港口航道致病性细菌检测微阵列基因芯片的研制
发布时间:2018-01-26 05:55
本文关键词: 致病性细菌 港口航道 基因芯片 检测 出处:《南方农业学报》2017年07期 论文类型:期刊论文
【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 r DNA通用引物和gyr B基因保守区引物进行PCR扩增,使用Allele ID 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。
[Abstract]:[objective] to prepare a set of microarray for detection of pathogenicity bacteria in port and waterway. This study laid a foundation for the research and application of gene chip detection in port and waterway. [methods] Genomic DNA of the target strain was extracted by routine method. 16s r DNA universal primers and gyr B gene conserved region primers were used for PCR amplification. Allele ID 6.0 and Array Designer 4.25 were used to design the oligonucleotide probe of the amplified target fragment, which was screened by PCR. The target probe was modified with amino and then was prepared on the aldehyde glass by chip spotting instrument. Optimization of the chip hybridization fixed conditions, and used for port waterway water sample detection. [results] the optimized hybridization conditions were as follows: probe sample concentration 10 渭 mol / L, UV crosslinking time 2.0 h, hybridization temperature 65 鈩,
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