Dataset Information

Development and Validation of PCR-RFLP Assay for Identification of Gambierdiscus species in the Greater Caribbean region.

ABSTRACT: The genus Gambierdiscus is a recognized group of marine epiphytic-benthic dinoflagellates that produce the toxins that cause ciguatera fish poisoning (CFP). To date, thirteen species and six ribotypes of Gambierdiscus have been identified, and multiple species commonly co-occur within a single site or epiphyte community. Toxicity can vary by species, thus it is important to be able to differentiate among species for research and monitoring purposes. Gambierdiscus species have very similar morphological characteristics and are difficult or impossible to distinguish using light microscopy. DNA sequencing has been an important tool in the definition of Gambierdiscus species, but it can be time-consuming and relatively expensive. To provide an alternative approach, a PCR-RFLP protocol was developed for efficient, rapid, and cost-effective identification of Gambierdiscus strains isolated from the Gulf of Mexico and Caribbean Sea, where CFP cases and Gambierdiscus spp. have been reported. The assay targets the D1-D2 hypervariable regions of the large subunit ribosomal RNA gene and uses a single restriction enzyme, BsrI. This method produces distinct RFLP banding patterns for the six species of Gambierdiscus reported from the Gulf of Mexico and Caribbean Sea, and also distinguishes them from four Pacific endemic species. This method was successfully used to type 465 clonal isolates of Gambierdiscus from the U.S. Virgin Islands and Akumal Beach - Mexico This BsrI PCR-RFLP method expands the tools available to researchers and managers engaged in monitoring activities and ecological studies.

SUBMITTER: Lozano-Duque Y

PROVIDER: S-EPMC6759227 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Development and Validation of PCR-RFLP Assay for Identification of Gambierdiscus species in the Greater Caribbean region.

Lozano-Duque Yesid Y Richlen Mindy L ML Smith Tyler B TB Anderson Donald M DM Erdner Deana L DL

Journal of applied phycology 20180507 6

The genus Gambierdiscus is a recognized group of marine epiphytic-benthic dinoflagellates that produce the toxins that cause ciguatera fish poisoning (CFP). To date, thirteen species and six ribotypes of Gambierdiscus have been identified, and multiple species commonly co-occur within a single site or epiphyte community. Toxicity can vary by species, thus it is important to be able to differentiate among species for research and monitoring purposes. Gambierdiscus species hav ...[more]

PMID: 31551649

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Project description:BackgroundFungal bloodstream infections (BSI) may be serious and are associated with drastic rise in mortality and health care costs. Candida spp. are the predominant etiological agent of fungal sepsis. The prompt and species-level identification of Candida may influence patient outcome and survival. The aim of this study was to develop and evaluate the CanTub-simplex PCR assay coupled with Tm calling and subsequent high resolution melting (HRM) analysis to barcode seven clinically relevant Candida species.MethodsEfficiency, coefficient of correlation and the limit of reliable detection were estimated on purified Candida EDTA-whole blood (WB) reference panels seeded with Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida dubliniensis cells in a 6-log range. Discriminatory power was measured on EDTA-WB clinical panels on three different PCR platforms; LightCycler®96, LightCycler® Nano, LightCycler® 2.0. Inter- and intra assay consistencies were also calculated.ResultsThe limit of reliable detection proved to be 0.2-2 genomic equivalent and the method was reliable on broad concentration ranges (106-10 CFU) providing distinctive melting peaks and characteristic HRM curves. The diagnostic accuracy of the discrimination proved to be the best on Roche LightCycler®2.0 platform. Repeatability was tested and proved to be % C.V.: 0.14 ± 0.06 on reference- and % C.V.: 0.14 ± 0.02 on clinical-plates accounting for a very high accuracy. Reproducibility was % C.V.: 0.11 between reference- and % C.V.: 0.12between clinical-panels which is highly acceptable.ConclusionOur assay demonstrates recent advances on Tm calling and HRM analysis for the molecular identification of relevant Candida species. This unique, simplex PCR assay may be capable to outperform conventional phenotypic methods by reducing time and providing accurate and reliable results directly from blood (2 h) or from whole blood culture bottles (12-24 h).

Project description:Molecular methods are increasingly used for both species identification of sandflies and assessment of their population structure. In general, they are based on DNA sequence analysis of targets previously amplified by PCR. However, this approach requires access to DNA sequence facilities, and in some circumstances, it is time-consuming. Though DNA sequencing provides the most reliable information, other downstream PCR applications are explored to assist in species identification. Thus, it has been recently proposed that the amplification of a DNA region encompassing partially both the cytochrome-B (cytb) and the NADH dehydrogenase 1 (nd1) genes followed by RFLP analysis with the restriction enzyme Ase I allows the rapid identification of the most prevalent species of phlebotomine sandflies in the Mediterranean region. In order to confirm the suitability of this method, we collected, processed, and molecularly analyzed a total of 155 sandflies belonging to four species including Phlebotomus ariasi, P. papatasi, P. perniciosus, and Sergentomyia minuta from different regions in Spain. This data set was completed with DNA sequences available at the GenBank for species prevalent in the Mediterranean basin and the Middle East. Additionally, DNA sequences from 13 different phlebotomine species (P. ariasi, P. balcanicus, P. caucasicus, P. chabaudi, P. chadlii, P. longicuspis, P. neglectus, P. papatasi, P. perfiliewi, P. perniciosus, P. riouxi, P. sergenti, and S. minuta), from 19 countries, were added to the data set. Overall, our molecular data revealed that this PCR-RFLP method does not provide a unique and specific profile for each phlebotomine species tested. Intraspecific variability and similar RFLP patterns were frequently observed among the species tested. Our data suggest that this method may not be applicable throughout the Mediterranean region as previously proposed. Other molecular approaches like DNA barcoding or phylogenetic analyses would allow a more precise molecular species identification.

Dataset Information

Development and Validation of PCR-RFLP Assay for Identification of Gambierdiscus species in the Greater Caribbean region.

Publications

Development and Validation of PCR-RFLP Assay for Identification of <i>Gambierdiscus</i> species in the Greater Caribbean region.

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