| BackgroundAnti-HBV therapy is the key to the treatment of chronic hepatitis B (CHB), and lamivudine (LAM) was the first effective hepatitis B virus (HBV) replication suppressive oral agent approved in the treatment of chronic hepatitis B. It can markedly reduce serum HBV DNA levels, enhance hepatitis B e antigen (HBeAg) seroconversion, and is associated with improvement in liver necroinflammatory activity. It can prevent the progress of liver diseases and reduce the incidence of HCC. It is likely to remain the first choice of therapy for economic reasons, particularly in the developing countries. However, viral resistance to Lamivudine with the emergence of YMDD variants during long-term Lamivudine therapy is a vital problem for optimal therapy. Genotypic resistant mutations have been detected in14-32%of patients after one year of treatment, which increases to38%,49%, and66%after2,3, and4years of treatment, respectively. Continued treatment with LAM in patients who have developed virologic resistance is of no benefit. To avoid clinical relapses that may be severe and occasionally life threatmenting, an appropriate rescue therapy should be initiated that has the most effective antiviral effect and minimal risk for selection of multidrug resistant (MDR) strains. Unfortunately, the inevitably emergence of YMDD mutant (rtM204V/I/S) after long-term lamivudine therapy, which will result in resistance to lamivudine. LAM resistance is associated with mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV polymerase C domain, more specifically, substitutions of methionine to either isoleucine (rtM204I) or valine (rtM204V). Although the mutation of HBV DNA polymerase activity is reduced, the replication ability lower than wild strain, but as the wild strains replicate by antiviral drugs inhibit, led to the mutant strains to advantage, eventually make lamivudine for mutant virus replication inhibitory effect is hard to play, the virus began to copy, HBV DNA and ALT levels rebounded. Therefore fast detection YMDD mutations, to avoid the occurrence of drug resistance, implement individualized treatment plan for patients is of great significance. Studies have reported that rtM204V variation and other antiviral drugs, such as Entecavir resistance, rtM204V/I variable selection will affect the follow-up treatment. Currently several methods have been used to detect lamivudine-resistant HBV, including the direct PCR sequencing, PCR products cloning sequencing method, polymerase restriction fragment length polymorphism (RFLP), reverse hybridization method, gene chip, and restriction fragment mass spectrometry polymorphism technology (RFMP). Sequencing remains the best approach for identifying new mutation patterns. However, because of its low sensitivity, this method can rarely dtect mixed populations of two or more HBV genotypes. Generally, sequencing cannot detect a minor subpopulation comprising less20%of a total viral population. So we need to establish a sensitive method to detect the mutation at an early time. PNA (peptide nucleic acid, PNA) was First designed and synthesized in1991by Nielsen Riso laboratory at the university of Copenhagen Denmark. It is known to be one of the most successful synthesized DNA or RNA analogues. PNAs are synthetic DNA analogs in which the phosphodiester backbone is replaced by repetitive units of N-(2-aminoethyl) gly-cine to which the purine and pyrimidine bases are attached via a methyl carbonyl linker [4]. The unique chemical makeup provides PNA with unique hybridization characteristics. Unlike DNA and RNA, the PNA backbone is not charged. Conse-quently, there is no electrostatic repulsion when PNA hybridizes to its target nucleic acid sequence, giving a higher stability to the PNA-DNA or PNA-RNA duplexes than the biological homo or heteroduplexes. This greater stability results in higher thermal melting temperature (Tm) values than is observed for DNA-DNA or DNA-RNA duplexes. An additional consequence of the polyam-ide backbone is that PNAs hybridize virtually independently of the salt concentration. Thus, the Tm of PNA-DNA duplex is barely affected by low ionic strength. This significantly facilitates the hybridization with the PNAs. The unnatural backbone of PNAs also means that PNAs are particularly resistant to protease and nuclease degradation. Because of this resistance to the enzyme degrada-tion, the lifetime of PNAs is extended both in vivo and in vitro. This high biostability increases the reliability of PNAs in diagnostic applications. As a further consequence of their synthetic backbone, PNA molecules are not recognized by polymerases and therefore cannot be copied or used directly as primers, nor can PNA oli-gomers be enzymatically incorporated into amplicons. PNAs hybridize to complementary DNA or RNA in a sequence dependent manner, according to the Watson-Crick hydrogen bond-ing scheme. In contrast to DNA, PNA can bind in either parallel or anti-parallel fashion, and hybridize with either single stranded DNA or double stranded DNA. However, the anti-parallel binding is favoured over the parallel one. Structural informations on the PNA binding modes have been obtained by nuclear magnetic resonance. PNA-DNA hybridization are significantly more affected by base mismatches than DNA-DNA hybridiza-tion. Using a15-mer PNA, all possible single mismatch combinations were tested in both PNA/DNA duplexes and corresponding DNA/DNA duplexes. In the PNA/DNA duplexes, the average Tm was15℃, whereas it was11℃ in the corresponding DNA/DNA duplexes.6,7Similar results were obtained for PNA/RNA duplexes.3This high level of discrimination at single base level indicates that short PNA probes could offer high specificity, and thus allow the further development of several PNA-based strategies for molecular investigations and diagnosis.Objectives1. For the early detection of drug resistance mutation in the process of antiviral treatment, based on PCR method and some properties of PNA, this study intends to establish a more sensitive method to detect HBV YMDD mutations of PNA-PCR method, the method to detect the contents of known viruses the different mixing ratio of plasmid mutations, compared with direct sequencing method, the sensitivity and specificity of the new method is validated.2. Based on the clinical nucleoside drugs treatment resistant test as well as patients without treatment of chronic hepatitis b patients with viral hepatitis of preexisting resistance test, validate the new method in the process of antiviral treatment resistant testing.Material and Methods1. PatientsThe first part chooses specimens of100cases of clinical sequencing a patient, the patients were treated with nucleoside drugs who were suspected clinical drug resistence or detection of drug resistance, and the second part of collecting outpatient service100cases without nucleoside analogues (acid) specimens of patients, the patients with HBeAg positive or negative, no HIV, HCV, HDV infection such as history, collect all of the patients serum, disease stored in-30℃.2. Extraction of HBV DNAHBV DNA was extracted from serum samples (200μl) by QIAamp DNA Blood Mini Kit, according to the manufacturer’s instruction.3. Amplification of HBV polymerase geneThe nucleotide sequences of HBV polymerase gene were determined using a nested polymerase chain reaction (PCR) and direct sequencing and PNA-PCR respectively. The nucleotide sequences of the PCR products were determined by Invitrogen Ltd while the result of the PNA-PCR method was directly observed by agarose gel electrophoresis.4. Statistical analysesFor categorical variabales, the Chi-Square test was performed. All data was analyzed using the statistical package SPSS (version13.0, SPSS Inc., Chicago, IL). Any P values below0.05were considered statistically significant (Two-tailed).Results1. Compare of the sensitivity and specificity of the method of drug resistance detection between PNA-PCR and direct-sequencing:In lower viral load,0.01%can be detected by the PNA-PCR, and direct sequencing method can detect10%of mutation, the sensitivity of the detection can be increased1000times, and in the case of the virus content is higher,0.001%can be detected using PNA-PCR method, while direct sequencing method can detect0.01%of mutation, the sensitivity of the detection can be increased increased10times, in total, compared with direct sequencing method PNA-PCR method resistant mutation detection sensitivity can be increased by10to1000times.2. The application of PNA-PCR in clinical drug resistance:(1)Detection of YMDD in patients use of nucleoside analogues (acid) therapy:Results in100patients with15patients negatie for PCR, the remaining85cases of patients with direct sequencing method to detect mutations results of35cases (YIDD of21cases, YVDD for11cases, YIDD+YVDD for2cases), the detection rate of40.0%. PNA-PCR method to detect mutations results of77cases (YIDD of40cases, YVDD for23cases, YIDD+YVDD for10cases), mutation detection rate was85.9%, compared with direct sequencing method, the sensitivity of detection increased significantly.(2) Detection of YMDD in patients none use of nucleoside analogues (acid) therapy: In100cases without nucleoside analogues (acid) therapy there are21patients were detected in patients with YMDD mutation in nature. Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity.Conclusions1. PNA-PCR method to detect HBV YMDD resistant mutant has higher sensitivity and specificity, especially in the low serum virus copies is, compared with direct sequencing method, PNA-PCR method has higher sensitivity, detection difference is obvious.2. PNA-one over ten thousand rtM204I or rtM204V PCR method to detect the resistant mutant strains, which can be detected early resistance must happen, for drug resistance in clinical antiviral treatment must occur to provide important indicators3. The natural mutant strains of YMDD exist in untreated patients with HBV infection and but drug-resistant mutant strains can exist for future patients after using lamivudine treatment, further research is needed. |
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