Abstract
BackgroundHuman adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control.MethodsWe developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays incorporating competitive internal controls for detection of HAdV 3 and HAdV 7, respectively. The assays were performed in a one-step in a single tube reaction at 39° for 20 min.ResultsThe analytical sensitivities of the duplex RAA assays for HAdV 3 and HAdV 7 were 5.0 and 14.8 copies per reaction, respectively (at 95% probability by probit regression analysis). No cross-reaction was observed with other types of HAdV or other common respiratory viruses. The duplex RAA assays were used to detect 152 previously-defined HAdV-positive samples. These results agreed with those obtained using a published triplex quantitative real-time PCR protocol.ConclusionsWe provide the first report of internally-controlled duplex RAA assays for the detection of HAdV 3 and HAdV 7. These assays effectively reduce the rate of false negative results and may be valuable for detection of HAdV 3 and HAdV 7 in clinical laboratories, especially in resource-poor settings.
Highlights
Human adenoviruses are a common group of viruses that cause acute infectious diseases
Singleplex recombinase-aided amplification (RAA) assay sensitivity and limit of detection The sensitivity of singleplex RAA assays for Human adenovirus (HAdV) 3 and HAdV 7 was determined using diluted recombinant standard plasmids
The detection limits of the singleplex RAA assays for HAdV 3 and HAdV 7 at 95% probability were 4.4 and 5.0 copies/reaction, respectively (Table 1)
Summary
Human adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control. Different serotypes of HAdV result in distinct clinical manifestations, including acute respiratory infections, conjunctivitis, pneumonia, gastroenteritis, myocarditis, and even central nervous system dysfunction [1, 2]. Previous studies reported that HAdV 3 and HAdV 7 were the main genotypes causing outbreaks of infection in many countries [3], including Singapore, the United States, the United Kingdom, Korea [4] and Canada [2, 5]. It is critical to develop sensitive and timely detection methods for HAdV 3 and HAdV 7, which may facilitate clinical diagnosis and epidemiological studies
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