多重寡核苷酸连接-聚合酶链式反应-通用基因芯片检测食源性致病菌方法的建立

发布时间：2018-01-13 15:14

本文关键词：多重寡核苷酸连接-聚合酶链式反应-通用基因芯片检测食源性致病菌方法的建立　出处：《卫生研究》2017年02期 　论文类型：期刊论文

【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。
[Abstract]:Objective to establish a general gene chip method for the detection of foodborne pathogenic bacteria based on multiplex oligonucleotide ligation-polymerase chain reaction (MOL-PCR). A pair of upstream and downstream detection probes were designed between their specific primers. Multiple PCR was used to enrich the target sequence and serve as template. The reaction was detected by multiple ligases and general asymmetric PCR markers were detected. A large number of fluorescent labeled single-stranded products containing complementary tag sequence Anti-tagged were obtained. The method can be used to detect single and multiple target bacteria infection by hybridization with tag sequence fixed on chip. The detection sensitivity of pure culture is 1.1% and 8.5%. 96 samples of food poisoning and clinical diarrhea were detected. The results of microarray were consistent with those of routine isolation, biochemical identification and fluorescence quantitative PCR. Conclusion this method provides a new detection platform for rapid, accurate, sensitive and high-throughput identification of pathogenic bacteria from foodborne diseases.
【作者单位】：西安市疾病预防控制中心;
【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)]
【分类号】：R155.3
【正文快照】： 食源性疾病已成为当前世界范围内一个主要公共卫生问题[1],在我国细菌性食物中毒最为普遍且影响最严重[2]。由于能够引起细菌性食物中毒的致病菌种类很多,并且不同致病菌常引发类似的临床症状,因此,建立一种快速、准确、高通量的食源性致病菌检测方法是有效控制和预防细菌性食

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