Analysis of Bacterial Communities Present in Agaricus bisporus Phase II Compost Using Amplified Ribosomal DNA Restriction Analysis (ARDRA)(Ⅰ)——Construction of a Bacterial 16S rDNA Library

An efficient and cost effective method for obtaining good quality DNA from composts containing high levels of organic matter has been developed.The protocol consisted of sample washing with sodium phosphate and EDTA,cell wall disruption using combined chemical and enzyme treatment prior to extraction,removal of humic acid and other inhibitors in high salt buffer containing polyvinyl polypyrrolidone (PVPP),and precipitation of DNA with polyethylene glycol (PEG-8000).The method was used to extract 16S ribosomal DNA from four samples taken at different stages in the preparation of PhaseⅡcompost used for Agaricus bisporus cultivation.Extracted DNA was sufficiently free of contaminants to allow PCR amplification of products derived from bacteria,actinomycetes and fungi.Purified products derived from PCR amplification of the extracted 16S rDNA using primers F27 and R1498 were used to construct 16S rDNA libraries.PCR amplification of ten randomly selected positive clones using primer M13 revealed that the 16S rDNA insertion rate was higher than 90%.