不同方法检测食源性致病菌的对比研究  

Comparative study of different methods for detecting foodborne pathogens

作　　者：罗宇鹏[1] 

机构地区：[1]成都市第五人民医院检验科,成都温江区611130

出　　处：《国际检验医学杂志》2015年第7期918-919,922,共3页International Journal of Laboratory Medicine

摘　　要：目的探讨基因芯片技术和多重PCR技术用于检测和筛查食源性致病菌的可行性。方法通过设计靶细胞引物序列,采用生物素标记反向引物5′端,氨基基团标记寡核苷酸探针5′端。将探针在固相载体上点样,制备基因芯片,PCR产物与芯片点制探针区域进行杂交,并对PCR杂交反应的体系进行优化。结果基因芯片技术可以同时检测志贺氏菌、沙门氏菌、肺炎克雷伯菌、布鲁氏菌、奇异变形菌、金黄色葡萄球菌和空肠弯曲菌等多种病原菌,操作简便,特异性强。细菌纯培养物灵敏度为5.0×102 CFU/mL,DNA检测灵敏度为0.1pg,检测分离菌株符合率为100%。利用引物建立和优化了PCR检测体系,分别确定了Mg2+浓度和退火温度Tm值为1.5mmol/L和56℃,检测灵敏度达到10pg,此灵敏度下可以扩增出全部特异性引物条带。结论通过基因芯片技术和多重PCR技术可以有效检测食源性致病菌,为高通量筛查检测病原菌提供了新思路,值得在食品安全领域推广应用。Objective To investigate the feasibility of the gene chip technique and multiplex PCR technique for detecting and screening foodborne pathogens.Methods The primer sequences were designed to target cells,the biotin was adopted to label the reverse primer 5′end and the amino group was adopted to label oligonucleotide probe 5′end.The probe spotted on a solid support for preparing microarray,PCR product was hybridized with microarray probe region,PCR and hybridization reaction system was optimized.Results The microarray technique could simultaneously detect multiple pathogens of Shigella,Salmonella,Klebsiella pneumoniae,Brucella,Proteus mirabilis,Staphylococcus aureus,Campylobacter jejuni,etc.,which was easy to operate and had strong specificity.The sensitivity of bacterial pure cultures was 5.0×10^2 CFU/mL,the sensitivity of DNA detection was 0.1pg,the coincidence rate for detecting isolated bacteria was 100 %.The PCR detection system was established and optimized by using primers,the concentration of Mg^2＋and the annealing temperature Tm value of 1.5mmol/L and 56 ℃ were determined,the detection sensitivity reached to 10 pg,all the specific primers amplified bands could be amplified under this sensitivity.Conclusion The gene chip technique and multiplex PCR technique can effectively detect foodborne pathogens,which provide a new idea for detecting pathogens with the high-throughput screening and are worth popularization and application in the field of food safety.

关 键 词：食品安全 食源性致病菌 多重PCR 基因芯片 

分 类 号：R446.5[医药卫生—诊断学]