CSFV-PRRSV-JEV联合共检基因芯片的研制与初步应用

Preliminary application of simultaneous detection microarray for CSFV-PRRSV-JEV

对猪瘟病毒（CSFV）、猪繁殖与呼吸障碍综合征病毒（PRRSV）和猪流行性乙型脑炎病毒（JEV）联合共检基因芯片的初步应用进行了研究。选用夹缝针,BaiOR点样缓冲液,确定靶基因质量浓度为200mg/L,各样点中心间距300μm,样点直径100μm,在相对湿度为50%~60%、8~30℃条件下用晶芯？SmartArrayerTM48点样仪在氨基化基片上接触式点样,成功制备了CSFV-PRRSV-JEV检测基因芯片。将标记后的探针基因与PRRSV-CSFV-JEV基因芯片经预杂交、杂交、洗涤干燥后,用晶芯？LuxScanTM10K微阵列芯片扫描仪扫描观察结果。结果表明：制备的芯片特异性强,检测灵敏度可达0.295μg/L,4℃条件下可保存120d;用制备的芯片对临床67份疑似繁殖障碍性疾病的病料进行检测,与RT-PCR检测结果符合率均在94%以上,为猪繁殖障碍性疾病的检测提供了理想的检测方法。

A simultaneous detection microarray for classical swine {ever virus （CSFV）, porcine re- productive and respiratory disorders （PRRSV） and swine Japanese encephalitis virus （JEV） was developed in this paper. The crevice needle and BaiOR sample buffer were selected in this experi- ment. And the target gene concentration was determined 200 mg/L,each point center spacing was 300 μm, point diameter was 100μm. The target genes were spotted on aminated slides with Mi- croarray Printing System at a relative humidity of 50% to 60% and a temperature of 8-30℃. Then CSFV-PRRSV-JEV detection microarray was successfully prepared. Labeled gene probes were pre- hybridizated,hybridizated,washed and dried with CSFV-PRRSV-JEV mieroarray and observed at Microarray Scanner Scanning System. The results showed that the microarray specificity was strong,sensitivity up to 0. 295 μg/L and can be stored at 4℃ for 120 d. Sixty seven samples from suspected clinical reproductive disorders were detected with the prepared microarray and RT-PCR. And their results coincidence rate was all above 94%. This research provides an ideal means for the detection of porcine reproductive disorders.