应用不对称PCR提高PRRSV-CSFV-JEV共检芯片的杂交效率  

Enhancing the hybridization efficiency of the PRRSV-CSFV-JEV microarray by asymmetric PCR

作　　者：张仙[1] 邓静 常晓霞[1] 杨国淋 马锐[1] 滑翔 赵玉佳[1] 欧阳达[1] 文心田[1,2] 黄小波[1,2] 曹三杰[1,2] 文翼平[1,2] 伍锐[1,2] 

机构地区：[1]四川农业大学动物医学院,动物传染病与基因芯片实验室,雅安625014 [2]四川农业大学人兽共患病研究室与猪病防治研究中心,成都611130 [3]四川华神兽用生物制品有限公司,四川畜科生物制品有限公司,成都610299

出　　处：《中国人兽共患病学报》2016年第1期51-55,64,共6页Chinese Journal of Zoonoses

基　　金：国家公益性行业(农业)科研专项(No.201203056)~~

摘　　要：目的比较采用普通RT-PCR和不对称PCR分别进行样品的直接荧光标记,应用于cDNA芯片杂交,分析其对芯片杂交效率的影响。方法以本实验室建立保存的重组质粒和病毒为模板,进行不对称RT-PCR引物浓度优化,应用普通RT-PCR与不对称RT-PCR进行直接荧光标记,将标记的产物与制备的cDNA芯片杂交,在相同条件下完成杂交后芯片的洗涤与扫描,记录扫描图片与数据。结果与普通RT-PCR标记方法相比,应用不对称RT-PCR技术标记单链靶基因,能特异有效提高芯片的杂交效率。结论应用不对称RT-PCR技术对样品进行荧光标记后,与cDNA芯片杂交能提高芯片的杂交效率,有利于cDNA芯片的检测应用。Microarray is wildly used in every field of biology as a tool of molecular biology research, with advantages of accuracy, fast and high-throughput. The hybridization efficiency is the critical factor of the microarray technology. We studied the hybridization efficiency of two fluorescence labeling methods by microarray hybridization. On the basis of our laboratory research, we selected T/PRRS-PSg, T/Yll, T/CSF1, T/PSS, T/PRM, and T/JEV as template, to optimize the concentration of asymmetric RT-PCR primer. The templates were come from recombinant plasmids which were established and saved in our laboratory. The templates would be amplified by traditional PCR and the amplification would be the probes of the cDNA microarrays. The samples used to be labeled by fluorescence, we amplified and labeled the samples from clinic by means of asymmetric PCR and traditional PCR labeling technology, they hybridized with the cDNA mieroarrays we prepared respectively, fol lowed by washing, scanning and analysis. Results indicated that compared to the traditional RT-PCR labeling method, the asymmetric RT-PCR labeling method showed superior results with enhance hybridization efficiency. The hybridization efficiency could be enhanced by asymmetric PCR labeling method, which improving the detection applicability of microarray technology.

关 键 词：不对称PCR CDNA芯片 标记 

分 类 号：Q789[生物学—分子生物学]