针对鼻咽癌的检测，我们开发并验证了一种新的以鼻咽拭子为基础的Epstein-Barr病毒C启动子甲基化定量分析方法。
Development and analytical validation of a novel nasopharynx swab-based Epstein-Barr virus C promoter methylation quantitative assay for nasopharyngeal carcinoma detection.
发表日期：2023 Aug 04
作者：
Zhi-Cong Wu, Ke-Na Lin, Xue-Qi Li, Xin Ye, Hua Chen, Jun Tao, Hang-Ning Zhou, Wen-Jie Chen, Dong-Feng Lin, Shang-Hang Xie, Su-Mei Cao
来源：
Cellular & Molecular Immunology
摘要：
依据文献报道，喉咽癌（NPC）细胞上域-巴尔病毒（EBV）C启动子（Cp）高甲基化是EBV潜伏感染喉咽上皮细胞的关键因素，并被确认为NPC检测的有希望的生物标志物。本研究开发了一种新的EBV Cp甲基化定量（E-CpMQ）检测方法，并评估了其在NPC检测中的诊断性能。该检测方法是通过结合改良的MethyLight和QASM技术开发的一种新的qPCR方法，同时定量检测甲基化和非甲基化的EBV Cp，使用创新的校准物提高检测准确性和一致性。本研究使用鼻咽拭子样本和合成标准物验证了E-CpMQ的分析性能，同时在137例NPC患者和137例非NPC对照中验证了该检测方法的诊断效能。E-CpMQ检测方法可以在10个拷贝的条件下检测EBV Cp的甲基化比率，并且具有100％的识别特异性，与焦磷酸测序的相关系数超过0.99。经校准的E-CpMQ检测降低了变异系数的平均值55.5％，总方差低于0.06单位标准偏差（SD）。线性甲基化比率检测范围为4.76％至99.01％。其敏感度和特异度分别为96.4％（95％CI：91.7-98.8％）和89.8％（95％CI：83.5-94.3％）。所开发的带有校准物的E-CpMQ检测方法可以准确可重复地定量EBV Cp的甲基化比率，为NPC的早期检测提供了一种敏感、特异、经济实用的方法。© 2023 Walter de Gruyter GmbH, Berlin/Boston.
Epstein-Barr virus (EBV) C promoter (Cp) hypermethylation, a crucial factor for EBV latent infection of nasopharyngeal epithelial cells, has been recognized as a promising biomarker for nasopharyngeal carcinoma (NPC) detection. In this study, we develop a novel EBV Cp methylation quantification (E-CpMQ) assay and evaluate its diagnostic performance for NPC detection.A novel qPCR assay for simultaneous quantification of methylated- and unmethylated EBV Cp was developed by the combinational modification of MethyLight and QASM, with an innovative calibrator to improve the detection accuracy and consistency. The NP swab samples and synthetic standards were used for the analytical validation of the E-CpMQ. The diagnostic efficacy of the developed E-CpMQ assay was validated in 137 NPC patients and 137 non-NPC controls.The E-CpMQ assay can detect the EBV Cp methylation ratio in one reaction system under 10 copies with 100 % recognition specificity, which is highly correlated to pyrosequencing with a correlation coefficient over 0.99. The calibrated E-CpMQ assay reduces the coefficient of variation by an average of 55.5 % with a total variance of less than 0.06 units standard deviation (SD). Linear methylation ratio detection range from 4.76 to 99.01 %. The sensitivity and specificity of the E-CpMQ respectively are 96.4 % (95 % CI: 91.7-98.8 %), 89.8 % (95 % CI: 83.5-94.3 %).The developed E-CpMQ assay with a calibrator enables accurate and reproducible EBV Cp methylation ratio quantification and offers a sensitive, specific, cost-effective method for NPC early detection.© 2023 Walter de Gruyter GmbH, Berlin/Boston.