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.
Kikuchi
T
T
Department of Bioresources Sciences, School of Agriculture, Ibaraki University, 3-21-1 Ami-machi, Inashiki, Ibaraki-ken 300-0393, Japan.
Iwasaki
K
K
Nishihara
H
H
Takamura
Y
Y
Yagi
O
O
D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005618:Fresh Water / Q000382:microbiology*; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D020560:Proteobacteria / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014241:Trichloroethylene / Q000378:metabolism*; D014874:Water Pollutants, Chemical / Q000378:metabolism*
D004269:DNA, Bacterial; D014874:Water Pollutants, Chemical; D014241:Trichloroethylene; D010105:Oxygenases; C021555:methane monooxygenase