解脲脲原体（Ureaplasma urealyticum，UU）在健康人群的泌尿生殖道存在普遍定植现象，通常被认为是人类泌尿生殖道的正常菌群，这使得研究者往往容易忽视其致病性，并且很少去关注对其进行常规检测。然而，由于在健康人群和患病人群的泌尿生殖道均可分离出UU，其也被认为是一种机会性致病菌。关于UU的致病性，越来越多的研究表明其与人类生殖健康密切相关，尤其是在妇产科门诊病人和不育夫妇中，推荐进行UU的常规筛查。目前，基于UU DNA检测的核酸扩增试验（nucleic acid amplification tests，NAATs）和基于RNA检测的NAATs已广泛应用于临床样本的检测中，并且国家药品监督管理局（National Medical Products Administration，NMPA）已经批准了多款UU核酸检测试剂，虽然关于这些试剂的分析性能在先前的研究中已多有报导，但是在临床应用中，其实际检测能力还处于未知水平。因此，本研究的第一部分开展了一项关于UU核酸检测的室间质量评价（External quality assessment，EQA）研究，旨在评估临床实验室的UU检测现状以及不同试剂的检测性能，为提高UU的检测质量和标准化提供有效的建议。在此项EQA研究中，样本盘由9支阳性样本和3支阴性样本组成，阳性样本由灭活的UU培养液制备而成，阴性样本为生理盐水。阳性样本进一步分为三组：高浓度、中浓度和低浓度样本。共有365家实验室报名参加了此次EQA活动，最后收集到由338家实验室回报的360份结果，其中合格的实验室为324家（95.86%，324/338），合格结果为346份（96.11%，346/360）。对于样本盘中的高浓度和中浓度样本，实验室检测结果的阳性符合率（positive percentage agreement，PPA）均大于97.5%，而对于低浓度样本，实验室检测结果的PPA差异显著，随着样本浓度的降低，PPA从86.94%降至51.94%。此外，对于低浓度样本，使用RNA检测试剂的实验室比使用DNA试剂的实验室展现出更高的PPA，但这一结果仅限于本研究中使用的灭活UU样本。总体而言，大部分参评实验室能够正确检测高浓度和中浓度样本，而实验室对低浓度样本的检测能力相对较弱的情况需要进一步提高和优化其检测方法和流程。

病毒核酸标准物质是保证测量结果准确性和可溯源性的重要计量“器具”，也是保证在不同实验室、不同检测试剂和方法间检测结果具有可比性的物质基础。本研究的第二部分基于成熟的MS2噬菌体包装外源性RNA技术，研制了与真实病毒颗粒在成分组成以及形态结构上相似，但无生物传染性风险的流感病毒（甲型H5N6）假病毒标准物质。本项目研制过程参照国家计量技术规范JJF1343-2022《标准物质的定值及均匀性、稳定性评估》文件以及相关技术文件执行，对标准物质进行了均匀性、稳定性研究，并由在测定标准物质特性值方面具有必备条件以及同等技术能力和经验的9家单位，采用数字PCR方法对标准物质M基因、HA基因以及NA基因浓度特性值进行测量，确定其标准值。均匀性评估结果显示，本标准物质序列间和单元间无统计学差异，表明标准物质均匀性良好。稳定性评估表明本标准物质在（2～8）℃、（20~25）℃以及37 ℃环境中可稳定7天；在（-20±5）℃环境中可稳定6个月。综合考虑均匀性、稳定性以及定值过程引入的不确定度评定本项目标准物质的总不确定度。最终，本项目标准物质M基因、HA基因、NA基因的标准值±扩展不确定度分别为：（2.7 ± 0.7）×10² copies/µL，（7.4 ± 1.6）×10² copies/µL，（2.7 ± 0.7）×10² copies/µL。本项目标准物质可作为测量标准用于流感病毒（甲型H5N6）核酸定性、定量检测，适用于检测方法验证、产品开发与评价，及实验室质量控制等领域。

综上所述，本研究首次开展了全国UU核酸检测室间质量评价研究，评估了目前我国实验室对于UU核酸检测的现状以及不同检测试剂的分析性能，为提高UU的检测质量提供了有效的措施和建议，有利于推进UU核酸检测的标准化。此外，本研究成功研制了流感病毒（甲型H5N6）假病毒标准物质，丰富了甲型流感病毒H5N6亚型毒株标准物质的储备，为实验室开展甲型H5N6流感病毒核酸定性、定量检测提供了测量标准，为核酸检测试剂的开发与评价、检测方法验证、实验室质量控制等领域奠定了研究基础。

Ureaplasma urealyticum (UU) commonly colonizes the urogenital tract of healthy individuals and is generally regarded as a part of the normal human urogenital flora. Consequently, researchers often underestimate its pathogenicity and rarely prioritize routine testing. However, since UU can be isolated from both healthy and diseased individuals, it is also considered an opportunistic pathogen. Mounting evidence links the pathogenicity of UU to human reproductive health, particularly in obstetrics and gynecologic outpatients and infertile couples, for whom routine UU screening is strongly recommended. Currently, DNA-based nucleic acid amplification tests (NAATs) and RNA-based NAATs for UU detection have been extensively applied in clinical settings, and many assays have been approved by the National Medical Products Administration (NMPA). Although previous studies have evaluated their analytical performance, the actual testing capabilities of laboratories using these reagents in clinical applications remains unclear. Therefore, the first part of this study conducted an external quality assessment (EQA) scheme to evaluate the performance of molecular detection of UU among clinical laboratories, and to provide recommendations for improving the quality and the standardization of UU testing. The EQA panel included 9 positive samples (inactivated UU culture supernatants) and 3 negative samples (sterile saline). The positive samples were further divided into high, moderate, and low concentration groups. A total of 365 laboratories participated in this EQA scheme, with 360 results collected from 338 participants. Out of the 360 datasets, the performance was deemed competent in 96.11% (346/360) of the analyses, which submitted from 95.86% (324/338) laboratories. For high and moderate concentration samples, the positive percentage agreement (PPA) of all participants were exceeded 97.5%, whereas PPA for low concentration samples varied significantly, decreasing from 86.94% to 51.94% as the sample concentration declined. Moreover, for low concentration samples, the participating laboratories using RNA-based NAATs demonstrated higher PPA compared to DNA-based NAATs, but this finding was specific to UU supernatants in this study. Overall, the majority of participants can reliably detect UU samples with high and moderate concentrations, while the poor analytical performance for low concentration samples requires further improvement and optimization.

Viral nucleic acid reference materials (RMs) are crucial metrological tools for ensuring the accuracy and traceability of measurement results. They also serve as a fundamental basis for guaranteeing the comparability of detection results across laboratories, reagents and methodologies. In the second part of this study, a non-infectious pseudovirus RM of influenza A (H5N6) virus, closely mimicking authentic viral particles in composition and morphology, was developed using a well-established MS2 bacteriophage-based exogenous RNA packaging technique. The production process adhered to the guidelines outlined in the National Metrological Technical Specification JJF 1343-2022, “Characterization, Homogeneity and Stability Assessment of Reference Materials”, and other relevant technical documents. The homogeneity and stability of the RM were validated, and certified values for matrix (M), hemagglutinin (HA), and neuraminidase (NA) genes were determined via droplet digital PCR by 9 qualified laboratories. Homogeneity tests revealed no statistically significant differences between or within the units, indicating satisfactory homogeneity. Stability tests demonstrated that the RM remained stable for 7 days at (2–8) °C, (20–25) °C, and 37 °C, and for 6 months at (-20±5) °C. A comprehensive evaluation, considering the uncertainties introduced during the homogeneity, stability, and characterization processes, was conducted to determine the overall uncertainty of the RM. The final certified values ± expanded uncertainty for the M, HA and NA genes of the RM were determined to be (2.7 ± 0.7) × 10² copies/μL, (7.4 ± 1.6) × 10² copies/μL, (2.7 ± 0.7) × 10² copies/μL, respectively. This RM can serve as a measurement standard for qualitative and quantitative detection of influenza A (H5N6) virus, and is applicable to method validation, reagent development and evaluation, and laboratory quality control.

In summary, this study represents the first nationwide EQA for molecular detection of UU in China, assessing the current laboratory performance and testing capabilities among laboratories, as well as the analytical performance of commercially available kits. The findings provide recommendations for enhancing the quality of UU testing, thereby promoting the standardization of molecular detection of UU. Furthermore, this research successfully developed a candidate pseudovirus RM for influenza A (H5N6) virus, expanding the available resources for the H5N6 subtype. This RM provides a critical measurement standard for laboratories conducting qualitative and quantitative detection of influenza A (H5N6) virus, and establishes a foundational resource for reagents development and evaluation, assay validation, and laboratory quality assurance.