Department of Large Animal Sciences, University of Copenhagen, Taastrup, Denmark.
Vet Parasitol. 2013 Jan 16;191(1-2):191-6. doi: 10.1016/j.vetpar.2012.08.018. Epub 2012 Sep 5.
Strongyle and trichostrongyle parasites are ubiquitous nematodes of grazing livestock. Several molecular diagnostic tests are based upon measuring and quantifying DNA obtained from parasite eggs. It is well known that such eggs undergo development during storage, but it remains unknown to which extent developmental stages can affect the variation of diagnostic test results. This study investigated the influence of developmental stages of strongyle eggs on the variation real-time polymerase chain reaction (PCR) results. Mixed species strongyle eggs were obtained from the faeces of a naturally infected horse. Eggs were isolated and placed in microtiter plates with demineralized water. A total of 25 wells containing 100 eggs each were set up and kept refrigerated for up to five days. Once daily, five wells were examined on an inverted microscope at 100× magnification, where the developmental stages of the eggs were noted, and then eggs harvested for DNA extraction. The protocol was repeated three times. Genomic DNA was extracted using a commercial kit previously validated for strongyle type eggs. PCR reactions were performed with a primer set specific for the ribosomal DNA region for all strongyle type parasites (NC1, NC2). SYBR Green Real-Time PCRs were performed in triplicates. Results revealed a statistically significant increase in PCR yield after three days, which was statistically associated with beginning embryonation of the eggs. In conclusion, storage time and developmental stage of strongyle eggs are significant sources of error in studies based on quantitative real-time PCR analysis. This study suggests that for refrigerated storage of more than three days, eggs should be inactivated and preserved for further analysis.
马消化道线虫是广泛存在于放牧家畜中的寄生线虫。一些分子诊断检测是基于测量和定量从寄生虫卵中获得的 DNA。众所周知，这些卵在储存过程中会发育，但发育阶段会在多大程度上影响诊断检测结果的变化尚不清楚。本研究调查了马消化道线虫卵发育阶段对实时聚合酶链反应（PCR）检测结果变化的影响。混合种马消化道线虫卵从自然感染马的粪便中获得。将卵分离出来，放置在含有去矿物质水的微量滴定板中。共设置了 25 个孔，每个孔包含 100 个卵，并冷藏保存长达五天。每天一次，在倒置显微镜下以 100×的放大倍数检查五个孔，记录卵的发育阶段，然后取出卵进行 DNA 提取。该方案重复了三次。使用先前针对马消化道线虫卵类型验证过的商用试剂盒提取基因组 DNA。使用针对所有马消化道线虫类型寄生虫核糖体 DNA 区域的引物组（NC1、NC2）进行 PCR 反应。SYBR Green 实时 PCR 重复进行三次。结果显示，三天后 PCR 产量呈统计学显著增加，这与卵开始胚胎发育有统计学关联。总之，马消化道线虫卵的储存时间和发育阶段是基于定量实时 PCR 分析的研究中出现误差的重要来源。本研究表明，对于冷藏储存超过三天的情况，应使卵失活并保存以进行进一步分析。
