环介导逆转录等温扩增法在快速检测人呼吸道合胞病毒中的应用-现代医学

环介导逆转录等温扩增法在快速检测人呼吸道合胞病毒中的应用

作者：杨海玉¹ 宋冬梅¹ 唐妮娜¹ 吴学琴¹ 王艳¹ 许佳² 胡道来² 宋健锋² 杭佳² 朱伯林²

单位：1. 泰州市中医院中心实验室, 江苏泰州 225300;
2. 中华人民共和国泰州海关综合技术服务中心, 江苏泰州 225300

分类号：R331

出版年·卷·期（页码）：2025·53·第三期（475-479）

摘要：

目的：建立适用人呼吸道合胞病毒(RSV)核酸检测的逆转录等温扩增法(RT-Lamp)，为基层实验室和口岸现场的快速诊断提供时效性、便捷性服务。方法：对RSV靶基因保守区用在线软件设计并筛选出一套环介导特异性引物，采用RT-Lamp技术对RSV核酸进行恒温扩增，优化反应体系和条件，并对灵敏度、特异度和试剂稳定性等指标进行验证。结果：该方法能检测出RSV-A和RSV-B两种亚型。与荧光RT-PCR法比对，RT-Lamp灵敏度为50 copies·mL^-1，特异度检测发现流感病毒、人冠状病毒、人偏肺病毒、人腺病毒、人鼻病毒、肠道病毒EV71、肠道病毒CoxA16均无扩增反应。加速稳定性试验显示，反应体系3周内仍然稳定。临床验证中，扩增反应判读结果时间跨度在35 min以内，总体符合率为91.7%(55/60)，与RT-PCR方法比较差异无统计学意义(P=0.65)。结论： RSV RT-Lamp检测方法准确性良好，且快速简便，为进一步开展大样本诊断准确性研究提供了基础。

Objective: To establish a reverse transcription isothermal amplification method(RT-Lamp) suitable for human respiratory syncytial virus(RSV) nucleic acid detection, providing timely and convenient services for rapid diagnosis in grassroots laboratories and port sites. Methods: An online software was used to design and screen a set of mediated specific primers for the conserved region of RSV target genes. RT-lamp technology was used to perform isothermal amplification of RSV nucleic acid, optimize the reaction system and conditions, and verify indicators such as sensitivity, specificity, and reagent stability. Results: This method can detect two subtypes of RSV, RSV-A and RSV-B. Compared with fluorescence RT-PCR, RT-Lamp has a sensitivity of 50 copies·mL^-1 and no amplification reaction for influenza virus, human coronavirus, human partial lung virus, human adenovirus, human rhinovirus, enterovirus EV71 and enterovirus CoxA16 in specificity testing. The accelerated stability test showed that the reaction system remained stable within 3 weeks. In clinical validation, the time span of amplification reaction interpretation results was within 35 minutes, with an overall conformity rate of 91.7%(55/60) and no statistically significant difference compared with fluorescence RT-PCR(P=0.65). Conclusion: The RSV RT-lamp detection method has good accuracy and is fast and simple, it provides a basis for further research on the accuracy of large sample diagnosis.

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