Neuberger-Castillo Lorie, Hamot Gaël, Marchese Monica, Sanchez Ignacio, Ammerlaan Wim, Betsou Fay
Integrated BioBank of Luxembourg (IBBL), Dudelange, Luxembourg.
Biopreserv Biobank. 2020 Apr;18(2):102-116. doi: 10.1089/bio.2019.0112. Epub 2020 Jan 30.
A formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A previously optimized stool processing protocol was validated for fitness-for-purpose for downstream microbiome analysis. DNA extraction from human stool was validated with various collection tubes, stabilizing solutions and storage conditions in terms of fitness-for-purpose for downstream microbiome analysis, robustness, and sample stability. Acceptance criteria were based on accurate identification of a reference material, homogeneity of extracted samples, and sample stability in a 2-year period. The automated DNA extraction using the chemagic™ Magnetic Separation Module I (MSM I) extracted 8 out of 8 bacteria in the ZymoBIOMICS Microbial Community Standard. Seven tested stabilizing solutions (OMNIgene•GUT, RNA, AquaStool™, RNAssist, PerkinElmer SEB lysis buffer, and DNA Genotek's CP-150) were all compatible with the chemagic MSM I and showed no significant difference in microbiome alpha diversity and no significant difference in the overall microbiome composition compared to the baseline snap-frozen stool sample. None of the stabilizing solutions showed intensive polymerase chain reaction (PCR) inhibition in the SPUD assay. However, when we take into account more stringent criteria which include a higher double-stranded DNA yield, higher DNA purity, and absence of PCR inhibition, we recommend the use of OMNIgene•GUT, RNA, or AquaStool as alternatives to rapid freezing of samples. The highest sample homogeneity was achieved with RNA- and OMNIgene•GUT -stabilized samples. Sample stability after a 2-year storage in -80°C was seen with OMNIgene•GUT -stabilized samples. We validated a combination of a stool processing method with various collection methods, suitable for downstream microbiome applications. Sample collection, storage conditions and DNA extraction methods can influence the microbiome profile results. Laboratories and biobanks should ensure that these conditions are systematically recorded in the scope of accreditation.
在实验室和生物样本库的认证背景下，缺乏对生物样本处理的正式方法验证。先前优化的粪便处理方案针对下游微生物组分析的适用性进行了验证。就下游微生物组分析的适用性、稳健性和样本稳定性而言，对使用各种收集管、稳定溶液和储存条件从人类粪便中提取DNA进行了验证。验收标准基于参考物质的准确鉴定、提取样本的均匀性以及两年内的样本稳定性。使用chemagic™ 磁分离模块I（MSM I）进行的自动化DNA提取在ZymoBIOMICS微生物群落标准中提取了8种细菌中的8种。七种经过测试的稳定溶液（OMNIgene•GUT、RNA、AquaStool™、RNAssist、珀金埃尔默SEB裂解缓冲液和DNA Genotek的CP-150）均与chemagic MSM I兼容，与基线速冻粪便样本相比，微生物组α多样性无显著差异，整体微生物组组成也无显著差异。在SPUD分析中，没有一种稳定溶液显示出强烈的聚合酶链反应（PCR）抑制作用。然而，当我们考虑更严格的标准，包括更高的双链DNA产量、更高的DNA纯度和无PCR抑制时，我们建议使用OMNIgene•GUT、RNA或AquaStool作为样本快速冷冻的替代方法。RNA和OMNIgene•GUT稳定的样本实现了最高的样本均匀性。在-80°C下储存两年后，OMNIgene•GUT稳定的样本显示出样本稳定性。我们验证了一种粪便处理方法与各种收集方法的组合，适用于下游微生物组应用。样本收集、储存条件和DNA提取方法会影响微生物组谱结果。实验室和生物样本库应确保在认证范围内系统记录这些条件。
