Tsai Y L, Palmer C J, Sangermano L R
Environmental Sciences Laboratory, County Sanitation Districts of Orange County, Fountain Valley, California 92728.
Appl Environ Microbiol. 1993 Feb;59(2):353-7. doi: 10.1128/aem.59.2.353-357.1993.
A method in which the polymerase chain reaction (PCR) was used was developed to amplify either a uidA gene fragment or a 16S rRNA gene fragment from Escherichia coli in sewage and sludge. Because of interference caused by humic acidlike substances, crude DNA extracts were purified with a Sephadex G-200 spun column before the PCR was begun. A Southern analysis in which a nonradioactive chemiluminescent method was used was performed to confirm the presence of PCR products. The sensitivity of detection for PCR products when the chemiluminescent method was used was determined to be 30 ag of E. coli genomic DNA template. In seeded sludge, the PCR amplified the target DNA from 80 E. coli cells per g of sludge and 50 Shigella dysenteriae cells per g of sludge. Because only 0.05 aliquot of a sludge extract was used for the PCR, we deduced that the PCR detected target DNA equivalent to the DNA of 2.5 to 4 cells in the extract. The PCR amplified the uidA fragment from diluted sewage influents and effluents containing E. coli cells. Therefore, the PCR performed with a chemiluminescent gene probe can be used to detect the presence of potentially pathogenic microorganisms in sewage and sludge. This technique can be expanded to permit direct detection of pathogenic microorganisms in water samples, thus leading to enhanced public health protection.
开发了一种使用聚合酶链反应（PCR）的方法，用于从污水和污泥中的大肠杆菌扩增uidA基因片段或16S rRNA基因片段。由于腐殖酸样物质造成的干扰，在开始PCR之前，用Sephadex G - 200旋转柱对粗DNA提取物进行了纯化。采用非放射性化学发光法进行Southern分析，以确认PCR产物的存在。使用化学发光法时，对PCR产物的检测灵敏度确定为30 ag大肠杆菌基因组DNA模板。在接种污泥中，PCR从每克污泥中的80个大肠杆菌细胞和每克污泥中的50个痢疾志贺氏菌细胞中扩增出目标DNA。由于仅将污泥提取物的0.05份用于PCR，我们推断PCR检测到的目标DNA相当于提取物中2.5至4个细胞的DNA。PCR从含有大肠杆菌细胞的稀释污水进水和出水中扩增出uidA片段。因此，用化学发光基因探针进行的PCR可用于检测污水和污泥中潜在致病微生物的存在。该技术可以扩展，以允许直接检测水样中的致病微生物，从而加强公共卫生保护。
