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

【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.