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

Yuanyuan Qiu

doi:10.4172/2155-9929.s5-006

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.

Highlights

Cyanobacteria, named blue-green algae, are prokaryotic photosynthetic microorganisms that grow in warm, eutrophic surface waters
Very good sensitivity of the Microcystin synthetase gene E (mcyE) gene quantitation was revealed in a linear log-range from 2.0E+00 to 2.0E+09 copies per reaction when selected primers/probes were used in the Real-time Quantitative PCR (rt-QPCR) reaction (Figure 1)
No amplification of mcyE gene was observed in non-toxic Microcystis CPCC124 and Chlorella protothecoids using the rt-QPCR assay with mcyE primers and probe targeting to the Microcystis, while 4.1E+07 copies/ml of mcyE gene in CPCC299 and 9.78E+05 copies/ml of mcyE gene in CPCC300 were detected respectively (Table 1)

Summary

Introduction

Cyanobacteria, named blue-green algae, are prokaryotic photosynthetic microorganisms that grow in warm, eutrophic surface waters. It has been reported that on average 59% of the cyanobacterial blooms contain toxins [1]. Microcystin is the most common and prevalent cyanobacterial toxin and is produced mainly by species of Microcystis, Planktothrix and Anabaena. Microcystin is a small monocyclic heptapeptide with seven amino acids and is soluble and stable in water [2]. The amino acids D-glutamate and Add a play important roles in interaction with protein phosphatases and are critical for the toxicity of microcystin [3,4]. The mcy gene cluster contains 10 genes, namely mcy A to mcy J, which have been fully sequenced and characterized in species Microcystis, Planktothrix and Anabaena [10,11,12,13]

Objectives

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Conclusion