鳗弧菌膜芯片检测条件的优化

Optimization of the detection protocol for membrane chip of Vibrio anguillarum

中文关键词: 基因芯片  尼龙膜  鳗弧菌  毒力基因

英文关键词: Gene chip  Nylon membrane  Vibrio anguillarum  Virulence gene

中文摘要:

      采用带正电的尼龙膜作为片基，利用手动芯片点样仪将鳗弧菌6个毒力基因的寡核苷酸探针点样于膜基底上，然后进行紫外交联，制备膜芯片。制备好的膜芯片与6个毒力基因的带有地高辛标记的PCR产物进行杂交，通过观察杂交信号来判断是否含有该基因。通过对膜芯片点样浓度、杂交时间及洗膜步骤进行优化，确定所设计膜芯片最适杂交时间为30 min、点样探针最适浓度50 μmol/L、洗膜过程只需要3步，时间缩短了45 min。

英文摘要:

      Gene chip was prepared with the oligonucleotide probes of 6 virulence genes of Vibrio anguillarum dot-applied on a piece of positively charged nylon membrane using the microcaster manual arrayer, followed by cross-linkage under the ultraviolet light. The detection of V. anguillarum virulence genes was performed by hybridizing with the DIG-labeled PCR products on the membrane chip. Here we report the optimization of the concentration of the probe, the time course of hybridization, and the process of washing steps. After optimization, the hybridization time was reduced to 30min, the concentration of the probe as 50μmol/L, and the process of washing was shortened to 3 steps of only 45 min.