Study On DNA Sequencing And INNO-LiPA HBV DR V.3 For Detection Of Drug-resistance Mutation Of HBV P Gene

The incidence of hepatitis B virus (HBV) infection is dramaticly high in China and severely threatening human healthy. The hepatitis B surface antigen(HBsAg) positive patients over the world are about 350 million. In China it is about 120 million,among which 30 million are chronic hepatitis patients. According to statistics there are about 800 thousand patients were died of serious hepatitis,liver cirrhosis and hepatocellular carcinoma caused by HBV infection. Therefore it is the most important task for us to prevent HBV infection and cure HBV-related diseases.In clinics,The main srtategy of chronic hepatitis B therapy is anti-viral treatment. The nucleoside/necleotide analogues drugs are convenient, less side-effect, and their antivirual are quick and potent, so they are used most commonly. Such drugs include lamivudine(LAM), adefovir(ADV), telbivudine(LdT), entecavir(ETV) and so on. However, the issue of drug-resistance will be more and more serious after long-term therapy,leading to hepatitis B recurrence and deterioration. Therefore, the drug resistance is a problem that must be prevented and solved during anti-HBV therapy. Clinical and experimental studies indicate that the persistent exist and continual replication of HBV are the key factor for the development of disease. Moreover,the HBV P gene reverse transcriptase (RT) region dose not have the correcion function by itself, so it is easy to bring on the mutation. In recent years, a variety of methods of detection of drug resistance,with their own characteristics and limitations, have been estabolised, so it is vital to establish a high specificity and sensitivity and convenient method for detection the drug resistance .The purpose of our study is to establish and optimize the PCR amplification method of the HBV P gene from serum of the patients with chronic hepatitis B, and to compare the accuracy of detecting mutations by direct DNA sequencing and INNO-LiPA HBV DR v.3, so that we can offer important means for individual therapy.1. The change of HBV biochemical indicatorsDetection of the HBV DNA,ALT,TBiL in chronic hepatitis B patients at the process of the therapy. It is found that HBV DNA and ALT were obvious lower after the therapy (P<0.05),however, the HBV DNA was significantly increased at the stages of 13～24 months in the LAM group and combination group(P<0.05).2. To establish the nested PCR amplification method of the HBV P geneAccording to the GenBank,the HBV sequences we selected mostly were the adr subtype.The nested PCR primers were designed with the help of the software Oligo 6.67. After repeated comparisons and experiments,we selected the suitable primers.The product is 725bp,including the structural domain A,B,C,D and E of HBV polymerase.It is confirmed by agarose gel electrophoresis.3. DNA sequencing The work is done by Shanghai Invirtogen Biotechnology CO.,Ltd. 86 samples were successfully sequenced,including 2 samples with HBV DNA <1×103copies/mL. The results were analyzed basing on Bioedit6.0.5,SeqmanTMⅡ,EditSeq TM ,MegAlign program. Among the 93 samples, totally 113 amino acid mutation sites were detected. In addition to the known mutation sites,we found some other mutation sites,such as V84I,S85C,A181S,L229W,N238H,N238A和N238T.4. Detected by line probe reverse hybridization assay40 samples were randomly seleced from the 93 samples,and deteced by INNO-LiPA HBV DR v3.It can detect 11 known mutation sites, including the sites of 80,173,180,181,184,194,202,204,233,236,250 in the HBV DNA polymerase.After amplification, hybridization, elution,and coloration, the result was appeared purple or brown on the belt. In 40 samples, we deteced mutations in the form as follows: L180M+M204V, V173L+L180M+M204V, M204I, L80I+M204I, L80I,L80V,A181T, A181V,N236, and only 5 samples were different from the results of DNA sequencing.Conclusions:①The nucelos(t)ide analogues drug resistance mostly appeared after 12 months.In addition to the known mutation sites(L80M/I,L180M,M204I/V,A181T,N236T,S184G,S202I,M250V),we found some other mutation sites,such as V84I,S85C,A181S,L229W,N238H,N238A and N238T.②DNA direct sequencing basing on nested PCR has a similar sensitivity with INNO-LiPA, but the result is consistent with that by INNO-LiPA HBV DR v.3 and can offer all gene mutation information of HBV P gene. In addition, it is not so expensive. Taken together, DNA seqeuncing is more suitable to Chinese patients.