实时荧光定量PCR对瘤胃纤维分解菌定量方法的构建

试验构建了白色瘤胃球菌、黄色瘤胃球菌及产琥珀酸丝状杆菌等3种瘤胃纤维分解菌实时荧光绝对定量PCR的标准品及标准曲线,以用于纤维分解菌的定量测定。提取瘤胃微生物总DNA,以各菌特异性引物进行PCR扩增,回收纯化PCR产物,与pMD18-T Vector连接并转化到大肠杆菌。用氨苄青霉素培养基筛选阳性重组质粒,提取含目的片段质粒DNA,通过PCR及测序鉴定重组质粒。根据OD值确定浓度,将梯度稀释的质粒作为模板,进行荧光定量PCR反应做出标准曲线。结果表明:所构建的3条标准曲线具有很高的相关性(R2>0.999),并获得了高扩增效率产物(白色瘤胃球菌为101%、黄色瘤胃球菌为98.0%、产琥珀酸丝状杆菌为97.7%),说明成功构建了瘤胃纤维分解菌实时绝对定量PCR的标准品和标准曲线,为定量研究瘤胃纤维分解菌奠定基础。

Development of a Real-time Absolute Quantitative PCR for Quantification of Cellulolytic Bacteria in Rumen

To quantify the population sizes of Ruminococcus albus,Ruminococcus flavefaciens and Fibrobacter succinogenes in rumen,the standard plasmids and curves of these species were constructed using real-time absolute quantitative PCR.Ruminal microbial total DNA was isolated from rumen content in sheep,linked with pMD18-T Vector to construct recombinant plasmid and transfected into Escherichia coli.Target plasmids in white colonies were selected by ampicillin screening,and their specificity was identified by direct PCR and DNA sequencing.Plasmid DNA was extracted from positive recombinant plasmids and the DNA concentration was measured spectrophotometrically.The serial gradient concentrations of plasmid DNA were used as standard to plot standard curves for each species,respectively.The determination coefficient of the three standard curves were higher than 0.999 and amplication efficiency was high,101.0 % for R.albus、98.0 % for R.flavefaciens and 97.7 % for F.succinogenes,respectively.It is indicated that the standard plasmids and curves were constructed successfully and can be applied to quantify the population sizes of unknown cellulolytic bacteria in rumen samples using real-time PCR.