Real Time PCR Assessment of HPV Infection in Saliva

Objectives: Persistent human papillomavirus (HPV) infection causes 70% of oropharyngeal cancers in the United States. However, there is no report of the oral HPV infection prevalence in Shelby County. Tetraspanin CD9 is a newly identified key cellular factor for HPV16 infection.

We aim to collect preliminary data on oral HPV infection in the Shelby County area and establish protocols and logistics for long-term study of oral HPV infection in the Shelby County area population; To test the correlation between CD9 expression and HPV E6/E7 expression.This study has been approved by UTHSC IRB # 17-05389-XP.
Methods: There were no exclusion criteria for this HPV prevalence study. Genomic DNA was isolated from an oral rinse or resting saliva using Ready-Lyse™ Lysozyme Solution (Epicentre). The quality and quantity of DNA were quantified using NanoDrop One. HPV E6 and E7 was detected using real time polymerase chain reaction (qRT-PCR) performed on a LightCycler 480 (Roche) real time PCR system. CD9 protein expression was detected using Western blot.
Results: We tested 29 samples. Among them, HPV E6 was detectable in 27 subjects. HPV E7 was detectable in 14 samples, with very low copy number (30Conclusions: HPV E6/E7 is detectable using real time PCR, but detection by PCR does not equal clinically defined HPV positivity. It is necessary to refine the protocol with a positive control and a copy number cutoff to reduce false positives and improve the specificity for oral HPV infection in the population of the Shelby County area.