4种腹泻病病原菌的可视化微孔板芯片检测方法  

The detection of four common diarrhea-causing bacteria by a visible microplate chip

作　　者：李永进[1] 顾大勇[2] 潘颖 

机构地区：[1]长江大学生命科学学院,湖北荆州434025 [2]深圳出入境检验检疫局 [3]珠海庄氏康大生物科技有限公司

出　　处：《现代预防医学》2013年第24期4545-4548,共4页Modern Preventive Medicine

摘　　要：目的构建一种针对4种常见腹泻病病原菌(霍乱弧菌、痢疾杆菌、出血性大肠杆菌O157、空肠弯曲菌)的可视化微孔板芯片检测方法。方法针对4种常见的腹泻病病原菌,设计特异性的PCR引物(5’端生物素标记)和检测探针。利用探针制备仪将探针按5×5点阵模式点置于N3微孔板芯片内,经紫外交联固定制成基因检测芯片。通过建立多重PCR扩增体系实现多重靶标的同时扩增,扩增形成的生物素化PCR产物与制备的微孔板芯片杂交,并进一步应用链霉亲和素-碱性磷酸酶和显色底物NBT/BCIP的显色反应来检测特异性的PCR杂交产物,检测结果阳性的样品同时以传统微生物分离鉴定方法鉴定。结果制备的基因检测芯片对标准菌株的测定展示了良好的特异性和稳定的实验结果。在对6份样本的测定中,检出痢疾杆菌感染1例,出血性大肠杆菌O157感染1例,空肠弯曲菌感染1例,检测结果与传统的微生物分离鉴定结果一致。结论本研究建立的可视化微孔板芯片检测技术可同时检测4种腹泻病病原菌,具有快速、准确及简便易行的特点,适用于临床的快速诊断及通关口岸的快速筛查。OBJECTIVE The objective of the study was to establish a method for the detection of four common pathogenic bacteria responsible for diarrhea （including Vibrio cholerae, Shigella dysenteriae, Enterohemorrhagic E. coli 0157, and Campylobacterjejuni） by using a visible microplate chip. METHODS Specific PCR primers （marked with biotin at the 5＂- end） and detection probes were designed to detect the four common diarrhea-causing bacteria. A probe preparation device was used to spot the probes onto a N3 microplate chip in a 5x5 lattice, followed by crosslinking immobilization by UV lights to create a gene detection chip. A multiplex PCR amplification system was established for simultaneous amplification of multi- ple targets; the amplified biotinylated PCR products were hybridized to the microplate chip, and chromogenic assays using streptavidin-alkaline phosphatase and chromogenic substrates NBT/BCIP were then adopted to detect specific hybrid PCR products. Positive specimens were reconfirmed by conventional bacterial isolation and identification methods. RESULTS The gene detection chip exhibited good specificity in the detection of standard bacterial strains and produced stable test results. A- mong the 6 tested specimens, 1 Shigella dysenteriae infection, 1 Enterohemorrhagic E. coli 0157 infection, and 1 Campy- lobacter jejuni infection were detected. The results were consistent with those obtained by adopting conventional bacterial iso- lation and identification methods. CONCLUSION The detection technique using a visible microplate chip established in this study is capable of simultaneously detecting the four diarrhea-causing bacteria. The method is fast, accurate, and easy to op- erate, and is suitable for fast diagnosis in clinical medicine and rapid screening in customs and ports of entry.

关 键 词：微孔板芯片 多重PCR 腹泻病 

分 类 号：R446.5[医药卫生—诊断学]