摘要
参考沙门氏菌侵袭性基因invA的序列,设计合成一对引物扩增其中一段序列,建立了特异性检测沙门氏菌的PCR方法。引物两端分别加了Bam HI和EcoR I切点,扩增片段大小为300bp。对收集的50个血清型123株沙门氏菌及7种23株非沙门氏菌进行PCR检测,结果仅沙门氏菌有300bp的扩增产物,显示了很强的特异性,为下一步克隆而设计的两个酶切位点对引物的特异性没有影响。经琼脂糖凝胶电泳,PCR的检出极限是10pg染色体DNA和10~2 cfu的细菌。将扩增产物经slot—blot与辣根过氧化物酶(HRP)直接标记的invA基因探针杂交,经增强型化学发光反应(ECL)及化学发光自显影(CPD)检测,可提高检测的敏感性一个数量级,同时增加了特异性。为推广应用,本文试验了8种模拟食品样品对PCR反应的影响,结果除奶酪外,其余都得到较好扩增。对120份污水及食品样品进行检测,该检测系统检出70份阳性结果,检出率高于常规分离。初步应用显示,PCR检测方法敏感、特异、简便、快速,适合于食品卫生检验和临床标本检验的现场应用。
Amplification of nucleotide sequence with the inv A gene of Salmonella typlinuriion was evaluated as a detection method of Salmonella . A collection of 123 strains of salmonella comprising 50 serovars, including the 10 most prevalent serovars in food samples was examined. Controls consisted of 27 non - Salmonella strains comprising 7 genera of bacteria. The specific PCR product was a 300 bp DNA fragment which was visualzed in 2% agarose gels. All Sabmonella strains in the study were detected, in contrast, none of the non -Salmonella strains yielded the specific amplification product. Specificity of amplification was further confirmed by slot-blot hybridization with a HRP-conjugated probe. PCR was able to detct 10 pg of chromosomal DNA of S. new- port strain 50029 and as little as102 cfu of S. newport strain 50029. The specifity of the primers was not interfered by two restrict enzyme sites (Bam HI, Eco RI) , which were designed of cloning. 120 food and polluted water samples were detected by the PCR method for the presence of Salmonella, 70 got positive results. PCR showed higher sensitivity than conventional method. The results suggest that detection of Salmonella spp by PCR is sensitive, specific, convenient and rapid, most suitable for Salmonella identification in clinical application and food hygiene inspection.
