Potential use of dissolved cyanobacterial DNA for monitoring toxic Microcystis cyanobacteria in filtered water

作者：Mbukwa Elbert A; Boussiba Sammy; Wepener Victor; Leu Stefan; Yuval Kaye; Msagati Titus A M^*; Mamba Bhekie B

来源：Physics and Chemistry of the Earth, 2013, 66: 167-172.

DOI：10.1016/j.pce.2013.07.004

• Dissolved DNA
• PCR template
• Primers
• Mcy genes
• Toxic Microcystis sp.
• Water quality

Toxic and non-toxic Microcystis sp. are morphologically indistinguishable cyanobacteria that are increasingly posing health problems in fresh water systems by producing odours and/or toxins. Toxic Microcystis sp. produces toxicologically stable water soluble toxic compounds called microcystins (MCs) that have been associated with cases of aquatic life and wildlife poisoning and kills including some cases of human illnesses/deaths around the world. Thus, the need for rapid detection of toxic Microcystis sp. in surface water is imperatively a necessity for early mitigation purposes. Genomic DNA from potentially toxic Microcystis sp. comprises of ten microcystin synthetase (mcy) genes of which six major ones are directly involved in MCs biosynthesis. In Polymerase Chain Reaction (PCR) methodsmcy genes can be amplified from intracellular/extracellular genomic DNA using PCR primers. However, little is known about the limitations of sourcing genomic DNA templates from extracellular DNA dissolved in water. In this work, filtered water (0.45 mu M) from a Microcystis infested Dam (South Africa) was re-filtered on 0.22 mu M syringe filters followed by genomic DNA isolation and purification from micro-filtrates (9 mL). Six major mcy genes (mcyABCDEG) from the isolated DNA were amplified using newly designed as well as existing primers identified from literature. PCR products were separated by gel electrophoresis and visualized after staining with ethidium bromide. The limitation of using dissolved DNA for amplification of mcy genes was qualitatively studied by establishing the relationship between input DNA concentrations (10.0-0.001 ng/mu L) and the formation of respective PCR products. The amplification of mcyA gene using new primers with as little as 0.001 ng/mu L of DNA was possible. Other mcy gene sensitivities reached 0.1 ng/mu L DNA dilution limits. These results demonstrated that with appropriately optimized PCR conditions the method can provide accurate cost-effective tools for rapid detection of toxic Microcystis sp. in water giving early information for water quality monitoring against MC producing cyanobacteria.

• 出版日期2013