目的：

沙眼衣原体（Chlamydia trachomatis，CT）感染是全球范围内最常见的性传播感染（sexually transmitted infections，STIs）之一，可引发盆腔炎、宫颈炎及宫外孕等严重并发症，甚至导致不孕症。因此，早期、精准的检测对于CT感染的防控至关重要。世界卫生组织推荐采用高灵敏度、高特异性的核酸扩增检测（nucleic acid amplification tests，NAATs）作为CT感染的标准诊断方法。然而，CT NAATs方法的建立、性能验证、室内质控及室间质评均依赖于接近临床样本的高质量质控品（quality control materials, QCs）。目前市售的CT QCs在均一性、稳定性和可再生性方面存在局限性，在一定程度上影响了NAATs检测的可靠性和可比性。因此研发一种可互换、生物安全、可再生且长期稳定的CT QCs，对提升CT核酸检测的质量控制水平具有重要意义。成簇规律间隔短回文重复序列/CRISPR相关蛋白（clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins，CRISPR/Cas）基因编辑技术已广泛应用于临床突变基因分子检测质控品的制备，可精准地将特定靶标序列整合至野生型细胞系。本研究拟利用CRISPR/Cas9技术，构建稳定整合CT DNA序列的HEK293T细胞系，并基于该细胞系制备符合CT NAATs质量控制要求的质控品，以优化CT感染的实验室检测质量管理。

方法：

本研究通过设计特异性的单链引导RNA（single guide RNA，sgRNA），引导Cas9核酸酶在HEK293T细胞的粘蛋白4（mucin 4，MUC4）基因位点产生DNA双链断裂（double strand breaks，DSBs），并利用非同源末端连接机制（non-homologous end joining，NHEJ）插入包含CT隐蔽质粒全长及主要外膜蛋白（major outer membrane protein，MOMP）基因全长（9,136 bp）的CT DNA序列。通过流式细胞分选、单细胞培养及PCR鉴定等方法筛选阳性细胞克隆，并建立稳定整合CT DNA序列的细胞系。随后，采用实时荧光定量PCR（quantitative real-time PCR，qPCR）方法测定阳性克隆细胞内CT DNA序列的插入拷贝数，并优化细胞保存液以提高细胞质控品的存储稳定性。此外，对质控品的均一性、稳定性和通用性进行系统验证。

结果：

1. CRISPR/Cas9编辑构建CT DNA整合细胞系：通过流式细胞分选获得480个单细胞克隆，经两周培养后，其中167个克隆成功扩增形成单克隆细胞株。PCR结合琼脂糖凝胶电泳分析显示，4株单克隆细胞整合了CT DNA序列的5′端，1株整合了CT DNA序列的3′端及全长。最终，成功构建了一株稳定整合CT DNA序列的HEK293T细胞系。

2. qPCR测定CT DNA阳性克隆细胞系的插入拷贝数：通过测定CT基因和GAPDH基因的Ct值，并基于连续分子摩尔比的标准品构建标准曲线，分析CT基因与GAPDH基因的Ct值差与分子摩尔对数比值的关系（y = -0.98x + 0.58）。阳性克隆细胞系中CT基因和GAPDH基因的Ct值分别为24.44和23.26，计算表明CT DNA序列为单拷贝插入。

3. 优化细胞保存条件提升质控品稳定性：37℃初步稳定性评价显示，PBS组和DMEM组质控品稳定性较差，保存液组质控品可能因保存液组分而影响核酸扩增效率。优化组质控品在初步稳定性和冻融稳定性评价中均表现出较高的稳定性和可靠性。

4. 质控品的均一性、稳定性及通用性验证：均一性验证显示，质控品具有良好的同质性。稳定性验证显示，质控品在-20℃、4℃和25℃条件下可稳定保存至少2个月，在37℃条件下可稳定保存至少2周。通用性验证显示，质控品在8种国家药品监督管理局（national medical products administration，NMPA）批准的CT NAATs试剂盒及9家实验室的检测中均表现出良好的检测一致性。

结论：

本研究成功构建了一株稳定整合CT DNA序列的HEK293T细胞系，并基于此开发了一种新型CT核酸检测质控品。与现有质控品相比，该质控品在通用性、均一性和长期稳定性方面表现优异，同时具备可再生性和生物安全性。该质控品的应用有望提高CT核酸检测的可靠性，优化临床检测的质量控制体系，并为CT感染的准确诊断提供支持。

Objective:

Chlamydia trachomatis (CT) infection is one of the most prevalent sexually transmitted infections (STIs) worldwide, which can lead to severe complications such as pelvic inflammatory disease, cervicitis, ectopic pregnancy, and even infertility. Therefore, early and precise detection is essential for effective prevention and control of CT infection. The World Health Organization (WHO) recommends nucleic acid amplification tests (NAATs) with high sensitivity and specificity as the standard diagnostic method for CT infection. However, the establishment of methods, performance validation, internal quality control (IQC), and external quality assessment (EQA) for CT NAATs necessitate the utilization of quality control materials (QCs) that closely resemble clinical samples. Currently available commercial CT QCs exhibit limitations in homogeneity, stability, and reproducibility, which may compromise the reliability and comparability of CT NAATs. Therefore, developing a commutable, biologically safe, reproducible, and long-term stable CT QC is critical for improving the quality control of CT nucleic acid assays. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) gene-editing technology has been widely applied in introducing specific target sequences into wild-type cell lines for QCs preparation in mutant gene detection. In this study, we employed CRISPR/Cas9 technology to construct a HEK293T cell line that stably integrates CT DNA sequence. Based on this cell line, we developed a CT QC that meets the quality control requirements of CT NAATs, aiming to enhance the quality management of CT laboratory diagnostics.

Methods:

In this study, we designed specific single-guide RNA (sgRNA) to direct Cas9 to induce DNA double-strand breaks (DSBs) at the mucin 4 (MUC4) gene locus in HEK293T cells. Using a non-homologous end-joining (NHEJ) mechanism, CT DNA sequence containing the full-length cryptic plasmid and the full-length major outer membrane protein (MOMP) gene (9,136 bp) were inserted. Positive cell clones were screened and validated through flow cytometry, single-cell culture, and PCR identification, leading to the establishment of stable cell lines. Quantitative real-time PCR (qPCR) was performed to determine the insertion copy number of CT DNA sequence in the positive clones. To improve the storage stability of cellular QCs, an optimized preservation solution was developed and evaluated. Additionally, homogeneity, stability, and commutability assessments were conducted to validate QCs performance.

Results:

1. Generation of CT DNA-integrated cell lines via CRISPR/Cas9 editing: A total of 480 single-cell clones were obtained by flow sorting, and 167 expanded into monoclonal cell lines after two weeks of culture. PCR and agarose gel electrophoresis analysis confirmed CT DNA sequence integration in monoclonal cell lines: four integrating the 5′ end sequence and one integrating the 3′ end and full-length sequence. Finally, a HEK293T cell line stably integrating the CT DNA sequence was successfully established.

2. Determination of insertion copy number in positive clones: A standard curve was generated to assess the relationship between the Ct value difference of CT and GAPDH genes and the molecular molar logarithmic ratio (y = -0.98x + 0.58). The Ct values for CT and GAPDH genes in positive clones were 24.44 and 23.26, respectively, indicating a single-copy integration of the CT DNA sequence.

3. Optimization of cell preservation to enhance QCs stability: Preliminary stability assessment at 37℃ demonstrated that QCs stored in PBS and DMEM exhibited poor stability, and QCs stored in the preservative solution demonstrated potential nucleic acid amplification inhibition. In contrast, the optimized group of QCs showed excellent stability and reliability in both the preliminary stability and freeze-thaw stability assessments.

4. Validation of QCs homogeneity, stability, and commutability: homogeneity verification showed that the QCs exhibited good consistency. Stability verification showed that the QCs could be stably stored for at least 2 months at -20℃, 4℃ and 25℃, and for at least 2 weeks at 37℃. Commutability verification showed that the QCs performed good assay consistency in eight China National Medical Products Administration (NMPA)-approved kits and nine laboratories.

Conclusion:

This study successfully established a HEK293T cell line stably integrating CT DNA sequence and developed a novel QC for CT NAATs. Compared with existing QCs, the newly developed QC exhibits superior commutability, homogeneity and long-term stability, as well as reproducibility and biosafety. Its application is expected to enhance the reliability of CT nucleic acid testing, optimize the quality control system for clinical diagnostics, and support the accurate diagnosis of CT infection.