摘要
为建立同时能鉴别甲型H1N1和猪流感病毒常见亚型的新型基因芯片检测方法,根据GenBank中已发表的甲型流感病毒MP的基因序列和甲型H1N1(2009)和猪流感病毒H1N1、H3N2亚型的基因序列,设计、筛选并合成7对特异性引物和1对通用引物;根据扩增的靶序列,设计并合成14条特异性探针和3条质控探针,制备了甲型H1N1(2009)流感病毒和猪流感病毒H1N1、H3N2亚型基因芯片;并进行了特异性试验、敏感性试验和田间样品的检测。结果显示,该芯片检测方法与猪细小病毒(PPV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)等猪常见病毒无交叉反应;对猪H1N1、猪H3N2和甲型H1N1(2009)流感病毒而言,最低可检测到105、104和105稀释的病毒株。结果证实,该方法特异性强、敏感性高,是一种高通量的甲型H1N1和猪流感常见亚型筛查方法。
Seven pairs of primers specific for different subtypes and a pair of universal primers were carefully designed based on the genomic sequences of A/H1N1 and swine influenza virus retrieved from GenBank database.Several multiplex RT-PCR methods were then developed.Further 14 oligonucleotide probes specific for A/H1N1 and swine influenza virus were designed according to the published gene in target cDNA domains.Then a microarray for A/H1N1 and swine influenza virus was developed with its specificity and sensitivity validated by using swine influenza virus strains and samples from different areas.The results showed that all the subtypes of swine influenza virus and A/H1N1 virus could be identified simultaneously on this microarray with high sensitivity,which could reach to 105 dilute viruses.Furthermore,there was no cross reactions with PPV,CSFV and PRRSV.Therefore the microarray is a useful diagnostic method with high specificity and sensitivity,and could be used for A/H1N1 and swine influenza surveillance.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2011年第12期1234-1241,共8页
Chinese Veterinary Science
基金
质检公益性行业科研专项(200910132)
基本科研业务费专项资金资助项目(2009JK022)
国家重点基础研究发展计划项目(2011CB504704-X)
