摘要： 基于多重PCR技术,建立鸡传染性贫血病病毒(CAV)、禽网状内皮增生症病毒(REV)、禽白血病病毒禽(ALV)A、C、D亚群的基因芯检测方法.根据NCBI收录的鸡传染性贫血病毒(CIAV)、禽网状内皮增生症病毒(REV)与禽白血病病毒(ALV)A、C、D亚群参考毒株的序列,设计合成特异性扩增引物及探针,将下游引物进行Cy3荧光标记,制备基因芯片;分别提取几种病毒的基因组DNA,进行PCR扩增后与探针进行杂交,荧光检测仪扫描并分析结果.结果表明:制备的芯片能够同时检测CIAV、REV与ALV A、C、D亚群,该方法对病毒的最低检测限为10⁶ copies/mL,特异性试验结果表明,该方法与NDV、MDV和IBDV均无交叉反应,可快速鉴别ALV、REV、CIAV.为今后在临床应用中快速鉴别诊断CIAV、REV与ALV A、C、D亚群提供了可行性.

关键词: 鸡传染性贫血病毒, 禽网状内皮增生症病毒, 禽白血病病毒, 基因芯片

Abstract: To develop a gene chip assay for the detection of Chicken infectious anemia virus,Reticuloeotheliosis virus,Avian leucosis virus subgroups A,C and D based on the specific gene fragment amplified by multiplex PCR.The references sequence of Chicken infectious anemia virus,Reticuloeotheliosis virus and Avian leucosis virus subgroups A,C and D were obtained from GenBank;which were used to design the specific primers and probes.The downstream primers were labeled with Cy3,and gene chip was subsequently prepared.The total DNA was extracted from the three viruses.The product amplified by using PCR with the labeled primers was hybridized with the gene chip.The hybridization signal was detected and analyzed by a fluorescence scanner.The results showed that:the chip was able to simultaneously detect Chicken infectious anemia virus,Reticuloeotheliosis virus and Avian leucosis virus subgroups A,C and D.This method with high sensitivity,which could reach to 10⁶ copies/mL.Furthermore,there was no cross reactions with NDV,MDV and IBDV.This study might provide potential method for rapid surveillance and differential diagnosis of Chicken infectious anemia virus,Reticuloeotheliosis virus and Avian leucosis virus subgroups A,C and D.

Key words: CIAV, REV, ALV, Gene chip

中图分类号: 

• S432.4