应用ddPCR检测甲型H1N1 pdm09流感病毒H275Y突变研究

Detection of H275Y mutation in influenza A H1N1 pdm09 virus by droplet digital PCR assay

目的评价微滴式数字PCR(ddPCR)检测甲型H1N1 pdm09流感病毒神经氨酸酶(NA)H275Y突变的方法。方法根据甲型H1N1 pdm09流感病毒NA片段包含275氨基酸位点的序列信息设计引物和双探针,优化ddPCR退火温度,建立甲型H1N1 pdm09流感病毒H275耐药敏感基因和H275Y耐药突变基因检测方法。采用检出限比较ddPCR和荧光定量PCR(qPCR)的灵敏度,通过检测甲型H1N1 pdm09、季节性H1N1、甲型H3N2流感病毒等14种呼吸道病毒样本比较ddPCR和q PCR的特异性。检测64份临床标本和5株流感病毒株,获得拷贝数,计算H275Y突变丰度,并将5株毒株的突变丰度与二代测序结果进行比较。结果确定62.2℃为最优退火温度。采用ddPCR和qPCR检测甲型H1N1 pdm09 H275耐药敏感质粒和H275Y耐药突变质粒,ddPCR检出限分别为5.28(95%CI:4.28~7.45)和6.51(95%CI:5.25~9.37)拷贝/反应;qPCR检出限分别为5.70(95%CI:4.83~7.45)和7.06(95%CI:5.92~9.40)拷贝/反应。ddPCR和qPCR均能检出甲型H1N1 pdm09样本中的H275耐药敏感质粒和H275Y耐药突变质粒,其他11种呼吸道病毒均未检出,两种方法检测结果一致。64份临床标本中,ddPCR检出1例感染甲型H1N1pdm09的重症肺炎患者的3份咽拭子标本存在H275Y突变,使用磷酸奥司他韦治疗第4天采集的标本突变丰度最高,为53.37%。ddPCR检测分离自该病例治疗第2、4、5天标本的流感病毒H275Y突变丰度分别为0.63%、88.93%和1.27%;二代测序检测治疗第4天标本的H275Y突变丰度为89.46%,未检测到治疗第2、5天标本存在H275Y突变。结论ddPCR的灵敏度和特异性较qPCR高,对低频突变的检测灵敏度较二代测序高,可定量检测甲型H1N1 pdm09流感病毒NA片段H275Y突变。

Objective To evaluate the effectiveness of droplet digital PCR(ddPCR)assay for detection of neuraminidase(NA)H275Y mutations in influenza A H1N1 pdm09 virus.Methods The primers and dual probes were designed based on the sequence of the H1N1 pdm09 NA gene fragment which contained 275 amino acid sites,and the annealing tem⁃perature of ddPCR assay was optimized to establish a method for detection of H275 drug-sensitive genes and H275Y drug-resistant genes in H1N1 pdm09 virus.The sensitivity of ddPCR assay and fluorescent quantitative PCR(qPCR)as⁃say was compared using the detection limit,and the specificity of ddPCR and qPCR assays was compared for detection of 14 respiratory virus samples.In addition,64 clinical samples and 5 influenza isolates were tested to calculate the abundance of H275Y mutations,and the mutation abundance of 5 influenza isolates was compared with next-generation sequencing results.Results The optimal annealing temperature was 62.2℃.The detection limits of ddPCR assay were 5.28(95%CI:4.28-7.45)copies/reaction for H1N1 pdm09 H275 drug-sensitive plasmids and 6.51(95%CI:5.25-9.37)copies/reaction for H1N1 pdm09 H275Y drug-resistant plasmids,and the detection limits of qPCR assay were 5.70(95%CI:4.83-7.45)copies/reaction for H1N1 pdm09 H275Y drug-sensitive plasmids and 7.06(95%CI:5.92-9.40)cop⁃ies/reaction for H1N1 pdm09 H275Y drug-resistant plasmids.Both ddPCR and qPCR assays detected H275 and H275Y drug-resistant plasmids in H1N1 pdm09 viral samples but did not detect H275 and H275Y drug-resistant plasmids in other 11 respiratory virus samples,and these two assays showed consistent results.Of the 64 clinical samples,ddPCR assay detected H275Y mutation in three pharyngeal swab specimens from a severe pneumonia patients infected with H1N1 pdm09 virus,and the greatest mutation abundance was detected in samples collected on day 4 post-treatment with oseltamivir phosphate(53.37%).ddPCR assay detected 0.63,88.93%%and 1.27%H275Y mutation abundance in samples collected on days 2,4 and 5 post-treatment with oseltamivir phosphate,and next-generation sequencing detect⁃ed 89.46%H275Y mutation abundance in samples collected on day 4 post-treatment with oseltamivir phosphate;howev⁃er,no H275Y mutation was detected in samples collected on days 2 or 5 post-treatment with oseltamivir phosphate.Conclusions ddPCR presents a higher sensitivity and specificity than qPCR assay for detection of H275Y mutations in H1N1 pdm09 virus,and presents a higher sensitivity than next-generation sequencing for detection of low-frequency mu⁃tations,which is effective for quantitative detection of H275Y mutations in the NA fragment of the H1N1 pdm09 virus.