Development of a real-time fluorescent quantitative PCR method for the detection of toxic gene copy number of microcystin algae

Abstract

Objective To develop a real-time fluorescent quantitative PCR method for the detection of toxic gene copy number of microcystin algae, so as to realize warning detection of microcystin algae. Methods With the primers based on microcystin synthetase gene mcy A, mcy D and partial Microcystis-specific 16S rDNA, the target gene was inserted into pMD® 18-T Vector for the preparation of plasmid standards to establish the standard curve of real-time fluorescent quantitative PCR method and through the standard curve to calculate the toxin-producing gene copy number of the unknown sample. Results The exponential curve of the initial template was linear with the Ct value and the related coefficient r2 was all higher than 0.995 and had good reproducibility. The established method could yield the same result as the recommended international standard method. Conclusions With good accuracy, sensitivity, specificity and practicability, this method could be used for predictive detection of water-borne toxin-produced microcystin algae. Key words: Microcystin; Mcy A; Mcy D; Real-time fluorescent quantitative PCR