Andreas Benkens , Claudia Buchholz , Bernadette Pogoda , Carsten Georg Harms , Carlos Saavedra
{"title":"直接多重PCR-NALFIA为海洋保护提供信息:使用一种创新的诊断工具来检测毛竹Ostrea edulis幼虫","authors":"Andreas Benkens , Claudia Buchholz , Bernadette Pogoda , Carsten Georg Harms , Carlos Saavedra","doi":"10.1051/alr/2021023","DOIUrl":null,"url":null,"abstract":"<div><div>The European oyster <em>Ostrea edulis</em> played a key role in the North Sea by providing several ecosystem functions and services. Today, <em>O. edulis</em> is classified as severely degraded or functionally extinct in Europe. Marine conservation is focusing on biogenic reef restoration, namely the restoration of <em>O. edulis</em> in Natura 2000 sites of the North Sea. The identification of oyster larvae related to natural spatfalls of restored reefs and monitoring of larval drift is a key aspect of marine protected area management. Morphological identification and distinction from other abundant bivalve larvae using microscopy is difficult. Existing molecular biological methods are expensive and bound to stationary laboratory equipment, or are inadequate in the visualization. In this study, we identified nucleic acid lateral flow immunoassay (NALFIA), a well-established tool in human pathogen diagnostics, as an efficient approach for point-of-care (POC) testing in marine monitoring. Based on the genetic sequence of the mitochondrial cytochrome b of <em>O. edulis</em>, forward and reverse primers were developed. The reverse primer was labelled with fluorescent dye FITC, forward primer with biotin. Reaction on the lateral flow stripe could be realized with a single <em>O. edulis</em> larva in direct PCR with multiplex primers in a portable PCR-cycler. The established NALFIA system can distinguish <em>O. edulis</em> larvae from <em>Crassostrea gigas</em> and <em>Mytilus edulis</em> larvae, respectively. This method offers new approaches in POC testing in marine research and monitoring. It gives quick and clear results, is inexpensive, and could be easily adapted to other species of interest.</div></div>","PeriodicalId":55491,"journal":{"name":"Aquatic Living Resources","volume":"34 ","pages":""},"PeriodicalIF":1.5000,"publicationDate":"2021-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Direct multiplex PCR-NALFIA to inform marine conservation: Use of an innovative diagnostic tool for the detection of Ostrea edulis larvae\",\"authors\":\"Andreas Benkens , Claudia Buchholz , Bernadette Pogoda , Carsten Georg Harms , Carlos Saavedra\",\"doi\":\"10.1051/alr/2021023\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>The European oyster <em>Ostrea edulis</em> played a key role in the North Sea by providing several ecosystem functions and services. Today, <em>O. edulis</em> is classified as severely degraded or functionally extinct in Europe. Marine conservation is focusing on biogenic reef restoration, namely the restoration of <em>O. edulis</em> in Natura 2000 sites of the North Sea. The identification of oyster larvae related to natural spatfalls of restored reefs and monitoring of larval drift is a key aspect of marine protected area management. Morphological identification and distinction from other abundant bivalve larvae using microscopy is difficult. Existing molecular biological methods are expensive and bound to stationary laboratory equipment, or are inadequate in the visualization. In this study, we identified nucleic acid lateral flow immunoassay (NALFIA), a well-established tool in human pathogen diagnostics, as an efficient approach for point-of-care (POC) testing in marine monitoring. Based on the genetic sequence of the mitochondrial cytochrome b of <em>O. edulis</em>, forward and reverse primers were developed. The reverse primer was labelled with fluorescent dye FITC, forward primer with biotin. Reaction on the lateral flow stripe could be realized with a single <em>O. edulis</em> larva in direct PCR with multiplex primers in a portable PCR-cycler. The established NALFIA system can distinguish <em>O. edulis</em> larvae from <em>Crassostrea gigas</em> and <em>Mytilus edulis</em> larvae, respectively. This method offers new approaches in POC testing in marine research and monitoring. It gives quick and clear results, is inexpensive, and could be easily adapted to other species of interest.</div></div>\",\"PeriodicalId\":55491,\"journal\":{\"name\":\"Aquatic Living Resources\",\"volume\":\"34 \",\"pages\":\"\"},\"PeriodicalIF\":1.5000,\"publicationDate\":\"2021-04-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Aquatic Living Resources\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://www.sciencedirect.com/org/science/article/pii/S1765295221000143\",\"RegionNum\":4,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"FISHERIES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Aquatic Living Resources","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/org/science/article/pii/S1765295221000143","RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"FISHERIES","Score":null,"Total":0}
Direct multiplex PCR-NALFIA to inform marine conservation: Use of an innovative diagnostic tool for the detection of Ostrea edulis larvae
The European oyster Ostrea edulis played a key role in the North Sea by providing several ecosystem functions and services. Today, O. edulis is classified as severely degraded or functionally extinct in Europe. Marine conservation is focusing on biogenic reef restoration, namely the restoration of O. edulis in Natura 2000 sites of the North Sea. The identification of oyster larvae related to natural spatfalls of restored reefs and monitoring of larval drift is a key aspect of marine protected area management. Morphological identification and distinction from other abundant bivalve larvae using microscopy is difficult. Existing molecular biological methods are expensive and bound to stationary laboratory equipment, or are inadequate in the visualization. In this study, we identified nucleic acid lateral flow immunoassay (NALFIA), a well-established tool in human pathogen diagnostics, as an efficient approach for point-of-care (POC) testing in marine monitoring. Based on the genetic sequence of the mitochondrial cytochrome b of O. edulis, forward and reverse primers were developed. The reverse primer was labelled with fluorescent dye FITC, forward primer with biotin. Reaction on the lateral flow stripe could be realized with a single O. edulis larva in direct PCR with multiplex primers in a portable PCR-cycler. The established NALFIA system can distinguish O. edulis larvae from Crassostrea gigas and Mytilus edulis larvae, respectively. This method offers new approaches in POC testing in marine research and monitoring. It gives quick and clear results, is inexpensive, and could be easily adapted to other species of interest.
期刊介绍:
Aquatic Living Resources publishes original research papers, review articles and propective notes dealing with all exploited (i.e. fished or farmed) living resources in marine, brackish and freshwater environments.
Priority is given to ecosystem-based approaches to the study of fishery and aquaculture social-ecological systems, including biological, ecological, economic and social dimensions.
Research on the development of interdisciplinary methods and tools which can usefully support the design, implementation and evaluation of alternative management strategies for fisheries and/or aquaculture systems at different scales is particularly welcome by the journal. This includes the exploration of scenarios and strategies for the conservation of aquatic biodiversity and research relating to the development of integrated assessment approaches aimed at ensuring sustainable and high quality uses of aquatic living resources.