【摘要】 目的:建立利用实时荧光定量PCR法检测人粪便中双歧杆菌浓度的方法,为检测人肠道内菌群浓度提供有效手段。方法:提取60例儿童粪便样本中细菌总DNA。选择细菌种属的特异性基因16SrRNA作为扩增区,依据双歧杆菌的16SrRNA序列共设计3条引物,以常规PCR扩增出的16SrRNA部分基因片段制作标准品,应用SYBR GreenⅠ双链嵌合染料,对连续稀释的标准品及待测样本进行分析,建立绝对定量的标准曲线,同时计算出待测样本双歧杆菌浓度;根据可检测到的标准品最低拷贝数计算反应灵敏度;分析PCR产物熔解曲线评价反应特异性;重复检测梯度稀释的标准品,用相同浓度标准品的Ct值变异系数(CV)评价反应稳定性。结果:常规PCR扩增出双歧杆菌16SrRNA部分基因片段长度约为613bp,测序结果正确,成功构建了双歧杆菌实时荧光定量PCR反应中的标准品;生成的标准曲线R2=0.999,最低检测限度为每个反应1.48×102个拷贝;实时荧光定量PCR产物的熔解曲线为单峰。对待测样本分批进行荧光定量PCR检测,各组间每微升1.48×103~1.48×107个拷贝浓度标准品Ct值的变异系数为2.94%、3.39%、3.54%、3.08%和3.34%。60份儿童粪便样本双歧杆菌浓度对数值为7.77±0.86(拷贝数·g-1湿便)。结论:本研究所建立的实时荧光定量PCR法敏感性高、特异性强,且重复性好,适用于检测人粪便中双歧杆菌的浓度。更多还原

【Abstract】 Objective To establish the real-time fluorescence quantitative PCR method for the detection of bifidobacteria in human fecal samples,and to provide an effective means for measuring intestinal bacteria. Methods Total DNA of bacteria was extracted from 60cases of children’s fecal samples.Three primers of bifidobacteria based on the 16Sribosomal RNA(16SrRNA)which possessed specialities of bacteria as amplified region were designed.The part of amplified 16SrRNA gene sequences was used as standard production.The serial dilution of standard was analyzed to build an absolute quantitative standard curve with SYBR GreenⅠ dye method, and the bifidobacterium contents in sixty human fecal samples were calculated.The sensitivity of the reaction was calculated by detecting the lowest detectable standard which determined the sensitivity of the reaction.The PCR products’ melting curve was used to evaluate the specificity.The coefficient of variation(CV)of different batches of standard with the same concentration was used to evaluate the stability of reaction.Results The length of PCR product fragment which was used to build the standard curve was about 613bp,the sequencing result was consist with the goals,and the standard sample of bifidobacteria was successfully established in real-time fluorescence quantitative PCR.The standard curve showed a good linear relationship with R2=0.999.The minimum detection value was 1.48×102 copies per reaction.The melting curve of real-time fluorescence quantitative PCR was a single peak.The test samples were batched and then examined by fluorescence quantitative PCR.The CV of standards’ Ct values which calculated from the standard(1.48×103-1.48×107copies·μL-1)were 2.94%,3.39%, 3.54%,3.08%,and 3.34%,respectively.The contents of bifidobacteria in fecal from 60children was 7.77± 0.86(copies·g-1 wet fecal)transformed by logarithmic.Conclusion The established real-time fluorescence quantitative PCR method has high sensitivity,strong specificity and good repeatability,which is suitable for detection of human fecal bifidobacteria content.更多还原