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.