Establishment of a method for rapid detection of influenza A virus with multienzyme isothermal rapid amplification-CRISPR/Cas13a in combination with magnetic bead-quantum dot sensors
Objective To establish a method for rapid detection of influenza A virus(IAV)by combining multienzyme isothermal rapid amplification(MIRA),clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 13(Cas13)and magnetic bead-quantum dot(MBQD)sensors.Methods MIRA amplification primers,CRISPR RNA(crRNA),fluorescent re-porter probes and connected probes were designed and screened targeting the M gene of IAV,and the reaction conditions were optimized to establish a MIRA-CRISPR/Cas13a-MBQD system.The detection sensitivity and specificity of the system were evaluated.In addition,the agreement between this system and real-time quantitative reverse-transcription PCR(RT-qPCR)assay was examined for detection of clinical samples.Results TheMIRA-CRISPR/Cas13a-MBQD system detected IAV within 60 min.The sensitivity of the system for detection of IAV was 1 copy RNA per reaction,and no cross-reaction was found with SARS-CoV-2,influenza virus B(IBV),respiratory syncytial virus(RSV),human metapneumovirus(HMPV),parainfluenza virus(PIV1),and three(HPIV3),adenovirus(ADV)or Mycoplasma pneumonia(MP).In addition,there a high agreement between the MIRA-CRISPR/Cas13a-MBQD system and RT-qPCR assay for detection of clinical samples(Kappa=1.00).Conclusions The established MIRA-CRISPR/Cas13a-MBQD system is rapid,sensitive and specific for detection of IAV.
Multienzyme isothermal rapid amplificationCRISPR/Cas 13aQuantum dot sensorInfluenza A virusRapid detection
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