Genotyping of human adenoviruses using a PCR-based reverse line blot hybridisation assay

Objectives: Human adenoviruses are common pathogens associated with a broad spectrum of disease. There is a growing clinical interest in typing clinical isolates since it is becoming increasingly clear that individual serotypes are associated with different disease spectra, virulence, severity of consequences, and outbreaks. Current methods cannot detect all known adenoviruses simultaneously and rapidly. We designed a practical adenovirus typing method with polymerase chain reaction (PCR)-based reverse line blot hybridisation assay (RLB) using hypervariable region-7 (HVR-7) in the hexon gene. Methods: A PCR-RLB assay was developed based on HVR-7 in the hexon region for potentially genotyping 51 adenovirus serotypes by hybridisation of 62 genotype-specific probes using amplicons generated from one genus-specific primer pair. Single PCR and sequencing were performed for confirmation of RLB results. Eighty-seven previously serotyped clinical isolates (representing 28 serotypes) were studied. Results: Thirty-two different genotypes were detected by RLB from 87 adenovirus isolates, of which 82 isolates showed consistent results with sequencing. Another five isolates revealed evidence by RLB of co-infection, and were confirmed with a combination of genotype-specific single PCR and sequencing. Conclusions: In comparison to sequencing and serological methods, the advantages of the RLB assay include: (1) rapid genotyping of multiple samples in a single run; (2) successful detection of co-infection; (3) detection of subgenotype variants. This will allow rapid and inexpensive characterisation of adenovirus infections and outbreaks.