Development And Evaluation Of Recombinase-aided Amplification Assays For Detection Of Human Adenovirus Serotypes 3 And 7

Human adenoviruse is a common group of viruses that cause acute infectious diseases.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.We developed and evaluated duplex real-time recombinase-aided amplification(RAA)assays incorporating competitive internal controls for detecting of HAdV-3 and HAdV-7,respectively.By exploring and optimizing the condition,a real-time fluorescence RAA assay for detecting HAdV-3 without nucleic acid extraction was established.By combining high-temperature rapid lysing method,RAA technology and LFD(lateral flow dipstick),we developed and evaluated the LFD-RAA assay for detecting HAdV-7 without extracting nucleic acids.The major contents were as follows:In Chapter 2 of this paper,we developed and evaluated duplex real-time RAA assays incorporating competitive internal controls fordetecting of HAdV-3 and HAdV-7,respectively.Assays were performed in a one-step in a single tube reaction at 39° for 20 min.The analytical sensitivities of the duplex RAA assays for HAdV-3 and HAdV-7 were5.0 and 14.8 copies per reaction,respectively(at 95% probability by probit regression analysis).No cross-reaction was detected 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(tq-PCR)protocol.We provide the first report of internally-controlled duplex real time RAA assays for detection of HAdV-3 and HAdV-7,which reduced the false negative rate effectively.In Chapter 3 of this paper,by exploring and optimizing the condition and using rapid lysis of nucleic acids at high temperature,we developed and evaluated a real-time fluorescence RAA assay for detecting HAdV-3without extracting nucleic acids.The sensitivity of the real-time fluorescence RAA method was as high as that of tq-PCR in the detection of 10 series diluted HAdV-3 strains.The highest corresponding Ct value of tq-PCR was 36.87.There was no cross-reaction with other common types of respiratory viruses.The two methods were used to detect 72previously-defined HAdV-positive samples parallelly,and the results were completely consistent.We provided the first report ofextraction-free real time RAA assays for the detection of HAdV-3,which simplified the step of nucleic acid extraction.In Chapter 4 of this paper,by combining high-temperature rapid lysing method,RAA technology,and LFD,we developed and evaluated the LFD-RAA assay for detecting HAdV-7 without extracting nucleic acids.The entire process took only 40 minutes to complete the detection of clinical samples as this assay requires no extraction of nucleic acids nor special detection equipment.The sensitivity of the LFD-RAA method was a little bit lower than tq-PCR in the detection of 10 series diluted HAdV-7 strains.The strains of which corresponding Ct value of 31.33 were difficult to detect with our method.No cross-reaction with other common types of respiratory viruses was detected.The two methods were used to detect 72 previously-defined HAdV-positive samples parallelly.The Kappa vaule was 0.83 and the results were highly consistent.We provided the first report of combined extraction-free and LFD-RAA technology for the detection of HAdV-7,which greatly reduced the entire detection process time.