Development of an hcp-based assay for the quantification of picocyanobacteria by real-time PCR. (c2012)

Abstract

Picocyanobacteria form most of the autotrophic picoplankton (APP) and are the major contributors to primary production in both freshwater ecosystems and oligotrophic oceans. Two related APP genera forming the picocyanobacterial clade are Synechococcus sp. and Prochlorococcus sp. Several methods have been used for the classification and quantification of picocyanobacteria including flow cytometry and analysis of photosynthetic pigments. However, more recently, quantitative real-time PCR (qPCR) has become the method of choice for quantification purposes as well as the assessment of biodiversity in picoplanktonic environments, including studies on cyanobacterial prevalence. Lately, both marine and freshwater picocyanobacteria were found to harbor a gene, hcp, 100% conserved among all the strains isolated and therefore representing a valuable target for quantification of picocyanobacteria by qPCR, which is the aim of the present study. The approach used is summarized by the creation of a plasmid that harbors the gene (based on one strain) to construct a standard curve for qPCR, followed by qPCR of environmental samples. We have carried out sequence alignment of hcp for Synechococcus sp. strains LS 0504, KD3a, ARC-11 and ARC-21. Based on the sequence alignment, we have designed primers for part of the hcp gene (186 bp). PCR reactions on both freshwater and marine DNA samples from strains LS 0504, KD3a, ARC-11, ARC-21 and WH 8102 verified the successful primer design. hcp, amplified from LS 0504 strain was then cloned and transformed into plasmid DNA and absolute quantification of samples collected from the Sargasso Sea, USA and from an artificial lake at Annaya, Lebanon was performed by qPCR using an external standard curve based on serial dilutions of the plasmid. The numbers of picocyanobacteria from marine samples were in the range of 103 cells/mL and numbers from freshwater samples were in the range of 105 cells/mL. We have thus successfully designed an hcp-based assay that can be applied to quantify picocyanobacteria in local freshwater and marine environments.