PCR-based detection of mcy genes in blooms of Microcystis and extracellular DNA of pond water

Abstract

Microcystin from Microcystis aeruginosa is the most widely studied hepatotoxin which is synthesized by the ~55-kb microcystin synthetase (mcy) gene cluster. In this study, employing polymerase chain reaction (PCR) amplification of six genes of the mcy genes cluster (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), the presence of the toxic cyanobacterium, Microcystis has been demonstrated in seven eutrophicated ponds of Varanasi. Unlike the DNA recovered from blooms or whole cells, extracellular DNA present in pond water was used as template which showed amplification of all the desired mcygenes. Additionally, amplification of five genes namely, mcyB, C, D, E and G was noted in a single reaction by multiplexing of desired primers. Our findings suggest that, (i) extracellular DNA present in water may be directly used as template, and (ii) multiplex PCR may be routinely employed for the monitoring of mcy genes. This study seems important especially for those ponds where blooms of Microcystis may be visibly absent but water does contain microcystin-LR. To our knowledge detection of mcy genes in any pond water using extracellular DNA as template in PCR assay has not been reported so far. Key words: Microcystis, cyanobacteria, microcystins, pond water, multiplex-PCR, extracellular DNA.