共检猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪轮状病毒的cDNA芯片的技术优化  

Optimization of the cDNA Microarray for Simultaneous Detecting PEDV-TGEV-PoRVA

作　　者：胡靖飞 尹人杰 黄小波 刘志鹏[1] 赵玉佳 曹三杰 文心田 文翼平[1] 赵勤 伍锐 HU Jingfei;YIN Renjie;HUANG Xiaobo;LIU Zhipeng;ZHAO Yujia;CAO Sanjie;WEN Xintian;WEN Yiping;ZHAO Qin;WU Rui(Research Center of Swine Disease,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Science-observation station Veterinary Drugs and Veterinary Diagnostic Technology,Ministry of Agriculture,Chengdu 611130,China;National Animal Experiment Teaching Demonstration Center,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院猪病研究中心,成都611130 [2]农业部兽用药物与兽医诊断技术四川科学观测实验站,成都611130 [3]四川农业大学国家级动物类实验教学示范中心,成都611130

出　　处：《四川农业大学学报》2019年第4期542-549,578,共9页Journal of Sichuan Agricultural University

基　　金：国家重点研发计划(2016YFD0500700);动物疫病共检测服务平台建设项目(D171100002117002)

摘　　要：【目的】对前期构建的猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和A型猪轮状病毒(PoRVA)共检cDNA芯片进行技术创新和关键参数优化。【方法】根据靶基因PEDV(S和M)、TGEV(N和S)以及PoRVA的(VP7和NSP4)分别设计6对特异性引物,扩增探针制备芯片阵列。重点改进了样品标记技术,引物直接标记与不对称PCR扩增结合,确定了不对称PCR扩增体系;并对点样缓冲液、水合时间、探针浓度、杂交时间和温度、清洗和干燥方式等条件进行优化。【结果】当博奥和百傲两种缓冲液比例为1∶1,水合时间为4 s,探针浓度为600 ng/μL,不对称PCR上下游引物比为1∶40,在48℃杂交3 h,用0.2%SDS清洗,1 000 r/min离心,该cDNA芯片效果最佳。【结论】本研究确定了PEDV-TGEV-GAR共检cDNA芯片的关键技术参数,为开展基因芯片的标准化制备和应用奠定了基础。【Objective】This study was aimed to optimize the technical innovations and key parameters based on the previous constructed cDNA microarrays simultaneously detecting the porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV) and porcine rotavirus type A(PoRVA).【Method】Specific primers were designed based on the sequence of S and M genes of PEDV,N and S genes of TGEV,and VP7 and NSP4 genes of PoRVA. The probes were amplified from recombinant plasmid. The sample labelling was specially optimized. Primer labelled directly combined with asymmetric PCR amplification. The key parameters of microarrays preparation such as the print buffer,hydration time,probes concentration,time and temperature of hybridization,methods of washing and drying were optimized.【Result】The results demonstrated that the ratio of two sample liquids of Capital Bio and BaiO Company was 1∶1. The hydration time was 4 s. The concentration of probes is 600 ng/μL.The asymmetric PCR with 1∶40 proportion could amplified the target genes. The conditions of hybridization was 48 ℃ for 3 h. At last,centrifuging for 1 000 r/min immediately after washing with 0.2% SDS.【Conclusion】In this research,determination of the key parameters of microarray preparation and reaction laid the foundation for standardized preparation and application of the microarrays.

关 键 词：猪腹泻病毒 CDNA芯片 直接标记法 不对称PCR 优化 

分 类 号：Q939.94[生物学—微生物学]