猪瘟病毒基因芯片检测技术的建立与应用

Establishment and application of oligo-microarray to detect classical swine fever virus in naturally infected pigs

应用寡聚核苷酸基因杂交技术建立一种快速可靠的检测猪瘟病毒的方法。在CSFV病毒保守序列5′非编码区设计了两对特异性引物，在此引物之间设计了特异的核苷酸探针（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CSFV检测。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。并利用该方法对87例临床样品进行了检测鉴定，结果分析显示，可准确鉴别CSFV病毒，灵敏度与琼脂糖凝胶检测接近，可检测到的质粒最低浓度为0．4pg／μl。结果表明寡核苷酸基因芯片技术可有效地应用于CSFV临床诊断和分子流行病学调查。

A rapid and reliable method for the identification of classical swine fever virus （CSFV） based on ohgonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides （22 - 30 mer） immohilized on the surface of glass slides were selected to bind to the multiple target sites within the 5′ no-coding area of CSFV conserved sequences. DNA targets labeled by Cy5 were amplified in a PCR with primers specific to CSFV. The identification of CSFV was based on hybridization with several individual CSFV-specific oligonucleotides. This approach combines the high semitivity of PCR with the selectivity of DNA-DNA hybridization. The utility and feasibility of oligonucleotide microarray hybridization were evaluated by testing standard and 87 clinical isolates. Analysis of the specimens showed that this microarray-based method was capable of unambiguous identification of CSFV and as sensitive as gel eleetrophoresis. Our results indieate that the oligonucleotide array is useful in the identification and discrimination of CSFV from chnieal isolates and spee- imens and molecular epidemiological analysis.