环境水体中肠道病原细菌的定量PCR检测

采用通用引物,通过实时荧光定量PCR方法(QPCR),对西安市5处地表水体中肠道病原细菌的细胞密度进行4个月的连续检测,并将QPCR检测结果与滤膜法测得的大肠菌群CFU值进行比较分析.结果显示, QPCR法可信度为94%,最小检测值为每管未稀释DNA提取物含2.7个大肠杆菌细胞.5个水体(N=60)检测结果表明, QPCR检测结果是大肠菌群CPU的2.2～5倍.病原细菌的几何平均值范围, QPCR法在25～67 000 CCE/100 mL之间, MF法大肠菌群为3～45 000 CFU/100 mL之间.2种方法的离散和回归分析表明, QPCR检测结果与大肠菌群CFU显著正相关,秩相关系数为r=0.983.

Detection of Enteric Pathogenic Bacteria from Surface Waters by Quantitative Polymerase Chain Reaction (QPCR)

A rapid quantitative polymerase chain reaction (QPCR) analysis method with universal primers was developed to detect cell densities of the enteric pathogenic bacteria from 5 surface water of Xi'an City for 4 months continuously. And the detection results by QPCR method were compared with counts of coliforms colony-forming units (CFU) determined by membrane filter (MF) analysis. The results showed that QPCR method had an estimated 94% confidence, and detection limit was 2.7 Escherichia coli cells per sample in undiluted DNA extracts. For five surface waters (N = 60), the geometric mean of pathogenic bacteria concentration determined by QPCR was 2.2-5 times of corresponding coliform CFU determined by MF analysis. Using QPCR analysis, these geometric means of pathogenic bacteria concentration ranged from 25 CCE/100 mL to 67 000 CCE/100 mL. Using MF culture analysis, coliforms ranged from 3 CFU/100 mL to 45 000 CFU/100 mL. Regression analysis showed that there was a significant positive correlation between pathogenic bacteria determined by QPCR method and coliforms determined by MF method, the correlation coefficient (r) was 0.983.