联合共检四种猪腹泻病毒的寡核苷酸芯片的构建及应用  

Construction and Application of Oligonucleotide Microarray for the Simultaneous Detection of Four Porcine(Sus scrofa) Diarrhea Viruses

作　　者：胡靖飞 滑翔 黄小波 赵玉佳[1] 曹三杰 文心田 文翼平[1,2,3] 伍锐 赵勤[1,2] HU Jing-Fei;HUA Xiang;HUANG Xiao-Bo;ZHAO Yu-Jia;CAO San-Jie;WEN Xin-Tian;WEN Yi-Ping;WU Rui;ZHAO Qin(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：《农业生物技术学报》2020年第2期349-359,共11页Journal of Agricultural Biotechnology

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

摘　　要：生产中常见仔猪(Sus scrofa)腹泻,造成严重经济损失。本研究建立了联合检测猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus, TGEV)、猪轮状病毒A群(Porcine rotavirus A group, PRoV A)与猪δ冠状病毒(Porcine deltacoronavirus, PDCoV) 4种猪肠道病毒的寡核苷酸芯片并进行了临床应用。根据PEDV的纤突蛋白(spike, S)、囊膜蛋白(membrane,M)基因、TGEV的S、核衣壳蛋白(nucleocapsid, N)基因、PRoV A的外衣壳蛋白(outer capsid protein, VP7)、非结构蛋白(non-structural protein, NSP4)基因与PDCoV的囊膜蛋白M、N基因设计8对特异性引物,再根据每对引物扩增基因的保守区域设计寡核苷酸探针,并用Cy-3荧光直接标记下游引物5’端进行多重PCR扩增标记样品。研究确定最佳制备和反应参数:100μmol/L探针原液与点样缓冲液比例为1∶2,水合处理10 h,杂交温度为42℃,杂交时间为120 min,以信噪比值(Signal-to-noise ratio, SNR)≥2或信号值>1 300判为阳性。该芯片灵敏性试验表明最低检测量为106copies/μL;特异性实验表明对非目标病原无交叉反应,保存期试验表明制备的芯片置于4℃下可保存至少60 d。用该芯片检测209份临床腹泻样品,结果显示PEDV、TGEV、PoRVA、PDCoV的阳性率分别为68.42%、4.30%、0.95%和2.87%,检测结果与普通逆转录PCR (reverse transcription PCR)一致。本研究构建的寡核苷酸基因芯片为4种仔猪腹泻病原的高通量筛查提供了新技术。Diarrhea is common in piglets and causes serious economic losses. In this study, an oligonucleotide microarray was developed for simultaneous the detection of Porcine epidemic diarrhea virus(PEDV), Transmissible gastroenteritis virus(TGEV), Porcine rotavirus group A(PRoV A) and Porcine deltacoronavirus(PDCoV). Specific primers were designed according to the genes of PEDV spike gene(S),membrane gene(M), TGEV spike gene(S) nucleocapsid gene(N), PoRV A outer capsid protein 7 gene(VP7),non-structural protein 4 gene(NSP4) and PDCoV membrane gene(M), nucleocapsid gene(N), and the corresponding oligonucleotide probes were designed according to the conservative region of each target gene fragment. The 5’ end of downstream primers was labelled Cy3 fluorophores directly for multiple PCR. The microarray reaction parameters of each reaction step were optimized, which were as follows: The dilution ratio of 100 μmol/L original probe and sample buffer was 1∶2, the optimal hybridization condition was 46 ℃ for120 min. Signal-to-noise ratio(SNR)≥2 or signal value >1 300 was defined as positive. The sensitivity of chip was 105 copies/μL. No cross reaction was detected with other non-target pathogens. The shelf life test showed that the microarray can be stored at 4 ℃ for at least 120 d. The detection of the 209 clinical samples by oligonucleotide microarray showed the positive rates of PEDV, TGEV, PDCoV and PoRV A were 68.42%,4.30%, 0.95% and 2.87%, respectively, which was consistent with the results by Reverse transcription PCR. In conclusion, the constructed oligonucleotide microarray provides a new alternative method for high-through screening of the 4 porcine enteric diarrheic viruses.

关 键 词：猪流行性腹泻病毒(PEDV) 猪传染性胃肠炎病毒(TGEV) 猪轮状病毒(PoRV) 猪δ冠状病毒(PDCoV) 寡核苷酸芯片 

分 类 号：S855.2[农业科学—临床兽医学]