Development and analytical validation of a novel nasopharynx swab-based Epstein-Barr virus C promoter methylation quantitative assay for nasopharyngeal carcinoma detection
Author:
Wu Zhi-Cong12ORCID, Lin Ke-Na3, Li Xue-Qi124, Ye Xin124, Chen Hua12, Tao Jun5, Zhou Hang-Ning12, Chen Wen-Jie12, Lin Dong-Feng12, Xie Shang-Hang12, Cao Su-Mei12
Affiliation:
1. Department of Cancer Prevention , Sun Yat-sen University Cancer Center , Guangzhou , P.R. China 2. State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy , Sun Yat-sen University Cancer Center , Guangzhou , P.R. China 3. School of Public Health , Guangdong Medical University , Dongguan , Guangdong , P.R. China 4. School of Public Health , Sun Yat-sen University , Guangzhou , P.R. China 5. School of Public Health, Li Ka Shing Faculty of Medicine , University of Hong Kong , Hong Kong SAR , P.R. China
Abstract
Abstract
Objectives
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.
Methods
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.
Results
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 %).
Conclusions
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.
Funder
The national Key Research and Development Program of China/Award Number National Natural Science Foundation of China Natural Science Foundation of Guangdong Province
Publisher
Walter de Gruyter GmbH
Subject
Biochemistry (medical),Clinical Biochemistry,General Medicine
Reference45 articles.
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