Construction Of A System For Rapid Detection Of HAV By Multienzyme Isothermal Rapid Amplification Combined With Lateral Flow Dipstick

Objective:Hepatitis A virus(HAV)infection is one of the causes of viral hepatitis,and as a food-borne virus,HAV is mainly transmitted through the fecal-oral route.The rapid and accurate diagnosis of HAV infection is important for the prevention and treatment of hepatitis A.In this study,we used Multienzyme isothermal rapid amplification(MIRA)combined with Lateral flow dipstick(LFD)and immunomagnetic bead enrichment method to detect HAV.This method is convenient,sensitive,and economical,especially for detection in resource-limited areas,and is important for early diagnosis and outbreak control of HAV.Methods:Primers were designed for HAV,HAV plasmids were synthesized,primers were evaluated using MIRA basic reaction and agarose gel electrophoresis,and the minimum detection limit was determined in combination with LFD.Plasmid DNA was subjected to10-fold gradient dilution for MIRA basic reaction and MIRA-LFD reaction,and sensitivity was compared with quantitative real-time PCR(qPCR).The specificity of this reaction was tested by performing MIRA basic reaction using multiple pathogens.RT-MIRA basic reaction and RT-MIRA-LFD reaction were performed by applying live attenuated HAV vaccine to enrich hepatitis A virus in water using immunomagnetic bead method to evaluate the enrichment effect.Results:After a minimum detection limit of 0.1 copies/μL for the MIRA-LFD reaction and 1 copy/μL for the RT-MIRA-LFD reaction,the qPCR assay showed a minimum detection limit of 10 copies/μL All other pathogen reactions showed no bands and negative results for LFD.Testing using clinical samples was consistent with qPCR results.The enrichment efficiency of HAV using the immunomagnetic bead method was 91.38%when the HAV concentration was 8.16×10~4 copies/μL,59.44%when the concentration was8.16×10~3 copies/μL,34.07%when the concentration was 8.16×10~2 copies/μL and 81.6copies/μL,the enrichment efficiency was 18.84%.Comments:1.RT-MIRA reaction can successfully detect HAV in agarose gel electrophoresis.2.RT-MIRA combined with LFD reaction is more sensitive and specific than qPCR.3.Immunomagnetic bead enrichment method can effectively enrich low concentration of HAV in water.