【摘要】 以大肠杆菌和粪肠球菌作为研究对象,研究建立了一种逆转录定量PCR(reverse transcription q quantitative PCR,RT-qPCR)方法,以选择性检测水中活性病原菌.研究结果表明,细菌体内的RNA经过RT-PCR逆转录成cDNA后利用qPCR可定量目的基因拷贝数,对处于稳定生长期的大肠杆菌(培养6～18 h)和粪肠球菌(培养10～38 h)体内的RNA含量进行检测分别为1 copies.CFU-1和7.98×102copies.CFU-1,以此作为细菌定量的依据,达到准确定量检测水体中目的基因RNA拷贝数而确定活性细菌含量的目的.通过3种方法(培养法、qPCR、RT-qPCR)检测热灭活后的大肠杆菌和粪肠球菌,结果表明与qPCR相比,RT-qPCR能够区分1.43 lg copy(大肠杆菌)与2.5 lg copy(粪肠球菌)的非活性菌,进而选择性检测活性病原菌.实际水样底物基质对RT-qPCR方法的影响不大,RT-qPCR与培养法之间有较好的线性相关性(大肠杆菌,R2=0.930,粪肠球菌,R2=0.948),本研究建立的方法可以用于实际水样活性病原菌的检测.更多还原

【Abstract】 A reverse transcription q quantitative PCR(RT-qPCR) assay method was established,which can quantify the copy numbers of RNA in pathogenic bacteria of E.coli and Enterococcus faecium.The results showed that cDNA was generated with the RT-PCR reagents,target gene was quantified with the qPCR,the copy numbers of RNA were stable at about 1 copies·CFU-1 for E.coli and 7.98×102 copies·CFU-1 for Enterococcus faecium respectively during the stationary grow phase for the both indicator bacteria [E.coli(6-18 h) and Enterococcus faecium(10-38 h)].The established RT-qPCR method can quantify the numbers of viable bacteria through detecting bacterial RNA targets.Through detecting the heat-treated E.coli and Enterococcus faecium by three methods(culture method,qPCR,RT-qPCR),we found that the qPCR and RT-qPCR can distinguish 1.43 lg copy non-viable E.coli and 2.5 lg copy non-viable Enterococcus faecium.These results indicated that the established methods could effectively distinguish viable bacteria from non-viable bacteria.Finally we used this method to evaluate the real effluents of the secondary sedimentation of wastewater treatment plant(WWTP),the results showed that the correlation coefficients(R2) between RT-qPCR and culture method were 0.930(E.coli) and 0.948(Enterococcus faecium),and this established RT-PCR method can rapidly detect viable pathogenic bacteria in genuine waters.更多还原