摘要： 为定量测定瘤胃脂肪分解菌,试验构建了脂肪分解菌实时荧光定量PCR的标准品及标准曲线。提取瘤胃微生物总DNA,以脂肪分解菌16S rDNA特异性引物进行PCR扩增,回收PCR产物,与 PMD19-T Vector 连接并转化大肠杆菌。阳性重组质粒经PCR和测序鉴定后,将提取的质粒DNA进行梯度稀释并作为模板,利用荧光定量PCR反应做出标准曲线。结果显示:PCR产物与目的基因的相似性大于99%,以不同稀释度的重组质粒为模板获得的荧光定量PCR扩增曲线差异明显,构建了相关系数接近1的标准曲线,且熔解曲线峰值单一。试验建立的实时荧光定量PCR方法可用于瘤胃脂肪分解菌的定量检测,为进一步研究该菌在反刍动物瘤胃发酵中的分子机理奠定了基础。

关键词: 瘤胃脂肪分解菌, 实时荧光定量PCR, 标准品质粒, 标准曲线

Abstract: To quantify the population of Anaerovibrio lipolytica in rumen, the standard plasmids and curves was constructed by using quantitative Real-time PCR.Total DNA of rumen microbial was isolated from rumen liquid in the Inner Mongolia sheep, linked with pMD19-T Vector to construct recombinant plasmid and transformed into Escherichia coli.White colonies including target plasmids were selected by ampicillin screening, and the specificity was identified by PCR and DNA sequencing.Plasmid DNA was extracted and the concentration of DNA was measured.The concentration of gradient plasmid DNA was used to construct standard curve for Anaerovibrio lipolytica.Similarity of PCR products and purpose gene is more than 99%, correlation coefficient of the standard curve is close to 1, and the melting peak showed single curve.It was shown that the standard plasmids and curves were successfully constructed and could be applied to quantify the population of unknown Anaerovibrio lipolytica in rumen samples using Real-time PCR.

Key words: Ruminal Anaerovibrio lipolytica, Real-time quantitative PCR, Standard plasmid, Standard curve

中图分类号: 

• S826.8