H6N1亚型禽流感病毒GeXP检测方法的建立

Establishment of a GeXP assay for detection of the H6N1 subtype avian influenza virus

根据基因库中H6亚型禽流感病毒（AIV） HA基因、N1亚型AIV NA基因和所有亚型AIV M基因序列,分别设计筛选出3对特异性引物,优化反应条件,建立了H6N1亚型AIV GeXP检测方法。该法对H6亚型AIV的检测出现194 bp （H6）、164 bp （AIV通用检测） 2个目的峰,对N1亚型AIV的检测出现249 bp （N1）、164 bp 2个目的峰,对H6N1亚型AIV的检测出现194 bp、249 bp和164 bp 3个目的峰,对其他亚型AIV的检测只出现164 bp通用检测目的峰,对常见禽病病原体均未出现任何检测峰;该法对H6亚型和N1亚型AIV检测下限为102拷贝/μL。本研究建立的H6亚型和N1亚型AIV高通量GeXP检测方法特异性强、灵敏度高,可同时快速鉴别检测H6亚型、N1亚型和所有亚型AIV,为H6N1亚型AIV的检测提供了一种简便、快速和有效的方法。

According to the sequences of HA, NA and M genes of H6, N1 and all subtypes avian influenza （AIV） in GenBank, three pairs of specific primers were designed respectively, and the reaction conditions were optimized to develop a GeXP assay for detection H6N1. The results were that the H6 subtype AIV could generate two corresponding peaks at 194 bp （H6-AIV） and 164 bp （universal detection for AIV） by this GeXP assay. The samples containing the N1 subtype AIV could generate two corresponding peaks at 249 bp （N1-AIV） and 164 bp. The samples containing the H6N1 subtype AIV could generate three corresponding peaks at 194 bp, 249 bp and 164 bp. The samples containing the other subtype AIVs could only generate one corresponding peak at 164 bp. No peak was generated from the other avian patho- genic viruses. The limit of detection for the H6 and N1 subtype AIVs was 102copies/μL. In conclusion, this high-throughput GeXP assay was a specific, sensitive method for detection of the H6, N1 and all the subtype AIVs in the same tube. It could be applied to rapid differen- tial diagnosis of the H6 subtype, N1 subtype and all the subtype AIV, and also provide a rapid, simple and effective tool for determining the infection of the H6N1 AIV.