实时荧光定量PCR方法检测幼兔粪便双歧杆菌的实验研究

Real-time quantitative PCR procedure for quantification of bifidobacteria in fecal samples from infant rabbits

目的:应用实时荧光定量PCR技术对实验幼兔粪便内双歧杆菌进行定量分析．方法:依据双歧杆菌16S rDNA序列设计属特异性引物,以常规PCR产物经克隆后的质粒 DNA为标准品,经光谱定量、梯度稀释后制备标准曲线．抽提正常对照组和双歧杆菌喂饲组幼兔粪便内的细菌基因组DNA,用实时荧光定量PCR技术定量分析样品中双歧杆菌数量．结果:两组幼兔粪便内双歧杆菌测定结果均成阳性,双歧杆菌喂饲组0．05 g湿粪内菌量的对数值较对照组显著升高(6．37±0．58 vs 5．18 ±0．98,P=0．004)．结论:实时荧光定量PCR方法可正确定量实验兔粪便内双歧杆菌数量．

AIM： To quantitatively detect the bifidobacteria in the fecal samples from infant rabbits by realtime quantitative polymerase chain reaction （RTQ-PCR）. METHODS： Genus-specific primer of bifidobacteria was designed for 16S rDNA-targeted PCR. The standard curve of RTQ-PCR was generated from a grade-dilution of plasmid DNA which was cloned from PCR products and spectroscopically quantified. Bacterial genome DNA in fecal samples was extracted from the bifidobacteriafed rabbits and the controls for analysis by RTQ- PCR, respectively. RESULTS： Bifidobacteria was positive in both groups of rabbit fecal samples. The level of bifidobacteria in the bifidobacteria-fed rabbits was significantly higher than that in the control group （logarithm for the numbers of bifidobacteria per 0.05 g feces： 6.37 ± 0.58 vs 5.18 ± 0.98, P = 0.004）. CONCLUSION： Real-time quantitative PCR can accurately quantify the bifidobacteria in fecal samples from infant rabbits.