Dataset Information

Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7.

ABSTRACT: BACKGROUND: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 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. METHODS:We 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. RESULTS:The 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. CONCLUSIONS:We 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.

SUBMITTER: Wang RH

PROVIDER: S-EPMC6604330 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7.

Wang Rui-Huan RH Zhang Hong H Zhang Yi Y Li Xin-Na XN Shen Xin-Xin XX Qi Ju-Ju JJ Fan Guo-Hao GH Xiang Xing-Yu XY Zhan Zhi-Fei ZF Chen Zi-Wei ZW Ma Xue-Jun XJ

Virology journal 20190701 1

<h4>Background</h4>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 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.<h4>Methods</h4>We developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays ...[more]

PMID: 31262315

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Project description:The avian influenza virus (AIV), a member of the type A influenza virus group, is harbored by avian hosts. The highly pathogenic H5 subtype of AIV has inflicted significant financial damage on the poultry industry and also presents a possible pandemic hazard to the worldwide human population. There is an urgent need for a detection method that is both rapid and accurate in identifying H5-AIV. In this study, two assays based on reverse transcription recombinase-aided amplification technology (RT-RAA) have been developed to detect H5-AIV, namely, real-time fluorescence and reverse transcription recombinase-aided amplification (RF-RT-RAA) and reverse transcription recombinase-aided amplification combined lateral flow dipstick (RT-RAA-LFD). The completion of the two methods can be achieved in 30 min, and there was no cross-reaction with the nucleic acid of other avian pathogens. The results indicated that the lowest detectable limit of RF-RT-RAA and RT-RAA-LFD for H5-AIV detection was 1 copy/μL, which was 100 times higher than that of RT-PCR (102 copies/μL). A total of 376 samples, consisting of 350 clinical and 26 experimental samples, underwent testing utilizing virus isolation, RF-RT-RAA and RT-RAA-LFD methods, respectively. The results demonstrated a high degree of consistency between those assays. In summary, both the RF-RT-RAA and RT-RAA-LFD methods demonstrated excellent specificity and sensitivity, making them ideal for use in both laboratory and field settings.IMPORTANCEAvian influenza virus (AIV) subtype H5 is a highly contagious zoonotic disease and a serious threat to the farming industry and public health. Traditional detection methods, including virus isolation and real-time PCR, require tertiary biological laboratories and are time-consuming and complex to perform, making it difficult to rapidly diagnose H5 subtype avian influenza viruses. In this study, we successfully developed two methods, namely, RF-RT-RAA and RT-RAA-LFD, for rapid detection of H5-AIV. The assays are characterized by their high specificity, sensitivity, and user-friendliness. Moreover, the results of the reaction can be visually assessed, which are suitable for both laboratory testing and grassroots farm screening for H5-AIV.

Project description:Newcastle disease is an acute and highly contagious disease of poultry caused by Newcastle disease virus infection, which does great harm to the poultry industry all over the world. To diagnose the disease simply and quickly, 2 detection methods were established based on reverse transcription recombinase-aided amplification (RT-RAA) technology. One is reverse transcription recombinase-aided amplification-lateral flow dipstick (RT-RAA-LFD) that is to combine RT-RAA with lateral flow dipstick; the other is real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) that is the combination of RT-RAA and exo probe. In this study, the reaction conditions such as reaction temperature and reaction time of the 2 methods were optimized, and their specificity and sensitivity were tested. The results showed that the RT-RAA-LFD method could be used to complete reaction within 23 min, and its lowest detectable limit was 102 copies/?L, 10 times higher than that of the conventional PCR method (103 copies/?L); the RF-RT-RAA method could be used to complete reaction within 26 min, and its lowest detectable limit was 10 copies/?L, 100 times higher than that of conventional PCR method (103 copies/?L), and it was as sensitive as real-time fluorescence-based quantitative PCR (10 copies/?L). The 2 methods had no cross reaction to the nucleic acid of other avian pathogens and showed good specificity. A total of 86 clinical samples suspected of the Newcastle disease virus were tested by conventional PCR, real-time fluorescence-based quantitative PCR, RT-RAA-LFD, and RF-RT-RAA. Based on the commonly used conventional PCR method, the other 3 detection methods had a coincidence rate of higher than 93%. In summary, RT-RAA-LFD and RF-RT-RAA had high specificity, sensitivity, and efficiency, which were suitable for clinical and laboratory diagnosis, respectively, and provided technical support for the prevention and control of Newcastle disease.

Dataset Information

Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7.

Publications

Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7.

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