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 mcy
genes. 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.