Cohen H J, Mechanda S M, Lin W
Laboratory Services Division, Food Production and Inspection Branch, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
Appl Environ Microbiol. 1996 Dec;62(12):4303-8. doi: 10.1128/aem.62.12.4303-4308.1996.
The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.
本研究的目的是评估通过聚合酶链反应(PCR)扩增fimA基因作为检测沙门氏菌菌株的特异性方法的适用性。鼠伤寒沙门氏菌和肠杆菌科的其他致病成员会产生形态和抗原相关的、纤细的、聚集性的1型菌毛。单个基因fimA编码主要的菌毛单位。为了获得更高的特异性,我们选择了一系列位于fimA基因序列内部的引物,并开发了一种用于检测沙门氏菌菌株的PCR方法。从加拿大动物和人类中分离出的376株沙门氏菌菌株(包括80多个血清型)用于评估该PCR方法。同样的程序也对40株非沙门氏菌菌株进行了检测。通过将单个菌落的细菌直接接种到含有一对针对fimA基因的特异性引物的PCR混合物中来筛选培养物。特异性PCR产物是一个85 bp的片段,通过聚丙烯酰胺凝胶电泳和溴化乙锭染色进行可视化。所有沙门氏菌菌株通过PCR均给出阳性结果。被沙门氏菌菌株污染的饲料和牛奶样品也通过该程序进行了检测。对所有测试的沙门氏菌菌株的检测以及未能从非沙门氏菌菌株中扩增出片段,证实fimA基因包含沙门氏菌菌株特有的序列,并表明该基因是用于检测食品样品中沙门氏菌菌株的合适PCR靶标。