Muiños-Bühl Anixa, González-Recio Oscar, Muñoz María, Óvilo Cristina, García-Casco Juan, Fernández Ana I
Departamento de Mejora Genética Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), 28040, Madrid, Spain.
Departamento de Mejora Genética Animal, Centro I+D Cerdo Ibérico INIA, 06300, Zafra, Badajoz, Spain.
Curr Microbiol. 2018 Jun;75(6):651-657. doi: 10.1007/s00284-017-1429-1. Epub 2018 Jan 9.
There is a growing interest in understanding the role of the gut microbiome on productive and meat quality-related traits in livestock species in order to develop new useful tools for improving pig production systems and industry. Faecal samples are analysed as a proxy of gut microbiota and here the selection of suitable protocols for faecal sampling and DNA isolation is a critical first step in order to obtain reliable results, even more to compare results obtained from different studies. The aim of the current study was to establish in a cost-effective way, using automated ribosomal intergenic spacer analysis technique, a protocol for porcine faecal sampling and storage at farm and slaughterhouse and to determine the most efficient microbiota DNA isolation kit among those most widely used. Operational Taxonomic Unit profiles were compared from Iberian pig faecal samples collected from rectum or ground, stored with liquid N, room temperature or RNAlater, and processed with QIAamp DNA Stool (Qiagen), PowerFecal DNA Isolation (Mobio) or SpeedTools Tissue DNA extraction (Biotools) commercial kits. The results, focused on prokaryote sampling, based on DNA yield and quality, OTU number and Sørensen similarity Indexes, indicate that the recommended protocol for porcine faecal microbiome sampling at farm should include: the collection from porcine rectum to avoid contamination; the storage in liquid N or even at room temperature, but not in RNAlater; and the isolation of microbiota DNA using PowerFecal DNA Isolation kit. These conditions provide more reliable DNA samples for further microbiome analysis.
为开发用于改进生猪生产系统和产业的新实用工具，人们对了解肠道微生物群在牲畜生产性能和肉质相关性状方面的作用兴趣日益浓厚。粪便样本作为肠道微生物群的替代物进行分析，在此，选择合适的粪便采样和DNA提取方案是获得可靠结果的关键第一步，尤其是为了比较不同研究所得结果。本研究的目的是以经济高效的方式，使用自动核糖体基因间隔区分析技术，建立一种在农场和屠宰场进行猪粪便采样和储存的方案，并在最广泛使用的试剂盒中确定最有效的微生物群DNA提取试剂盒。比较了从伊比利亚猪直肠或粪便采集的粪便样本的操作分类单元图谱，这些样本分别储存在液氮、室温或RNAlater中，并用QIAamp DNA Stool（Qiagen）、PowerFecal DNA Isolation（Mobio）或SpeedTools Tissue DNA extraction（Biotools）商业试剂盒进行处理。基于DNA产量和质量、OTU数量和 Sørensen相似性指数的结果，聚焦于原核生物采样，表明农场猪粪便微生物群采样的推荐方案应包括：从猪直肠采集以避免污染；储存在液氮中甚至室温下，但不要储存在RNAlater中；使用PowerFecal DNA Isolation试剂盒分离微生物群DNA。这些条件可为进一步的微生物群分析提供更可靠的DNA样本。
