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目的建立一种基于多重寡核苷酸连接-聚合酶链式反应(MOL-PCR)的食源性致病菌通用基因芯片检测方法。方法针对细菌性食物中毒8种常见致病菌,在各自特异性引物之间设计一对首尾相接的上、下游检测探针。采用多重PCR富集靶序列并作为模板,通过多重连接酶检测反应及通用不对称PCR标记,获得大量含标签互补序列(Anti-tag)的荧光标记单链产物,可与芯片上固定的标签序列(Tag)进行杂交检测。结果该方法能特异性地鉴别单一和多重目标菌感染。纯培养物的检测灵敏度为(1.1~8.5)×10~2CFU/mL。对96份食物中毒和临床腹泻样本检测,芯片结果与常规分离与生化鉴定及荧光定量PCR结果一致。结论该方法为快速、准确、灵敏、高通量地鉴定食源性疾病的病原菌提供了一种新型检测平台。
Objective To establish a universal gene chip for food-borne pathogens based on multiple oligonucleotide-linked polymerase chain reaction (MOL-PCR). Methods Eight kinds of common pathogenic bacteria of bacterial food poisoning were designed, and a pair of upper and lower detection probes were designed between their specific primers. The multiplex PCR was used to enrich the target sequence and serve as a template for the detection of a large number of fluorescently labeled single-stranded products containing the tagged anti-tag by multiplex ligase detection reaction and general asymmetric PCR, which can be used in combination with on-chip fixed tag sequences (Tag) hybridization test. Results The method can specifically identify single and multiple target bacterial infections. The detection sensitivity of pure culture was (1.1 ~ 8.5) × 10 ~ 2CFU / mL. 96 samples of food poisoning and clinical diarrhea detection, chip results and conventional separation and biochemical identification and fluorescence quantitative PCR results. Conclusion This method provides a new platform for the rapid, accurate, sensitive and high-throughput identification of pathogenic bacteria in food-borne diseases.