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

Optimizing Epstein-Barr Virus DNA Testing in Nasopharyngeal Carcinoma: A Systematic Evaluation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 10 February 2026 V1 Latest version Share on Optimizing Epstein-Barr Virus DNA Testing in Nasopharyngeal Carcinoma: A Systematic Evaluation Authors : Guojing Wang , Jing Wang , Quanquan Gao , Yuzhao Jiang , Baojun Wei , Xinxiong Zeng , and Wei Cui [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.177069999.98992344/v1 206 views 74 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract 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. Title: Optimizing Epstein-Barr Virus DNA Testing in Nasopharyngeal Carcinoma: A Systematic Evaluation Authors: Baojun Wei 1# | Xinxiong Zeng 1,2# | Quanquan Gao 1 | Yuzhao Jiang 1 | Jing Wang 1 | Guojing Wang 1 | Wei Cui 1 * 1 Department of Clinical Laboratory, State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100021, China. | 2 Beijing Huairou Hospital, No.9, Yongtai North Street, Beijing, 101400, China # Baojun Wei and Xinxiong Zeng are contributed equally to this work and are regarded as co-first authors *Correspondence :Wei Cui 1 *（ [email protected] ） Funding ：This work was supported by the National Key Research and Development Program of China 2024YFC2419105. Keywords： Nasopharyngeal carcinoma | EBV DNA | Real-time PCR. ABSTRACT 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. 1 | Introduction Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor arising from the nasopharyngeal mucosa. NPC is highly prevalent in southern China and Southeast Asia, accounting for more than 70% of global cases [1–3].In southern China, more than 95% of undifferentiated non-keratinizing NPC cases are associated with Epstein–Barr virus (EBV) infection [4,5].Given the diagnostic challenges of early-stage NPC due to subtle clinical symptoms and its deep anatomical location, a large prospective cohort study by Chan et al. [6] demonstrated that quantitative plasma EBV DNA testing shows high sensitivity (97.1%) and specificity (98.6%) for early NPC detection.These findings indicate that plasma EBV DNA detection is currently one of the most clinically valuable screening tools for NPC. However, the clinical application of EBV DNA testing remains limited by insufficient standardization. Multiple studies have reported substantial variability in sensitivity (53%–96%) across different experimental designs [7,8].Comparative analyses suggest that this variability primarily arises from inconsistencies in testing procedures, including sample type, storage conditions, nucleic acid extraction methods, and PCR target genes.To improve the homogeneity of EBV DNA detection, the establishment of a unified and standardized workflow is urgently needed.This study systematically evaluated key aspects of EBV DNA detection including sample type, storage conditions, nucleic acid extraction methods, and PCR target genes to explore an optimized protocol, which establishes an experimental basis for developing future standardized testing protocols. 2 | Materials and Methods 2.1 | Study Population and Samples Samples were collected from between March 2024 and February 2025 including EDTA-anticoagulated plasma samples from 175 patients with NPC, paired serum samples from 42 of these patients, and EDTA-anticoagulated plasma samples from 41 healthy individuals. The resulting plasma or serum was aliquoted and stored at 4 °C or −20 °C according to the requirements of different experimental stages. Sample testing was conducted in three parts. Part 1 evaluated sample type, storage temperature, and storage duration using paired plasma and serum samples from 42 patients. Part 2 involved the random selection of 30 plasma samples from patients with NPC for parallel comparison of two nucleic acid extraction methods. Part 3 used 63 NPC plasma samples and 41 control plasma samples to preliminarily assess the sensitivity and specificity of single-copy and multi-copy targets. Subsequently, 20 additional distinct clinical samples with low copy numbers were included for each target to validate the 95% LoD explored in this study. 2.2 | Materials and Equipment The magnetic bead–based nucleic acid extraction reagents were purchased from Xi’an Tianlong Technology Co., Ltd., whereas the column-based nucleic acid extraction reagents were obtained from Tiangen Biochemical Co., Ltd. Real-time quantitative PCR was performed using the ABI 7500 real-time PCR system (Applied Biosystems, USA).The real-time quantitative PCR (qPCR) detection kit targeting the BamHI-W region was obtained from Shengxiang Biotechnology Co., Ltd., whereas the qPCR detection kit targeting the EBNA-1 region was obtained from Guangzhou Da’an Gene Co., Ltd. And the qPCR detection kit targeting the EBNA-LP region from Xi’an Tianlong Biotechnology Co., Ltd.was used. The EBV reference material used was the WHO International Standard for EBV (NIBSC code: 09/260).This standard contains the full-length viral genome of EBV strain B95-8 (type 1) at a fixed concentration of 6.7 log10 IU/mL (5 × 10⁶ IU/mL).For use, the standard was reconstituted in 1.0 mL of nuclease-free water to achieve a final concentration of 6.7 log10 IU/mL, in accordance with the manufacturer’s instructions. 2.3 | Pre-test Variable Optimization A total of 42 plasma samples and 42 paired serum samples were collected from patients with pathologically diagnosed NPC at our hospital.All paired serum and plasma samples were tested on the day of collection, and the results were recorded as baseline (Day 1) data. Subsequently, serum and plasma samples from each patient were aliquoted into six portions (200 μL per portion). Each set of aliquots was assigned according to storage temperature, with three portions stored at 4 °C and three portions stored at −20 °C. On days 3, 5, and 7 of storage, one aliquot from each temperature group was tested for nucleic acid extraction and amplification, and the results were recorded. EBV DNA extraction and amplification were performed using the instruments and supporting reagents from Xi’an Tianlong Technology Co., Ltd. All procedures were performed strictly in accordance with the manufacturer’s instructions for both the instruments and the kits.The EBV DNA decay rate was calculated using the formula below, and statistical analysis was performed to compare the differences in decay rates. Formula: Decay rate\(\text{=}\frac{\text{log10（}\text{concentration\ t₁}\text{）-log10（}\text{concentration\ tn}\text{）}}{\text{t₁}\text{-tn}}\) Where t₁ is the starting time (Day 1) and tn is the ending time (Day 3, Day 5, Day 7), enabling analysis of each factor’s influence. 2.4 | Optimal Nucleic Acid Extraction Method Selection Based on the optimal sample storage conditions identified in Phase I, plasma samples from 30 patients with NPC diagnosed were collected and stored for subsequent analysis. EBV DNA extraction was performed on all samples using two commonly used nucleic acid extraction methods (magnetic bead–based and column-based methods). The column-based method employed a genomic DNA extraction kit from Tiangen Biochemical Technology Co., Ltd. with fully manual operation, whereas the magnetic bead–based method employed a genomic DNA extraction kit from Xi’an Tianlong Biotechnology Co., Ltd. with an automated nucleic acid extraction system. Subsequent nucleic acid amplification was performed using qPCR reagents from Xi’an Tianlong Biotechnology Co., Ltd. The analytical limit of detection of the amplification reagent (500 copies/mL) was used as the cutoff value; results below this threshold were classified as negative, whereas those equal to or above this threshold were classified as positive. Differences between the two extraction methods were assessed by comparing their positive detection rates. Additionally, for samples testing positive by both methods, EBV DNA levels (expressed as log10 copies/mL) were further compared to facilitate a comprehensive evaluation of the relative performance of each method. 2.5 | EBV Genomic Target Evaluation Integrating the optimized results from Phases I and II, a three-part assessment was conducted for the two EBV genomic targets, BamHI-W and EBNA-1: (1) detection in 104 clinical samples; (2) exploration of the 95% LoD for both targets; and (3) clinical validation of the lower 95% LoDs. For clinical performance evaluation, residual plasma samples from 63 patients with confirmed NPC were included as the NPC group, and residual plasma samples from 41 healthy individuals undergoing routine physical examinations were included as the control group. All 104 samples underwent EBV DNA detection, with values > 0 copies/mL considered positive. Accuracy, sensitivity, specificity, and AUC were calculated and compared for both targets based on the results. The 63 NPC samples were further stratified by viral load concentration to analyze changes in sensitivity, specificity, and AUC across different concentration ranges, thereby evaluating detection performance at varying viral load levels. For 95% LoD exploration, EBV-negative plasma was diluted with the WHO International Standard for EBV. Both targets (BamHI-W and EBNA-1) shared the same serial dilution series: 400, 200, 100, 50, 25, 12.5, and 6.25 IU/mL.Each dilution level was tested in 10 replicates, with 0 copies/mL as the cutoff for positive and negative determination. The positive detection rate at each concentration was calculated, and the 95% LoD for both targets was estimated using Probit regression analysis. For clinical validation of the 95% LoDs, 20 plasma samples from patients with NPC with viral loads near the corresponding 95% LoD were selected for each target (original concentrations were obtained at the initial clinical visit using Tianlong reagents). Each sample was assayed repeatedly for 4 times. Results > 0 copies/mL were considered positive. The stability of the assay 95% LoD was evaluated based on the frequency of results approximating the original concentrations. For Tianlong reagents,unit conversion was performed using the manufacturer- provided conversion factor (1 IU = 3.5 copies) for clinical sample selection. 2.6 | Statistical Analysis Statistical analyses were performed using GraphPad Prism version 9.0.The Wilcoxon signed-rank test was used to compare differences in EBV DNA decay rates across sample types (serum vs. plasma), storage durations (days 1–3, 1–5, and 1–7), and storage temperatures (4 °C vs. −20 °C), with the level of statistical significance defined as a P value < 0.05.McNemar’s chi-square test was used to assess differences in positive detection rates between the two nucleic acid extraction methods (column-based vs. magnetic bead–based).The chi-square (χ²) value was calculated, and the exact P value was reported.Probit regression analysis was used to estimate the 95% LoD for both targets. 3 | Results 3.1 | Optimization of Pre-Analytical Variables Effect of Sample Type on Nucleic Acid Stability: Under identical storage temperatures (4°C or −20°C), no significant difference was observed in the decay rate of EBV DNA load between plasma and serum samples over the 1–7-day storage period (Figure 1A,1B).These findings suggest that sample type (serum vs. plasma) is not an independent factor affecting nucleic acid stability, and that the two sample types exhibit equivalent performance under clinical storage condition. Effect of Storage Temperature on Nucleic Acid Stability: For plasma samples, the EBV DNA load decay rate over the entire1-7 days storage period was significantly higher in the 4°C group than in the −20°C group (P < 0.05).These results demonstrate that storage at −20°C effectively inhibits plasma nucleic acid degradation and represents an optimized condition for long-term plasma storage. For serum samples, during short-term storage (1-3 days), the EBV DNA load decay rate was significantly higher in the 4°C group than in the −20°C group (P = 0.012). However, during longer storage intervals (1-5 days and 1-7 days), no statistically significant difference was observed between the two groups (P > 0.05).These findings indicate that the storage advantage of −20°C for serum samples is limited to a relatively short time duration (Figure 2A,2B). 3.2 | Optimal Nucleic Acid Extraction Method Among the 30 patients with pathologically confirmed NPC, the magnetic bead–based method showed an EBV DNA positivity rate of 86.7% (26/30), compared with 73.3% (22/30) for the column-based method. McNemar’s chi-square test indicated no statistically significant difference in positive detection rates between the two nucleic acid extraction methods (χ² = 2.250, P = 0.125). Overall analysis suggested that the magnetic bead–based method did not exhibit a statistically significant overall advantage over the column-based method. In this study, a cutoff value of 500 copies/mL was used as the clinical threshold for EBV DNA positivity, and EBV DNA load values were obtained for all samples. Further analysis showed that in 93.3% (28/30) of samples, the magnetic bead–based method detected higher EBV DNA loads than the column-based method (Figure 3). Among low-load samples (<10³ copies/mL), the column-based method identified 12 samples (40.0%, 12/30), of which 7 (58.3%, 7/12) fell below the positivity threshold (500 copies/mL). In contrast, the magnetic bead–based method identified 7 low-load samples (23.3%, 7/30), among which 4 (57.1%, 4/7) were below this threshold. Among the 12 low-load samples identified by the column-based method, 5 (41.7%, 5/12) showed EBV DNA loads ≥10³ copies/mL when tested using the magnetic bead–based method. This finding is consistent with the overall upward shift observed in EBV DNA values obtained using the magnetic bead–based method, suggesting that this method may offer greater nucleic acid enrichment efficiency in samples with low viral loads. Although no statistically significant difference was observed in overall clinical applicability between the two methods, the magnetic bead–based method showed greater detection capability for samples within the threshold range (500–1000 copies/mL), which may provide more reliable technical support for EBV DNA detection in patients with NPC and low viral loads. Taken together, these results suggest that magnetic bead–based detection may offer certain clinical advantages in specific clinical scenarios. 3.3 | EBV Genomic Target Evaluation 3.3.1 | Comparison of Detection Results in 104 Clinical Samples The BamHI-W target demonstrated 96.1% accuracy and 95.2% sensitivity; EBNA-1 target accuracy was 92.3% with sensitivity of 87.3% (Table 1A and Table 1B ). In accuracy and sensitivity comparisons, the BamHI-W target outperformed the EBNA-1 target. However, EBNA-1 target specificity reached 100%, slightly higher than the BamHI-W target’s 97%. Although both targets demonstrated advantages in sensitivity and specificity, the AUC value for the BamHI-W target (0.976) was higher than that for the EBNA-1 target (0.937), indicating superior overall discriminative capability for the BamHI-W target. In concentration-group analysis, the BamHI-W target demonstrated a more pronounced sensitivity advantage in the low-concentration E0–E1 group. However, no significant difference in detection capability was observed between the two targets in the high-load E2 and above groups. This result suggests that the qPCR detection method based on the BamHI-W target holds high application value for early screening of NPC with low viral load. Overall, BamHI-W target detection values consistently exceeded those of the EBNA-1 target (Figure 4), further validating its superiority in clinical sample testing. Table 1A | Comparative analysis of NPC concentration groups for BamHI-W region Group TP FP TN FN Accuracy Sensitivity Specificity AUC E1 14 1 40 3 93.1% 82.3% 97% - E2 18 0 41 0 100% 100% 100% - E3 15 0 41 0 100% 100% 100% - E4 10 0 41 0 100% 100% 100% - E5 3 0 41 0 100% 100% 100% - Total 60 1 40 3 96.1% 95.2% 97% 0.976 Table 1B | Comparative analysis of NPC concentration groups for EBNA-1 region Group TP FP TN FN Accuracy Sensitivity Specificity AUC E1 9 0 41 8 86.2% 52.9% 100% - E2 18 0 41 0 100% 100% 100% - E3 15 0 41 0 100% 100% 100% - E4 10 0 41 0 100% 100% 100% - E5 3 0 41 0 100% 100% 100% - Total 55 0 41 8 92.3% 87.3% 100% 0.937 Note: EBV DNA testing using conventional Tianlong reagents has been completed for 63 NPC samples. Samples were stratified into groups based on post-test concentration values, and each NPC concentration group was combined with a healthy control group to form a subgroup for analysis. Samples with detection values greater than 0 copies/ml were judged as positive. TP: Number of true positives; FP: Number of false positives; TN: Number of true negatives; FN: Number of false negatives. 3.3.2 | Exploration of the Minimum Detection Limit for EBV Genomic Targets and Clinical Sample Validation Probit regression analysis showed that the 95% LoD for the BamHI-W target was 7.4 IU/mL, which was markedly lower than that for the EBNA-1 target (119 IU/mL) (Table 2A and Table 2B ). These results indicate that the multicopy BamHI-W target has higher analytical sensitivity for early-stage detection, facilitating the identification of samples with low EBV DNA concentrations. As routine testing at our center uses Tianlong reagents and both targets adopt IU/mL as the 95% LoD unit, clinical samples were selected at concentrations approximating the theoretical 95% LoD values for validation based on the manufacturer’s conversion factor (1 IU = 3.5 copies): 20 blood samples at approximately 25.9 copies/mL for the BamHI-W target and 20 blood samples at approximately 427 copies/mL for the EBNA-1 target. Each sample underwent 4–5 replicate tests, except for a few samples with insufficient residual volume, which were tested in four replicates only. Validation results showed that 19 of the 20 clinical samples were positive in the BamHI-W target group, while all 20 samples were positive in the EBNA-1 target group (Table 3A and Table 3B). According to the statistical definition of the 95% LoD, one negative result is permissible among 20 tested samples. Therefore, the validation results for both the BamHI-W target (19/20) and the EBNA-1 target (20/20) met the theoretical criteria for the 95% LoD, thereby confirming the reliability of the 95% LoD estimates in clinical samples. Table 2A | Evaluation of the minimum detection limits for BamHI-W region BamHI-W region Theoretical concentration 200 IU/ml 100 IU/ml 50 IU/ml 25 IU/ml 12.5 IU/ml 6.25 IU/ml Total number of tests 10 10 10 10 10 10 Number of detections 10 10 10 10 10 9 95%minimum detection limit 7.4 IU/ml Table 2B | Evaluation of the minimum detection limits for EBNA-1 region EBNA-1 region Theoretical concentration 200IU/ml 100 IU/ml 50 IU/ml 25 IU/ml 12.5 IU/ml 6.25IU/ml Total number of tests 10 10 10 10 10 10 Number of detections 10 9 7 6 5 4 95% minimum detection limit 119 IU/ml Table 3A | Clinical sample validation of the minimum detection limits for BamHI-W region Sample Original concentration BamHI-W concentration Sample Original concentration BamHI-W concentration 1 20.25 25.8 11 20.42 28.9 19.6 8.02 28.7 46.5 29.1 11.5 2 45.01 50.5 12 21.54 62.1 34.7 45.1 44.2 146 57.6 27.9 3 34.83 18.2 13 20.79 49.4 53.5 3.52 8.13 26.4 52.8 17.5 4 32.51 47.8 14 30.84 18.2 51.3 45 77.9 41.1 78.7 13.8 5 23.47 76.6 15 34.41 93.5 83.3 52.7 99.7 33.8 35.5 54.8 6 22.44 60.2 16 31.67 20.2 59.6 9.94 37.9 24.1 22.9 15.8 7 29.48 23.3 17 21.89 0 37.2 0 83.9 15.6 11.3 12.1 8 32.62 97.6 18 27.99 126 88.8 113 125 92.1 27 34.3 9 24.16 109 19 22 37.3 43 16.1 79.1 8.69 40.3 8.99 10 20.19 148 20 29.04 15.1 209 20 113 13.7 37.1 19 Table 3B | Clinical sample validation of the minimum detection limits for EBNA-1 region Sample Original concentration EBNA-1 concentration Sample Original concentration EBNA-1 concentration 1 320.76 59.3 11 394.82 131 54.6 158 115 127 56.9 89.1 2 334.83 252 12 338.88 140 445 240 184 255 165 209 3 327.95 394 13 410 91.1 358 105 503 99.5 338 123 4 352.57 137 14 486.84 191 128 311 204 100 105 105 5 320.61 67.1 15 369.99 78.3 164 118 40.8 97 78.8 136 6 321.99 254 16 457.76 320 201 134 174 69.6 95.9 266 7 494.34 26.9 17 386.15 314 91.5 91.5 40.4 72.2 58.3 60.9 8 331.62 10.9 18 348.6 341 36.6 291 5.67 256 25.2 173 9 462.94 360 19 482.93 106 248 91.1 283 103 275 187 10 410.46 151 20 373.8 250 150 246 158 274 146 128 4 | Discussion As a key biomarker for EBV associated diseases, the sensitivity and specificity of quantitative EBV DNA detection are critical for clinical decision-making. However, substantial heterogeneity in detection protocols across laboratories and studies leads to wide variation in reported sensitivities and markedly compromises inter-laboratory reproducibility [7,9]. Comprehensive analyses indicate that variability in quantitative EBV DNA detection protocols primarily arises from four key factors: sample type, storage conditions, nucleic acid extraction methods, and PCR target gene selection. Among these factors, the choice of PCR target genes (multicopy versus single-copy) has attracted increasing attention because of its substantial impact on detection performance [10]. Meanwhile, emerging technologies such as digital PCR offer potential for assay optimization by enabling absolute quantification without the need for calibration curves. However, current studies report inconsistent findings when comparing digital PCR with qPCR [11,12], suggesting that the technical readiness of digital PCR for routine clinical application still requires further validation.In this context, the present study focuses on key sources of variability in the EBV DNA detection workflow to systematically optimize procedures, ultimately aiming to provide a scientific foundation for a future unified laboratory testing system. Studies on pre-analytical variables indicate that storage at −20 °C significantly preserves EBV DNA stability. Compared with storage at 4 °C, storage at −20 °C markedly reduces the rate of nucleic acid degradation in both plasma and serum samples. This effect is closely associated with the suppression of nuclease activity at low temperatures. Notably, the protective effect in serum samples showed marked time dependence: during short-term storage (≤3 days), storage at −20 °C significantly inhibited nucleic acid degradation compared with 4 °C (P = 0.0124); however, beyond 3 days of storage, no statistically significant difference was observed between the two temperature groups (P > 0.05). In contrast, for plasma samples, storage at −20 °C consistently exerted a significant protective effect on EBV DNA stability across all tested time points. This phenomenon can be attributed to two major factors. First, the synergistic protective effect of EDTA as an anticoagulant, which indirectly inhibits metal dependent nuclease activity by chelating Ca²⁺ and Mg²⁺ [13] and suppresses Fe²⁺ participation in the Fenton reaction, thereby reducing oxidative DNA damage mediated by free radicals [14]. Second, it may be related to the cumulative physical damage caused by ice crystal formation. Thus, serum samples not only lack the protective effects conferred by EDTA but also exhibit a gradual attenuation of the cold induced inhibitory advantage due to progressive ice crystal related damage over time. Consequently, the difference in degradation between the two temperature groups becomes insignificant during medium to long term storage. Therefore, plasma samples should be prioritized for routine clinical testing, with strict adherence to −20 °C cold chain storage to overcome the time limited protective effect observed in serum samples. In this comparative evaluation of nucleic acid extraction methods for EBV DNA detection in clinical samples, the magnetic bead–based method achieved a positivity rate of 86.7% (26/30), which was higher than the 73.3% (22/30) observed with the column-based method. However, McNemar’s test revealed no statistically significant difference in positivity rates between the two methods (P = 0.125). In contrast, Zheng Yuhong et al. [15] reported a significantly higher positivity rate for the magnetic bead method (95%) than for the column-based method (84%)，with a statistically significant difference (P < 0.05). These findings are inconsistent with the results of the present study. This discrepancy may be attributable to differences in sample size. Specifically, the study by Zheng Yuhong et al [15] . included 100 samples, whereas the present study included only 30 samples. The smaller sample size may have reduced the statistical power of the chi-square test, thereby limiting the ability to detect a statistically significant difference. Increasing the sample size may help clarify whether a statistically significant difference exists between the two methods with respect to positivity rates. Although no significant difference in positivity rates was observed between the two methods in this study, indicating comparable overall detection performance, the magnetic bead–based method demonstrated clear advantages in operational efficiency. This method enables high-throughput and automated extraction, significantly reducing processing time and minimizing the risk of manual error, thereby making it more suitable for large-scale clinical testing. Furthermore, EBV DNA load values measured using the magnetic bead–based method were generally higher than those obtained with the column-based method, particularly in samples with low viral loads. Compared with the traditional column-based method, the magnetic bead–based method utilizes nanoscale magnetic beads. The markedly increased specific surface area of these beads effectively enhances nucleic acid adsorption and enrichment. At the same time, this approach avoids nucleic acid damage caused by mechanical shear forces associated with centrifugation, thereby further improving extraction efficiency in low-concentration samples [16,17]. Taken together, these advantages in high-throughput processing, automation, and nucleic acid enrichment confer broader potential for the magnetic bead–based method in routine clinical testing and large-scale research applications. Evaluation of 104 clinical samples showed that the BamHI-W target exhibited high accuracy and sensitivity but relatively lower specificity compared with the EBNA-1 target. Moreover, across the entire concentration range, the detection values obtained for the BamHI-W target were generally higher than those for the EBNA-1 target. This difference primarily arises from the inherent copy number difference between the two targets within the EBV genome. Unlike the single-copy EBNA-1 gene, the BamHI-W target is located within the EBNA-LP region of the EBV genome as a tandem repeat sequence [18]. During qPCR, the higher number of initial templates for the BamHI-W target provides a stronger amplification basis, facilitating earlier attainment of the amplification threshold. Consequently, amplification signals are detected at earlier cycle numbers, resulting in higher analytical sensitivity. However, the number of BamHI-W repeat units varies among EBV strains [19], which may lead to quantitative inaccuracies when this target is applied across different EBV infected populations. Despite its relatively lower specificity and potential quantitative inaccuracy, the BamHI-W target retains excellent detection performance, particularly in samples with low viral loads, owing to its high sensitivity. Accordingly, it has substantial clinical utility in early screening for NPC and in monitoring high risk populations. In contrast, as a highly conserved single-copy gene, the EBNA-1 target is less affected by copy number variation and allows more accurate quantitative measurement; therefore, the EBNA-1 target is more suitable for precise monitoring of dynamic viral load changes during treatment. Using WHO reference materials, the 95% LoD of the multicopy BamHI-W target was found to be significantly lower than that of the single-copy EBNA-1 target. So the BamHI-W target has superior analytical sensitivity of. In clinical validation based on the theoretical 95% LoD, one sample was undetected by the BamHI-W target, whereas all samples were detected by the EBNA-1 target. This discrepancy indicates that, as a multicopy target, BamHI-W exhibits increased variability in detection performance near the 95% LoD when applied to clinical samples of diverse origins and matrix backgrounds, reflecting relatively limited stability. Previous studies using digital PCR have reported elevated coefficients of variation and poorer precision for the BamHI-W target in low-to-medium concentration samples [20], which is consistent with the present clinical validation findings. Comprehensive analysis of 104 clinical samples and 95% LoD experiments indicates that, although the BamHI-W target provides an extremely low detection threshold and high sensitivity, its multicopy nature is also associated with inherent limitations, including reduced quantitative accuracy, limited stability near the 95% LoD, and an increased likelihood of weak, nonspecific amplification signals arising from multiple viral templates derived from repetitive sequences in healthy individuals or non-malignant infections, which may increase false-positive rates and thereby reduce detection specificity. In contrast, the single-copy nature of the EBNA-1 target confers superior quantitative accuracy and higher specificity. In EBV DNA molecular diagnostics, target selection requires a balanced consideration of sensitivity, specificity, and quantitative accuracy. The clinical value of EBV DNA detection relies not only on the high detection rate provided by the BamHI-W target but also on precise quantification achieved by the EBNA-1 target for risk stratification and treatment response assessment. Therefore, EBV DNA detection should not be confined to a single target. Instead, targets should be flexibly selected according to specific clinical scenarios to fully leverage their respective strengths while mitigating limitations, thereby achieving comprehensive optimization of detection performance. In summary, based on systematic optimization across three key components of EBV DNA detection, the following recommendations are proposed for the first two components. Specifically, plasma samples are recommended for routine clinical testing and large-scale cohort studies. Samples should be promptly separated and stored at −20 °C after collection to minimize to the greatest extent the impact of nucleic acid degradation on analytical reliability. With respect to nucleic acid extraction, the magnetic bead–based method is recommended because of its high throughput, automation, and strong nucleic acid enrichment capacity, making it particularly suitable for large scale testing. In the third section concerning target optimization and system evaluation, we emphasize that EBV DNA testing should not focus solely on identifying a single optimal target gene, but must consider the practical clinical value of different targets in guiding decision making.Guided by clinical application needs, the advantages and limitations of different targets should be comprehensively evaluated to enable appropriate target selection. At present, it is essential to clarify the appropriate clinical indications for different targets, thereby enabling EBV DNA testing to support disease diagnosis, treatment, and comprehensive management. This will ultimately facilitate the transition from optimization of molecular detection techniques into improved clinical practice. To refine the methodological protocol for EBV DNA detection, key variables，including sample type, storage conditions, nucleic acid extraction methods, and PCR target genes were systematically evaluated. The generalizability of the findings, however, may be constrained by the relatively limited sample size. Future work should therefore aim to expand the cohort to encompass a broader spectrum of clinical profiles, such as newly diagnosed patients, individuals with different clinical subtypes, and post-treatment cases, to enhance the reliability of the results and facilitate evaluation of both targets across disease stages. These collective efforts are anticipated to establish a more robust and widely applicable evidence base, thereby improving clinical decision-making for nasopharyngeal carcinoma. Author Contributions Wei Cui, Baojun Wei, and Xinxiong Zeng designed and refined the study. Xinxiong Zeng performed data collection and analysis and wrote the initial draft. Baojun Wei made substantial revisions to the draft. Quanquan Gao, Yuzhao Jiang, Jing Wang, and Guojing Wang assisted with sample collection and certain experimental procedures. Wei Cui supervised the study. All authors have read and approved the final manuscript. Acknowledgments We would like to express our gratitude to the teachers and colleagues who provided valuable technical guidance during the experimental procedures. Our thanks also go to the colleagues who contributed to the sample collection process, which was essential for completing this study. Ethics Statement This study was approved by the Ethics Committee of National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing,China. Conflicts of Interest The authors declare no conflicts of interest. Data Availability Statement The data supporting the findings of this study are available from the corresponding author upon reasonable request. References 1. Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019 Jul 6;394(10192):64-80. 2. Hsu C, Shen YC, Cheng CC, Hong RL, Chang CJ, Cheng AL. Difference in the incidence trend of nasopharyngeal and oropharyngeal carcinomas in Taiwan: implication from age-period-cohort analysis. Cancer Epidemiol Biomarkers Prev. 2006,15(5):856-61. 3. Tang LL, Chen WQ, Xue WQ, He YQ, Zheng RS, Zeng YX, Jia WH. Global trends in incidence and mortality of nasopharyngeal carcinoma. Cancer Lett. 2016,374(1):22-30. 4. Coghill AE, Hildesheim A. 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Keywords blood dna extraction epidemiology epstein-barr virus research and analysis methods virus classification Authors Affiliations Guojing Wang Chinese Academy of Medical Sciences State Key Laboratory of Molecular Oncology View all articles by this author Jing Wang Chinese Academy of Medical Sciences State Key Laboratory of Molecular Oncology View all articles by this author Quanquan Gao Chinese Academy of Medical Sciences State Key Laboratory of Molecular Oncology View all articles by this author Yuzhao Jiang Chinese Academy of Medical Sciences State Key Laboratory of Molecular Oncology View all articles by this author Baojun Wei Chinese Academy of Medical Sciences State Key Laboratory of Molecular Oncology View all articles by this author Xinxiong Zeng Chinese Academy of Medical Sciences State Key Laboratory of Molecular Oncology View all articles by this author Wei Cui [email protected] Chinese Academy of Medical Sciences State Key Laboratory of Molecular Oncology View all articles by this author Metrics & Citations Metrics Article Usage 206 views 74 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Guojing Wang, Jing Wang, Quanquan Gao, et al. 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