MALDI-TOF analysis of polymerase chain reaction products from methanotrophic bacteria.
Polymerase chain reaction (PCR) assays were designed to amplify 56- and 99-base regions of the pmoA gene from Methylosinus trichosporium OB3b and Methylomicrobium albus BG8, two species of methanotrophic bacteria that are of interest for monitoring bioremediation activity. The PCR product sizes are in a mass range that is accessible to analysis by MALDI-TOF mass spectrometry. A rapid purification procedure using commercially available reversed-phase cartridges was applied prior to MALDI-TOF analysis. A small aliquot (1.5%, 1.5 microL) from a single 100-microL PCR reaction was sufficient for reliable detection. No cross-amplification products were observed when primers designed for one bacterial species were used with genomic DNA of the other species. The methodology described here has potential to allow less expensive and faster characterization of the ability of microbial populations to destroy pollutants in groundwater and soil at contaminated industrial sites.
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Tennessee 37831, USA. hurstgb@orn1.gov
G B
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Hurst
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Weaver
M J
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Doktycz
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Buchanan
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Costello
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Lidstrom
D001673:Biodegradation, Environmental; D004269:DNA, Bacterial / Q000032:analysis; D005798:Genes, Bacterial; D008772:Methylococcaceae / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012988:Soil Microbiology; D012989:Soil Pollutants / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014871:Water Microbiology; D014874:Water Pollutants, Chemical / Q000032:analysis
D004269:DNA, Bacterial; D012989:Soil Pollutants; D014874:Water Pollutants, Chemical