Key Laboratory of Aquatic Product Processing; Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture and Rural Affairs; South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510300, China.
Shanghai Ocean University, Shanghai, 201306, China.
Parasit Vectors. 2019 Jul 24;12(1):360. doi: 10.1186/s13071-019-3624-3.
Perkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37-42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1-10 target DNA copies in less than 20 minutes.
We report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye.
The LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20-25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field.
Our LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.
由原生动物 Perkinsus 引起的 Perkinsosis 病导致了全球许多软体动物物种的大规模死亡。快速、早期和准确地检测 Perkinsus 感染对于应对疫情和管理疾病传播是必要的。目前用于 Perkinsus spp. 的诊断方法既费时又需要专业设备和经验丰富的人员，因此不适合现场应用。重组聚合酶扩增（RPA）检测是一种高度敏感和选择性的等温扩增技术，在 37-42°C 的温度下运行，需要最少的样本准备，并且能够在不到 20 分钟内扩增低至 1-10 个目标 DNA 拷贝。
我们报告了一种新的 RPA 检测方法，该方法扩增了北海派琴虫的内部转录间隔区（ITS）区域，随后通过侧流（LF）条带快速检测扩增子，通过肉眼轻松可视化结果。
LF-RPA 检测方法成功地使用一组 20-25bp 长的引物扩增了北海派琴虫 DNA。在 37°C 孵育 25 分钟后，在 LF 条带上通过肉眼在 5 分钟内读取结果。我们的 LF-RPA 检测方法与 qPCR 检测方法一样敏感，能够检测到低至 26 个北海派琴虫 DNA 拷贝。与其他两种 Perkinsus 物种 P. olseni 和 P. chesapeaki 发生交叉扩增，但与培养环境中的其他潜在病原体无关。我们比较了 LF-RPA、常规 PCR 和 qPCR 检测方法在 60 个牡蛎样本上的性能。虽然 LF-RPA 检测方法的灵敏度为 86.2%，特异性为 77.4%，与常规 PCR 结果基本一致，但它们的灵敏度更高（93.3%），特异性（86.7%）更高，与 qPCR 检测结果更吻合。未来的研究应集中开发简单的 DNA 提取方法，这些方法不需要专业实验室和复杂的提取程序，以促进 LF-RPA 检测方法在现场的应用。
我们的 LF-RPA 检测方法为检测 Perkinsus 物种提供了一种快速有效的方法。这种新的检测方法有可能在现场应用。
