基于重组酶介导等温扩增技术检测人腺病毒4型方法的建立与评价

doi:10.6040/j.issn.1671-7554.0.2023.0640

摘要/Abstract

摘要： 目的基于重组酶介导等温扩增技术(RAA)拟建立一种简单快速的人腺病毒4型(HAdV-4)检测方法。方法以HAdV-4的六邻体(Hexon)基因为靶标设计特异性引物与探针,利用构建的阳性质粒标准品,在39 ℃、40 ℃、42 ℃条件下进行重组酶介导等温扩增,选出最佳反应温度,并对该方法进行灵敏度、重复性及特异性检测。使用实时荧光RAA测定法检测100份临床样本,并与实时荧光PCR检测结果进行比较,评估HAdV-4实时荧光RAA测定法在临床样本中的检测性能。结果最终确定了实时荧光RAA扩增最适温度为42 ℃,该方法检测限为10¹ copies/μL。当质粒标准品浓度为10¹~10⁴ copies/μL时,批内的变异系数均<5%,重复性良好。特异性检测结果显示,该方法可以准确检测HAdV-4而不与其他亚型和其他病原体发生交叉反应。100例临床样本分别使用实时荧光PCR和实时荧光RAA测定法进行检测,两种方法的符合率为100%。结论本研究开发了一种特异且简便的HAdV-4快速检测方法。

关键词: 人腺病毒4型, 重组酶介导等温扩增技术, Hexon基因, 快速检测, 分子诊断技术

Abstract: Objective To establish a simple and rapid method for human adenovirus type 4(HAdV-4)detection with recombinase-aided amplification(RAA). Methods The specific primers and probes were designed based on the Hexon gene of HAdV-4. The constructed positive plasmid standard was used for RAA under different temperatures(39 ℃, 40 ℃, 42 ℃)to select the best reaction temperature, and the sensitivity, repeatability, and specificity of the method were tested. A total of 100 clinical samples were detected with real-time RAA, and the results were compared with those of real-time PCR to evaluate the detection performance of real-time RAA. Results The optimal temperature for real-time RAA was 42 ℃, and the detection limit was 10¹ copies/μL. When the concentration of the plasmid standard was 10¹-10⁴ copies/μL, the intra-assay coefficient of variation was less than 5%, showing good repeatability. The results of specificity showed that the method accurately detected HAdV-4 without cross-reactivity with other subtypes or other pathogens. Real-time PCR and real-time RAA of the 100 samples showed the coincidence rate was 100%. Conclusion A specific, simple and rapid assay for HAdV-4 detection is developed.

Key words: Human adenovirus type 4, Recombinase-aided amplification, Hexon gene, Rapid detection, Molecular diagnostic techniques

中图分类号:

R446.5

刘晴晴,王宁宁,成军,周华君,车飞虎,孙青阳,王月,张英杰,戴玉柱. 基于重组酶介导等温扩增技术检测人腺病毒4型方法的建立与评价[J]. 山东大学学报 (医学版), 2023, 61(10): 74-82.

LIU Qingqing, WANG Ningning, CHENG Jun, ZHOU Huajun, CHE Feihu, SUN Qingyang, WANG Yue, ZHANG Yingjie, DAI Yuzhu. Establishment and evaluation of a recombinase-aided amplification for the detection of human adenovirus type 4[J]. Journal of Shandong University (Health Sciences), 2023, 61(10): 74-82.

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