Rapid Detection and Quantitation of Microcystin-Producing Microcystis Using Real-Time PCR

Abstract

This study is to develop and validate a real-time quantitative PCR (rt-QPCR) assay for rapid quantitation of microcystin-producing Microcystis using unprocessed surface water samples collected from Alberta lakes. Microcystin synthetase gene E (mcyE) was targeted for microcystin-producing Microcystis and 16S rRNA was used to measure blooming level of total cyanobacteria. The assay was optimized and validated with 20 reference samples collected in 2011. The limit of detection (LOD) of rt-QPCR was 50 copies/ml for both mcyE and 16S rRNA. An excellent precision was observed in 24 replicates [coefficient of variation (cv)=1.12% for 1.0E+05 and = 0.79% for 1.0E+03 copies]. The rt-QPCR assay was applied for detection of mcyE in 527 water samples collected from 45 lakes during the open-water season of 2012 in Alberta and 369 samples were mcyE positive. Microcystin-producing Microcystis was detected in 41 out of the 45 lakes in which, relatively high copy numbers of mcyE (≥ 1.0E+05 copies/ ml) were determined in 9 lakes. Cyanobacteria were present in all 45 lakes determined by 16S rRNA. The rt-QPCR assay developed with specific target to mcyE is sensitive, specific and robust for rapid detection and differentiation of toxic Microcystis from non-toxic cyanobacteria in surface water.

Assessment of the Appearance and Toxin Production Potential of Invasive Nostocalean Cyanobacteria Using Quantitative Gene Analysis in Nakdong River, Korea

“…Additionally, INC could be detected down to the level of 1 cell mL −1 in field samples. Quantitative gene analyses in most prior studies targeted toxin genes of cyanobacteria that proliferate in large numbers, such as those in the genus Microcystis [ 38 , 39 ]. A few quantitative gene analysis studies were specifically conducted on C. raciborskii , for which a species-specific primer was developed [ 27 , 28 , 40 ].…”

Democratizing water monitoring: Implementation of a community-based qPCR monitoring program for recreational water hazards

“…Toxigenic (mcyE gene) cyanobacteria monitoring. The mcyE gene targeting qPCR assay was performed as described in Qiu et al (2013) [22] and Sipari et al (2010) [23] (T S1 Table). The LOD 95 of this technique is 6.25 copies/5μL.…”

Enviromental Risk Factors Associated with Primary Liver Cancer in Western Kenya: A Mini-review

“…Limited studies have been done to explore the impact of pesticides exposure to human in regards to hepatocellular carcinoma development. As a results, no accurate data has been provided to support the significant association of DDT and its related compounds with development of hepatocelluar carcinoma [35]. For instance , one study in USA reported that farmers were at an increased risk for HCC development compared to non-farmers [36] while other studies report a non-significant increased risk for HCC among farmers [29].…”

“…These toxins are mainly stored inside the cyanobacterial cell and are released into the water during cell lysis [17], potentially leading to high toxin concentrations in water bodies [55]. The toxins thus pose a substantial health risk to livestock, humans and aquatic animal species who rely on such water for drinking, sanitization or as food source [35] .Various environmental parameters affects MCs production within freshwater ecosystems, including pH, nitrogen, phosphorus and water temperature [56]. MCs contain a unique molecular substructure, 3-amino9-methoxy-2, 6, 8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda) [30] (Fig.…”