禽白血病病毒B、E和J亚群基因芯片检测方法的建立

Development of a Gene Chip Assay for the Detection of Avian Leucosis Virus Subgroups B,E and J

目的基于多重PCR技术,建立禽白血病病毒(ALV)的B、E、J亚群的基因芯片分型和检测方法。方法根据NCBI已收录的ALV三个亚群的参考毒株cDNA序列,在各亚群特异性基因突变区两端选取其保守区域,设计合成三个亚群的通用上游引物1条,以及B、E亚群的通用下游引物和J亚群下游引物各1条,将上述引物用Cy3标记,建立多重PCR体系;参考靶序列内部的三个亚群各自的保守区域,选择亚群之间基因突变位点多的区域,设计合成5条寡核苷酸探针,制作寡核苷酸探针基因检测芯片;以寄主细胞DF-1中提取传代ALV的cDNA,以及合成NCBI收录的各亚群参考毒株的cDNA序列作为检测模板;利用Cy3标记的PCR扩增产物,与基因芯片进行杂交反应,扫描结果。结果芯片准确检测并分型三个亚群的参考毒株,其检测灵敏度能够达到102个基因拷贝,且与禽类常见的四种病毒均无交叉反应。结论本研究结果证明,基因芯片技术是一种ALV的B、E和J亚群进行检测和分型的有效方法,且具有较高的特异性和灵敏度,为今后在临床应用中快速鉴别诊断ALV等免疫抑制病提供可行性。

Objective To develop a gene chip assay for the detection of avian leucosis virus subgroups B, E and J based on the specific gene fragment amplified by multiplex PCR. Methods By aligning multiple sequences of ALV subgroups B, E and J in GenBank, a conservative gene segment with multiple internal site variations was chosen as detection target. One forward primer for subgroups B, E and J, one reverse primer for subgroups B and E, and one reverse primer for subgroup J were designed to amplify the target gene in the three subgroups of ALV. The primers Cy3-1abeled were used to amplify the target gene fragments from the template DNA by multiplex PCR. Five oligonucleotides from the internal variations of the target gene were synthesized and printed on the amino-modified slides as the probes. The template cDNA was obtained by either synthesizing based on the representative sequences of subgroups B, E and J of ALV in GenBank or reversely transcripting the extracted cDNA from ALV-infected DF-1 cell. The amplified products were hybridized to the oligonucleotide probes immobilized on the glass slides. Results After hybridization, the gene chips were scanned, and the hybridization pattern agreed perfectly with the known location of each probe from subgroups B, E and J. The detection sensitivity could reach to 102 copies of gene. No cross-hybridization could be detected in the 4 other common avian viruses. Conclusion The results demonstrated that the gene chip assay was an effective way to distinguish ALV subgroups B, E and J simultaneously with good specificity and sensitivity. The method may provide potential for rapid surveillance and differential diagnosis of ALV or other immunosuppressive diseases in poultry.