Evaluation of Saliva Collection and DNA Extraction Methods for Practical Application of Salivary Human Herpesvirus 6 and 7 Assays

Salivary human herpesvirus 6 and/or 7 (HHV-6/7) have recently attracted attention as microbiological markers of physiological fatigue in laborers and athletes. However, the accuracy and efficiency of the HHV-6/7 assays can be improved for practical application. We conducted three experiments to identify suitable saliva collection and DNA extraction methods for practical salivary HHV-6/7 assays. The main experiment compared the data, including template DNA or HHV-6/7 concentrations, among three saliva collection methods (cotton, synthetic, and no swabs) and two DNA extraction methods (magnetic bead-based and silica column-based). It showed that using swabs had adverse effects: lower template DNA concentration, lower HHV-6/7 detection rates, higher coefficient of variation values, and lower concentrations. Moreover, magnetic bead-based methods resulted in higher HHV-6/7 detection rates and lower coefficient of variation values. Sub-experiment 1 examined practical saliva collection methods and demonstrated that the stimulated spitting method could collect saliva in a shorter time with lower subjective stress than the unstimulated spitting and stimulated swabbing methods. Sub-experiment 2 investigated diurnal variation in salivary HHV-6/7 levels but did not show diurnal variation. These findings suggest that (1) the combination of stimulated spitting saliva collection and magnetic bead-based DNA extraction is most suitable for practical salivary HHV-6/7 assays, and (2) saliva collection can be conducted whenever needed.