Real-Time PCR based test for the early diagnosis of Haplosporidium pinnae affecting fan mussel Pinna nobilis

Autor: Amalia Grau, José Rafa García-March, Gaetano Catanese, M. López-Sanmartín, José María Valencia, J.I. Navas
Rok vydání: 2019
Předmět:
2417.05 Biología Marina
Oyster
Haplosporida
Artificial Gene Amplification and Extension
Polymerase Chain Reaction
Mediterranean Basin
Oysters
Database and Informatics Methods
Critically endangered
Medicine and Health Sciences
Mussels
IUCN Red List
DNA extraction
Protozoan Infections
Animal
Protozoans
0303 health sciences
Multidisciplinary
biology
Mediterranean Region
Eukaryota
04 agricultural and veterinary sciences
3105.12 Ordenación y Conservación de la Fauna Silvestre
Medicine
Sequence Analysis
Research Article
Bivalves
Death Rates
Bioinformatics
Science
Zoology
Conservation
Research and Analysis Methods
Real-Time Polymerase Chain Reaction
DNA
Ribosomal
Sensitivity and Specificity
Pinna nobilis
03 medical and health sciences
Extraction techniques
Population Metrics
Species Specificity
biology.animal
Parasitic Diseases
Mediterranean Sea
Animals
Parasities
Molecular Biology Techniques
Molecular Biology
030304 developmental biology
Population Biology
2409 Genética
Organisms
Biology and Life Sciences
Correction
Molluscs
Sequence Analysis
DNA
DNA
Mussel
biology.organism_classification
Invertebrates
Parasitic Protozoans
Bivalvia
Molecular biology techniques
Threatened species
040102 fisheries
0401 agriculture
forestry
and fisheries
Sequence Alignment
Zdroj: PLoS ONE
PLoS ONE, Vol 14, Iss 2, p e0212028 (2019)
ISSN: 1932-6203
Popis: All sequence files are available from the Genbank database (accession numbers MK142774-MK142779). The protocol is available from protocols.io (DOI: dx.doi.org/10. 17504/protocols.io.xmyfk7w). Noble pen shell or fan mussel, Pinna nobilis Linnaeus (1758), protected since 1992, was incorporated into the Spanish Catalogue of Threatened Species (Category: Vulnerable, Royal Decree 139/2011). The status is presently in the process of being catalogued as critically endangered, pending approval by Spanish Government (https://www.mapama.gob.es/ es/biodiversidad/participacion-publica/Borrador_OM_situacion_critica.aspx). The International Union for the Conservation of Nature (IUCN) alerted the countries of the Mediterranean basin to the “emergent situation” due to serious mortality events suffered by the fan mussel, putting it in serious risk of extinction. Thus, emergency actions have been implemented by Spanish authorities in which several research institutes from all over the country are involved. The parasite, Haplosporidium pinnae, was recently characterized by histology, TEM, SEM and molecular biology techniques and it was considered responsible for the mass mortality of P. nobilis in the Mediterranean Sea. In this context, the aim of this study has been to develop species-specific quantitative PCR (qPCR) protocol carrying out a fast, specific and effective molecular diagnose of H. pinnae. In this sense, the detection limit for qPCR was equal to 30 copies of SSU rDNA / ng of DNA using plasmid alone and when 100ng DNA of non-infected oyster were added. The qPCR assay revealed that 94% of the 32 analysed mantle tissues of fan mussel were infected by H. pinnae, showing a high sensitivity and specificity for its detection (100% if we don’t consider negative and too much degraded samples). This technique will allow us to make quicker follow-ups of the disease, allowing us to get a better understanding of its evolution in order to help in the rescue of P. nobilis populations Project MAPAMA ref. 28-5310 LIFE IPE INTERMARES(LIFE15 IPE ES 012) IFAPA-UCV 014/2018 and co-funded by PHENOFISH Project State Plan for Scientific and Technical Research and Innovation 2013-2016, MINECO, ref. PTA2015-14709-I Ministerio de Economía, Industria y Competitividad, Gobierno de España, INIA CCAA (DOC INIA 8-2013) Project BF/HEM 15-1662 Ciencias del Mar
Databáze: OpenAIRE
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