Abstract：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 temperature 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) assay 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) copies/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 detected 89.46% H275Y mutation abundance in samples collected on day 4 post-treatment with oseltamivir phosphate; however, 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 mutations, which is effective for quantitative detection of H275Y mutations in the NA fragment of the H1N1 pdm09 virus.
楼秀玉, 颜浩, 孙逸, 王欣莹, 陈寅, 茅海燕. 应用ddPCR检测甲型H1N1 pdm09流感病毒H275Y突变研究[J]. 预防医学, 2022, 34(11): 1139-1144.
LOU Xiuyu, YAN Hao, SUN Yi, WANG Xinying, CHEN Yin, MAO Haiyan. Detection of H275Y mutation in influenza A H1N1 pdm09 virus by droplet digital PCR assay. Preventive Medicine, 2022, 34(11): 1139-1144.
[1] HUANG W J,CHENG Y H,LI X Y,et al.Neuraminidase inhibitor susceptibility profile of human influenza viruses during the 2016-2017 influenza season in Mainland China[J].J Infect Chemother,2018 ,24(9):729-733. [2] CHAUDHRY A,BASTIEN N,LI Y,et al.Oseltamivir resistance in an influenza A(H3N2)virus isolated from an immunocompromised patient during the 2014-2015 influenza season in Alberta,Canada[J].Influenza Other Respir Viruses,2016,10(6):532-535. [3] 包佳琪,王晓晨,邹迁达,等.流感病毒对神经氨酸酶抑制剂耐药性的研究进展[J].中华临床感染病杂志,2018,11(6):473-481. BAO J Q,WANG X C,ZOU Q D,et al.Research progress on drug-resistance of influenza virus to neuraminidase inhibitors[J].Chin J Clin Infect Dis,2018,11(6):473-481. [4] NGUYEN H T,ALICIA M F,GUBAREVA L V.Neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods[J]. Antivir Ther,2012,17:159-173. [5] 刘译利,高晶.数字聚合酶链反应在病原微生物检测中的应用进展[J].国际检验医学杂志,2020,41(21):2684-2688. LIU Y L,GAO J.The application of digital polymerase chain reaction in pathogen detection[J].Int J Lab Med,2020,41(21):2684-2688. [6] WHALE A S,BUSHELL C,GRANT P R,et al.Detection of rare drug resistance mutations by digital PCR in a human influenza a virus model system and clinical samples[J].J Clin Microbiol,2016,54(2):392-400. [7] MITCHELL S L,CHANG Y C,FEEMSTER K,et al.Implementation of a rapid influenza A/B and RSV direct molecular assay improves emergency department oseltamivir use in paediatric patients[J].J Med Microbiol,2018,67(3):358-363. [8] BOSMAN K J,NIJHUIS M,HAM P V,et al.Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir[J/OL].Sci Rep,2015,5[2022-09-16].http://doi.org/10.1038/srep13811. [9] 韩冰,吴翠萍,赵芯,等.数字PCR和荧光定量PCR诊断急性期布鲁菌病的灵敏度比较初步研究[J].传染病信息,2019,32(4):312-316. HAN B,WU C P,ZHAO X,et al.A preliminary study on the sensitivity of digital PCR and fluorescence quantitative PCR in the diagnosis of acute brucellosis[J].Infect Dis Inf,2019,32(4):312-316. [10] JEANNOT E,BECETTE V,CAMPITELLI M,et al.Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma[J].J Pathol Clin Res,2016,28,(4):201-209. [11] 孙婷婷,姜艳芳,宫平,等.应用数字PCR检测环境中微量新型冠状病毒残留[J].科学通报,2021,66(13):1653-1662. SUN T T,JIANG Y F,GONG P,et al.Detection of microamounts of novel coronavirus residues in environment by digital PCR[J].Chin Sci Bull,2021,66(13):1653-1662. [12] 王晓晨. H1N1 pdm09流感重症肺炎患者耐药位点筛查及H275Y突变与奥司他韦耐药相关性研究[D].杭州:浙江大学,2020. WANG X C.Screening of drug-resistant sites in patients with severe H1N1 pdm09 influenza pneumonia and research of the association between H275Y mutation ans oseltamivir-resistance [D].Hangzhou:Zhejiang University,2020.
