Sensitive Detection Of Hepatitis C Virus Using A Catalytic Hairpin Assembly Coupled With A Lateral Flow Immunoassay

Background:Research background:Hepatitis C caused by hepatitis C virus（HCV）infection is an infectious disease that causes liver damage through blood,skin and mucous membranes and sexual contact.At present,more than 71 million people have been infected worldwide,and most of the infected people are prone to develop from mild liver injury to chronic hepatitis,and then develop liver cirrhosis or hepatocellular carcinoma;Therefore,rapid and accurate detection of HCV is of great significance for early stage and treatment.At present,the"gold standard"method for the detection of HCV-RNA in clinical laboratories is mainly RT-PCR,but RT-PCR has certain shortcomings,such as long detection time,complicated operation,expensive equipment,and certain requirements for detection technicians,etc.In recent years,point-of-care testing（POCT）has shown the advantages of short testing time and miniaturization of instruments in clinical testing work.To this end,we plan to develop a POCT detection technology for the detection of HCV-RNA to provide a high-efficiency and high-throughput detection method for clinic.Objective:Isothermal enzyme-free amplification system based on catalytic hairpin self-assembly（CHA）,isothermal amplification of HCV RNA specific target detection sequences,and detection of CHA amplification by coupled with a lateral flow immunoassay（LFIA）test strip.A fast,efficient and simple HCV RNA detection method was constructed.Methods:First,query the HCV genome,select its conserved regions and specific sequences as the target detection sequences in the subsequent detection through multiple sequence alignment;secondly,design hairpin probes according to the target detection sequences,and construct two different HCV respectively.1.CHA fluorescence sensor:The probes were labeled with fluorescein FAM and BHQ-1quenching groups,respectively,and the feasibility of CHA was verified according to the fluorescence quenching effect.Subsequently,the experimental conditions required for the reaction were optimized,and the sensitivity and specificity of the system for detecting HCV-RNA were further investigated.2.CHA-coupled with a lateral flow immunoassay（CHA-LFIA）:The designed probes were labeled with digoxigenin and biotin,and the feasibility of the method was verified by native polyacrylamide gel electrophoresis;the system was optimized the optimal reaction conditions were obtained and the sensitivity and specificity of the detection were obtained.Finally,the clinical diagnostic efficiency of the system was evaluated by the detection of clinical samples.Compared with the CHA fluorescence sensor to explore the effect of two different sensing systems on the detection of HCV-RNA.Results:According to the NCBI website,the whole HCV genome was queried,and the specific sequence fragments selected after aligning the screened sequences were used to design hairpin probes on the NUPACK online website,so as to construct the CHA system.1.CHA fluorescence sensing system:In the verification of fluorescence quenching efficiency,the reaction curve gradually decreased,and the fluorescein FAM was effectively quenched by the BHQ-1 group,indicating that the hairpin probes were hybridized and bound to each other driven by the target sequence.A double-stranded molecule is formed.Through optimization of experimental conditions,it was found that the optimal reaction conditions were the ratio of probe concentration of 1:3,the reaction temperature of 37°C,and the reaction time of 30 min.Sensitivity and specificity were further analyzed,and the results showed that CHA fluorescence sensing had a detectable lowest limit of detection（LOD）of 10 pM,and showed high specificity for the target sequence.2.CHA-LFIA:First,the feasibility of the constructed CHA system was verified by native polyacrylamide gel electrophoresis,and it was found that the two designed hairpin probes achieved the CHA signal under the trigger of the specific target sequence.Amplified;then,the optimal experimental conditions were found to be the probe H1:H2 ratio of 1:3,the reaction temperature of 37°C,the reaction time of 30 min,and the optimal concentration of Mg²⁺in the buffer solution of 5 mmol/L.The CHA-LFIA method showed high sensitivity with an LOD of1 f M and showed a good linear range from 10 n M to 10 p M with a linear correlation equation Y=-8013.7x+32644（R²=0.9743）.However,the detectable LOD of the constructed CHA fluorescence sensor is 10 p M,and the sensitivity is two orders of magnitude lower than that of CHA-LFIA.In terms of specificity,CHA-LFIA also showed high specific recognition ability for the target sequence.The designed four mutated sequences once again proved that the designed method does not interfere with the detection of the target sequence.Finally,the clinical diagnostic efficiency of the CHA-LFIA system was evaluated by clinical HCV-RNA negative and positive samples.The results showed that the clinical sample detection sensitivity was100%,specificity was 100%,negative predictive value was 100%,and positive predictive value was 100%.Conclusions:We have constructed a fast,simple,highly sensitive and specific signal amplification system that can efficiently amplify under isothermal and enzyme-free conditions without expensive instruments and complicated procedures.After a rational design,CHA triggers the hybridization between probes by the target sequence,and detects its reaction product by means of fluorescence sensing and coupling with LFIA test strips.The reaction conditions are further optimized,and HCV can be detected efficiently.The sensing system developed in this study has the potential to realize the POCT of HCV RNA detection,and can be further applied to HCV clinical screening,diagnosis and drug prognosis detection in the future.