Establishment And Clinical Application Of A Direct Sequencing Method Of PCR Products For Detecting Of Drug-resistant Mutations Within HBV Reverse Transcriptase Region

Chronic hepatitis B virus(HBV)infection affects approximately 257 million people worldwide and is the main cause of liver diseases such as liver cirrhosis and hepatocellular carcinoma.Nucleos(t)ide analogs are often used clinically to inhibit virus replication,but the selection of drug-resistant virus strains often leads to treatment failure and disease progression.The development of drug resistance begins with mutations in the HBV polymerase gene,followed by an increase in viral load and serum alanine aminotransferase levels several weeks to months later.Detection of anti-HBV resistance mutations is thus critical in prompt decision making for new therapeutic regimes.Part one Establishment and evaluation of a method for detection ofdrug-resistant mutations within HBV reverse transcriptase region ObjectiveThis study explored the establishment of a direct sequencing method of PCR products for detection of 10 classical resistance sites within HBV reverse transcriptase region.Method1 Establishment of a direct sequencing method of PCR products for detecting drug-resistant mutations within HBV reverse transcriptase regionAccording to common mutation sites within HBV reverse transcriptase region,universal primers covering the rt169 to rt250 sites within the reverse transcriptase regions of HBV genotype B/C were designed by Primer Premier6.0 software.Serum HBV DNA was used as DNA template for PCR amplification,and then the PCR products were identified by both agarose gel electrophoresis and Sanger sequencing analysis to verify whether the target gene was successfully amplified.2 Construction of a standard plasmid containing the rt169 to rt250 sites of HBV reverse transcriptase regionSerum HBV DNA template was amplified by primers incorporated Eco RI and Hind III restriction sites.The purified PCR products and p UC19 cloning vector were then digested with the restriction enzymes Eco RI and Hind III,respectively.After the phospheodiester bond of these two cleaved DNA fragments joined together using T4 DNA ligase,the next step was to transform the recombinant plasmid DNA into E.coli strain DH5α competent cells.Then colony PCR screening and plasmid DNA sequencing analysis were performed to determine whether the recombinant plasmid standard template was successfully constructed.3 Evaluation of the direct sequencing method of PCR productsThe constructed recombinant strains were expansively cultured and then the extracted plasmid DNA was quantified and serial diluted.The standard plasmid of each concentration gradient was used as the template for PCR amplification and repeated three times.And the PCR products were then identified by 1.5% agarose gel electrophoresis.The upper and lower limits of detection of this method were defined as the maximum and minimum template concentrations where the detection rates of the target bands were both 100%.The rt M204 I mutation sample verified by Sanger sequencing was repeatedly detected by the established PCR method for ten days and then the detection rate of the above mutation site was analyzed to evaluate the repeatability of this method.Construct the rt M204 I mutant recombinant plasmid according to the molecular cloning method,and mix the mutant plasmid DNA into the wild-type plasmid according to a certain ratio.Each mixed DNA sample was used as the template for PCR amplification,and the sequencing results were then compared and analyzed by Mutation Surveyor software to evaluate the sensitivity of this method for the detection of drug-resistant mutants.Serum DNA samples from normal subjects and patients infected with human cytomegalovirus,human papillomavirus or Treponema pallidum were isolated and then used as the template for PCR amplification,as well as distilled water and standard plasmid DNA served as blank and positive controls.The target bands in gel after electrophoresis was then identified to evaluate the specificity of this method.Result(1)By optimizing the PCR amplification system and conditions,a standardized PCR protocol was established to detect the drug-resistant mutation sites within HBV reverse transcriptase region.The 50 μL PCR reaction mix as follows: distilled water 33 μL,20 mmol/L 10×PCR Buffer(Mg2+ plus)5 μL,d NTP mixtures(each d NTP 2.5 mmol/L)4 μL,Ta Ka Ra Ex Taq polymerase(5 U/μL)1 μL,upstream primer(10 μmol/L)1 μL,downstream primer(10 μmol/L)1 μL,template DNA 5 μL(<500 ng).PCR amplification conditions were pre-denaturation at 95°C for 5 min;40 cycles of denaturation at 94°C for 10 s,primer annealing at 55°C for 20 s,and primer extension at 72°C for 1 min;and an additional step of final extension at 72°C for 5 min.(2)The wild-type standard plasmid containing the rt169 to rt250 sites of HBV reverse transcriptase region was successfully constructed by molecular cloning technique.(3)The detection limit of this method ranged from 0.36 copies/μL to2.88×1011 copies/μL,and the sensitivity of this method combined with Mutation Surveyor software in the detection of drug-resistant HBV mutants was as low as 5%.The rt M204 I mutation detection rate of the same sample in ten repeated trials was 100%.And no target bands were detected in both blank control and HBV DNA negative samples.ConclusionThe direct sequencing method of PCR products established in our study can simultaneously detect the drug-resistant mutation sites between rt169 and rt250 within HBV reverse transcriptase region.It has the characteristics of low detection limit,good repeatability,high specificity,simple and fast.And this method coupled with Mutation Surveyor software can increase the detection sensitivity for low-proportion resistant mutants.Part two The clinical application of direct sequencing method of PCR products to detect HBV resistance mutations ObjectiveThe drug resistance of HBV originates from mutations within HBV reverse transcriptase(RT)region during the prolonged nucleos(t)ide analogs(NAs)antiviral therapy.So far,the characteristics of how these mutations distribute and evolve in the process of therapy have not been clarified yet.Therefore,this study aimed to detect HBV resistance mutations and genotypes in untreated and NAs-treated CHB patients through the above established direct sequencing method of PCR products and to analyze whether mutations in the RT region of CHB patients were affected by different genotypes.MethodThe resistance mutations within HBV reverse transcriptase of 450 patients with chronic hepatitis B were analyzed through direct sequencing method of PCR products,and the corresponding HBV genotypes was then identified by constructing a phylogenetic tree.ResultThe HBV reverse transcriptase gene sequences in 408 patients were successfully amplified.Phylogenetic analysis showed that HBV genotype B was dominant.Sequences analysis of 408 patients showed that classical resistance mutations were detected only in patients treated with NAs,and the resistance rate was high(46.90%,106/226).Only rt A181T/V mutation rate was significantly different between patients with HBV genotype B and gentotype C infection,and there was no significant difference in the mutation rate of other classical drug resistance sites.In terms of the mutation rates of other sites within HBV RT region,nine genotype-related amino acid polymorphism sites(rt191,rt214,rt221,rt222,rt223,rt224,rt226,rt229 and rt238)were found,and most of which showed higher mutation rates in untreated patients(P<0.05).ConclusionThere is a certain degree of genetic variation between HBV genotypes B and C,but the role of HBV genotypes in driving drug resistance changes during the use of antiviral drugs is limited.