应用聚合酶链反应快速检测沙门氏菌

利用聚合酶链反应（ＰｏｌｙｍｅｒａｓｅＣｈａｉｎＲｅａｃｔｉｃｎ，ＰＣＲ）技术检测沙门氏菌。根据沙门氏菌鞭毛素Ｉ相抗原基因，设计了一对各长２０个碱基的引物，扩增２６９ｂｐ的片段。并用８个标准标沙门氏菌株和３个阴性菌株检查引物特异性，结果完全相符。采用５０μＬ体积，ＮＴＰｓ２００μｍｏｌ，引物各１μｍｏｌ，ｍｇ＋＋３ｍｍｏｌ，Ｔａｑ酶２Ｕ。循环温度为９７℃７ｍｉｎ予变性后；９４℃１ｍｉｎ，６０℃１ｍｉｎ，３５个循环后再延伸７２℃７ｍｉｎ。经电泳分析，灵敏度可达每毫升检出１０４个细胞，经二次扩增后，灵敏度还可提高。实验共检查了３０个可疑样品，检出率明显高于生化检查和免疫学方法检查。

DETECTION OF SALMONELLA BY POLYMERASE CHAIN REACTION

One set of oligonucleotide primer was used in the polymerase chain reaction (PCR) to detect salmonella species. According to HI gene of salmonella, we synthesized a pair of primer. It targeted a 269 bp region of H 1 gene which is specific to salmonella species. It was tested with eight stamdard salmonella species and three negative strains for speaficity of primer: The standard PCR was typically done in a 50 μL volume. It contained 3 0 m mol·L -1 MgCl 2, lμ mol·L -1 of each primer, 200 μ mol L· -1 of each deoxynucleotide trisphate, 50 m mol·L -1 KC l and 2 units of Taq polymerase. The condition: predenature at 97℃ for 7 min, and then an additional 35 cycles with denature at 94℃ 1 min, annealing at 55℃ 40 sec, and extension 1 min, final extansion at 72℃ for 7 min. The sensibility of RCR amplification reached to detect out 10 4 cells per mL, 30 samples were analyzed in this experiment, RCR method was superior to serological and biochemical method.