Establishment And Assessment For Reverse Hybridization Assay Of HBV Drug-resistant Mutations

Hepatitis B virus has been threatening the health of humanity. Nucleoside analogues as one of the main drugs have made many significant achievements in anti-HBV infection. However, HBV drug resistance can occur in varying degrees with long-term treatment, which will restrict the choice of the follow-up drugs and their efficacy seriously. Thus, it is important to detect drug resistance in the early stage. Currently, most methods detecting drug-resistant mutations are limited to experimental research. Only direct sequencing, fluorescence-quantitative PCR and line probe assay are applied in the clinical trial. Compared with other two methods, line probe assay based on reverse hybridization principle is a simple and rapid method which can detect multiple sites and has broad application prospect. Lamivudine, adefovir, and entecavir are the first batch of drugs applied in clinical treatment. Based on reverse hybridization principle, the aim of this study was to establish a method which can detect HBV resistant mutants related to these three drugs simultaneously. In order to optimize reaction system, this study also constructed HBV drug-resistant standard strain. Subsequently, methodological assessment and clinical evaluation are used to analyze this detection system.Objective: To construct HBV drug-resistant standard strain and establish a specific and sensitive method for detecting HBV resistance related to three kinds of nucleoside analogues by using HBV wild-type and drug-resistant standard strain, and finally to assess the feasibility of this testing system.Methods: 1. a 267bp amplified mutation fragment for mutagenic primer (rt169T, rt173L, rt180M, rt181T, rt184G, rt202I, rt204V, rt215S, rt236T, rt250V), HBV wild-type standard strain as a template, HBV drug-resistant standard strain was obtained by PCR.2. HBV wild-type and drug-resistant standard strains were used to optimize the reverse hybridization conditions and screen ideal probes at ten codon positions related to three-drug resistance (I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T, M250V/I/L).3. Performance of this reverse hybridization system are analyzed by methodological assessment and clinical evaluation. HBV wild-type and drug-resistant standard strains were tested respectively in order to analyze the specificity of the reaction system; the sensitivity analysis was finished though mixed wild-type and drug-resistant strains in the serial proportion and diluting PCR products to the lowest concentration detected by this method. To confirm clinical feasibility of this method, 10 samples from 10 nucleoside-na?ve patients and 30 samples from 30 patients received nucleoside analogue therapy was detected by this method and direct sequencing. In the study, the results between them were analyzed in order to observe the accuracy of detection and the lowest DNA concentration detected by this method.Results: 1. HBV drug-resistant standard strain (rt169T, rt173L, rt180M, rt181T, rt184G, rt202I, rt204V, rt215S, rt236T, rt250V) was constructed, and its accuracy was confirmed by direct sequencing.2. The reaction system was established and ideal probes were screened. This method can amplify signal difference and detect resistant mutations at 10 positions associated with three drugs.3. The results of methodological assessment and clinical evaluation confirmed that this method is a specific and sensitive approach for analysis of drug resistance. Detection signals appear when the amount of PCR products reaches 10ng/μl; HBV wild-type or mutants can be detected when they account for 5% of the virus population. The results of clinical samples using the method were in accordance with that Of DNA sequencing, and it can be detected by this method when HBV DNA concentration was≥104copies / ml.Conclusion: HBV drug-resistant standard strain was constructed rapidly and accurately by the way which mutagenic primers substituted for the corresponding sequence of HBV wild-type standard strain. This strategy provides a new methodology for constructing other mutants. In the second part, depending on wild-type and drug-resistant standard strains, this method was established which can detect HBV resistant mutations associated with three kinds of nucleoside analogues simultaneously. In order to distinguish HBV mutants from the virus population and amplify signal difference, a lot of meaningful attempts have been done, among which selecting hybridization solution including TEACL, introducing poly-C modification in the ends of probes, using three-digoxigenin labeled primers have achieved good results. Finally, methodological evaluation and clinical feasibility analysis confirmed that the established system is a specific and sensitive method which can be used to detect HBV drug-resistant mutations.