DNA提取前处理方法对萨拉米香肠中细菌种群结构的影响

通过比较不同DNA提取前处理方法在高通量测序中对萨拉米香肠细菌种群结构的影响,为建立规范的高通量测序的操作流程优选出更适宜的前处理方法.采用直接提取法(M0)、拍击均质法(Ml)和振荡珠磨法(M2)3种常用前处理方法提取萨拉米香肠中细菌DNA,利用MiSeq高通量测序技术分析萨拉米香肠中细菌16S rRNA V3-V4...

Effect of Pretreatment Methods for DNA Extraction on the Bacterial Community Structure in Salami

This study aimed to compare the effect of different pretreatment methods for DNA extraction on the bacterial community structure in salami for the purpose of selecting appropriate pretreatment methods for a standardized high-throughput sequencing process. We extracted bacterial DNA from salami by three common pretreatment methods: direct extraction (M0), tapping homogenization (M1), and bead beating (M2). We performed MiSeq high-throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene and analyzed the bacterial community structure based on operational taxonomic units (OTUs). The results showed that a total of 319, 206, and 253 OTUs were observed with M0, M1, and M2, respectively; the number of OTUs shared by the three methods was 129, accounting for 31.85% of the total OTUs. For the three methods, the Chao1 index was 177.93 ± 31.02, 120.76 ± 28.60, and 166.96 ± 15.63, respectively, and the Shannon index was 2.79 ± 0.22, 2.95 ± 0.31, and 3.25 ± 0.30, respectively. At the phylum and family level, the bacterial community structure of the samples obtained by different pretreatment methods was similar despite differential abundance; however, different pretreatment methods affected the results of subsequent community structure analysis at the genus level. M0 increased the abundance and diversity of dominant bacteria, and could be applied to detect bacteria in maturing salami samples. M1 and M2 removed free DNA, leaving only bacterial cells with a complete structure, which could better reflect the bacterial community structure in samples. The bacterial community structure and abundance observed with M1 and M2 for homogeneous samples tended to be consistent. In conclusion, this study shows that different pretreatment methods can affect the results of bacterial community structure analysis. A standardized procedure for high-throughput sequencing should be established as soon as possible to ensure data reliability and the comparability of results across studies.