Establishment Of A New Visual Detection Method For Influenza Virus H1N1 Based On HCR Technology

Highly infectious influenza viruses,especially that of influenza A,can cause large-scale outbreak of worldwide influenza,causing huge economic loss and posing long-term threat to the health and safety of humans and animals.Therefore,rapid and accurate detection of influenza virus is a key link in the prevention and control of influenza epidemic.In order to achieve rapid and convenient detection of influenza virus,we established three visualization methods to detect influenza virus H1N1.Based on hybridization chain reaction(HCR)nucleic acid amplification technology combined with the signal output mode of colloidal gold nucleic acid test strip,the simple and rapid visual detection has been realized of influenza virus H1N1.Specifically the methods are as follows:1.The method of visual detection of influenza virus H1N1 was established by using HCR signal amplification technique combined with colloidal gold nucleic acid test strip.The capture probe(CP)and detect probe(DP)labeling Carboxyfluorescein(FAM)has been designed to cope with the specific DNA fragment of H1N1.When the RNA of H1N1 is present,the HCR products labeling the biotin and the nucleic acid polymers labeling FAM-CP will be formed.The two kinds of products will be captured by the anti-FAM on the test line,when combined with the test strip testing.Then because of the specific affinity between the streptavidin(SA)and Biotin,the coating SA red colloidal gold particles(Au NPs)is fixed to the text line,the accumulation of Au NPs on the test line showing the red band of the biosensor,thus enabling the visual detection of H1N1.The detection limit of specific DNA was as low as 2.96 p M.2.Molecular beacons(MBs)nucleic acid probe technology were combined with HCR signal amplification technology to realize double amplification of the signal.When the detection target,H1N1 RNA,exists,it will combine with MBs,changing the MBs conformation,which will lead to the hybridization between two auxiliary probes(Helper A and Helper B)and MBs,forming a new double-stranded DNA and replacing the target H1N1 RNA.In this way,H1N1 RNA combines with other MBs in a circular manner,realizing the first amplification of the signal.The exposed stem of MBs is designed to be complementary to the probe DP,which is capable of efficient binding to the HCR product labeling Biotin,which thereby achieves the double signal amplification.Then combined with test strip sensor,visual detection of influenza virus H1N1 has been realized.The detection limit of specific DNA was as low as 21.9 f M.3.The duplex specific nuclease(DSN)combined with HCR signal amplification technique were used to design a capture probe(CP),which labels Biotin and 6-carboxyfluorescein(FAM)simultaneously.When H1N1 RNA was present,CP and H1N1 RNA hybrid formed double-strands.Due to the characteristics of the enzyme DSN,it digested the complementary DNA in the DNA-RNA hybrid double-strand,while RNA can be recycled and combined with the other CP to achieve the first amplification of the signal.The short DNA released by the CP and the H1,H2 labeling Biotin triggered the HCR reaction,thus achieving double amplification of the signal.Then combined with the test strip sensor,visual detection of influenza virus H1N1 has been realized.The detection limit of specific DNA was as low as 3.12 f M.In summary,the three visualization methods to detect influenza virus H1N1 established in this study can achieve quick and convenient target detection,save detection time and cost,and support us with an alternatively valuable assay for influenza prevention and control at the grass-roots level and large-scale screening.