在五个欧洲实验室中组织了一项环形试验,以达到人畜共患寄生虫弓形虫的微卫星(MS)分型的一致性。根据先前公开的基于15个MS标记的片段长度多态性的方法,由每个实验室循环和分析三个样品组。第一样品组比较了一般的分型结果并集中于DNA浓度的影响;第二样品组集中于可以区分相同原型谱系内的弓形虫菌株的多态性指纹标记;和第三样品组集中于非原型基因型。实验室之间的方法差异,包括用于确定MS片段长度的软件程序,使用问卷进行了整理。总的来说,谱系水平的分型结果达成了高度一致,尤其是在DNA浓度最高的样本中。然而,观察到特定标志物的实验室特异性差异.片段长度的主要中位数差异,多达6个碱基对,与用于标记片段特异性引物的荧光团有关。此外,从不同供应商获得的具有相同序列的引物对产生不同长度的片段。此外,测序图谱评估和解释方式的差异可能导致片段长度测定的结果偏差.MS打字的协调,例如,通过使用相同的荧光团或通过对所确定的片段长度进行数值调整,可以提高实验室结果的一致性。这是第一次实验室间比较,为优化该技术提供指导(作为补充)。
A ring
trial among five European laboratories was organized to reach consistency in microsatellite (MS) typing of the zoonotic parasite Toxoplasma gondii. Three sample sets were circulated and analyzed by each laboratory following a previously published method that is based on fragment length polymorphism of 15 MS markers. The first sample set compared typing results in general and focused on effects of DNA concentration; the second sample set focused on the polymorphic fingerprinting markers that can differentiate T. gondii strains within the same archetypal lineage; and the third set focused on non-archetypal genotypes. Methodological variations between laboratories, including the software programs used to determine MS fragment length, were collated using a questionnaire. Overall, lineage-level typing results reached a high level of agreement, especially in samples with the highest DNA concentrations. However, laboratory-specific differences were observed for particular markers. Major median differences in fragment length, of up to 6 base pairs, were related to the fluorophore used to label fragment-specific primers. In addition, primer pairs with identical sequences obtained from different suppliers resulted in fragments of differing length. Furthermore, differences in the way the sequencing profiles were assessed and interpreted may have led to deviating results in fragment length determination. Harmonization of MS typing, for example, by using the same fluorophores or by numerical adjustments applied to the fragment-lengths determined, could improve the uniformity of the results across laboratories. This is the first interlaboratory comparison, providing guidelines (added as a supplement) for the optimization of this technique.