目的: 建立一种覆盖率高,敏感、特异的针对苛养型人腺病毒（HAdV）Hexon基因的TaqMan探针实时荧光定量PCR检测方法,对人源生产用细胞基质和原材料进行苛养型HAdV检测。方法: 针对Hexon基因保守序列设计合成引物、探针及质粒对照品,将以腺相关病毒（AAV）为载体的HAdV（AAV-HAdV）假病毒作为阳性对照样品,含HAdV的F亚属（HAdV-F）基因序列的质粒作为扩增对照,全面验证该检测方法的线性、灵敏度、特异性、耐用性和重复性,确保其可应用于生物制剂及其原材料的苛养型HAdV安全评价。结果: 该检测方法对病毒毒株覆盖率高,可覆盖100%的HAdV-F毒株（94个分离株）;质粒对照品在20~2×108 copies · μL-1具有良好的线性关系,R2可达1.000;检测灵敏度可达到4 copies · μL-1;具有良好的特异性,对人腺病毒5型和人腺病毒2型不能进行特异性扩增;具有良好的耐用性,使用2款不同型号的仪器检测,标准曲线R2分别为0.999和1.000,灵敏度循环阈值（Ct）分别为37.22±0.62和36.50±0.58,阳性对照样品Ct分别为21.67±0.03和20.61±0.18,各项检测结果无明显差异;使用2种不同货号试剂检测,标准曲线R2均为1.000,灵敏度Ct分别为37.99±1.19和38.31±0.97,阳性对照样品Ct分别为19.69±0.09和20.58±0.05,各项检测结果无明显差异;在不同人员或不同仪器的条件下进行中间精密度验证,4次实验HAdV-F质粒扩增对照的检测结果分别为1.29×105、1.54×105、1.73×105、1.37×105 copies · μL-1,RSD为13.2%,中间精密度良好;AAV-HAdV假病毒作为阳性对照样品对人间充质干细胞样品进行适用性验证,阳性对照样品检测结果为137 copies · μL-1,适用性样品检测结果为133 copies · μL-1,样品加标回收率为97.1%。结论: 该检测方法具有良好的线性、灵敏度、特异性、耐用性和重复性,且对苛养型HAdV毒株的覆盖率高,可用于生产用细胞基质样品中苛养型HAdV检测。
董莹莹
,
陈晓菲
,
张峒
,
刘明月
,
张瑞瑞
,
曹译丹
,
李慧婷
,
付欣悦
,
王艳辉
,
王新乐
,
崔梦姗
,
庞琳
,
饶春明
. 苛养型人腺病毒实时荧光定量PCR检测方法的建立*[J]. 药物分析杂志, 2025
, 45(7)
: 1122
-1129
.
DOI: 10.16155/j.0254-1793.2024-1338
Objective: To establish a high coverage, sensitive and specific TaqMan probe real-time fluorescence quantitative PCR method for the detection of Hexon gene of fastidious human adenovirus (HAdV), and to determine the fastidious HAdV in human-derived cell substrates and raw materials. Methods: In this study, primers, probes, and plasmid controls were designed and synthesized based on the conserved sequences of Hexon genes. AAV-HAdV pseudovirus was used as a positive control, the plasmid containing the gene sequence of the F subgenus of human adenovirus (HAdV-F) was used as an amplification control. The linearity, sensitivity, specificity, robustness, and reproducibility of the detection method were fully verified to ensure that it could be applied to the safety evaluation of fastidious HAdV in biologics and their raw materials. Results: A high coverage rate for virus strains was achieved by this detection method, and 100% of HAdV-F strains (94 isolates) were covered. A good linear relationship was exhibited by the plasmid controls within the range of 20-2×108 copies · μL-¹, and the R² value could reach 1.000. The detection sensitivity was able to reach 4 copies · μL-¹. Good specificity was demonstrated by this method, as specific amplifications for human adenovirus type 5 and human adenovirus type 2 could not be performed. Good robustness was also possessed by this method. When two different models of instruments were used for detection, the R2 values of the standard curves were 0.999 and 1.000 respectively, the cycle threshold (Ct) values for sensitivity were 37.22±0.62 and 36.50±0.58 respectively, and the Ct values for the positive controls were 21.67±0.03 and 20.61±0.18 respectively. No significant differences were found in the results of various detections. When two reagents of different article numbers were used for testing, the R2 values of the standard curves were both 1.000. The Ct values for sensitivity were 37.99±1.19 and 38.31±0.97 respectively, and the Ct values for the positive controls were 19.69±0.09 and 20.58±0.05 respectively. No significant differences were observed in the results of various detections. The intermediate precision was verified under the conditions of different personnel or different instruments. The detection results of the HAdV-F plasmid amplification control in four experiments were 1.29×105 copies · μL-¹, 1.54×105 copies · μL-¹, 1.73×105 copies · μL-¹, and 1.37× 105 copies · μL-¹ respectively. The relative standard deviation was calculated to be 13.2%, indicating good intermediate precision. The AAV-HAdV pseudovirus was used as a positive control for the applicability verification in human mesenchymal stem cell samples. The detection result of the positive control was determined to be 137 copies · μL-¹, and the detection result of the applicability sample was found to be 133 copies · μL-¹. The spiked sample recovery rate was calculated as 97.1%. Conclusion: The assay has good linearity, sensitivity, specificity, robustness, and reproducibility, and has high coverage of the exacting HAdV strain, which can be used for the detection of exacting HAdV in cell matrix samples for production.
[1] WATANABE M,NISHIKAWAJI Y,KAWAKAMI H,et al.Adenovirus biology,recombinant adenovirus,and adenovirus usage in gene therapy[J].Viruses,2021,13(12):2502
[2] DAVISON AJ,BENKŐ M,HARRACH B.Genetic content and evolution of adenoviruses[J].J Gen Virol,2003,84(Pt 11):2895
[3] MENNECHET FJD,PARIS O,OUOBA AR,et al.A review of 65 years of human adenovirus seroprevalence[J].Expert Rev Vaccines,2019,18(6):597
[4] HENDRICKX R,STICHLING N,KOELEN J,et al.Innate immunity to adenovirus[J].Hum Gene Ther,2014,25(4):265
[5] KOSULIN K.Intestinal HAdV infection:tissue specificity,persistence,and implications for antiviral therapy[J].Viruses,2019,11(9):804
[6] 王瑞欢,陈丽丽,马学军.人腺病毒分子生物学分型检测方法的研究进展[J].实用预防医学,2021,28(3):382
WANG RH,CHEN LL,MA XJ.Research progress on molecular biological typing detection methods of human adenovirus[J].Pract Prev Med,2021,28(3):382
[7] MACNEIL KM,DODGE MJ,EVANS AM,et al.Adenoviruses in medicine:innocuous pathogen,predator,or partner[J].Trends Mol Med,2023,29(1):4
[8] GBD 2016 Diarrhoeal Disease Collaborators.Estimates of the global,regional,and national morbidity,mortality,and aetiologies of diarrhoea in 195 countries:a systematic analysis for the global burden of disease study 2016BD 2016 Diarrhoeal Disease Collaborators.Estimates of the global,regional,and national morbidity,mortality,and aetiologies of diarrhoea in 195 countries:a systematic analysis for the global burden of disease study 2016[J].Lancet Infect Dis,2018,18(11):1211
[9] JACOBSSON PA,JOHANSSON ME,WADELL G.Identification of an enteric adenovirus by immunoelectroosmophoresis(IEOP)technique[J].J Med Virol,1979,3(4):307
[10] KIDD AH,MADELEY CR.In vitro growth of some fastidious adenoviruses from stool specimens[J].J Clin Pathol,1981,34(2):213
[11] UHNOO I,SVENSSON L,WADELL G.Enteric adenoviruses[J].Baillieres Clin Gastroenterol,1990,4(3):627
[12] 中华人民共和国药典 2020年版.三部[S].2020:11
ChP 2020.Vol Ⅲ[S].2020:11
[13] Quality of biotechnological products:viral safety evaluation of biotechnology products derived from cell lines of human or animal origin.ICH Harmonised Tripartite Guideline[J].Dev Biol Stand,1998,93:177
[14] Food and Drug Administration.Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals[S].1993
[15] Characterization and Qualification of Cell Substrates and Other Biological Materials used in the Production of Viral Vaccines for Infectious Disease Indications[S].2010
[16] SARKAR A,SAHA S,PAUL A,et al.Understanding stem cells and its pivotal role in regenerative medicine[J].Life Sci,2021,273:119270
[17] NEMBO EN,HESCHELER J,NGUEMO F.Stem cells in natural product and medicinal plant drug discovery-an overview of new screening approaches[J].Biomed Pharmacother,2020,131:110730
[18] LIN X,SUN Y,DONG X,et al.IPSC-derived CAR-NK cells for cancer immunotherapy[J].Biomed Pharmacother,2023,165:115123
[19] YAMANAKA S.Pluripotent stem cell-based cell therapy-promise and challenges[J].Cell Stem Cell,2020,27(4):523
[20] 干细胞制剂质量控制及临床前研究指导原则(试行)[S].2015
Guidelines for Quality Control of Stem Cell Preparations and Preclinical Research(Trial)[S].2015
[21] SHAHRAJABIAN MH,SUN W.The significance and importance of dPCR,qPCR,and SYBR Green PCR kit in the detection of numerous diseases[J].Curr Pharm Des,2024,30(3):169
[22] 安春艳,井良义,陈卓,等.《中国药典》与《美国药典》 《欧洲药典》 《日本药典》中聚合酶链式反应法的比较研究[J].生命科学仪器,2022,20(5):46
AN CY,JING LY,CHEN Z,et al.Comparative study of polymerase chain reaction among Chinese Pharmacopeia,United States Pharmacopeia,European Pharmacopeia,and Japanese Pharmacopeia[J].Life Sci Instrum,2022,20(5):46
[23] LU X,ERDMAN DD.Quantitative real-time PCR assays for detection and type-specific identification of the endemic species C human adenoviruses[J].J Virol Methods,2016,237:174
[24] LU X,TRUJILLO-LOPEZ E,LOTT L,et al.Quantitative real-time PCR assay panel for detection and type-specific identification of epidemic respiratory human adenoviruses[J].J Clin Microbiol,2013,51(4):1089
[25] ZHAO S,WU X,TAN Z,et al.Generation of human embryonic stem cell-derived lung organoids for modeling infection and replication differences between human adenovirus types 3 and 55 and evaluating potential antiviral drug[J].J Virol,2023,97(5):e0020923
