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Abstract：
Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSVA) and B (HRSVB) of HRSV. Next, the Pyrococcus furiosus Argonaute (PfAgo) protein utilizes small 5\'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/μL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSVA and HRSVB infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings.
摘要：
人呼吸道合胞病毒（HRSV）是导致婴儿和幼儿急性呼吸道感染（ARTI）的最常见病原体，并可能导致大量的财务和医疗费用。这里,我们开发了一个同时，双基因和超灵敏的检测系统，用于在60分钟内分型HRSV，只需要最少的实验室支持。简而言之，以从鼻咽拭子中提取的病毒RNA为模板，进行多重整合逆转录重组酶聚合酶扩增（RT-RPA），以扩增HRSV亚型A（HRSVA）和B（HRSVB）的特定区域。接下来,愤怒的热球菌Argonaute（PfAgo）蛋白利用小的5'-磷酸化DNA指导切割靶序列并产生荧光团信号（FAM和ROX）。与传统的金标准(RT-qPCR)和直接免疫荧光法(DFA)相比,该方法具有操作简便的附加优点，效率和灵敏度，检测限(LOD)为1拷贝/μL。在临床样本验证方面，HRSVA和HRSVB感染测定方法的诊断准确率大于95%.该技术为儿童HRSV引起的ARTI的诊断和暴发管理提供了可靠的现场护理（POC）测试。尤其是在资源有限的环境中。
