Analysis of virulence factors in Salmonella species

目前，已有超过2000种沙门氏菌的血清型被记录下来，细菌分离物来自不同的实验室，那里有针对不同血清型沙门氏菌的抗血清。然而，分离细菌的鉴定是非常费力的，甚至在已经建立良好的实验室中也需要训练有素的人员。在发展中国家，很难有一套完整的生化和血清学材料来鉴定沙门氏菌。为了解决这一问题，我们构建了采用聚合酶链反应（PCR）方法检测沙门氏菌毒力的快速诊断系统。从临床标本和食品样品中分离出58个血清型的567株沙门氏菌用于PCR系统。利用stn101和stn111肠毒素基因引物扩增样品中的互补靶DNA。用sin106和sin112引物进行肠侵基因PCR时，除从肠胃炎患者中分离的1株Rissen血清型沙门氏菌外，其余菌株的PCR产物均显示出预期的378bp的DNA片段。这些特异的肠毒素和肠侵入基因引物对包括肠道细菌在内的30种细菌的224株均无阳性反应。利用合成的寡核苷酸探针对PCR产物进行southern blot杂交试验，发现stn和invA操纵子对应的位置明显呈阳性反应，表明与stn和sin引物混合反应得到的PCR产物中存在特异性的小片段DNA。制备了兔抗沙门氏菌肠毒素血清，并建立了免疫印迹检测系统。少

At present, over 2,000 serovars of Salmonella species have been recorded with bacterial isolates from various laboratories where the antisera against different serovars of Salmonella are available. The identification of bacterial isolates, however, has been so laborious and well-trained personnel are needed even in the already well-established laboratories. In developing countries, it is hard to have a complete set of biochemical and serological materials for the identification of Salmonella. To solve this problem, we constructed the rapid diagnosis system for the detection of virulent Salmonella species by using the polymerase Chain Reaction (PCR) method.Five hundred sixty seven strains of Salmonella with 58 serovars isolated form clinical specimens and food samples were dedicated to the PCR system. As the stn101 and stn111 primers for enterotoxin gene were used to amplify the complementary target DNA in the samples. It was found that all strains have shown the 260 bp specific DNA fra … More gment in the PCR products as demonstrated by 0.0% agarose gel-electrophoresisWhen PCR were performed by using sin106 and sin112 primers for enteroinvasive gene, the expected 378bp DNA fragment were also found in PCR products of all these Salmonella isolates except for one strain of Salmonella serovar Rissen isolated from the patient with gastroenteritis. These specific primers for enterotoxin and enteroinvasive genes did not show any positive result with the other 224 strains of 30 different bacterial species including enteric bacteria. Confirmation of the PCR products by southern blot hybridization test using synthetic oligonucleotide probes which positions corresponded to stn and invA operons have clearly revealed positive reaction and thus, indicated the specific small DNA fragments in the PCR products obtained from the reaction mixture with stn and sin primers.We also produced anti rabbit-serum against Salmonella enterotoxin and constructed the western blotting system for the detection of it. Less