【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。更多还原

【Abstract】 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 mer)immobilized on the surface of glass slides were selected to bind to the multiple target sites within the replication gene that are conserved among individual PCV genotypes.Cy5 labeled DNA targets were amplified in a PCR with primers common to both genotypes.The identification of PCV genotype was based on hybridization with several individual genotype-specific oligonucleotides.This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization.The utility and feasibility of oligonucleotide microarray 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 specimens in a clinical laboratory.更多还原