摘要
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 develop a multiplex oligonucleotide ligation-polymerase chain reaction（ MOL-PCR） based universal microarray assay for multiplexed detection of foodborne pathogens. Methods Eight common foodborne pathogens causing bacterial food poisoning were selected as detection models. An upstream and downstream adjacent detection probes were designed within specific primer pair for each of eight pathogens.Target fragments of the eight pathogens were enriched by multiplex PCR and used as ligation templates. Abundant fluorescently labeled single-stranded amplicons containing anti-tag sequences were gained by multiplex ligase detection reaction and asymmetric PCR labeling with universal primers. The products could be detected by hybridization with corresponding tag sequences immobilized on DNA microarrays. Results The results indicated that the assay could specifically identify all eight pathogens in single and multiple infections. The detection limits were（ 1. 1- 8. 5） × 10~2 CFU /mL of pure bacterial cultures. The microarray results for 96 food poisoning and clinical diarrheal samples were consistent with that of traditional culture,biochemical identification and real-time PCR. Conclusion The assay provides a novel platform for rapid,accurate,sensitive and high-throughput detection of pathogenic bacteria of foodborne diseases.
出处
《卫生研究》
CAS
CSCD
北大核心
2017年第2期225-231,共7页
Journal of Hygiene Research
基金
陕西省自然科学基础研究计划项目(No.2015JM3112)
西安市社会发展引导计划-医学研究项目[No.SF1516(2)]
关键词
食源性致病菌
多重寡核苷酸连接-聚合酶链式反应
通用基因芯片
foodborne pathogens
multiplex oligonucleotide ligation-polymerase chain reaction（MOL-PCR）
universal DNA microarrays
