Optimizing Epstein-Barr Virus DNA Testing in Nasopharyngeal Carcinoma: A Systematic Evaluation

This study explores optimized Epstein-Barr Virus (EBV) DNA detection protocols by evaluating key factors such as sample type, storage conditions, nucleic acid extraction methods, and PCR targets to provide a scientific foundation for standardized testing. EBV DNA decay rates were compared using 42 paired plasma–serum samples, assessing the effect of different storage conditions (4 °C vs. −20 °C). Nucleic acids were extracted from 30 NPC plasma samples using both magnetic bead–based and column-based methods, comparing their positive detection rates. 104 clinical samples were tested for the BamHI-W and EBNA-1 targets, comparing accuracy, sensitivity, specificity, and AUC. The 95% LoD for both targets was assessed using WHO standards and validated with 20 additional samples. Plasma samples stored at −20 °C showed significantly lower EBV DNA decay rates compared to those stored at 4 °C (P < 0.05). In serum samples, the advantage of −20 °C storage was limited to 3 days. Magnetic bead–based extraction had a higher positive detection rate (86.7%) compared to column-based extraction (73.3%), but no statistically significant difference was observed (P=0.125). For PCR targets, the BamHI-W target showed an accuracy of 96.1%, sensitivity of 95.2%, specificity of 97.0%, AUC of 0.976, and 95% LoD of 7.4 IU/mL. The EBNA-1 target had an accuracy of 92.3%, sensitivity of 87.3%, specificity of 100%, AUC of 0.937, and 95% LoD of 119 IU/mL. Both targets showed over 95% concordance in clinical sample validation.Optimizing EBV DNA detection requires refinement across all procedural stages. Plasma sample storage at −20°C is recommended to minimize nucleic acid degradation. The magnetic bead–based extraction method, due to its automation and high throughput, is suitable for routine clinical use and large-scale studies. The BamHI-W target, with high sensitivity and lower detection limits, is ideal for screening high-risk populations, while the EBNA-1 target, with higher specificity, is more suitable for efficacy assessment and risk stratification. PCR target selection should be flexible based on specific clinical needs rather than confined to a single target.