通过比较不同核酸提取方法对EB病毒（EBV）-DNA检测的影响，探讨EBV-DNA检测的优化方案。

收集16例全血EBV-DNA阳性标本（EBV-DNA浓度>1000 copies/ml），分别用手工提取法、A品牌核酸提取仪（A机）和B品牌核酸提取仪（B机）提取全血核酸，检测各样本核酸浓度及A₂₆₀/A₂₈₀比值，分析不同提取法所得核酸质量，再采用荧光定量PCR检测EBV-DNA浓度，比较3种核酸提取法检测EBV-DNA的差异。为探究全血与血浆检测EBV-DNA的差异，对19例手工提取后检测结果为阳性的全血标本，用试剂盒配套的血浆提取方法手工提取其血浆核酸，采用荧光定量PCR检测EBV-DNA浓度。

16例全血EBV-DNA阳性样本经3种不同方法提取核酸后，手工提取核酸浓度均数为624 ng/μl，A机提取核酸浓度均数为141 ng/μl，B机提取核酸浓度均数为273 ng/μl。2种仪器提取法结果与手工提取法比较，均差异有统计学意义（A机P<0.05，B机P<0.01）。荧光定量PCR检测EBV-DNA浓度显示，手工提取全部为阳性，A机有4例未检测出阳性，B机有5例未检测出阳性。结果提示，低浓度EBV标本如采用该研究中的仪器提取核酸可能发生漏检。在同时检测全血与血浆的19例EBV-DNA阳性标本中，只有1例血浆阳性，全血样本EBV-DNA阳性率高于血浆样本，差异具有统计学意义（P<0.01）。经过以上验证，该单位EBV-DNA均使用全血标本通过手工提取法提取核酸进行检测。2021年1月至2022年12月共检测7945例EBV-DNA样本，阳性1160例，阳性率14.60%，与文献报告的阳性率基本符合。

不同核酸提取方法及标本类型对EBV-DNA检测结果存在明显影响。因此，在项目应用于临床之前应做好方法学比对，与临床协商选择合适的标本类型进行检测。项目进行过程中应对检测结果进行定期评价，为临床提供准确、有效的检测结果。

To compare the effects of different nucleic acid extraction methods on the detection of Epstein-Barr virus (EBV)-DNA, and to provide evidence for optimizing the existing methods.

16 whole blood samples positive for EBV-DNA (EBV-DNA concentration>1000 copies/ml) were collected, and the whole blood nucleic acid was extracted by manual extraction method and 2 kinds of nucleic acid extractors respectively. The nucleic acid concentration and A₂₆₀/A₂₈₀ ratio of each sample were detected, and the quality of nucleic acid obtained by different extraction methods was analyzed. The EBV-DNA concentration was detected by fluorescence quantitative PCR, and the differences in EBV-DNA detection between the 3 nucleic acid extraction methods were compared. To investigate the differences in EBV-DNA detection between whole blood and plasma, the plasma nucleic acid was manually extracted from 19 whole blood specimens that tested positive after manual extraction using the kit-matched plasma extraction method, and the concentration of EBV-DNA was detected by quantitative fluorescence PCR.

The results showed that the average concentration of nucleic acid extracted by manual was 624 ng/μl, the average concentration of nucleic acid extracted by machine A was 141 ng/μl, and the average concentration of nucleic acid extracted by machine B was 273 ng/μl. There were statistically significant differences between the results of the two instrument extraction methods and the manual method (machine A: P<0.05, machine B: P<0.01). The EBV-DNA concentration detected by fluorescence quantitative PCR revealed that all samples were positive using manual extraction, while 4 samples were not detected as positive using machine A and 5 were not detected using machine B. These results suggested that instrument-based nucleic acid extraction methods may result in missed detections for low-concentration EBV specimens. Among the 19 EBV-DNA-positive specimens tested for both whole blood and plasma, only 1 plasma sample was positive. The positive rate of EBV-DNA in whole blood samples was higher than in plasma samples, with a statistically significant difference (P<0.01). Based on the above validation, all EBV-DNA testing in this facility is conducted using whole blood specimens with nucleic acid extracted manually. From January 2021 to December 2022, a total of 7945 EBV-DNA samples were tested, with 1160 positive cases and a positive rate of 14.60%, which is basically consistent with the positive rates reported in the literature.

Different nucleic acid extraction methods and specimen types have significant effects on EBV-DNA results. Therefore, the methodology should be compared and the appropriate specimen type should be selected in consultation with the clinic before applying the project clinicall. The test process should be evaluated periodically for accurate and valid results.