Sanders Justin L, Kent Michael L
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA.
Dis Aquat Organ. 2011 Sep 9;96(2):145-56. doi: 10.3354/dao02375.
The zebrafish Danio rerio is an increasingly important biological model in many areas of research. Due to the potential for non-protocol-induced variation, diseases of zebrafish, especially those resulting in chronic, sub-lethal infections, are of great concern. The microsporidium Pseudoloma neurophilia is a common parasite of laboratory zebrafish. Current methods for detection of this parasite require lethal sampling of fish, which is often undesirable with poorly spawning mutant lines and small populations. We present here an improved molecular-based diagnostic assay using real-time polymerase chain reaction (PCR), and including sonication treatment prior to DNA extraction. Comparisons of several DNA extraction methods were performed to determine the method providing the maximum sensitivity. Sonication was found to be the most effective method for disrupting spores. Compared to previously published data on PCR-based assay using a dilution experiment, sensitivity is increased. This shows that our assay, which includes sonication, is capable of detecting parasite DNA at 1 log higher dilution than the conventional PCR-based assay, which does not include sonication. Furthermore, we demonstrate the application of this method to testing of water, eggs, and sperm, providing a potential non-lethal method for detection of this parasite in zebrafish colonies with a sensitivity of 10 spores 1(-1) of water, 2 spores per spiked egg sample, and 10 spores microl(-1) of spiked sperm sample.
斑马鱼（Danio rerio）在许多研究领域中已成为越来越重要的生物学模型。由于存在非实验方案诱导变异的可能性，斑马鱼疾病，尤其是那些导致慢性、亚致死感染的疾病，备受关注。微孢子虫嗜神经假瘤虫（Pseudoloma neurophilia）是实验室斑马鱼的常见寄生虫。目前检测这种寄生虫的方法需要对鱼进行致死性取样，这对于产卵不佳的突变品系和小群体来说往往是不可取的。我们在此展示一种改进的基于分子的诊断检测方法，该方法使用实时聚合酶链反应（PCR），并在DNA提取前进行超声处理。我们对几种DNA提取方法进行了比较，以确定能提供最大灵敏度的方法。结果发现超声处理是破坏孢子最有效的方法。与之前发表的关于基于PCR检测方法的稀释实验数据相比，灵敏度有所提高。这表明我们的检测方法（包括超声处理）能够在比不包括超声处理的传统基于PCR的检测方法高1个对数的稀释度下检测到寄生虫DNA。此外，我们证明了该方法在水、卵和精子检测中的应用，为斑马鱼群体中这种寄生虫的检测提供了一种潜在的非致死性方法，其灵敏度为每毫升水10个孢子、每个加标卵样本2个孢子以及每微升加标精子样本10个孢子。
