Sydney School of Veterinary Science, Faculty of Science, University of Sydney, Camden, NSW, Australia.
J Appl Microbiol. 2019 Aug;127(2):429-444. doi: 10.1111/jam.14326. Epub 2019 Jun 7.
This study evaluated methods to sample and extract nucleic acids from Pacific oysters to accurately determine the microbiome associated with different tissues.
Samples were collected from haemolymph, gill, gut and adductor muscle, using swabs and homogenates of solid tissues. Nucleic acids were extracted from fresh and frozen samples using three different commercial kits. The bacterial DNA yield varied between methods (P < 0·05) and each tissue harboured a unique microbiota, except for gill and muscle. Higher bacterial DNA yields were obtained by swabbing compared to tissue homogenates and from fresh tissues compared to frozen tissues, without impacting the bacterial community composition estimated by 16S rRNA gene (V1-V3 region) sequencing. Despite the higher bacterial DNA yields with QIAamp® DNA Microbiome Kit, the E.Z.N.A. Mollusc DNA Kit identified twice as many operational taxonomic units (OTUs) and eliminated PCR inhibition from gut tissues.
Sampling and nucleic acid purification substantially affected the quantity and diversity of bacteria identified in Pacific oyster microbiome studies and a fit-for-purpose strategy is recommended.
Accurate identification of Pacific oyster microbial diversity is instrumental for understanding the polymicrobial aetiology of Pacific oyster mortality diseases which greatly impact oyster production.
本研究评估了从太平洋牡蛎中采样和提取核酸的方法，以准确确定与不同组织相关的微生物组。
使用拭子和固体组织的匀浆从血淋巴、鳃、肠道和闭壳肌采集样本。使用三种不同的商业试剂盒从新鲜和冷冻样本中提取核酸。不同方法之间的细菌 DNA 产量存在差异（P<0.05），除了鳃和肌肉外，每个组织都存在独特的微生物群。与组织匀浆相比，拭子法和从新鲜组织中提取的细菌 DNA 产量更高，而不会影响通过 16S rRNA 基因（V1-V3 区）测序估计的细菌群落组成。尽管 QIAamp® DNA Microbiome 试剂盒的细菌 DNA 产量较高，但 E.Z.N.A. Mollusc DNA 试剂盒鉴定的操作分类单元（OTUs）数量是前者的两倍，并且消除了肠道组织的 PCR 抑制。
采样和核酸纯化极大地影响了太平洋牡蛎微生物组研究中鉴定的细菌数量和多样性，建议采用适合目的的策略。
准确识别太平洋牡蛎的微生物多样性对于理解太平洋牡蛎死亡疾病的多微生物病因至关重要，这些疾病对牡蛎生产有重大影响。
