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首页> 外文OA文献 >Entwicklung von Testsystemen auf der Basis der 'Loop Mediated Isothermal Amplification (LAMP)' Methode zum Nachweis von Yersinia ruckeri, dem Erreger der Rotmaulseuche (ERM) und von Renibacterium salmoninarum, dem Erreger der bakteriellen Nierenkrankheit (BKD) der Salmoniden
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Entwicklung von Testsystemen auf der Basis der 'Loop Mediated Isothermal Amplification (LAMP)' Methode zum Nachweis von Yersinia ruckeri, dem Erreger der Rotmaulseuche (ERM) und von Renibacterium salmoninarum, dem Erreger der bakteriellen Nierenkrankheit (BKD) der Salmoniden

机译：基于“环介导等温扩增（Lamp）”方法检测鲁氏耶尔森氏，Rotmaulseuche（ERm）和Renibacterium鲑，细菌性肾脏病的鲑鱼的病原体（BKD）的致病原的测试系统的研制

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Loop-mediated isothermal amplification (LAMP) is a powerful, innovative gene amplification technique which is emerging as an easy to perform and rapid diagnostic tool for detection and identification of microbial diseases. Early and accurate detection is of paramount importance concerning the diagnosis of the highly contagious bacteria Yersinia ruckeri and Renibacterium salmoninarum. An easy to perform diagnostic technique is also required if assays should be carried out in field inquiries. The method provides a single step, reaction tube assay only requiring a temperature-controlled water bath. In the experiments of the presented study, LAMP assays were conducted for Y. ruckeri (the pathogen causing Enteric Redmouth Disease, ERM) and R. salmoninarum (the pathogen causing Bacterial Kidney Disease, BKD). In the case of ERM, the amplified target was a sequence stretch of the gene yruI/yruR encoding the quorum sensing system which controls the expression of virulence genes. In the case of BKD, a sequence stretch of the gene encoding the major soluble antigen protein (p57) in R. salmoninarum was amplified. This protein indicates an active infection because it is the predominant cell surface-associated and secreted protein by the bacterium.The newly established LAMP assays for ERM and BKD enabled amplification of a stretch of each target gene at a temperature of 63°C in less than one hour, with no need of thermal cycling. Assays are carried out with a reaction mix containing four specific primers, the sample and Bst DNA polymerase. Amplification products were detected by visual inspection, agarose gel electrophoresis, and in real-time using a turbidimeter. Assays specificity were demonstrated using DNAs from other related bacteria yielding no amplification product, and by restriction analysis with HphI and EcoRV enzymes producing a specific bands´ pattern of the amplified products. Compared to regular PCR-based detection methods, the developed LAMP assays were consistently faster and ten-fold more sensitive. A safe detection of the specific sequence stretches with high specificity and efficiency was possible using DNA isolated both from bacterial extracts and from clinical fish specimens. These findings showed that LAMP assays are more sensitive than other detection methods such as time consuming culture methods and PCR assays. In conclusion, for the first time LAMP assays developed and optimised to detect Y. ruckeri and R. salmoninarum were introduced as diagnostic tools. In comparison with the performance of already established diagnostic methods, LAMP assays are superior in sensitivity, rapidness, specificity, and cost-efficiency. Both assays are highly appropriate for application in field inquiries to monitor the spread of ERM and BKD.
机译：环介导的等温扩增（LAMP）是一种功能强大的创新基因扩增技术，它正在作为一种易于执行且快速的诊断工具，用于检测和鉴定微生物疾病。对于高传染性细菌耶尔森氏菌和沙门氏菌，早期和准确的检测至关重要。如果应在现场查询中进行测定，还需要一种易于执行的诊断技术。该方法提供了仅需温度控制的水浴的一步反应管测定。在本研究的实验中，对L. ruckeri（引起肠红嘴病，ERM的病原体）和R.鲑鱼（引起细菌性肾脏病，BKD的病原体）进行了LAMP分析。在ERM的情况下，扩增的靶标是yruI / yruR基因的序列延伸，其编码控制毒力基因表达的群体感应系统。在BKD的情况下，在鲑鱼沙门氏菌中编码主要可溶性抗原蛋白（p57）的基因的序列延伸被扩增。该蛋白是一种活跃的感染，因为它是细菌与细胞表面相关的主要分泌蛋白。新建立的针对ERM和BKD的LAMP测定法能够在低于63°C的温度下扩增每个靶基因的一部分。一小时，无需热循环。用含有四种特异性引物（样品和Bst DNA聚合酶）的反应混合物进行测定。通过目视检查，琼脂糖凝胶电泳并使用浊度计实时检测扩增产物。使用其他相关细菌的DNA不会产生扩增产物，并通过使用HphI和EcoRV酶进行限制性分析来产生扩增产物的特定条带模式，从而证明了测定的特异性。与常规的基于PCR的检测方法相比，已开发的LAMP测定始终更快且灵敏度提高了十倍。使用从细菌提取物和临床鱼标本中分离的DNA，可以以高特异性和效率安全检测特定序列片段。这些发现表明，LAMP分析比其他检测方法（如费时的培养方法和PCR分析）更灵敏。总之，首次开发并优化了用于检测Ruckereri和R.salmoninarum的LAMP检测方法作为诊断工具。与已经建立的诊断方法的性能相比，LAMP分析在灵敏度，快速性，特异性和成本效率方面均优越。两种检测方法都非常适合在现场调查中监测ERM和BKD的传播。

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    Saleh Mona;

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  • 年度 2009
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