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Nat Rev Urol:科研人看这里!尿石症微生物组研究的标准化研究方案怎么做?

2021-07-05 MedSci原创 MedSci原创

微生物群在健康和疾病方面的作用越来越受到关注和研究。美国肠道计划(American Gut Project)等大型项目从数千个人中取样微生物群,其中包含大量的元数据和多种疾病的状态。然而,这种大规模的

微生物群在健康和疾病方面的作用越来越受到关注和研究。美国肠道计划(American Gut Project)等大型项目从数千个人中取样微生物群,其中包含大量的元数据和多种疾病的状态。然而,这种大规模的项目由于缺乏对特定疾病的详细了解而受阻,例如,在与美国肠道项目相关的524个元数据术语中,一个术语涉及肾脏疾病,三个术语涉及糖尿病,没有一个术语涉及尿石症或其他泌尿系统疾病,尽管有许多报道称肠道微生物群和泌尿系统健康之间存在潜在联系。

尿石症的患病率从1976年的3.2%上升到2016年的10.1%,在过去的25年里经历了流行病学的快速转变——发病年龄正在降低,性别差距正在缩小。这种疾病给美国的医疗保健系统造成了100亿美元的负担。

考虑到泌尿系结石的复杂性,确定微生物组是否以及如何导致结石的发生需要一种细致入微的方法。关于尿石症的大量宏基因组相关性研究(MWAS)已经发表,发现了微生物组和尿石症之间的潜在相互作用。然而,由于实验技术的差异和该领域缺乏标准化,这些数据的再现性、适用性和生理相关性仍然存在问题。

为了满足这一需求,第一个泌尿系结石疾病微生物组国际联盟MICROCOSM成立了,共识小组成员被要求参加一个,以制定微生物组研究的标准化方案。特定研究的方案被修订,直到达成共识。该共识组为典型临床微生物组尿石症研究管道的每一步制定了标准化方案,并可通过安全的在线服务器公开获得。

这种标准化为未来的研究创造了基准,以促进结果的一致解释,并总体上探索有效的干预措施,以预防尿石症的发生,也将有助于对其他泌尿系统疾病的微生物组研究感兴趣的研究者。该研究结果近日发表在Nature Reviews Urology期刊上。

1.元数据收集

这些元数据分为样本变量、患者变量、结石变量、抗生素变量、既往病史等,为了方便元数据的收集,MICROCOSM开发了一个结构化的问卷,其中包含了大部分无法从患者的医疗记录中轻易获得的变量,并创建了模板文件来记录这些元数据。

2.样品采集

与任何临床研究一样,首先应该进行分析以确定研究目标所需的样本量,如为微生物组研究专门开发的evidence 23和msWaldHMP24等统计软件包。专家小组构建了几个样本收集方案,并达成一致,这些方案已在先前的微生物组学研究中得到验证。协商一致的协议包括收集中段排尿和大便样本的技术(由患者自己执行),以及收集结石和导管或上尿路(UUT)尿液的协议(由医生执行)。

3.样品的储存、保存和加工。

尿液和结石样本应在4℃保存在防腐剂中(计划培养时用硼酸保存,计划测序时用AssayAssure保存31),然后在收集后24小时内在80℃保存。对于尿液样本,如果尿液样本在室温下保存时间超过1小时,AssayAssure可最大限度地减少微生物群落的变化;如果尿液样品在1h内冷冻,则不需要AssayAssure。

4.DNA提取试剂盒

对于所有的DNA提取,每一组样本都应包括包含无菌水和所有提取试剂的阴性对照;阳性对照应包括商业标准的混合微生物群落样品,在每个测序批次中运行。随后,专家组建议,所有提取都应使用凝胶电泳进行验证,并使用纳米滴分光光度计或量子荧光计对浓度进行定量(Thermo Scientific)。

5.测序与数据分析

测序建议采用对端测序,可以使用16S rRNA基因的V4区,也可以使用Illumina MiSeq或HiSeq进行散弹枪宏基因组测序;对于后者,Illumina的NextSeq或Novaseq也有可能。Illumina平台能产生更长的、更准确的读取,比其他当代平台具有更高的吞吐量,是微生物组研究中最广泛使用的平台。

从尿液和肾结石中分离细菌的培养条件

这项协商一致的协议和专家组的建议对于目前尚不具备进行这类研究所需的设备或专门知识的调查人员特别有用,因为议定书提供了一种方法,可以持续收集样本并将其运送到装备有进行MWA的实验室,以及为对泌尿学其他领域微生物组学研究感兴趣的人提供了指南。

这项工作确立了该领域的基准,并为进一步基于微生物组学的研究提供了未来的资源,同时也促进了多机构合作推进结石病微生物组学的研究。

参考文献:Kachroo, N., Lange, D., Penniston, K.L. et al. Standardization of microbiome studies for urolithiasis: an international consensus agreement. Nat Rev Urol 18, 303–311 (2021). https://doi.org/10.1038/s41585-021-00450-8

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    2021-07-05 Avigo

    学习学习

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