Development of a quantitative PCR for detection and quantification of Prorocentrum donghaiense

Abstract

Prorocentrum donghaiense is a dinoflagellate with a high frequency of bloom formation in the East China Sea. These blooms harm coastal ecosystems, marine fisheries, aquatic environments, and public health. Therefore, new and rapid methods that accurately process and specifically detect this alga are crucial to facilitate long-term monitoring or to provide timely warnings of P. donghaiense blooms. We report the development of a quantitative real-time PCR (qPCR) method to identify and detect P. donghaiense. The partial large subunit (LSU) rDNA D1-D2 was cloned and sequenced to design specific amplification primers. The specificity of the primers was tested using regular PCR and fluorescent PCR against a wide range of microalgae widely distributed along the Chinese coast. The qPCR detection protocol was based on two standard curves. Both curves were constructed from standard samples of tenfold serially diluted solutions of the recombinant plasmid containing the LSU D1-D2 fragment and crude DNA extracts with a known number of target cells. A quantitative relationship between the cell numbers and their corresponding plasmid copy numbers was established; this relationship can be used to determine the target cell number of unknown samples in combination with a standard curve that was generated from tenfold-diluted plasmid solutions and the determined C t value of target DNA. The effectiveness of the developed protocol was tested with a series of simulated and field samples. The developed qPCR had a detection sensitivity of up to 3.45 cells. The performance of qPCR was not affected by nontarget DNA. The detection test with a series of samples fixed for 40 days showed that qPCR is competent for long-term monitoring programs that require the quantitative analysis of fixative-preserved samples. qPCR can identify the target cells in the field samples within 3-4 h. No significant differences in the quantitative results of the target cells were observed between qPCR and light microscopy. Overall, the established qPCR method is specific, sensitive, rapid, accurate, and promising for the field detection of P. donghaiense in natural samples.

Colorimetric DNA-based assay for the specific detection and quantification of Ostreopsis cf. ovata and Ostreopsis cf. siamensis in the marine environment

“…Herein, genomic DNA from cultures has been used to construct calibration curves, although the use of plasmids or cells from cultures has also been described (Andree et al, 2011;Nishimura et al, 2016;Zhang et al, 2016). Since the rDNA copy number per microalgae cell may vary depending on the species, strains, growth phase and/or environmental conditions, it is challenging to provide cell quantifications in field samples using molecular methods (Galluzzi et al, 2010;Perini et al, 2011).…”

Rapid detection of Chattonella marina by PCR combined with dot lateral flow strip

“…Since the 1980s, nucleic acid-based molecular techniques have been gradually applied to detect harmful algae. Several molecular detection techniques for HABs have been developed so far, including fluorescence in situ hybridization (FISH) (Miller and Scholin 2000;Liu et al 2020), sandwich hybridization (SH) (Diercks et al 2008;Zhu et al 2012), polymerase chain reaction (PCR) (Lyu et al 2017;Elleuch et al 2020), isothermal amplification (IA) (Toldrà et al 2019a;Zhang et al 2019), microarrays (Taylor et al 2013;Noyer et al 2015), biosensors (Diercks-Horn et al 2011;Toldrà et al 2019b), and real-time qPCR (Nishimura et al 2016;Zhang et al 2016). These molecular detection techniques have, to a certain extent, overcome the disadvantages of traditional morphological identification methods, greatly improved the detection efficiency, and provided new ideas for the monitoring and early warning of HABs.…”