Ifremer, RBE-SG2M-LGPMM, Station de La Tremblade, Avenue de Mus de Loup, F-17390 La Tremblade, France.
Ifremer, RBE-SG2M-LGPMM, Station de La Tremblade, Avenue de Mus de Loup, F-17390 La Tremblade, France.
Prev Vet Med. 2020 Oct;183:105126. doi: 10.1016/j.prevetmed.2020.105126. Epub 2020 Aug 19.
Aquaculture including shellfish production is an important food resource worldwide which is particularly vulnerable to infectious diseases. Marteilia refringens, Bonamia ostreae and Bonamia exitiosa are regulated protozoan parasites infecting flat oysters Ostrea edulis that are endemic in Europe. Although some PCR assays have been already developed for their detection, a formal validation to assess the performances of those tools is often lacking. In order to facilitate the diagnosis of flat oyster regulated diseases, we have developed and evaluated a new multiplex Taqman® PCR allowing the detection of both M. refringens and Bonamia sp. parasites in one step. First part of this work consisted in assessing analytical sensitivity and specificity of the new PCR assay. Then, diagnostic performances were assessed by testing a panel of field samples with the new real-time PCR and currently recommended conventional PCR methods for the detection of M. refringens and Bonamia sp. Samples were collected from the main flat oyster production sites in France (N = 386 for M. refringens and N = 349 for B. ostreae). In the absence of gold standard, diagnostic sensitivity and specificity of the new PCR were estimated through Bayesian latent class analysis (DSe 87,2% and DSp 98,4% for the detection M. refringens, DSe 77,5% and DSp 98,4% for the detection of Bonamia sp.). Those results suggest equivalent performances for the detection of Bonamia sp. and an improved sensitivity for the detection of M. refringens compared to commonly used conventional protocols. Finally, the new PCR was evaluated in the context of an inter-laboratory comparison study including 17 European laboratories. Results revealed a very good reproducibility with a global accordance (intra-laboratory precision) >96% and a global concordance (inter-laboratory precision) >93% for both targets, demonstrating that this new tool is easily transferable to different laboratory settings. This is the first assay designed to detect both Marteilia refringens and Bonamia sp. in a single step and it should allow reducing the number of analysis to monitor both diseases, and where relevant to demonstrate freedom from infection.
水产养殖包括贝类养殖，是全球重要的食物资源，特别容易受到传染病的影响。海栖玛俐菌、牡蛎海栖纤毛虫和牡蛎海栖纤毛虫 exitiosa 是感染欧洲特有的平牡蛎 Ostrea edulis 的受监管原生动物寄生虫。尽管已经开发了一些用于检测它们的 PCR 检测方法，但通常缺乏对这些工具性能的正式验证。为了便于诊断平牡蛎受监管的疾病，我们开发并评估了一种新的多重 Taqman®PCR，允许在一步中同时检测海栖玛俐菌和 Bonamia sp.寄生虫。这项工作的第一部分是评估新 PCR 检测方法的分析灵敏度和特异性。然后，通过使用新的实时 PCR 检测和目前推荐的用于检测海栖玛俐菌和 Bonamia sp.的常规 PCR 方法检测来自法国主要平牡蛎生产地点的样本组来评估诊断性能。从法国的主要平牡蛎生产地点采集了样本（用于海栖玛俐菌的 N = 386，用于牡蛎海栖纤毛虫的 N = 349）。在没有金标准的情况下，通过贝叶斯潜在类别分析估计新 PCR 的诊断灵敏度和特异性（用于检测海栖玛俐菌的 DSe 为 87.2%，DSp 为 98.4%，用于检测牡蛎海栖纤毛虫的 DSe 为 77.5%，DSp 为 98.4%）。这些结果表明，在检测 Bonamia sp.方面具有等效性能，并且与常用的常规方案相比，检测海栖玛俐菌的灵敏度有所提高。最后，在包括 17 个欧洲实验室的实验室间比较研究中评估了新的 PCR。结果显示，对于两个目标，均具有非常好的重现性（实验室内精密度）>96%，一致性（实验室间精密度）>93%，证明该新工具易于转移到不同的实验室环境中。这是第一个设计用于在单个步骤中同时检测海栖玛俐菌和 Bonamia sp.的检测方法，它应该可以减少监测这两种疾病所需的分析数量，并在相关情况下证明无感染。
