Establishment And Preliminary Application Of RPA-LFD Detection Method For Avian Influenza Virus/Riemerella Anatipesifer

Avian influenza(AI)is a major avian infectious disease caused by Avian influenza virus(AIV)and is listed as a class of animal diseases in China.Riemerella anatipestifersis is a major infectious disease caused by Riemerella anatipesifer(RA),which is harmful to the duck industry.It is listed as a second-class animal disease in China.At present,the detection methods of AIV and RA have the disadvantages of lengthy process,poor sensitivity,easy cross-contamination or limited by instrument and equipment.Therefore,it is necessary to establish a rapid,accurate,ultra-sensitive and simple detection method to better diagnose,monitor and prevent these two diseases.Recombinase ploymerase amplification(RPA)is a novel nucleic acid isothermal amplification technology.This technology can efficiently and rapidly amplify a target fragment in a micronucleic acid sample without relying on complex instruments.Lateral flow dipstick(LFD)is a Lateral flow chromatographic assay(LFCA),Immune colloidal gold technique(GICT)and Biotin-avidin system(BAS)combined with rapid molecular detection technology,which has the advantages of simple operation,intuitive results and easy interpretation.The fully enclosed target nucleic acid rapid detecting device is a disposable nucleic acid detecting device formed by placing a nucleic acid detecting test strip into a palm plastic box,and the device has good sealing property,and can avoid sample cross-contamination and nucleic acid aerosol diffusion.In this study,RPA technology and LFD technology were combined to establish AIV and RA RPA-LFD detection methods.Based on the AIV matrix protein(M)gene sequence,specific primers and probes are designed and special tags are added.After the initial establishment of the AIV RPA-LFD detection method,the reaction conditions were optimized.AIV RPA-LFD detection method was used to detect N1,N2 and N6 subtypes AIV,and the results were all positive,indicating that the AIV RPA-LFD detection method has wide applicability.The specific study results of AIV RPA-LFD detection methods show that other common viral infectious diseases such as Infectious bursal disease virus(IBDV),Newcastle disease virus(NDV),infection The results of Infectious laryngotracheitis virus(ILTV)were negative,indicating that the specificity of the AIV RPA-LFD assay was good.The sensitivity of the AIV RPA-LFD assay showed that the detection limit of AIV c DNA by AIV RPA-LFD was 8 pg,and the detection limit of AIV c DNA by PCR was 6.25 pg.The detection sensitivity of the two detection methods for AIV c DNA was consistent;The detection limit of AIV RPA-LFD for recombinant plasmid carrying AIV M gene was 10~3copies.The detection limit of recombinant plasmid carrying AIV M gene was 10~4copies.The sensitivity of AIV RPA-LFD to plasmid detection was 10 times that of PCR for plasmid detection.It indicates that the AIV RPA-LFD detection method has high sensitivity.The AIV RPA-LFD detection method was initially applied to clinical disease(50 parts)detection.The results showed that the AIV RPA-LFD detection results were consistent with the PCR detection results,and the coincidence rate of the two was 100%.It indicates that the AIV RPA-LFD detection method has certain clinical application value.Specific primers and probes are designed and specifically labeled according to the RA gyrase B subunit(gyr B)gene sequence.After the RA RPA-LFD detection method was initially established,the reaction conditions were optimized.The specificity of the RA RPA-LFD assay showed that the detection results of other common pathogens such as Escherichia coli(E.coli),Pasteurella,and Salmonella were negative,indicating that the RA RPA-LFD assay method Good specificity.The sensitivity of RA RPA-LFD assay showed that the detection limit of RA RPA-LFD for RA DNA was 48 fg,the detection limit of RA DNA for PCR was 5.9 pg,and the sensitivity of RA RPA-LFD for RA DNA detection was PCR.The sensitivity of RA DNA detection was 100 times;the detection limit of RA RPA-LFD for recombinant plasmid carrying RA gyr B gene was 10~2copies,and the detection limit of recombinant plasmid carrying RA gyr B gene was 10~4copies.The sensitivity of RA RPA-LFD to plasmid detection was PCR is 100 times more sensitive to plasmid detection.It shows that the RA RPA-LFD detection method has high sensitivity.The RA RPA-LFD detection method was initially applied to clinical simulation sample detection.The results showed that the RA RPA-LFD detection results were consistent with the PCR detection results.It shows that the RA RPA-LFD detection method has certain clinical feasibility.The AIV/RA RPA-LFD detection method established in this study was used to extract the pathogenic nucleic acid and reacted at 37°C for 20 min.The terminal detection was performed by using the fully-closed target nucleic acid rapid detection device,and the detection result could be directly read.In summary,the AIV/RA RPA-LFD detection method is efficient,sensitive,specific,and supports on-site rapid detection,so that it can be promoted and applied at the grassroots level.