摘要
根据GenBank上蓝舌病病毒(BTV)、O型口蹄疫病毒(FMDV)、山羊痘病毒(GPV)、绵羊痘病毒(SPPV)和牛病毒性腹泻病毒(BVDV)等5种病毒的特异性保守序列,分别设计多重荧光标记引物和相应寡核苷酸探针。使用芯片点样液稀释各探针至终浓度30μmol/L,点样制备11×11阵列芯片。核酸杂交后,建立并优化基因芯片检测方法。结果显示,使用470mL/L甲酰胺杂交液,42℃摇转杂交4h为最佳基因芯片杂交条件。建立的基因芯片检测方法与伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、禽流感病毒(AIV)和新城疫病毒(NDV)等无交叉反应,检测敏感性可达20拷贝病毒核酸。制备的基因芯片稳定,保存6个月可用。对151份临床样品进行基因芯片和商业化PCR试剂盒平行检测,两者的符合率为100%。
Using the specific conservative sequences of BTV, FMDV/O,GPV,SPPV and BVDV which were searched in GenBank, we designed multiple PCR primers and corresponding specific oligo probes respectively. The probes were diluted to 30 μmol/L with dilution buffer and spotted on substrate-coated chip to 11 X 11 array. Followed by hybridization, the gene chip method was developed and optimized. The results showed that the effiident hybridization conditions were rotated 2 hours in 470 mL/L formamide at 42℃. The gene chip method had no cross-reactivity with PRV, PRRS, AIV and NDV. The gene chip stored for six months had a stable result and the sensitivity was up to 20 copies. The gene chip method and commer- cial PCR kit were used to detect the 151 clinical samples parallelly, and the coincidence was 100%.
出处
《动物医学进展》
CSCD
北大核心
2013年第4期27-32,共6页
Progress In Veterinary Medicine
基金
中国检科院基本科研业务费专项资金资助项目(2009JK010)
关键词
皮毛
动物病毒
基因芯片
检测
animal fur
animal viruses
gene chip
detection