摘要

       随着高通量测序技术在污水处理厂抗生素耐药基因分析中的应用日益广泛，需要对样品预处理和DNA提取方法进行比较，以实现实验室间的标准化比较。在两个污水处理厂的进水、活性污泥和出水中应用了三种广泛使用的DNA提取方法（FastDNA®土壤旋转试剂盒、PowerSoil®DNA分离试剂盒和ZR粪便DNA微制备），其中有和没有在50%乙醇中保存和冷冻，在香港和美国，从使用三个试剂盒提取的DNA获得的注释序列共享相似的分类学和ARG谱。总的来说，使用FastDNA旋转试剂盒对三种污水处理厂样品（进水、活性污泥、出水）进行土壤测试时，发现DNA的产量和纯度以及捕获的精氨酸的多样性都是最高的。16srrna基因的定量聚合酶链反应证实了与DNA提取率和代表性革兰氏阴性菌（大肠杆菌）回收率相同的趋势。此外，与新鲜样品相比，乙醇固定、深冷冻和海外运输对精氨酸分布没有明显影响。这一方法有助于为今后全球比较污水处理厂ARG分布提供信息。

       With the growing application of high-throughput sequencing-based metagenomics for profiling antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs), comparison of sample pretreatment and DNA extraction methods are needed to move toward standardized comparisons among laboratories. Three widely employed DNA extraction methods (FastDNA® Spin Kit for Soil, PowerSoil® DNA Isolation Kit and ZR Fecal DNA MiniPrep), with and without preservation in 50% ethanol and freezing, were applied to the influent, activated sludge and effluent of two WWTPs, in Hong Kong and in the USA. Annotated sequences obtained from the DNA extracted using the three kits shared similar taxonomy and ARG profiles. Overall, it was found that the DNA yield and purity, and diversity of ARGs captured were all highest when applying the FastDNA SPIN Kit for Soil for all three WWTP sample types investigated here (influent, activated sludge, effluent). Quantitative polymerase chain reaction of 16S rRNA genes confirmed the same trend as DNA extraction yields and similar recovery of a representative Gram-negative bacterium (Escherichia coli). Moreover, sample fixation in ethanol, deep-freezing and overseas shipment had no discernable effect on ARG profiles, as compared to fresh samples. This approach serves to inform future efforts toward global comparisons of ARG distributions in WWTPs.

        https://academic.oup.com/femsec/article/94/2/fix189/4781310