Stubner Stephan
Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, D-35043, Marburg, Germany.
J Microbiol Methods. 2002 Jul;50(2):155-64. doi: 10.1016/s0167-7012(02)00024-6.
Real-time PCR is a new and highly sensitive method for the quantification of microbial organisms in environmental samples. This work was conducted to evaluate real-time PCR with SybrGreen (SG) detection as quantification method for Desulfotomaculum lineage 1 organisms in samples of rice field soil. The method was optimized in several parameters like SG concentration. These allowed quantitative PCR with different primer combinations yielding PCR products with lengths up to 1066 bp and with sensitivities of 10(2) targets for all assays. The detection limit in environmental DNA extracts (rice bulk soil and rice roots) was 10(6) targets per gram dry weight according to the dilution of the DNA extracts necessary to overcome PCR inhibition of humic substances. A verification, that the fluorescence increase was due to specific PCR products, was done by agarose gel electrophoresis since melting curve analysis of the PCR products did not show a distinct peak in the first derivative, when the environmental DNA extracts were used in PCR. Amplification with a primer combination specific for Desulfotomaculum lineage 1 organisms showed an abundance of this group of approximately 2% and 0.5% of the eubacterial 16S rDNA targets in rice bulk soil and rice root samples, respectively. Approximately half of this number was obtained in both habitats with a PCR assay specific for a Desulfotomaculum sequence cluster obtained previously from rice field soil.
实时荧光定量PCR是一种用于环境样品中微生物定量分析的新型高灵敏度方法。本研究旨在评估以SybrGreen（SG）检测的实时荧光定量PCR作为稻田土壤样品中脱硫肠状菌系1微生物的定量方法。该方法在SG浓度等多个参数方面进行了优化。这些优化使得使用不同引物组合进行定量PCR成为可能，所产生的PCR产物长度可达1066 bp，且所有检测的灵敏度均为10²个靶标。根据克服腐殖质对PCR抑制所需的DNA提取物稀释度，环境DNA提取物（水稻土和水稻根）中的检测限为每克干重10⁶个靶标。由于当使用环境DNA提取物进行PCR时，PCR产物的熔解曲线分析在一阶导数中未显示明显峰值，因此通过琼脂糖凝胶电泳验证荧光增加是否归因于特异性PCR产物。用针对脱硫肠状菌系1微生物的引物组合进行扩增表明，该菌群在水稻土和水稻根样品中分别约占真细菌16S rDNA靶标的2%和0.5%。在这两个生境中，使用先前从稻田土壤中获得的针对脱硫肠状菌序列簇的PCR检测方法，得到的数量约为此数量的一半。
