Laboratory of Animal Diversity and Systematics, Katholieke Universiteit Leuven, Ch. Debériotstraat 32, 3000 Leuven, Belgium.
Infect Genet Evol. 2011 Aug;11(6):1413-8. doi: 10.1016/j.meegid.2011.05.006. Epub 2011 May 13.
Genotyping individual larval stages and eggs of natural parasite populations is complicated by the difficulty of obtaining reliable genotypes from low quantity DNA template. A suitable storage and extraction protocol, together with a thorough quantification of genotyping errors are therefore crucial for molecular epidemiological studies. Here we test the robustness, handling time, ease of use, cost effectiveness and success rate of various fixation (Whatman FTA(®) Classic and Elute Cards, 70% EtOH and RNAlater(®)) and subsequent DNA extraction methods (commercial kits and proteinase K protocol). None of these methods require a cooling chain and are therefore suitable for field collection. Based on a multiplex microsatellite PCR with nine loci the success and reliability of each technique is evaluated by the proportion of samples with at least eight scored loci and the proportion of genotyping errors. If only the former is taken into account, FTA(®) Elute is recommended (83% success; 44% genotyping error; 0.2 €/sample; 1h 20 m handling time). However, when also considering the genotyping errors, handling time and ease of use, we opt for 70% EtOH with the 96-well plate technology followed by a simple proteinase K extraction (73% success; 0% genotyping error; 0.2 €/sample; 15m handling time). For eggs we suggest (1) to pool all eggs per person in 1.5 ml tubes filled with 70% EtOH for transport and (2) to identify each egg to species level prior to genotyping. To this end we extended the Rapid diagnostic PCR developed by Webster et al. (2010) with a S. mansoni-specific primer to discriminate between S. mansoni, S. haematobium and S. bovis in a single PCR reaction. The success rate of genotyping eggs was 75% (0% genotyping error). This is the first study to incorporate genotyping errors through re-amplification for the evaluation of schistosome sampling protocols and the identification of error-prone loci.
对自然寄生虫种群的个体幼虫期和卵进行基因分型非常复杂，因为从低数量的 DNA 模板中获得可靠的基因型非常困难。因此，对于分子流行病学研究来说，合适的储存和提取方案以及对基因分型错误的彻底量化至关重要。在这里，我们测试了各种固定（Whatman FTA（®）Classic 和 Elute 卡、70%乙醇和 RNAlater（®））和随后的 DNA 提取方法（商业试剂盒和蛋白酶 K 方案）的稳健性、处理时间、易用性、成本效益和成功率。这些方法都不需要冷链，因此适合野外采集。基于九个性状的多重微卫星 PCR，通过至少有八个可评分基因座的样本比例和基因分型错误的比例来评估每种技术的成功率和可靠性。如果只考虑前者，则推荐使用 FTA（®）Elute（83%成功率；44%基因分型错误；0.2 欧元/样本；1h20m 处理时间）。然而，当同时考虑基因分型错误、处理时间和易用性时，我们选择 70%乙醇结合 96 孔板技术，然后进行简单的蛋白酶 K 提取（73%成功率；0%基因分型错误；0.2 欧元/样本；15m 处理时间）。对于卵，我们建议（1）将每人的所有卵放入装满 70%乙醇的 1.5ml 管中进行运输；（2）在基因分型之前，将每个卵鉴定到种水平。为此，我们在 Webster 等人开发的快速诊断 PCR（2010 年）中扩展了一个 S. mansoni 特异性引物，以便在单个 PCR 反应中区分 S. mansoni、S. haematobium 和 S. bovis。卵基因分型的成功率为 75%（0%基因分型错误）。这是第一项通过重新扩增评估血吸虫采样方案和鉴定易错基因座的基因分型错误的研究。
