Establishment And Application Of A Real-time PCR Method For Monitoring YMDD Motif Mutations Associated With Lamivudine Resistance And Hepatitic B Virus Genotypes

Fluorescence quantitative Real-time PCR method developed in recent years, has become increasingly important in a variety of application. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. Since the nucleic acid amplification and detection steps are performed in the same closed vessel, the risk for release of amplified nucleic acids into the environment, and contamination of subsequent analyses, is negligent compared with conventional PCR methods. Technically, quantitative real-time PCR is performed by the addition of standards which have known specified or calibrated levels of target nucleic acid. Three to five dilutions of a standard are included in each test run of each quantitative real-time PCR determination. Using the known copy level of the standard reagent, the software of the instrument generates a standard curve in a plot that relates fluorescence (measure of the amplified product) and the cycle number in which the nucleic acid target is detected. Quantitative detection of nucleic acid is determined by comparing the cycle number (crossover point or Ct) of the specimen with the standard curve generated with known levels of the target nucleic acid. excellent sensitivity and specificity, low contamination risk, ease of performance and speed, has made real-time PCR technology an appealing alternative to conventional methods .according to fluorescence marked difference ,it have fluorescent dyes and fluorescence probe. Three types of fluorescence probe detection methods have been used most frequently with real-time PCR test: 5 nuclease (TaqMan probes), molecular beacons, and FRET hybridization probes. Real-time PCR with TaqMan probes is the main object of this study. We established a real-time PCR method with TaqMan probe real-time PCR using the principle of primer selection, which can be applied to detect HBV YMDD mutants and TaqMan probe real-time PCR for determination of Hepatitis B Virus genotype. Part I Establishment and Application of a Real-time PCR Method for Monitoring YMDD Motif Mutations Associated with Lamivudine ResistanceLamivudine is an antiviral drug that is used to treat hepatitis B virus (HBV) infection. Long-term therapy does not completely suppress viral replication, and resistant mutants emerge.Resistance is mediated by changes in the tyrosine-methionine-apartate-aspartate (YMDD) motif in the catalytisite of the HBV polymerasegene.Until now,rather insensitive methods,such as nucleotidesequencing, Line probe assay, oligonucleotide chips, peptide nucleic acid clamping, PCR restriction fragment length polymorphism (RFLP), a real-time quantitative PCR method using attached universal template probe have been utilized to detect lamivudine-resistant. Owing to real-time PCR amplification instrument is popular in our country laboratory. We established a real-time PCR method with TaqMan probe real-time PCR able to detect HBV YMDD mutants in 187 serum sample from chronic HBV patients treated with lamivudine. To confirm the accuracy of this method, sequence analysis was conducted. Conclusion can be made, the real-time PCR method with TaqMan probe and selective primers is a rapid sensitive method for detection of lamivudine-resistant mutants.Part II Establishment and Application of a Real-time PCR Method for determination of Hepatitis B Virus genotype.a real-time PCR method with TaqMan probe real-time PCR detect HBV Genotype in 150 serum sample from chronic HBV patients, sequence analysis was conducted in 113 sample, which were HBV viral loads≥5000IU/ml and a sample which were HBV viral loads ranged from1000IU/ml to 5000IU/ml . The result of 114 samples were compared with the genotypes published byNCBI. (www.ncbi.nlm.nih.gov/ projects/ genotyping/ formpage.cgi) the method used can detect HBV Genotype B,Genotype C and mixed genotype B and C,The method has significant advantages:excellent sensitivity and specificity. the reliability of this assay should be considered when choosing a genotyping method for Hepatitis B genotypes in clinical practice.SummaryIn general, this technique has many advantages: sensitivity and specificity, low contamination risk, ease of performance and speed. Real-time PCR method has a great potential of applications for both chemical and biochemical analysis. This new and very efficient analytical technique is of growing interest for biochemical analysis and its use is likely to increase rapidly.