showed that the differentiation coefficient in batch and inner was all under 15%. There was a significantly difference (P < 0.01) in positive rate between two digestion-decontamination procedure with N-acetyl-cysteine-NaOH liquefacient (94%) and NaOH liquefacient (62%). The positive rate of the samples cultured one day (65%) was significantly higher than that of the samples without preculture (40%). The results for detection of clinical samples by two reagents, ours and BIOTEC Lab, were nearly the same.

Because its rapidity, simplicity, and sensitivity, PhaB assay can be used for rapid detection of MTB, but the condition of test is very important.