Genotyping Of Hepatitis C Virus By Isothermal Amplification

ObjectivesTo establish two isothermal amplification methods for detecting hepatitis C virus(HCV)genotyping,loop-mediated isothermal amplification(LAMP)method and real time helicase-dependent isothermal amplification(RT-HDA).and to perform performance analysis on clinical patients with HCV genotyping and exploring clinical performance evaluation.Method:1.Establish two isothermal amplification assays for genotyping of HCV.Based on the principle of LAMP,primers for each subtype were designed for HCV-1b,2a,3,6a subtypes.Based on the principle of helicase-dependent isothermal amplification(HDA).a set universal l/3 primers were designed as syetem ? for amplifying HCV-1b.3a,3b subtypes,other set universal 2/6 primers were designed as syetem ? for amplifying HCV-2a,6a.In syetem ? and ?,probes correlated with subtypes were designed to distinguish HCV subtypes2.According to the sequences of the 5'UTR-Core region of the whole HCV gene.the LAMP primer of different subtypes 1b.2a.3 and 6a was designed by Primer Explorer V.4 software,and LAMP reaction system was established and optimized.Universal 1/3 primers and 2/6 primers and Taqman probes based on HDA priciple were designed to distinguish HCV-1b,2a.3a.3b,6a subtypes3.Performance evaluation of LAMP method was explored.Primers reacted with templates to determine the primer specificity.Each HCV subtypes serum was separately diluted,and each diluted sample of the concentration was equally divided into 10 parts.Then RNA was extracted and detected by LAMP primers and HDA probes to determine limit of detection.The anti-interference ability of the two methods was determined by template extracted from HIV,HBV,HAV-positive serum reacted with LAMP primers and HDA probes4.The clinical application of HCV genotyping in LAMP methods.Each 20 cases of HCV-1b,2a,3 and 6a serum samples which confirmed by gene sequencing were detected by LAMP and real-time PCR methods simultaneously to compare the detecttion rate and correct rate.5.Primers designed by HDA principle were tested for the feasibility by conventional PCR reagents,then primers were verified by HDA reagents.The performance of the HDA method was evaluatedResults:1.Successfully establishing LAMP method for HCV genotyping,LAMP method reacted at 65?,60 min to distinguish HCV-1b,2a,3,6a subtypes.2.LAMP primers only reacted with corresponding template.The minimum detection limit of HCV-lb,3,6a was 1.5×103 IU/mL.the minimum detection limit of HCV-2b was 1.0×103 IU/mL.3.Sequencing 80 clinical specimens to determine HCV genotype(each subtypes of HCV-1b,2a,3,6a was 20).75 specimens were successfully detected and classified by LAMP method,HCV-1b,2a,3,6a respectively detects 20,17,18,20 by LAMP method.74 specimens were detected by RT-PCR method,HCV-1b,2a,3,6a respectively detects 20,18,19,17 by RT-PCR.4.HCV-1/3 universal primer designed by principle of HDA method can amplify HCV-1b,3a,3b subtypes by conventional PCR reagents,but by the HDA method it has not been successfulConclusions:The loop-mediated isothermal amplification method was established in this study.It can specifically and sensitively detect four subtypes of HCV-1b,2a,3 and 6a.The HCV 1/3 universal primer was designed to get the correct product with PCR kits,but with the Helicase-dependent isothermal amplification method,it has not been successful.