In 2022,a global outbreak of mpox was anticipated,with several cases reported in non-endemic countries in early May.Given the challenge of distinguishing the mpox virus(MPXV)from other pathogens based solely on sympto...In 2022,a global outbreak of mpox was anticipated,with several cases reported in non-endemic countries in early May.Given the challenge of distinguishing the mpox virus(MPXV)from other pathogens based solely on symptoms,there is an urgent need for prompt and reliable MPXV detection methods.In this study,we developed assays using recombinase-aided amplification(RAA)to identify MPXV and evaluated their applicability with clinical samples.The assays were designed to detect theN4R gene of MPXV.All assays demonstrated detection limits of 1 copy/μL within the reaction system and exhibited no cross-reactivity with ectromelia or the TianTan strain of vaccinia virus,confirming their high specificity.Our established assay provides results in less than 50 min.Furthermore,we evaluated our assay using clinical samples from laboratory-confirmed mpox patients and demonstrated that the RAA-based assay is valuable for diagnosing MPXV infections in field and clinic settings,especially in areas with limited laboratory resources.Overall,three RAA-based nucleic acid assays for MPXV were established,providing a powerful tool for efficient,rapid,and specific detection of MPXV infection.展开更多
Introduction:Since 2019,numerous variants of concern for severe acute respiratory syndrome virus 2(SARS-CoV-2)have emerged,leading to significant outbreaks.The development of novel,highly accurate,and rapid detection ...Introduction:Since 2019,numerous variants of concern for severe acute respiratory syndrome virus 2(SARS-CoV-2)have emerged,leading to significant outbreaks.The development of novel,highly accurate,and rapid detection techniques for these new SARSCoV-2 variants remains a primary focus in the ongoing efforts to control and prevent the coronavirus disease 2019(COVID-19)pandemic.Methods:Reverse transcription-recombinase polymerase amplification combined with the clustered regularly interspaced short palindromic repeatsassociated protein 12a(CRISPR/Cas12a)system was used to validate the detection of the Omicron BA.2,BA.4,and BA.5 variants of SARS-CoV-2.Results:Our results demonstrate that the CRISPR/Cas12a assay is capable of effectively detecting the SARS-CoV-2 BA.2,BA.4,and BA.5 variants with a limit of detection of 10,1,and 10 copies/μL,respectively.Importantly,our assay successfully differentiated the three SARS-CoV-2 Omicron strains from one another.Additionally,we evaluated 46 SARS-CoV-2 positive clinical samples consisting of BA.2(n=20),BA.4(n=6),and BA.5(n=20)variants,and the sensitivity of our assay ranged from 90%to 100%,while the specificity was 100%.Discussion:This research presents a swift and reliable CRISPR-based method that may be employed to track the emergence of novel SARS-CoV-2 variants.展开更多
基金supported by the Beijing Natural Science Foundation(7254390)the Youth Science Foundation of the Chinese Center for Disease Control and Prevention(2024A103)the National Key R&D Program of China(2022YFC2303400).
文摘In 2022,a global outbreak of mpox was anticipated,with several cases reported in non-endemic countries in early May.Given the challenge of distinguishing the mpox virus(MPXV)from other pathogens based solely on symptoms,there is an urgent need for prompt and reliable MPXV detection methods.In this study,we developed assays using recombinase-aided amplification(RAA)to identify MPXV and evaluated their applicability with clinical samples.The assays were designed to detect theN4R gene of MPXV.All assays demonstrated detection limits of 1 copy/μL within the reaction system and exhibited no cross-reactivity with ectromelia or the TianTan strain of vaccinia virus,confirming their high specificity.Our established assay provides results in less than 50 min.Furthermore,we evaluated our assay using clinical samples from laboratory-confirmed mpox patients and demonstrated that the RAA-based assay is valuable for diagnosing MPXV infections in field and clinic settings,especially in areas with limited laboratory resources.Overall,three RAA-based nucleic acid assays for MPXV were established,providing a powerful tool for efficient,rapid,and specific detection of MPXV infection.
基金Supported by the National Key Research and Development Program of China(2021YFC2300101,2021YFC0863300,2022YFC 2304101,2022YFC2303401).
文摘Introduction:Since 2019,numerous variants of concern for severe acute respiratory syndrome virus 2(SARS-CoV-2)have emerged,leading to significant outbreaks.The development of novel,highly accurate,and rapid detection techniques for these new SARSCoV-2 variants remains a primary focus in the ongoing efforts to control and prevent the coronavirus disease 2019(COVID-19)pandemic.Methods:Reverse transcription-recombinase polymerase amplification combined with the clustered regularly interspaced short palindromic repeatsassociated protein 12a(CRISPR/Cas12a)system was used to validate the detection of the Omicron BA.2,BA.4,and BA.5 variants of SARS-CoV-2.Results:Our results demonstrate that the CRISPR/Cas12a assay is capable of effectively detecting the SARS-CoV-2 BA.2,BA.4,and BA.5 variants with a limit of detection of 10,1,and 10 copies/μL,respectively.Importantly,our assay successfully differentiated the three SARS-CoV-2 Omicron strains from one another.Additionally,we evaluated 46 SARS-CoV-2 positive clinical samples consisting of BA.2(n=20),BA.4(n=6),and BA.5(n=20)variants,and the sensitivity of our assay ranged from 90%to 100%,while the specificity was 100%.Discussion:This research presents a swift and reliable CRISPR-based method that may be employed to track the emergence of novel SARS-CoV-2 variants.
