摘要目的:改良甲型H1N1和季节性H3N2流感病毒鸡胚分离培养方法，并对其分离效果进行评估。方法:随机挑取甲型H1N1(H1N1pdm09)和季节性H3N2(H3N2snl)流感病毒核酸阳性标本各80份，分别接种10日龄(传统法)和14日龄(改良法)鸡胚羊膜腔及尿囊腔使病毒适应鸡胚(E1～E2)，两种方法均接种10日龄尿囊腔扩增病毒(E2～E3)，比较两种方法最终病毒分离阳性率；用荧光定量PCR法检测羊膜腔和尿囊腔培养物中病毒核酸，评估不同接种部位的病毒增殖情况；根据原始标本核酸检测Ct值结果和改良法最终病毒分离阳性率，分析原始标本中病毒含量与分离阳性率的相关性。结果:改良法获得H1N1pdm09毒株42株，阳性率为52.5%( χ2＝38.571， P<0.01)；获得H3N2snl毒株54株，阳性率为67.5%( χ2＝40.921， P<0.01)。改良法接种鸡胚不同部位，在H1N1pdm09样本中有明显差异( χ2＝30.476， P<0.01)，在H3N2snl样本中见明显差异( χ2＝4.928， P＝0.026)。原始标本不同Ct值区间的改良法分离率未见明显差异( χH1N1pdm092＝10.226， χH3N2snl2＝3.764， P>0.05)。 结论:改良法接种14日龄鸡胚使病毒适应，最终获得毒株数量显著高于传统法接种10日龄鸡胚的，可显著提升H1N1pdm09和H3N2snl流感病毒的鸡胚分离阳性率。羊膜腔对于H1N1pdm09和H3N2snl流感病毒更为敏感，有助于病毒在鸡胚内的适应。不同Ct值区间的标本分离率和病毒分离总阳性率未见显著差异，需进一步验证评价标本质量的因素。

abstractsObjective:To improve the isolation and culture method of seasonal influenza virus in embryonated chicken eggs (ECEs), and evaluate their isolation efficiency.Methods:We randomly selected 80 positive samples of H1N1 (H1N1pdm09) and seasonal H3N2 (H3N2snl) influenza virus nucleic acid, and inoculated them into the amniotic and urinary sac cavities of 10-day-old (traditional method) and 14-day-old (improved method) ECEs respectively to adapt the virus to the ECEs (E1-E2). Both method were used to inoculate 10-day-old urinary sac amplification virus (E2-E3), and the final virus isolation positive rates of the two method were compared; using fluorescence quantitative PCR method to detect viral nucleic acids in the improved amniotic and urinary sac cultures, and evaluate the viral proliferation at different inoculation sites; we analyzed the correlation between virus content and isolation positivity rate in the original specimen based on the CT value of nucleic acid testing and the final virus isolation positivity rate using the improved method.Results:The improved method obtained 42 strains of H1N1pdm09 strain, with a positive rate of 52.5% ( χ2=38.571, P<0.01); obtained 54 strains of H3N2snl strain, with a positive rate of 67.5% ( χ2=40.921, P<0.01). Significant differences were observed in the isolation efficiency of H1N1pdm09 samples when the improved method was applied to different inoculation sites of chicken embryos ( χ2=30.476, P<0.01), and similar differences were noted for H3N2snl samples ( χ2=4.928, P=0.026). There was no significant difference in the isolation rate of different CT value intervals of the original samples ( χH1N1pdm092=10.226, χH3N2snl2=3.764, P>0.05). Conclusions:The improved method of inoculating 14-day old ECEs adapted the virus, and the final number of strains obtained was significantly higher than the traditional method of inoculating 10 day old ECEs, which can significantly improve the positive isolation rate of H1N1pdm09 and H3N2snl influenza virus in ECEs. The amniotic cavity is more sensitive to H1N1pdm09 and H3N2snl influenza viruses, which helps the virus adapt in ECEs. There was no significant difference in the sample isolation rate and total positive rate of virus isolation among different CT value ranges, and further verification is needed.