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Research Journal of Environmental Sciences
Year: 2007  |  Volume: 1  |  Issue: 5  |  Page No.: 229 - 236

Real Time PCR Assay for Polyphosphate Kinase Genes in Activated Sludge

S.B. Maqbool, A. Ahmad, M.B. Sticklen and S.A. Hashsham    

Abstract: 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.

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