Toxic Microcystis novacekii T20-3 from Phakalane Ponds, Botswana: PCR Amplifications of Microcystin Synthetase (mcy) Genes, Extraction and LCESI- MS Identification of Microcystins

Abstract

Treated water effluent from Phakalane waste water secondary maturation ponds in Gaborone City (Botswana) enters the Limpopo River via Notwane River. Effluent samples from these ponds were collected and investigated using molecular and analytical methods to determine presence of mcy genes and microcystins (MCs), respectively. It was observed that, potentially toxic algal blooms were present in this effluent and therefore algal toxins. Using Polymerase Chain Reaction (PCR) method; cyanobacterial 16S-rRNA and mcyA, -B, -C, -D, -E and -G genes were amplified and PCR products separated by gel electrophoresis and visualized after ethidium bromide staining. DNA sequence for mcyA gene was obtained using PCR products from a newly designed primer pair for the amplification of mcyA gene. BLAST results of the obtained DNA sequence were evaluated and aligned to NCBI database for species identification. The alignment gave the highest similarity (100%) in nucleotide sequence that was aligned to the DNA sequence of a toxigenic M. novacekii T20-3 based on the published data. Microcystin-RR, -YR, -LR and -WR were chromatographed, identified and quantified from M. novacekii T20-3 cell extracts using LC-ESI-MS technique after liquid-partitioning (LP) and solid-phase extraction (SPE) steps complementing PCR findings. Higher quantities of MC-RR, -LR and YR: 53.620 ± 0.063, 12.114 ± 0.024 and 5.280 ± 0.035 μg/g DW were observed, respectively.