Sensitive and rapid detection of microcystin synthetase E Gene (mcyE) by loop-mediated isothermal amplification: A new assay for detecting the potential microcystin-producing Microcystis in the aquatic ecosystem |
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| Affiliation: | 1. Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ningbo 315211, China;2. Ningbo Branch of National Engineering Research Center for Beijing Biochip Technology, Ningbo 315201, China;1. University of Sassari, Department of Architecture, Design and Urban Planning, Via Piandanna 4, 07100 Sassari, Italy;2. University of Cagliari, Department of Life and Environmental Sciences, via T. Fiorelli 1, 09126 Cagliari, Italy;3. EN.A.S. Ente Acque della Sardegna, Settore della limnologia degli invasi, Viale Elmas 116, 09122 Cagliari, Italy;1. Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616, USA;2. Cátedra de Bioquímica, Facultad de Química, Universidad de la República, Montevideo, Uruguay;3. Cátedra Inmunología, Facultad de Química, Universidad de la República, Instituto de Higiene, Montevideo, Uruguay;1. Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay;2. Ecología Funcional de Sistemas Acuáticos, CURE, Rocha, Universidad de la República, Uruguay;3. Sección Limnología, IECA, Facultad de Ciencias, Universidad de la República, Uruguay;4. Polo de Desarrollo Universitario, Modelización y Análisis de Recursos Naturales, CURE, Rocha, Universidad de la República, Uruguay;1. Biotechnology, Department of Biochemistry, University of Turku, Tykistökatu 6A 6th Floor, 20520 Turku, Finland;2. Biochemistry, Department of Biosciences, Åbo Akademi University, Tykistökatu 6A 3rd Floor, 20520 Turku, Finland;1. Biotechnology, Department of Biochemistry, University of Turku, Tykistökatu 6A 6th Floor, Turku 20520, Finland;2. Biochemistry, Department of Biosciences, Åbo Akademi University, Tykistökatu 6A 3rd Floor, Turku 20520, Finland |
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| Abstract: | In order to develop a new molecular technique that has the potential to assist with monitoring and management of water bodies for potential microcystin producing cyanobacterial species that occur in mixed populations in many regions of the world, we designed a new loop-mediated isothermal amplification (LAMP) assay based on microcystin biosynthesis genes. Four sets of primers were designed to recognize six distinct sequences on target the mcyE gene that encodes a protein (McyE) being responsible to catalyze the addition of d-glutamate to Adda. One set (MCYE2) was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for mcyE detection were determined. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 40 min at an isothermal temperature of 61 °C. For the sensitivity of LAMP, the detection limit was 8.5 pg/μl (approximately 17 pg) DNA. The eleven microcystin producing and four non-toxic cyanobacterial strains were selected for testing of specificity. The results of the amplification were positive with all microcystin-producing strains tested and not with four non-toxic strains, which showed that the primers had good levels of specificity. For testing the application of LAMP assay in the aquatic ecosystem, seven environmental samples from ponds and lakes in Ningbo City were also analyzed using the LAMP targeting the mcyE gene as well as an ELISA assay. Compared with these results of ELISA assay, LAMP assay is satisfied. All of these validated LAMP method being fast, simple and low in cost is a potentially valuable means for potential toxic of cyanobacterial blooms detection, especially for routine monitoring purposes in future. |
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| Keywords: | LAMP assay Rapid detection |
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