Development of a Rapid Isothermal Assay for Detection of Adenovirus Types Important in Respiratory Infections

Nucleic acid amplification tests (NAATs) for human adenoviruses (HAdVs) causing respiratory infections usually target the hexon gene. However, new HAdV types with substantial variations in the hexon gene may not be detected. Thus, we focus on NAATs based on a conserved region in the penton gene to detect all HAdV types causing respiratory infections. A highly conserved region at the 3' end of the penton gene was chosen as a target for NAAT. Primers and probes for quantitative polymerase chain reaction (qPCR) and isothermal recombinase polymerase amplification (RPA) were designed for the detection of all HAdV types causing respiratory infections. Two highly sensitive qPCR assays were established, one for the detection of HAdV-E4 and HAdV-B types and another for the detection of HAdV-C types (LOD < 10 standard DNA copies for both assays). Furthermore, a one-tube RPA with a universal RPA probe was developed for rapid detection of all HAdV types causing respiratory infections (LOD ≤ 244 standard DNA copies). All three assays were used for testing clinical nasopharyngeal swabs obtained from SARS-CoV-2-negative children with respiratory disease symptoms. Eight out of 243 samples tested were found to be HAdV positive by qPCR and by one-tube RPA, except for one sample with a very low viral load of 30 genome equivalents. Penton gene-based NAAT systems were developed and successfully used for the detection of HAdV in clinical samples. The newly developed one-tube RPA assay offers the possibility for rapid and simple detection of respiratory HAdV infections at the point of need.