Improved method for detection of methanotrophic bacteria in forest soils by PCR

A primer set was designed for the specific detection of methanotrophic bact eria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the alpha -subunit of the p articulate methane monooxygenase present in all methanotrophs. In control e xperiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specifi c for members of the genera Methylosinus, Methylocystis Methylomonas, Methy lobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing metha notrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the sam ples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acid s. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct s ize were amplified by PCR by use of the new primers. By sequencing of clone d PCR products, it could be confirmed that the PCR products represented par tial sequences with strong similarity to the pmoA gene. The sequence was mo st related to the pmoA sequence of a type II methanotroph strain isolated f rom the Ah layer of the investigated soils.