通用引物PCR方法在地表水病原菌检测中的应用

为了探索通用引物PCR方法在地表水病原细菌检测中的应用价值，利用16S rRNA基因的高度保守性，设计并合成细菌的通用引物，采用合成的引物扩增参考菌株及西安市区不同地表水样，并对PCR产物分别进行序列测定及序列同源性分析，同时检测水样中的细菌总数和粪大肠杆菌浓度.结果显示，通用引物扩增4种参考菌株，320 bp处均可得到清晰的电泳条带，其特异性通过序列测定及同源性分析得到进一步验证；通用引物PCR可检出250 ng/L的埃希氏大肠杆菌标准菌株DNA，其PCR检测的灵敏度可达0.275 CFU/mL；用通用引物对西安市区不同地表水样进行PCR扩增，其中■河、兴庆湖、大唐芙蓉园北湖、北石桥污水处理厂出水样品320 bp处都得到了清晰的电泳条带，而黑河水样没有条带；与之相对应的粪大肠杆菌群检测结果显示，只有北石桥污水处理厂出水和兴庆湖水样有粪大肠杆菌检出. 

Application of Pathogenic Bacteria Detection in Surface Water by Universal Primer PCR Method

In order to investigate the application values of polymerase chain reaction(PCR) technique in detection of pathogenic bacteria in surface water,the universal primers were designed and synthesized according to the high conversation of 16S rRNA gene.The DNA of bacteria from several water bodies and secondary effluent in the urban area of Xi'an City were extracted and then amplified with the primers,and sequencing and homology analyses of the PCR production were conducted.As a result,the PCR products of the 4 reference strains were identified at 320 bp on the electrophoregrams,and they were further verified by sequencing and homology analyses.The detection limit of the total DNA of E.coli reference strain was about 250(ng/L) and the sensibility of PCR detection was up to 0.275(CFU/mL).Regarding different water samples,limpid electrophoresis strips were identified at 320 bp for 1 river and 2 lakes in the urban area,as well as the secondary effluent from a domestic wastewater treatment plant,all of which are believed to be under the influence of domestic pollution,while no strip was identified for another river water which is the well protected source for potable water supply.Bacteria enumeration was also conducted by conventional culture method for comparison.Regarding fecal coliform,it was only detected from the secondary effluent and a polluted lake.