由病毒引起的呼吸道感染（respiratory tract infections，RTI）是临床上常见的对人类健康造成威胁的重要疾病，其可由多种病毒引起，具有相似的流行病学特征和相同的临床症状，给临床准确诊断带来了挑战。

以实时荧光聚合酶链反应（real-time polymerase chain reaction，real-time PCR）技术为代表的核酸检测方法被公认为呼吸道病毒感染诊断的“金标准”。基于实时荧光PCR技术的体外诊断产品主要集中在临床样本中单一病原体核酸序列的检测，即单重检测。随着技术的进步，也为了更广泛地满足临床检测的需要，能同时识别临床样本中多个病原体核酸序列的多重检测方法被开发出来并广泛用于病毒性RTI的诊断。

相较于单重检测，多重检测方法中反应组分的多样性和反应体系的复杂性对核酸样品、检测试剂以及检测平台有更严格的要求。鉴于多重检测方法在检测性能方面的潜在挑战，本研究使用13种常见的呼吸道病毒及其亚型的灭活病毒细胞培养上清液样本，包括新型冠状病毒Omicron BA.5变异株、甲型流感病毒H1N1亚型、甲型流感病毒H3N2亚型、乙型流感病毒Victoria谱系、呼吸道合胞病毒A型、呼吸道合胞病毒B型、人鼻病毒B72型、人腺病毒3型、人腺病毒7型、副流感病毒1型、副流感病毒2型、副流感病毒3型以及人偏肺病毒A2型，评估了我国医疗机构实验室最常使用的6款呼吸道病毒核酸多重检测试剂（圣湘三联检、圣湘六联检、伯杰二联检、伯杰六联检、硕世二联检、卓诚惠生六联检）的两个关键检测性能：分析敏感性和竞争干扰。同时，本研究还纳入了12款呼吸道病毒核酸单重检测试剂作为比较。

分析敏感性评价结果显示，同一多重检测试剂内不同毒株样本的计算检测限存在差异，同一毒株样本不同检测试剂间的计算检测限存在差异，其中甲型流感病毒H1N1亚型在不同检测试剂间的计算检测限差异较大（从1405.20 copies/mL到35887.75 copies/mL不等）；与单重检测试剂相比，多重检测试剂在检测临床常见病毒如新型冠状病毒、腺病毒、流感病毒以及呼吸道合胞病毒时的计算检测限相当或更低，在检测人鼻病毒、人偏肺病毒以及副流感病毒时的计算检测限则相对更高。竞争干扰评价结果显示，当样本中一种毒株的浓度较低而另一种毒株的浓度较高时，大多数多重检测试剂能成功识别这种特定浓度条件下的混合感染样本。

综上所述，本研究评估和比较了目前临床上常规用于呼吸道病毒感染诊断的实时荧光PCR试剂检测性能，希望能为呼吸道病毒核酸多重检测试剂的开发和应用提供有用信息。

Respiratory tract infections (RTIs) caused by viruses are prevalent and significant conditions in clinical settings. Viral RTIs can be caused by a variety of viruses with overlapping epidemic peaks and similar clinical symptoms, posing a challenge to accurate clinical diagnosis. Represented by real-time polymerase chain reaction (real-time-PCR), nucleic acid assays are recognized as the “gold standard” for the diagnosis of viral RTIs. In vitro diagnostic medical devices for real-time PCR assays primarily focused on the detection or quantitation of a singular nucleic acid sequence within a clinical specimen, called singleplex real-time-PCR assays. Advances in technology and clinical needs enable the development of multiplex real-time PCR assays tailored for the concurrent identification of numerous targets in a clinical sample. Since its introduction, multiplex real-time PCR has been successfully applied in syndromic diagnosis for RTIs. The diversity of reaction components and complexity of reaction systems in multiplex molecular tests may impose more stringent requirements for sample purity, input reagents, and platforms. Given the potential challenges in the detection performance of multiplex assays, this study used thirteen inactivated cell culture supernatants of viruses with major subtypes to evaluate the analytical sensitivity and competitive interference of the six most commonly used multiplex real-time PCR kits for the detection of respiratory viruses in China. The thirteen viruses with major subtypes included severe acute respiratory syndrome coronavirus 2 Omicron BA.5 strain (SARS-CoV-2 Omicron BA.5), pandemic influenza A (H1N1) 2009 virus (H1N1pdm09), influenza A (H3N2) virus (H3N2), Victoria lineage of influenza B virus (B/Victoria), respiratory syncytial virus subtypes A and B (RSVA and RSVB), human rhinovirus B72 strain (HRV-B72), human adenovirus types 3 and types 7 (HAdV-3 and HAdV-7), parainfluenza virus types 1, types 2, and types 3 (PIV-1, PIV-2, and PIV-3), human metapneumovirus A2 strain (hMPV-A2). The six multiplex real-time PCR kits are Sansure 3-plex kits, Sansure 6-plex kits, BioGerm 2-plex kits, BioGerm 6-plex kits, BioPerfectus 2-plex kits, and ABT 6-plex kits. Accordingly, twelve matching singleplex real-time PCR kits were used for comparison. The results of analytical sensitivity revealed that the calculated limits of detections (LoD) were variable across the viruses and kits. There were differences in the LoD for different strains within the same multiplex assay, as well as variations in the LoD for the same strain across different assays. The calculated LoD differed the most in the case of H1N1pdm09, ranging from 1405.20 to 35887.75 copies/mL according to the kit type. Most of the evaluated multiplex kits demonstrated comparable or enhanced analytical sensitivity compared with singleplex kits for clinically significant viruses, including Omicron BA.5, HAdV-3, HAdV-7, H1N1pdm09, H3N2, B/Victoria, RAVA, and RSVB, whereas multiplex kits showed relatively less analytical sensitivity for HRV-B72, hMPV-A2, PIV-1, and PIV-3. In addition, most multiplex kits successfully identified co-infections when one analyte was present at a low concentration and another analyte was present at a high concentration. In conclusion, this study evaluated and compared the detection performance of real-time PCR reagents currently routinely used in clinical settings for diagnosing viral RTIs, with the aim of providing valuable insights for the development and application of multiplex nucleic acid detection reagents targeting respiratory viruses.