摘要：Polyphosphate kinase gene ( ppk ) encodes the enzyme polyphosphate kinase (PPK) that is thought to be responsible for the synthesis of polyphosphate (poly-P) in phosphorus accumulating organisms (PAOs). Methods to detect and quantify ppk gene simultaneously may be useful to detect microbial communities containing PPK. The objective of this study was to develop a real time polymerase chain reaction (RLT-PCR) assay for the detection and quantification of ppk gene in activated sludge . RLT-PCR conditions were optimized to amplify a 1.2 kb fragment of known ppk gene sequences in a plasmid and in Pseudomonas aeruginosa using degenerate primers. The protocol was then modified to use with the ABI Prism 7700 sequence detection system using SYBR® Green I for real time quantification of the amplicons. Four activated sludge microbial communities expected to contain varying levels of PAOs were analyzed to quantify the abundance of ppk . The results indicated that communities enriched for PAOs contained 10- to 100-fold higher levels of ppk (103-105 copies of ppk gene per 100 ng of total DNA) than the non-enriched communities (102 or less copies per 100 ng of total DNA). RLT-PCR is indeed a promising method to monitor environmental samples of complex nature and high similarity.
