分子生物标志物与诊断杂志

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

Abstract

Yuanyuan Qiu, Tao Yuan, Tatiana Kon, Ron Zurawell, Yu Huang, Mark Graham, Stephan Gabos and Xiaoli Pang

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.