A high resolution melting method for the molecular identification of the potentially toxic diatom Pseudo-nitzschia spp. in the Mediterranean Sea

Laura Pugliese,Francesca Andreoni,Silvia Casabianca,Antonella Penna,Federico Perini

doi:10.1038/s41598-017-04245-z

Laura Pugliese, Francesca Andreoni + Show 3 more

Open Access

https://doi.org/10.1038/s41598-017-04245-z

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Abstract

The aim of this study was to develop and validate a high resolution melting (HRM) method for the rapid, accurate identification of the various harmful diatom Pseudo-nitzschia species in marine environments. Pseudo-nitzschia has a worldwide distribution and some species are toxic, producing the potent domoic acid toxin, which poses a threat to both human and animal health. Hence, it is important to identify toxic Pseudo-nitzschia species. A pair of primers targeting the LSU rDNA of the genus Pseudo-nitzschia was designed for the development of the assay and its specificity was validated using 22 control DNAs of the P. calliantha, P. delicatissima/P. arenysensis complex and P. pungens. The post-PCR HRM assay was applied to numerous unidentified Pseudo-nitzschia strains isolated from the northwestern Adriatic Sea (Mediterranean Sea), and it was able to detect and discriminate three distinct Pseudo-nitzschia taxa from unidentified samples. Moreover, the species-specific identification of Pseudo-nitzschia isolates by the HRM assay was consistent with phylogenetic analyses. The HRM assay was specific, robust and rapid when applied to high numbers of cultured samples in order to taxonomically identify Pseudo-nitzschia isolates recovered from environmental samples.

Highlights

Diatoms are among the most productive eukaryotic microalgae
The Pseudo-nitzschia spp. primers, designed to amplify the target sequence of the LSU rDNA region, were examined in silico using BLAST and they were found to be specific to P. calliantha, P. delicatissima, P. cf. arenysensis and P. pungens
A reliable, rapid and robust molecular qPCR high resolution melting (HRM) assay was developed in order to rapidly and accurately detect potentially harmful Pseudo-nitzschia species in cultured samples obtained from coastal water survey

Summary

Introduction

Diatoms (class Bacillariophyceae) are among the most productive eukaryotic microalgae. Molecular taxonomy studies based on different genetic markers, including ribosomal RNA gene (LSU) and internal transcribed spacers (ITS regions), cytochrome oxidase 1 (cox 1) and chloroplast genes of ribulose 1,5 biphosphate carboxylase (rbcl), have uncovered numerous cases of genetically distinct, and at times reproductively isolated, groups of strains or genetic lineages that could not be distinguished with light microscopy[26,27,28] Recent molecular approaches, such as qPCR13, 29, 30, ARISA31, microarray[32,33,34] and dot blot hybridization systems[35] have been used for specific and sensitive Pseudo-nitzschia species identification and/or quantification from clonal cultures and field samples[36]. The melting HRM profiles seem to be more specific than some probe signals, and because of the high throughput analysis, the HRM assay has lower costs than PCR and sequencing reactions

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