【摘要】 目的 探索基于拉曼光谱分析技术建立快速筛查高危型人乳头瘤病毒（HPV）感染的方法，并通过临床样本验证其可行性及有效性。方法 选取南京医科大学附属宿迁第一人民医院门诊就诊的300例女性患者，根据HPV DNA检测结果分为HPV阴性对照组（n=100）、HPV16/18阳性组（n=100）和其他高危型HPV阳性组（n=100）。采用枸橼酸钠还原法合成金纳米颗粒，用4-巯基苯甲酸（4-MBA）、4-氨基硫代苯酚（4-ATP）和2-萘硫醇（2-NT）三种拉曼报告分子标记，再偶联针对HPV16 L1蛋白、HPV18 L1蛋白和通用高危型HPV E6/E7蛋白的单克隆抗体，构建三种拉曼活性探针。采用共聚焦拉曼光谱仪采集样本光谱，通过支持向量机（SVM）建立HPV分型模型。对临床标本进行检测并与传统HPV DNA检测方法进行比较及性能评价。结果 三种拉曼活性探针光谱特性良好，特征峰无重叠。构建的SVM模型分类准确度为93.0%，对HPV16、HPV18及其他高危型HPV的分型准确度分别为95.2%、93.7%和91.4%。受试者工作特征（ROC）曲线分析显示，检测曲下面积（AUC）值分别为0.978、0.962和0.943。拉曼光谱检测方法的灵敏度、特异度、准确度分别为92.5%、94.3%、93.3%,Kappa系数为0.865。高危型HPV检测下限为0.1IU/ml，变异系数为4.3%；温度（4℃～37℃）稳定性测试无显著差异（P>0.05）；干扰试验显示，血红蛋白和白蛋白信号偏差分别控制在8%和6%以内，白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)信号偏差分别小于7%和6%。连续30天监测显示，探针拉曼信号强度衰减率为3.8%，稳定性良好。检测时间缩短至45.3±6.7min，成本降至每样本4元。结论 基于拉曼光谱分析技术的高危HPV检测方法具有良好的灵敏度、特异度和分型能力，与传统方法相比，具有检测时间短、成本低、特异度高等优势，具备广阔的临床应用前景。更多还原

【Abstract】 Objective To explore a rapid screening method for high-risk human papillomavirus(HPV) infection based on Raman spectroscopy analysis and validate its feasibility and effectiveness using clinical samples. Methods A total of 300 female outpatients from Suqian First People’s Hospital Affiliated to Nanjing Medical University were enrolled and divided into three groups according to HPV DNA test results: HPV-negative control group(n=100), HPV16/18-positive group(n=100), and another high-risk HPV-positive group(n=100). Gold nanoparticles were synthesized using the sodium citrate reduction method, labeled with three Raman reporter molecules: 4-mercaptobenzoic acid(4-MBA), 4-aminothiophenol(4-ATP), and 2-naphthyl mercaptan(2-NT), and then conjugated with monoclonal antibodies targeting HPV16 L1 protein, HPV18 L1 protein, and pan-high-risk HPV E6/E7 proteins to construct three Raman-active probes. Confocal Raman spectroscopy was used to collect sample spectra, and a support vector machine(SVM) was used to establish an HPV genotyping model. Clinical specimens were detected and compared and performance evaluated with conventional HPV DNA detection methods. Results The three Raman-active probes showed excellent spectral characteristics with no overlapping characteristic peaks. The SVM model achieved an overall classification accuracy of 93.0%, with genotyping accuracies of 95.2%, 93.7%, and 91.4% for HPV16, HPV18, and other high-risk HPV types, respectively. ROC curve analysis showed AUC values of 0.978, 0.962, and 0.943. The Raman spectroscopy method exhibited a sensitivity of 92.5%, specificity of 94.3%, accuracy of 93.3%, with a Kappa coefficient of 0.865. The detection limit for high-risk HPV was 0.1 IU/mL with good reproducibility(coefficient of variation: 4.3%). No significant difference was observed in temperature stability tests(4～37 ℃, P>0.05). Interference tests indicated signal deviations for hemoglobin and albumin were within 8% and 6%, respectively, while those for IL-6 and TNF-α were below 7% and 6%. Continuous 30-day monitoring indicated the Raman signal intensity decay rate was 3.8%, indicating good stability. The detection time was shortened to 45.3±6.7 minutes, and cost decreased to 4 yuan per sample. Conclusions The high-risk HPV detection method based on Raman spectroscopy has good sensitivity, specificity, and genotyping capability. Compared with traditional methods, it offers has advantages such as shorter detection time, lower cost, and higher specificity, presenting broad clinical application prospects.更多还原