Abstract:
Objective To develop a simple and rapid detection method for HAdV-11/55 by combining recombinase aided amplification technology with real-time fluorescent probe.
Methods Using the Hexon gene of HAdV-11/55 as the target sequence, multiple pairs of primers and probes were designed and synthesized, and the best combination of primer and probe was selected.Recombinase-mediated isothermal amplification was carried out at 39 ℃, 40 ℃ and 42 ℃ to select the best reaction temperature, and the sensitivity and specificity of the method were evaluated.
Results The optimal temperature of the method was 42 ℃.The minimum detection limit is 101 copies/μL.The specificity test results showed that the real-time fluorescent RAA detection method could detect HAdV-11/55, and there was no cross-reaction with other subtypes and other respiratory pathogens.The results of this method were 100% consistent with the results of real-time fluorescent PCR, which had excellent detection performance.
Conclusions The real-time fluorescent RAA assay developed in this study can be completed within 20 minutes with only simple thermostatic equipment.It is a rapid and simple method for detecting HAdV-11/55, which is suitable for point-of-care molecular diagnostic testing of HAdV-11/55.
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