Tsai Y L, Olson B H
County Sanitation Districts of Orange County, Fountain Valley, California 92728.
Appl Environ Microbiol. 1992 Jul;58(7):2292-5. doi: 10.1128/aem.58.7.2292-2295.1992.
The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.
聚合酶链反应（PCR）用于从腐殖质含量高的沉积物中扩增大肠杆菌16S核糖体基因片段。通过快速冻融法从1克接种或未接种大肠杆菌的样品中提取总DNA。采用了几种方法（使用Bio-Gel P-6和P-30以及Sephadex G-50和G-200柱，以及使用Stoffel片段）来减少对PCR的干扰。当使用用Tris-EDTA缓冲液（pH 8.0）饱和的Sephadex G-200旋转柱纯化含有腐殖质的粗DNA提取物时，获得了最佳结果。收集洗脱级分用于PCR分析。从每克含少于70个大肠杆菌细胞的接种沉积物中获得了扩增的DNA片段。由于每克含70个细胞的DNA提取物的洗脱级分中仅1/100用于PCR，因此检测灵敏度确定为小于1个大肠杆菌细胞。因此，DNA直接提取结合该技术以去除腐殖质的干扰，然后进行PCR，可成为检测含腐殖质环境样品中少量细菌细胞的有力工具。
