Quantitative Detection of Trichloroethylene-Degrading Mycobacterium sp. TA27 with a Real-time PCR Product Detection System

Abstract

We developed a method for enumerating trichloroethylene (TCE) degrading Mycobacterium sp. TA27 by means of PCR. The PCR primers and probe were designed based on the 16S rRNA sequences of Mycobacterium sp. TA27, compared with corresponding regions of 8 phylogenetically related strains. The total DNAs of strain TA27 and the other 8 mycobacteria were extracted and used for PCR amplification by a 5'-nuclease real-time PCR assay. The highest specificity for strain TA27 was observed at an annealing temperature of 62°C and at forward and reverse primer concentrations of 0.2 μM each. The other 8 mycobacteria were not detected under these conditions. The detection limits for the DNA and cells of strain TA27 were 0.05 pg/tube and 25 cells/tube, respectively. The utility of the PCR assay for the quantification of strain TA27 cells was demonstrated in TCE-polluted groundwater.