Bach H-J, Tomanova J, Schloter M, Munch J C
Institute for Soil Ecology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany.
J Microbiol Methods. 2002 May;49(3):235-45. doi: 10.1016/s0167-7012(01)00370-0.
Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed.Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay. The developed assays were applied for the quantification of bacteria in soil samples.
开发了实时定量PCR检测方法，用于对纯培养物和环境样品中不同细菌群体进行绝对定量。16S rRNA基因用作真细菌的标记，细胞外肽酶基因用作潜在蛋白水解细菌的标记。对于设计的16S rDNA TaqMan检测方法，证明了设计的引物-探针组合对真细菌具有特异性，在广泛的起始拷贝数范围内具有高扩增效率和高重现性。使用大肠杆菌的16S rDNA靶序列作为定量外标，通过TaqMan-PCR监测液体培养物中蜡样芽孢杆菌、枯草芽孢杆菌和荧光假单胞菌的细胞浓度。结果与平板计数和DAPI染色细胞的显微镜计数一致。讨论了16S rRNA操纵子多样性对细菌定量的意义。此外，测试了先前设计的用于靶向不同类别的细菌细胞外肽酶的三组引物对及其与探针一起用于基于TaqMan-PCR的蛋白水解细菌定量的适用性。由于探针的高度简并性不允许进行准确定量，因此在PCR过程中使用SybrGreen代替分子探针来可视化和定量PCR产物。对于所有三种肽酶基因类别，荧光与起始拷贝数之间的相关性与16S rDNA TaqMan检测方法的质量相同。蜡样芽孢杆菌的中性金属肽酶基因、枯草芽孢杆菌的枯草杆菌蛋白酶基因和荧光假单胞菌的碱性金属肽酶基因的检测量与通过16S rDNA靶向检测方法研究的细菌数量完全对应。所开发的检测方法用于土壤样品中细菌的定量。
