港口航道致病性细菌检测微阵列基因芯片的研制

摘要

[目的]制备出一套针对港口航道致病性细菌检测的基因芯片,为港口航道基因芯片检测技术的研究及应用打下基础.[方法]采用常规方法提取目标菌株基因组DNA,以16S rDNA通用引物和gyrB基因保守区引物进行PCR扩增,使用AlleleID 6.0和Array Designer 4.25对扩增获得的目的片段进行寡核苷酸探针设计,经PCR筛选验证,目标探针以氨基化修饰后通过芯片点样仪点制在醛基玻片上;优化芯片杂交固定条件,并用于港口航道的水样检测,以验证微阵列基因芯片的检测效果.[结果]优化后的芯片杂交固定条件为探针点样浓度10μmol/L、紫外交联时间2.0 h、杂交温度65℃,有效提高了基因芯片检测的灵敏度,与传统检测方法相比可实现快速、高通量、准确的目标.研制的微阵列基因芯片可特异性检测出港口航道中含有的霍乱弧菌(Vibrio cholerae)、阴沟肠杆菌(Emterobacter cloacae)、溶藻弧菌(V.alginolytivus)、哈氏弧菌(V.harveyi)、副溶血弧菌(V.parahemolyticus)、嗜水气单胞菌(Aeromonas hydrophila)和创伤弧菌(V.vulnificus)等7株致病性细菌,且均未出现非特异性杂交.[结论]针对港口航道致病性细菌建立的微阵列基因芯片检测技术具有特异性强、灵敏度高、快速便捷的特点,可用于港口航道及周边地区的海洋环境监测和海产品质量安全检测.%[Objective]Gene chip to detect pathogenic bacteria in port and waterway was developed in order to lay a foundation for gene chip detection technology targeting port and waterway. [Method]The genomic DNA of the target strains were extracted by conventional methods. 16S rDNA universal primer and conserved region primers of gyrB gene were used for PCR amplification. Oligonucleotide probe designed on target fragments obtained by amplification was carried out using AlleleID 6.0 and Array Designer 4.25. Through PCR screening and validation, amino-modified target probes were spotted on aldehyde slide via microarrayer. Chip hybridization conditions were optimized, and was applied to detection of water and sediment samples from port and waterway. In this way, detection effects of microarray gene chip were validated.[Result]The optimized chip hybridization conditions were: probe concentration 10 μmol/L, ultraviolet crosslinking time 2.0 h and hybridization temperature 65 ℃. It increased sensitivity of gene chip detection, and was of high speed, high throughput and accuracy compared with traditional detection method. The microarray gene chip could detect seven pathogenic strains from port and waterway: Vibrio cholerae, Enterobacter cloacae, V. alginolyticus, V. harveyi, V. parahemolyticus, Aeromonas hydrophila and V. vulnificus. No specific hybridization was detected. [Conclusion]Micro-array gene chip detection technology targeting pathogenic bacteria in port and waterway is highly specific, sensitive, rapid and convenient. It can be used for marine environmental monitoring and marine products quality and safety detection in port and their waterway and surrounding areas.