猪圆环病毒检测与分型寡核苷酸芯片的建立及应用  

Detection and genotyping of porcine circovirus in naturally infected pigs by oligo-microarray

作　　者：姜永厚[1] 商晗武[2] 陈伟杰 徐辉 丁先锋[1] 赵灵燕 方立[4] 黄怡君 

机构地区：[1]浙江理工大学生命科学学院,浙江杭州310018 [2]中国计量学院生命科学学院,浙江杭洲310018 [3]浙江省畜牧兽医局,浙江杭洲310020 [4]浙江大学,浙江杭洲310029

出　　处：《中国兽医学报》2008年第10期1174-1180,共7页Chinese Journal of Veterinary Science

基　　金：浙江省科技厅项目(2005C22032)

摘　　要：本研究应用寡聚核苷酸基因杂交技术建立了一种快速可靠的检测猪圆环病毒(PCV)基因型的方法。在PCV保守序列复制酶基因内设计了2对特异性引物,在此物之间设计了2种基因型特异的核苷酸探针(22～30mer)。通过PCR扩增Cy5标记的DNA片段与固定在玻片表面的探针杂交进行PCV基因分型。该技术结合了PCR方法的高度敏感性和DNA-DNA杂交技术的选择特异性。利用该方法对58例临床样品进行了检测鉴定。结果显示,该技术能准确鉴别PCV病毒基因型。且其灵敏度是凝胶电泳的5倍。试验结果表明寡核苷酸基因芯片技术可有效地应用于PCV临床诊断和分子流行病学调查。A rapid and reliable method for the identification of porcine circovirus（PCV）genotypes based of oligonucleotide microarray hybridization was developed. The genotype-specific oligonuleotides （22 - 30 ruer）immobilized on the surface of glass slides were selected to bind to the muhiple target sites within the replication gene that are conserved among individual PCV genotypes. Cy5 labeled DNA largets were amplified in a PCR with primers common to both genotypes. The identification of PCV genotype was based on hybridization with several individual genotypespecific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hy bridization. The utility and feasibility of oligonucleotide mieroarray hybridization was evaluated by testing standard and 58 clinical isolates. Analysis of the specimens showed that this microarray based method is capable of unambiguous identification of both genotypes and five-fold more sensitive than gel electrophoresis. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of PCV from clinical isolates and speci mens in a clinical laboratory.

关 键 词：猪圆环病毒 基因芯片 断奶仔猪多系统衰竭综合症 寡聚核苷酸探针 检测 

分 类 号：S852.65[农业科学—基础兽医学]