多孔板对B型流感病毒分离影响因素分析  

Analysis of influencing factors on Influenza B virus isolation by multi-well plates

作　　者：杜冬冬 刘斌 梁磊 成娉婷 黄纷纷 李海燕 刘万静 杨力 史伟[3] 屈娅荣 焦欢 DU Dongdong;LIU Bin;LIANG Lei;CHENG Pingting;HUANG Fenfen;LI Haiyan;LIU Wanjing;YANG Li;SHI Wei;QU Yarong;JIAO Huan(Ankang Center for Disease Control and Prevention,Ankang,Shaanxi 725000,China;不详)

机构地区：[1]安康市疾病预防控制中心,陕西安康725000 [2]安康市汉滨区江北中学 [3]陕西省疾病预防控制中心

出　　处：《医学动物防制》2023年第12期1213-1216,共4页Journal of Medical Pest Control

基　　金：陕西省自然科学基础研究计划资助项目(2020JM-700)。

摘　　要：目的研究B型流感病毒在不同多孔板中的生长情况,探索其合适的分离条件。方法对4株B型流感病毒进行血凝鉴定后将滴度调整到1∶32,稀释10000倍后接种于6孔板,按接种量(0.40ml、0.60ml、0.80ml和1.00ml)分为4组,吸附后每组加入2.00ml、2.50ml、3.00ml和3.50ml病毒维持液。接种于24孔板,按接种量(0.10ml、0.15ml、0.20ml和0.25ml)分为4组,吸附后每组加入0.80ml、1.00ml、1.20ml和1.40ml病毒维持液,每24h观察致细胞病变并进行血凝滴度(hemagglutination titer,HA)鉴定。采用χ^(2)检验对不同多孔板的HA进行分析。结果随时间增长,病毒滴度均增加,所有致细胞病变均有滴度,6孔板每24h检测各孔滴度均能达到1∶8以上,B/Yamagata和B/Victoria亚型滴度最高为1∶256,24孔板每24h检测各孔阴阳性不一,B/Victoria亚型的滴度最高为1∶16,B/Yamagata亚型的滴度最高为1∶32。不同多孔板的HA差异有统计学意义(χ^(2)=46.30,P<0.05)。结论B型流感病毒的分离与多孔板、维持液量、接种量和培养时间等有关。6孔板建议选择3.00~3.50ml+0.80ml组合,24孔板建议选择1.20~1.40ml+0.20~0.25ml组合,培养时间为72h。Objective To research the growth of Influenza B virus in different multi-well plates and explore its appropriate isolation conditions.Methods After hemagglutination identification of 4 strains of Influenza B viruses,the titer was adjusted to 1∶32,diluted 10000 times and then inoculated in 6-well plates,divided into 4 groups according to the inoculum size(0.40ml,0.60ml,0.80ml,and 1.00ml).After adsorption,2.00ml,2.50ml,3.00ml,and 3.50ml of virus maintenance solution were added to each group.Inoculated in 24-well plates,according to the inoculum size were divided into 4 groups(0.10ml,0.15ml,0.20ml,and 0.25ml).After adsorption,0.80ml,1.00ml,1.20ml and 1.40ml of virus maintenance solution was added to each group.The cytopathic effects were observed every 24 hours and the identification of hemagglutination titer(HA)was conducted.The chi-squared test was used to analyze HA of different perforated plates.Results With the increase of time,the viral titer all increased,and all cytopathic effects had titers.The titer of each well of the 6-well plates reached more than 1∶8 every 24 hour test.The highest titers of the B/Yamagata and B/Victoria subtypes were both 1∶256,and the detection of the 24-well plates were tested every 24h with different negatives and positives for each well.The highest titer of B/Victoria subtype was 1∶16 and of the B/Yamagata subtype was 1∶32.There were statistically significant differences in HA between the different multi-well plates(χ^(2)=46.30,P<0.05).Conclusion The isolation of Influenza B virus is related to the multi-well plates,the amount of maintenance liquid,the dose of inoculation and the culture time etcetera.The 6-well plates is recommended to choose the combination of 3.00-3.50ml+0.80ml and the 24-well plates is recommended to choose the combination of 1.20-1.40ml+0.20-0.25ml,and the culture time is 72 hours.

关 键 词：B型流感病毒 几何平均滴度 多孔板 病毒分离 培养 影响因素 分析 

分 类 号：R373.13[医药卫生—病原生物学]