增菌PCR法检测小鼠沙门氏菌

Enriched PCR for Detection of Salmonella spp. in Mice

根据沙门氏菌 inv A基因序列合成一对寡核苷酸引物 ,建立了增菌 PCR法检测实验动物粪便沙门氏菌的方法。首先以 7种常见的沙门氏菌及 3种非沙门氏菌标准菌株检验了 PCR法的特异性和灵敏度。 7种沙门氏菌扩增出 2 84bp的特异区带 ,非沙门氏菌皆无特异带扩增 ,检测灵敏度为 5 0个 CFU。比较了不同方法即差速离心集菌、玻璃粉吸附和选择性增菌处理粪便样品制备模板 DNA,相应于 3种方法的 PCR检测灵敏度分别为 :2× 1 0 4 ,5× 1 0 3,1 0个 CFU。然后 ,用增菌 PCR法与分离培养法对比检测了 1 0 5只实验动物。在 80只 KM小鼠中 ,分离培养法检出 1 0只阳性 ,PCR法检出 1 2只阳性 ,高于分离培养法。结果提示 :增菌

A pair of oligonucleotide primer was synthesized according to the invA gene nucleotide sequence within Salmonella specices. A new method of PCR was successfully developed by enrichment broth cultivation for the detection of Salmonella in laboratory animals.The DNA was extracted from 7 strains of Salmonella and 3 strains of non salmonella bacteria(by a rapid boiled lysate technique). All Salmonella yielded 284 bp specific band, while no specific product yielded in non salmonella bacteria. As few as 50 CFU of salmonella in pure culture could be detected by PCR. Three sample processing methods for mice feces, including low and high speed centrifugation, binding to glass powder, and selective enrichment cultivation, were used for the extraction of DNA template, and the corresponding sensitivities of the methods were 2×10 4,5×10 3,10 CFU, respectively. One hundred and five laboratory animals were detected for the salmonella by enriched PCR procedure and conventional culture technique. The results suggested that the enriched cultivation PCR have advangtages in rapid detection of Salmonella in a large number of laboratory animals