基于RPA-CRISPR/Cas12b的一管式H5亚型禽流感病毒快速检测方法的建立OA

Three relatively conserved regions of the H5 subtype avian influenza virus were selected for the design of sgRNAs and specific amplification primers for reverse transcription recombinase polymerase amplification(RT-RPA).A detection method based on RPA-CRISPR/Cas12b was established for the rapid identification of H5 subtype avian influenza virus.The sensitivity,specificity,repeatability,and feasibility of this method were systematically evaluated.The results showed that the detection limit of the recombinant plasmid positive standard was 1.0 copies/μL,representing a tenfold increase in sensitivity compared with conventional fluorescence RT-PCR.No cross-reactivity was observed with nucleic acids from H7 subtype avian influenza virus,H9 subtype avian influenza virus,Newcastle disease virus(NDV),or infectious bronchitis virus(IBV).When 50 simulated clinical samples were tested,a 100％ concordance rate was achieved between this assay and the nationally recommended real-time RT-PCR method.These findings indicate that the RPA-CRISPR/Cas12b detection method established in this study provides a reliable and rapid on-site screening tool for frontline personnel at ports of entry.