Rapid Molecular Diagnosis of Human Adenoviruses by Real Time PCR

Abstract

Abstract: : Purpose: To establish a simple, rapid, quantitative and qualitative real time PCR method (Light Cycler) to detect a partial hexon gene of human adenovirus (AdV). Methods: Specimens: Eye swabs were collected from the 26 patients with acute conjunctivitis. Remained portion of their multidose bottles were collected after used by them. Virus: The prototype strains of AdV-1 to AdV-51 were purchased from the American Type Culture Collection and used without passage in cell culture. DNA amplification: DNA was extracted from the prototype strains of AdV-1 to AdV-51. A set of primer was used to amplify a 554-bp of hexon gene of AdV. The virus gene was qualitated or quantitated by using Light Cycler PCR with SYBR Green. Results: The set of primers allowed amplification of 554-bp of hexon gene of 51 prototype strains. The hexon gene of AdV was detected from 13 out of 26 swabs and their nucleotide sequences were determined. The high copy numbers such as 4.2×10¹~3.8×10⁶/mL were detected in multidose bottles. The serotypes of AdVs were determined by phylogenetic analysis, along with 51 prototype strains. The phylogenetic tree showed that all nucleotide sequences from the swabs and multidose bottles formed a distinct cluster with each prototype strain, and thus allowed identification of each serotype. Interestingly the serotypes of AdV detected from the multidose bottles were coincident with those from the patient’s swab. Conclusions: We developed the molecular diagnostic method based on real time PCR and hexon-based phylogenetic analysis, which enable quantitative assay of viral concentration and identification of serotypes of adenovirus.The result suggests that the multidose bottles contaminated with AdV may play a role for transmission of the virus from a patient to another person through ophthalmic solution.

Keywords: adenovirus • conjunctivitis • clinical laboratory testing