Strand David A, Jinnerot Tomas, Aspán Anna, Viljamaa-Dirks Satu, Heinikainen Sirpa, Rolén Elin, Vrålstad Trude
Norwegian Veterinary Institute, Oslo, Norway.
National Veterinary Institute, Uppsala, Sweden.
J Invertebr Pathol. 2023 Nov;201:108008. doi: 10.1016/j.jip.2023.108008. Epub 2023 Oct 18.
The parasitic oomycete Aphanomyces astaci is the causative agent of crayfish plague, a devastating disease for European freshwater crayfish. Species specific quantitative real-time PCR (qPCR) can offer rapid detection of the pathogen. However, the well established A. astaci qPCR assay recommended by the World Organization for Animal Health (WOAH) amplifies the recently described Aphanomyces fennicus. Consequently, false-positive results may occur. This calls for the improvement of the established species specific A. astaci qPCR assay in order to avoid amplifying A. fennicus while screening for A. astaci. We developed an improved species specific A. astaci qPCR assay and validated the assay across three laboratories, using established procedures including different qPCR master mixes for each respective laboratory. Genomic DNA from A. astaci, A. fennicus and closely related Aphanomyces spp. was analysed and compared with both the improved and established assay. Additionally, DNA from crayfish tissue and environmental samples were analysed with both assays. The improved assay showed similar sensitivity with the established assay for all sample types, while proving highly specific for A. astaci avoiding amplification of A. fennicus and the other tested Aphanomyces spp. Environmental DNA (eDNA) samples collected at River Lierelva in Norway amplified with the established assay, but not with the improved assay indicating false positive. We were able to sequence a 530 bp fragment of the ITS region from these eDNA samples and the consensus sequence showed 99.9-100 % pairwise identity with A. fennicus and 97.2-98 % pairwise identity with A. astaci, suggesting that the occurrence of A. fennicus is not limited to Finland, where it was first discovered.
寄生卵菌小龙虾疫霉是小龙虾瘟疫的病原体，这是一种对欧洲淡水小龙虾具有毁灭性的疾病。物种特异性定量实时PCR（qPCR）可实现对该病原体的快速检测。然而，世界动物卫生组织（WOAH）推荐的成熟的小龙虾疫霉qPCR检测方法会扩增最近描述的芬兰疫霉。因此，可能会出现假阳性结果。这就需要改进现有的物种特异性小龙虾疫霉qPCR检测方法，以便在筛查小龙虾疫霉时避免扩增芬兰疫霉。我们开发了一种改进的物种特异性小龙虾疫霉qPCR检测方法，并在三个实验室中使用既定程序进行了验证，每个实验室使用不同的qPCR预混液。对来自小龙虾疫霉、芬兰疫霉和密切相关的疫霉属物种的基因组DNA进行了分析，并与改进后的检测方法和既定检测方法进行了比较。此外，还使用这两种检测方法对小龙虾组织和环境样本的DNA进行了分析。改进后的检测方法对所有样本类型显示出与既定检测方法相似的灵敏度，同时对小龙虾疫霉具有高度特异性，避免了芬兰疫霉和其他测试的疫霉属物种的扩增。在挪威利勒尔瓦河采集的环境DNA（eDNA）样本在既定检测方法中扩增，但在改进后的检测方法中未扩增，表明存在假阳性。我们能够对这些eDNA样本的ITS区域的530bp片段进行测序，共有序列与芬兰疫霉的成对同一性为99.9 - 100%，与小龙虾疫霉的成对同一性为97.2 - 98%，这表明芬兰疫霉的出现并不局限于其首次发现的芬兰。
