Quantitative and rapid detection of the trichloroethylene-degrading bacterium Methylocystis sp. M in groundwater by real-time PCR.

We developed a method based on real-time PCR for the specific and rapid enumeration of a trichloroethylene-degrading methanotroph, Methylocystis sp. M, with the aim of monitoring the strain in groundwater. A primer set designed from the nucleotide sequence of the mmoC gene of a soluble methane monooxygenase (sMMO) gene cluster from Methylocystis sp. M was specific to amplify the DNA region from the strain and no PCR products were amplified with the sMMO gene clusters from six other methanotroph strains. The real-time PCR reliably quantified Methylocystis sp. M over at least five orders of magnitude (5x10(6) to 5x10(2 )cells/PCR tube, or 2x10(8) to 2x10(4 )cells/ml). Five cells of Methylocystis sp. M per PCR tube (2x10(2 )cells/ml) were detectable when the cells were suspended in distilled water. The concomitant presence of other methanotrophs in samples did not affect the reliability of enumeration; and recovery of the cells with a membrane filter enabled us to quantify cells of the strain in groundwater. This quantification procedure was completed within 3 h, including preparation time of environmental samples. We conclude that real-time PCR using the mmoC primer set can be used practically to analyze the behavior of Methylocystis sp. M at bioremediation sites.