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

Abstract

Nowadays, cyanobacteria blooms and microcystins (MCs) pollution are threatening water safety and public health. In this study, a rapid detection method was established for detecting MCs producing cyanobacteria. The MC synthesis gene mcyG was measured through recombinase polymerase amplification combined with lateral flow strips (LF-RPA) technology. The target gene mcyG was amplified at a temperature range of 37–45 °C, and the amplification time to detect mcyG was only 15 min at 37 °C. The optimal reaction conditions were confirmed using single dependent variable experiments, suggesting that the best probe dosage for 50 μL of the reaction mixture was 0.2 μL, the best dilution ratio of products was 1/100, and the best loading volume was 10 μL. The specificity test proved that the LF-RPA assay could distinguish MCs producing cyanobacteria from nontoxic algae well. Within 35 min of amplification time, the detection limit of the LF-RPA assay was 103 copies/mL mcyG and 104 cells/mL Microcystis aeruginosa FACHB-905. Overall, the LF-RPA assay could detect MCs producing cyanobacteria in water samples quickly and accurately, and it has a great promise to be applied for monitoring the MCs producing cyanobacteria blooms in natural waters. HIGHLIGHTS A rapid detection method based on the LF-RPA assay was developed for detecting microcystins producing cyanobacteria.; The amplification time of mcyG was only 15–35 min at 37 °C in the LF-RPA assay.; Reducing the probe dosage and increasing the dilution ratio could effectively improve the accuracy of the test results.; The detection limit of the LF-RPA assay was similar to that of the PCR assay.;

Keywords

• lateral flow strip
• mcyg
• microcystins producing cyanobacteria
• rapid detection
• recombinase polymerase amplification