16SrRNA的分子生物学方法分析牛奶中的细菌菌群

Molecular Biological Analysis of Bacterial Community in Milk Based on 16S rRNA Gene

选择4个不同生产厂家的市售牛奶作为研究对象,提取牛奶中细菌的基因组DNA,利用聚合酶链式反应(PCR)-克隆-测序的方法获得牛奶中菌群的16S rRNA-V3区序列信息,运用生物信息学分析牛奶中细菌种类、菌群多样性和菌群结构;荧光定量PCR法测定样品中的细菌总数。结果表明:各品牌牛奶中均含有大量细菌的16S rRNA-V3区DNA序列;牛奶样品中测序共得到472条16S rRNA-V3区序列,其中有452条序列可以明确鉴定到属,包括3个革兰氏阳性菌属和14个革兰氏阴性菌属;20条序列可以鉴定到纲的水平。各品牌牛奶中细菌总数均处于相对较高水平;4个厂家牛奶间菌群分布不同,菌群多样性存在差异。

The number of bacteria in milk has gained growing attention as the new national dairy standard is implemented. However, few people are concerned about its bacterial species and flora. Bacterial communities in milk samples from 4 manufactures were analyzed. Bacterial genome DNA was extracted from milk and molecular segments of the V3 region of the 16S rRNA gene was obtained by PCR, cloning and sequencing. Bacterial species, diversity and flora were analyzed by bioinformatics. Bacterial counts of samples were measured by florescent real-time quantitative PCR. The results showed that molecular segments of the V3 region in the 16S rRNA existed in large quantities in milk. Among 472 clones of the V3 region in the 16S rRNA obtained from sequencing, 452 could be clearly classified to genera, including 3 Gram-positive （G＋） and 14 Gram-negative （G-） species, and 20 others could be classified to the class level. These results indicated that the bacterial counts were all at a high level in different milk. Samples from 4 manufactures differed in their bacterial communities, and the diversity of bacterial flora was obviously different.