RESUMO
Objective: Digital polymerase chain reaction (dPCR) assay is an advanced PCR technique that allows for the simultaneous detection and absolute quantification of diverse pathogens.Commercially validated kits available for detecting all subtypes of human adenovirus (HAdV) are limited. This study aimed to demonstrate the development of an in-house nanoplate-based dPCR assay with high sensitivity, even at low copy numbers. Materials and Methods: In this methodological study, the standardized HAdV DNA was prepared by amplifying the specific hexon gene region with real-time PCR and purifying the HAdV DNA using magnetic beads from HAdV-positive extractions. Dilutions were tested in triplicate during three independent runs to determine the dynamic range, the limit of detection (LoD), the limit of quantification (LoQ), precision, and reproducibility. The primer and probe sequences used in the study were selected based on a literature review to ensure the detection of all HAdV serotypes in a single run. The selected primers were verified using the US National Center for Biotechnology Information (NBCI) nBLAST tools, and the target sequence was determined using the BioEdit software. The DNA concentration of the stock solution was measured using a Qubit fluorometer. The estimated copy number of the stock solution per milliliter was calculated based on the length of the amplified base sequence and fluorometer measurement. Results: The dynamic range of the test was determined to be from 770.4 to 0.9476 cp/µl, with the LoD and LoQ values both being 0.9476 cp/µl. The coefficient of determination (r 2) value of the test was 0.9986. Conclusion: The results demonstrated that the dPCR method could be an ideal tool for the diagnosis and absolute quantification of human adenoviruses, especially in low copy numbers. In order to determine the reproducibility of the test and validate the method for field use, it needs to be developed and adapted in various laboratories and supported by clinical studies.
RESUMO
OBJECTIVE: To investigate the prevalence of tonsillar human papillomavirus infection in Istanbul, the most populous city of Turkey. METHODS: Tonsil specimens were obtained from 206 cadavers aged 18 to 89 years. Tonsillectomy was performed during routine autopsy for each subject in the 24 hours after death. After dissolution, tissues were processed with the polymerase chain reaction (PCR) method to identify HPV DNA. The data obtained from the DNA sequencer were processed in the database of GenBank®. RESULTS: One hundred sixty-six (80.6%) male and 40 (19.4%) female cadavers were included in the study. One case demonstrated HPV-16, one had HPV-82, one had HPV-55 and one had HPV-13. All four cases were male. Prevalence of tonsillar HPV was 1.94% and of HPV 16 was 0.48%. CONCLUSION: The prevalence of tonsillar HPV infection was found 1.94% and of HPV 16 0.48% in our study.
