Quantitative PCR assay for the simultaneous identification and enumeration of multiple Karenia species
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RESEARCH ARTICLE
Quantitative PCR assay for the simultaneous identification and enumeration of multiple Karenia species Jihen Elleuch 1
&
Mohamed Barkallah 1 & Kirsty F. Smith 2 & Idriss Ben Neila 3 & Imen Fendri 4 & Slim Abdelkafi 1
Received: 8 December 2019 / Accepted: 15 June 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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. Keywords Harmful algal blooms . Karenia species . TaqMan probe . ITS1–5.8S–ITS2 region . bivalve mollusk tissue
Introduction Harmful algal blooms (HABs) are common and natural toxic events that are the subject of growing interest worldwide because of their potential threat to public health and/or aquatic ecosystems (Vila et al. 2001; Glibert et al. 2018; Gobler 2020; Roegner et al. 2020). These natural phenomena are related to Responsible Editor: Vitor Manuel Oliveira Vasconcelos Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-020-09739-4) contains supplementary material, which is available to authorized users. * Jihen Elleuch [email protected] 1
Laboratoire de Génie Enzymatique et Microbiologie, Equipe Biotechnologie des Algues, Ecole Nationale d’Ingénieurs de Sfax, Université de Sfax, Sfax, Tunisia
2
Cawthron Institute, 98 Halifax Street East, Private Bag 2, Nelson 7042, New Zealand
3
Veterinary Research Center of Sfax, Sfax, Tunisia
4
Laboratory of Plant Biotechnology Applied to the Improvement of Cultures, Faculty of Sciences of Sfax, University of Sfax, Sfax, Tunisia
the increase of phytoplankton biomass including a wide range of microalgal species (Anderson 1997; Galluzzi et al. 2010; Sildever et al. 2019; Trainer et al. 2020; Müller et al. 2020; Wang et al. 2020). Among the most notorious are species from the class Dinophyceae, some of which produce potent phycotoxins causative agents of mortality and ichthyotoxicity phenomena (Landsberg and Steidinger 1998; Clément et al. 2001; Deeds et al. 2002; Kempton et al. 2002; Hallegraeff et al. 2010; Anderson et al.
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