Detection of microcystin-producing cyanobacteria in water samples using loop-mediated isothermal amplification targeting mcyB gene

Abstract

Microcystin toxin-producing cyanobacteria are known to have harmful effects on humans and animals. We have developed a loop-mediated isothermal amplification (LAMP)-based detection method by targeting the microcystin synthetase B gene (), the gene responsible for the production of microcystin. The sensitivity of the method was found to be 1 fg per reaction, and it was 1000-fold higher than the conventional PCR. The LAMP method was able to amplify the target gene with a minimum amount of dNTP (0.4 mM), which further reduces the cost of reaction. The improved LAMP assay could detect the presence of the toxin-producing cyanobacteria in water samples within 2 h of time, which demonstrates the rapidness of the method. Freshwater samples were screened using the developed LAMP, and seven water samples collected from lakes and a bird sanctuary tested positive for gene harboring cyanobacteria, and negative in all other drinking waters. Hence, the developed LAMP could be a possible alternative to the existing molecular methods for screening for microcystin in environmental samples with greater sensitivity.

Recent trends in the detection of freshwater cyanotoxins with a critical note on their occurrence in Asia

“…This technology has completely revolutionized the amplification of DNA due to the high primer specificity towards its target. Ramya et al, 2018, developed LAMP to identify microcystin synthetase B gene (mcyB) responsible for the production of MCs. The developed method was rapid (~2 h), and able to detect the toxin producing cyanobacteria from the seven water samples collected from the freshwater lakes and bird sanctuary from Chennai, Tamil Nadu, India [99].…”

Spatio-temporal variation of toxin-producing gene abundance in Microcystis aeruginosa from Poyang Lake

“…Thus, the sequence of the mcy gene can be used to determine the toxicity potential of Microcystis and non-toxic Microcystis. The mcyA gene is necessary for the biosynthesis of MCs [12], while the mcyB gene is a key element in the synthesis of polypeptide chains in the growth of MCs [13]. Therefore, we analyzed the gene abundances of mcyA and mcyB to detect the microcystins level in Poyang Lake.…”

Rapid detection ofmcyGgene of microcystins producing cyanobacteria in water samples by recombinase polymerase amplification combined with lateral flow strips

“…One is to detect MCs, including high-performance liquid chromatography (HPLC) (Hemmati et al 2019), liquid chromatography-mass spectrometry (LC-MS) (Palagama et al 2018), high-performance capillary electrophoresis (Massey et al 2020), enzyme-linked immunosorbent assay (ELISA) (Lu et al 2020a(Lu et al , 2020b, protein phosphatase inhibition assay, and biosensors (Moore et al 2016). The other is to detect MC synthesis genes (e.g., mcyA, mcyB, mcyE, and mcyG), such as polymerase chain reaction (PCR) (Manali et al 2019), quantitative PCR (qPCR; Otten et al 2017), and loop-mediated isothermal amplification (LAMP) (Ramya et al 2018). These methods can meet the need for highly sensitive detection of MCs producing cyanobacteria and have important reference significance for the detection of the MC synthesis genes.…”