A comparison of laboratory extraction techniques for the detection of Epstein-Barr virus in saliva of nasopharyngeal carcinoma patients

Abstract

Nasopharyngeal carcinoma (NPC) is common in southern Chinese. It is closely associated with Epstein-Barr virus (EBV). Recent studies suggest that tumor burden is proportional to the level of EBV DNA in blood of NPC patients and that rapid blood testing can be used to guide therapeutic intervention. Instead of collecting invasive blood samples, saliva may be a viable alternative. However, no studies have reported on the detection of EBV DNA in saliva of NPC patients. Moreover, different DNA extraction techniques for PCR in saliva samples have never been studied. This study aimed to compare the effectiveness of DNA extraction using a commercial extraction kit against the standard boiling technique for quantitative real time PCR (RT-PCR) assays in saliva of NPC patients. Stimulated whole saliva samples from 207 newly diagnosed NPC patients were screened by qualitative PCR for the presence of EBV DNA. Twenty-three positive samples then underwent DNA extraction followed by RT-PCR testing using primer/probe set for BALF5. Duplicate measurements were performed and reproducibility was assessed. The two DNA extraction methods were evaluated by Bland-Altman’s plot and linear regression. The relationship between salivary EBV DNA level and clinical variables was evaluated by t-test. The results of RT-PCR were reproducible for both groups. The two techniques were moderately correlated (r = 0.67, p < 0.05) and the degree of agreement was good. However, the mean EBV DNA level in the boiling group (3.02 ± 8.67 × 106 copies/μl) was significantly higher than the extraction kit group (1.15 ± 2.66 × 106 copies/μl) (p < 0.05). The EBV DNA level was marginally higher in patients of advanced overall stage (p = 0.05). This study showed that the performance of extraction kit was inferior to the simple boiling technique for the detection of salivary EBV DNA in NPC patients using real-time PCR. Further studies are required to enhance the detection rate of EBV DNA in saliva of NPC patients.