[1]刘晴晴,王宁宁,成 军,等.基于重组酶介导等温扩增技术建立人腺病毒14 型快速检测方法及初步应用评价[J].现代检验医学杂志,2023,38(06):13-18+29.[doi:10.3969/j.issn.1671-7414.2023.06.003]
　LIU Qingqing,WANG Ningning,CHENG Jun,et al.Establishment of A Rapid Detection Method for Human Adenovirus Type 14 Based on Recombinase-aided Amplification Technology and Preliminary Application Evaluation[J].Journal of Modern Laboratory Medicine,2023,38(06):13-18+29.[doi:10.3969/j.issn.1671-7414.2023.06.003]
点击复制

基于重组酶介导等温扩增技术建立人腺病毒14 型快速检测方法及初步应用评价()
分享到：

《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年06期
页码:
13-18+29
栏目:
论著
出版日期:
2023-11-15

文章信息/Info

Title:
Establishment of A Rapid Detection Method for Human Adenovirus Type 14 Based on Recombinase-aided Amplification Technology and Preliminary Application Evaluation
文章编号:
1671-7414（2023）06-013-07
作者:
刘晴晴1; 2; 王宁宁1; 2; 成 军2; 周华君2; 车飞虎2; 杨春利2; 孙青阳2; 王 月2; 戴玉柱2; 张英杰1
（1. 蚌埠医学院检验医学院，安徽蚌埠 233000；2. 中国人民解放军联勤保障部队第九〇三医院临床研究部，杭州 310013）
Author(s):
LIU Qingqing1; 2;  WANG Ningning1; 2;  CHENG Jun2;  ZHOU Huajun2;  CHE Feihu2;  YANG Chunli2;  SUN Qingyang2;  WANG Yue2;  DAI Yuzhu2;  ZHANG Yingjie1
（1.School of Laboratory Medicine, Bengbu Medical College，Anhui Bengbu 233000, China；2.Department of Clinical Research, the 903rd Hospital of the People’s Liberation Army, Hangzhou 310013，China）
关键词:
人腺病毒14 型; 重组酶介导的等温扩增技术; 快速分子诊断
分类号:
Q503；R373
DOI:
10.3969/j.issn.1671-7414.2023.06.003
文献标志码:
A
摘要:
目的 基于重组酶介导的等温扩增技术（recombinase-aided amplification, RAA）开发一种快速、准确的人腺病毒14 型检测方法。方法 根据人腺病素14 型（human adenovirus serotype 14, HAdV-14）的Hexon 基因序列，使用Primer 6.0 软件设计特异性引物及探针，并构建阳性质粒标准品，优化反应条件，建立一种快速RAA 检测方法，并对该方法的灵敏度及特异度进行评价。收集2017 ～ 2022 年中国人民解放军联勤保障部队第九〇三医院发热性呼吸综合征患者的咽拭子50 份，应用建立的实时荧光RAA 测定法进行检测，结果与实时荧光PCR 检测结果进行比较。结果 最终确定了最佳引物探针组合为HAdV14-F1-4，HAdV14-R1 和HAdV14-P；经反应条件优化，40℃进行RAA扩增效果最佳；该方法灵敏度为101 copies/μl，当质粒标准品浓度为104，103，102 和101 copies/μl 时，批内变异系数均<5%；50 例临床样本中HAdV-14 的检测结果与实时荧光PCR 一致。结论 建立了一种基于RAA 技术的HAdV-14 快速检测方法，可以在仅有简单恒温设备的条件下检测HAdV-14。
Abstract:
Objective To develop a rapid and accurate detection method for human adenovirus serotype 14(HAdV-14) based on recombinase-aided amplification (RAA) technology. Methods According to the Hexon gene sequence of HAdV-14, the specific primers and probes were designed by Primer 6.0 software, and the positive plasmid standard was constructed. The reaction conditions were optimized to establish a rapid RAA detection method，and the sensitivity and specificity of the method were evaluated. A total of 50 throat swabs were collected from patients with febrile respiratory syndrome in the 903rd Hospital of the People’s Liberation Army from 2017 to 2022. The established real-time fluorescent RAA assay was used for detection, and the results were compared with the results of the real-time PCR. Results The optimal primer and probe combination was determined as HAdV14-F1-4, HAdV14-R1 and HAdV14-P. After optimizing reaction conditions, RAA amplification at 40℃ was the best. The sensitivity of the method was 101 copies/μl. When the concentration of the plasmid standard was 104, 103, 102 and 101 copies/μl，the intra-assay coefficient of variation was less than 5%. The results of HAdV-14 in 50 clinical samples were consistent with those of real-time PCR. Conclusion A rapid detection method for HAdV-14 based on RAA technology was established, which could detect HAdV-14 with only simple thermostatic equipment.

参考文献/References:

[1] WU Tao, WU Haizhen, ZHAO Kangchen, et al. Rapid detection of human mastadenovirus species B by recombinase polymerase amplification assay[J]. BMC Microbiology, 2019, 19(1): 8.
[2] WANG Xinye, WANG Dawei, UMAR S, et al. Molecular typing of human adenoviruses among hospitalized patients with respiratory tract infections in a tertiary hospital in Guangzhou, China between 2017 and 2019[J]. BMC Infectious Diseases, 2021, 21(1): 748.
[3] RADKE J R, COVERT H J, BAUER F, et al. Adenovirus 14p1 immunopathogenesis during lung infection in the syrian hamster[J]. Viruses, 2020, 12(6):595.
[4] 庞晓燕, 冀云鹏, 周雪原, 等．2020 ～ 2021 年呼和浩特市儿童呼吸道腺病毒感染的流行病学和实验室检测及临床特征研究[J]. 现代检验医学杂志, 2023,38(2): 129-135． PANG Xiaoyan, JI Yunpeng, ZHOU Xueyuan, et al. Epidemiological,laboratory detection and clinical characteristics of respiratory adenovirus infection in children in hohhot from 2020 to 2021[J]. Journal of Modern Laboratory Medicine, 2023, 38(2): 129-135.
[5] ZHAO Mengchuan, GUO Yinghui, QIU Fangzhou, et al. Molecular and clinical characterization of human adenovirus associated with acute respiratory tract infection in hospitalized children [J]. Journal of Clinical Virology, 2020, 123: 104254.
[6] XU Deshun, CHEN Liping, WU Xiaofang, et al. Ji L.molecular typing and epidemiology profiles of human adenovirus infection among hospitalized patients with severe acute respiratory infection in Huzhou, China[J].PLoS One, 2022, 17(4): e0265987.
[7] ZHAO Shan, GUAN Wenyi , MA Kui , et al . Development and application of a fast method to acquire the accurate whole-genome sequences of human adenoviruses [J]. Frontiers in Microbiology,2021, 12：661382.
[8] STARR K, GRENINGER A L, MAKHSOUS N,et al. Comparison of three adenovirus quantitative PCR assays with ATCC reference strains and clinical samples[J]. Journal of Clinical Microbiology, 2019,57(11): e00735-19.
[9] 宁明哲, 陶月, 陈雨欣, 等．新型冠状病毒血清特异性抗体检测的假性问题分析和对策探讨[J]．现代检验医学杂志, 2020, 35(6): 129-131． NING Mingzhe, TAO Yue, CHEN Yuxin, et al. SARSCoV-2 serological antibody testing: problems of false detections and the solutions[J]. Journal of Modern Laboratory Medicine, 2020, 35(6): 129-131.
[10] QIU Fangzou, SHEN Xinxin, ZHAO Mengchuan,et al.A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2,3 and 7[J]. Virology Journal, 2018, 15(1): 81.
[11] WONG S S Y, YIP C C Y, SRIDHAR S, et al. Comparative evaluation of a laboratory-developed realtime PCR assay and RealStar? adenovirus PCR kit for quantitative detection of human adenovirus[J]. Virology Journal, 2018, 15(1): 149.
[12] WANG Ruihuan, ZHANG Hong, ZHANG Yi, et al. Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7[J]. Virology Journal, 2019, 16(1): 86.
[13] 陈昌国, 陈秋圆, 侯兵兵, 等. 基于LAMP 技术针对溶藻弧菌gyrB 基因快速检测方法的建立[J]. 现代检验医学杂志, 2019, 34(6): 6-9. CHEN Changguo, CHEN Qiuyuan, HOU Bingbing,et al. Establishment of the rapid detection method targeting to GyrB gene of vibrio parahaemolyticus based on LAMP technology[J]. Journal of Modern Laboratory Medicine, 2019, 34(6): 6-9.
[14] HOU Laiwang, LI Darong, ZHANG Ni, et al. Development of an isothermal recombinase-aided amplification assay for the rapid and visualized detection of Klebsiella pneumoniae[J]. Journal of the Science of Food and Agriculture, 2022, 102(9): 3879-3886.
[15] WU Shangyi, DUAN Hong, ZHANG Yingchao, et al. A salmonella microfluidic chip combining noncontact eddy heater and 3D Fan-Shaped mixer with recombinase aided amplification[J]. Biosensors Biosensors(Basel), 2022, 12(9): 726.
[16] WU Xiuhong, CHU Fengsheng, ZHANG Luxuan, et al. New rapid detection by using a constant temperature method for avian leukosis viruses [J]. Frontiers in Microbiology, 2022, 13: 968559.
[17] ZHENG Yuzhong, CHEN Jiangtao, LI Jian, et al. Reverse transcription recombinase-aided amplification assay with lateral flow dipstick assay for rapid detection of 2019 novel coronavirus [J]. Frontiers in Cellular and Infection Microbiology, 2021, 11: 613304.
[18] QI Juju, LI Xinna, ZHANG Yi, et al. Development of a duplex reverse transcription recombinase-aided amplification assay for respiratory syncytial virus incorporating an internal control[J]. Archives of Virology, 2019, 164(7): 1843-1850.
[19] MU Dan, ZHOU Donggen, XIE Guoyang, et al. The fluorescent probe-based recombinase-aided amplification for rapid detection of Escherichia coli O157:H7 [J]. Molecular and Cellular Probes, 2021, 60:101777.
[20] ZHANG Weiwei, FENG Yanling, ZHAO Hangqing, et al. A recombinase aided amplification assay for rapid detection of the Klebsiella pneumoniae carbapenemase gene and its characteristics in Klebsiella pneumoniae[J]. Frontiers in Cellular and Infection Microbiology,2021, 11: 746325.
[21] LI Jia, MACDONALD J, VON STETTEN F. Correction: review: a comprehensive summary of a decade development of the recombinase polymerase amplification[J]. The Analyst, 2020, 145(5): 1950-1960.
[22] FAN Xiaoxu, LI Lin, ZHAO Yonggang, et al. Clinical validation of two recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of african swine fever virus [J]. Frontiers in Microbiology, 2020, 11: 1696.
[23] PIEPENBURG O, WILLIAMS C H, STEMPLE D L,et al. DNA detection using recombination proteins[J].PLoS Biology, 2006, 4(7): e204.
[24] LIN Hongqing, LIANG Yuanhao, ZOU Lirong, et al. Combination of isothermal recombinase-aided amplification and CRISPR-Cas12a-mediated assay for rapid detection of major severe acute respiratory syndrome coronavirus 2 variants of concern [J].Frontiers Microbiolgy, 2022, 13: 945133.
[25] CUI Huan, TU Fei, ZHANG Cheng, et al. Real-Time reverse transcription recombinase-aided amplification assay for rapid amplification of the N gene of SARSCoV-2[J]. International Journal of Molecular Sciences,2022, 23(23): 15269.
[26] GHOSH P, CHOWDHURY R, HOSSAIN M E,et al. Evaluation of recombinase-based isothermal amplification assays for point-of-need detection of SARS-CoV-2 in resource-limited settings [J].International Journal of Infectious Diseases, 2022, 114:105-111.

备注/Memo

备注/Memo:
基金项目： 浙江省自然科学基金（批准号：LGF20H200009）：基于RAA 技术在ARID 病原快速检测中的应用研究；浙江省医药卫生科技规划项目（批准号：2019331539）：军营内呼吸道感染HADV-55 型的流行病学特征及迁徙分析；杭州市医药卫生科技规划项目（批准号：OO20190415）：军营内呼吸道感染HADV-55 型的流行病学特征及迁徙分析。
作者简介：刘晴晴（1999-），女，初级检验师，在读硕士， 研究方向：分子诊断与技术，E-mail:1669399941 @qq.com。
通讯作者： 戴玉柱（1985-），男，硕士，硕士生导师，副主任技师，研究方向：临床感染性疾病的分子诊断， E-mail:dyz5895@qq.com。
张英杰（1979-），男，副教授，硕士生导师，研究方向：肝纤维化分子机制的研究，E-mail:8592386 @qq.com。
更新日期/Last Update: 2023-11-15