Development and application of a real-time PCR assay for the sensitive detection of diarrheic toxin producer Prorocentrum lima

Abstract

Prorocentrum lima (P. lima) is a widely spread dinoflagellate in the Mediterranean Sea and it has become increasingly involved in harmful algal blooms. The purpose of this study is to develop a probe-based real-time polymerase chain reaction (PCR) targeting the ITS1-5.8S-ITS2 region for the detection and absolute quantification of P. lima based on linear and circular DNA standards. The results have shown that the quantitative PCR (q-PCR), using circular plasmid as a template, gave a threshold cycle number 1.79–5.6 greater than equimolar linear standards. When microalgae, commonly found in aquatic samples were tested, no cross-amplification was observed. The q-PCR brought about a good intra and inter-run reproducibility and a detection limit of 5 copies of linear plasmid per reaction. A quantitative relationship between the cell numbers and their corresponding plasmid copy numbers was attained. Afterwards, the effectiveness of the developed protocol was tested with 130 aquatic samples taken from 19 Tunisian sampling sites. The developed q-PCR had a detection sensitivity of up to 1 cell. All the positive samples were taken from three sampling sites of Medenine Governorate with cell abundances that ranged from 22 to 156,000 cells L−1 of seawater. The q-PCR assay revealed a high sensitivity in monitoring the aquatic samples in which the low concentrations of P. lima were not accurately detected by light microscopy. Indeed, this approach is at the same time rapid, specific and sensitive than the traditional microscopy techniques and it represents a great potential for the monitoring of P. lima blooms.