禽流感病毒核酸检测用定性标准样品的研制

Preparation of qualitative reference material for detection of nucleic acid of avian influenza A virus

本研究以禽流感病毒经典毒株A/turkey/Wisconsin/1/1966(H9N2)为原料,通过SPF鸡胚繁殖、外源病毒检测、灭活、冻干和分装等步骤,最终制备成功禽流感病毒核酸检测用定性标准样品。采用荧光定量RT-PCR方法对标准样品进行均匀性评估、稳定性监测和合作定值,并进行了初步临床试用。结果显示,所制备的标准样品纯净、均匀,无外源病毒污染;标准样品在-20℃、4℃、25℃、37℃环境下可分别稳定保存12个月以上、60 d、7 d和3 d;9家单位合作定值结果一致,均为禽流感病毒阳性;临床试用结果良好。上述结果表明,所制备的标准样品可用于禽流感病毒核酸检测用对照品、质量控制以及试剂盒检测方法评价等工作。

In this study,the classical H9 N2 subtype avian influenza A virus(AIV) strain A/turkey/Wisconsin/1/1966(H9 N2) was used as raw material for preparation of reference material(RM) for detection of nucleic acid of AIV.Toward this end,the H9 N2 virus was inoculated into SPF embryonated chicken eggs and the allantoic fluid was harvested at appropriate time.Subsequently,the amplified virus was examined for exogenous viruses,and then inactivated,lyophilized and aliquoted,resulting in RM.The RM was detected and evaluated for homogeneity and stability.In addition,the RM was collaboratively valued by9 qualified laboratories and used for clinical trial.Our results indicated that the RM was stable for more than 12 months,60 days,7 days and 3 days after it was stored at-20 ℃,4 ℃,25 ℃ and 37 ℃,respectively.AIV-positive results were consistently obtained by the 9 cooperating laboratories,and the clinical application of the RM demonstrated its good quality.The RM we developed could be used as positive control for detection of nucleic acid of AIV,quality control as well as evaluation of diagnostic kits/approaches.