Morin Nicole J, Gong Zhilong, Li Xing-Fang
Environmental Health Sciences, Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
Clin Chem. 2004 Nov;50(11):2037-44. doi: 10.1373/clinchem.2004.036814. Epub 2004 Sep 13.
Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi are pathogenic bacteria that can be found in contaminated water supplies throughout the world. No currently available assays can simultaneously detect and identify all three pathogens. Our aim was to develop a rapid and reliable technique for simultaneous detection of these pathogens.
Four unique genes were chosen as the targets of detection. Forward and reverse primers were designed to specifically amplify different sizes of these target genes: a 239-bp region of the E. coli O157 lipopolysaccharide (LPS) gene (rfbE); a 179-bp region of the H7 flagellin gene (fliC); a 419-bp region of the V. cholerae O1 LPS gene (rfbE); and a 329-bp region of Salmonella Typhi LPS gene (tyv). To ensure the detection of only viable replicating bacteria, RNA was extracted for analysis. After reverse transcription, cDNAs were simultaneously amplified in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. To characterize the assay we analyzed, in a blinded fashion, seven unknown RNA samples containing various combinations of total RNA from these bacteria as well as clinical isolates.
All seven unknown RNA samples were correctly identified. The assay was able to detect and identify as few as 30 cells of E. coli O157:H7 and Salmonella Typhi in clinical isolates, and the presence of other bacteria did not interfere with the analysis.
An assay combining reverse transcription with single-tube multiplex PCR was successfully developed and validated for simultaneous detection of viable E. coli O157:H7, V. cholerae O1, and Salmonella Typhi.
大肠杆菌O157:H7、霍乱弧菌O1和伤寒沙门氏菌是致病细菌，在世界各地受污染的水源中均可发现。目前尚无检测方法能同时检测和鉴定这三种病原体。我们的目标是开发一种快速可靠的技术，用于同时检测这些病原体。
选择四个独特的基因作为检测靶点。设计正向和反向引物，以特异性扩增这些靶基因的不同大小片段：大肠杆菌O157脂多糖（LPS）基因（rfbE）的239 bp区域；H7鞭毛蛋白基因（fliC）的179 bp区域；霍乱弧菌O1 LPS基因（rfbE）的419 bp区域；伤寒沙门氏菌LPS基因（tyv）的329 bp区域。为确保仅检测有活力的复制细菌，提取RNA进行分析。逆转录后，通过多重PCR在单个管中同时扩增cDNA。通过凝胶电泳分析多重PCR产物。为了表征我们所分析的检测方法，我们以盲法分析了七个未知RNA样本，这些样本包含来自这些细菌以及临床分离株的总RNA的各种组合。
所有七个未知RNA样本均被正确鉴定。该检测方法能够检测和鉴定临床分离株中低至30个大肠杆菌O157:H7和伤寒沙门氏菌细胞，其他细菌的存在不干扰分析。
成功开发并验证了一种将逆转录与单管多重PCR相结合的检测方法，用于同时检测有活力的大肠杆菌O157:H7、霍乱弧菌O1和伤寒沙门氏菌。
