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目的比较不同的双歧杆菌标准品制备法,筛选适用于实时荧光定量PCR检测的双歧杆菌标准品。方法以双歧杆菌标准菌株作为实验菌株,分别采用菌株DNA法、PCR产物扩增纯化法、质粒DNA法制备梯度浓度标准品,运用紫外分光光度计和实时荧光定量PCR技术进行检测,并对数据进行统计学处理。结果实时荧光定量PCR检测发现,3种不同的双歧杆菌标准品制备法的标准曲线均R2>0.990;不同制备法的双歧杆菌标准品DNA浓度和纯度检测比较,质粒DNA制备法所制的双歧杆菌标准品浓度和纯度较另外两种方法高,差异有统计学意义(P<0.01)。结论双歧杆菌质粒DNA制备法制备的标准品质量较高,适用于实时荧光定量PCR检测,可以为双歧杆菌分子生物学检测提供参考依据。
Objective To compare different preparation methods of Bifidobacterium standard and screen the Bifidobacterium standard which is suitable for real-time fluorescence quantitative PCR. Methods The standard strain of Bifidobacterium was used as the experimental strain. The DNA of the strain, PCR amplification and purification method were used respectively. The gradient concentration standard was prepared by the plasmid DNA method and detected by ultraviolet spectrophotometer and real-time fluorescence quantitative PCR. Statistical analysis. Results Real-time fluorescence quantitative PCR assay showed that the standard curves of the three different preparations of Bifidobacterium standard were all R2> 0.990. The DNA concentration and purity of the Bifidobacterium standard prepared by different preparation methods were compared with that of the standard DNA preparation method Bifidobacterium standard concentration and purity than the other two methods, the difference was statistically significant (P <0.01). Conclusion Bifidobacterium plasmid DNA preparation prepared by high quality standards for real-time fluorescence quantitative PCR detection, Bifidobacterium can provide a reference for the molecular biology tests.