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Karlodinium veneficum is a toxic benthic globally distributed dinoflagellate which has direct impacts on human health and the environment. Early and accurate detection of this harmful algal bloom-forming species could be useful for potential risks monitoring and management. In the present work, a real-time PCR targeting the internal transcribed spacer ribosomal DNA region for the specific detection and absolute quantification of K. veneficum was designed. Then, the assay conditions were adjusted and validated. The developed qPCR was highly specific for the target species and displayed no cross-reactivity with closely related dinoflagellates and/or other microalgal species commonly distributed along the Tunisian coast. Its lowest detection limit was 5 rDNA copies per reaction, which is often considered satisfying. qPCR assay enumeration accuracy was evaluated using artificially inoculated environmental samples. The comparison of the cell abundance estimates obtained by qPCR assay with the theoretical estimates showed no statistically significant difference across a range of concentrations. We suggest that the qPCR approach developed in the present study may be a valuable tool to investigate the distribution and seasonal dynamics of K. veneficum in marine environments.

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Gymnodinium catenatum is a dinoflagellate known to cause paralytic shellfish poisoning (PSP), commonly associated with human muscular paralysis, neurological symptoms, and, in extreme cases, death. In the present work, we developed a real-time PCR-based assay for the rapid detection of the toxic microalgal species, G. catenatum, in environmental bivalve mollusc samples as well as seawater samples. G. catenatum-specific primers and probe were designed on the ITS1-5.8S-ITS2 rDNA region. Hydrolysis probe qPCR assay was optimized. ITS1-5.8S-ITS2 rDNA region copy numbers per G. catenatum cell genome were estimated to be 122.73 ± 5.54 copies/cell, allowing cell quantification. The application of the optimized qPCR assay for G. catenatum detection and quantification in field samples has been conducted, revealing high sensitivity (detection of around 1.3105 cells/L of seawater samples. Thus, the designed hydrolysis probe qPCR assay could be considered an efficient tool for phytoplankton monitoring whilst ensuring accuracy and sensitivity and providing cost and time savings.

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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.

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Quantitative PCR (qPCR) is the method of choice for specific detection and quantification of harmful algal bloom (HAB) species. Development of qPCR assay for simultaneous enumeration of species that frequently co-exist in HABs is required. A high sensitivity TaqMan qPCR assay, using probe and primers, located at ITS1-5.8S-ITS2 rDNA region, detecting, specifically, Karenia selliformis, K. bidigitata, and K. mikimotoi, was designed. ITS1-5.8S-ITS2 rDNA region copy numbers per Karenia cell genome were estimated to 217.697 ± 67.904, allowing cell quantification. An application of the designed methodology in field samples has been conducted, and it showed high sensitivity (detection of around 10-1 cell/100 mg of bivalve mollusk tissue, equivalent to about 20 copies of the target sequence). We suggest that the optimized method could contribute to early detection of three closely related Karenia species in seafood cultivating areas to promote control quality, guarantee a fast and effective intervention, and improve public health prevention.

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The objective of this study was to develop and evaluate a SYBR Green real time PCR method for the specific detection of Salmonella spp using a novel target, the siiA gene. Primer specificity testing was done on a panel of 76 Salmonella strains and 32 non-Salmonella strains. The primers directed against the siiA gene amplified all Salmonella strains tested, while non-Salmonella strains were not amplified. The melting temperatures of the 107 bp amplicons were consistently specific as they gave melting peaks around 75.5°C. The precision of the assay, based on intra and inter-run variations, was shown to be widely acceptable. In the second part of this study, 45 Salmonella strains were screened for the presence of 6 virulence-associated genes (sopB, cat2, safC, sefB and SC1248) located in several Salmonella Pathogenicity Islands (SPIs) and the spvC gene from the Salmonella virulence plasmid. The prevalence of these genes ranged from 51% to 100%. Variable virulence gene profiles were obtained even within the same serotype.

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