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Establishment and Application of a Real-time PCR Method for Monitoring YMDD Motif Mutations Associated with Lamivudine Resistance and Hepatitic B Virus Genotypes

Author: SunYuZuo
Tutor: ShenZuoJun
School: Anhui Medical University,
Course: Clinical Laboratory Science
Keywords: Hepatitis B virus Polymerase chain reaction ( PCR ) Mutation Lamivudine Genotype
CLC: R450
Type: Master's thesis
Year: 2008
Downloads: 184
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Abstract

Real-time quantitative PCR (real-time quantitative PCR) is the rise in recent years, has become one of the important technologies of molecular biology. Real-time PCR is PCR system fluorophores, fluorescent signal accumulated real-time monitoring of the entire PCR process, and finally by the standard curve method of quantitative analysis of unknown template. With the PCR product was detected by the fluorescent signal, on the one hand to improve the sensitivity, on the other hand can collect data at each PCR cycle, the establishment of real-time amplification curves, and to accurately determine the number of cycles (CT value) was calculated starting copy number of the field value, do DNA quantification in real sense. Due to PCR amplification and product analysis process are carried out in a closed tube, greatly reducing the chance of contamination compared to conventional PCR methods, quantitative PCR with specific, rapid, sensitive, accurate, wide linear range operation simple, safe, and no post-PCR processing. The method is also different fluorescently labeled, divided into a fluorescent dye and fluorescence probe method. The fluorescence probe method based on different principles of probe design is divided into the 5 'nuclease probes (5'Nuclease Probes (TaqMan)), molecular beacons (molecular beacon) and FRET hybridization probes (FRET-hybridization probe). The real-time PCR TaqMan probe is the main object of study of this subject, the papers reported that application of the principle of the selection primer establish TaqMan probe real-time quantitative PCR method to for LAM treatment HBV process in YMDD mutation point mutation monitoring and TaqMan probe real-time PCR method for the detection of Hepatitis B virus genotype. The first part of the TaqMan probe primer real-time PCR used in the process of LAM treatment of HBV YMDD mutation point mutation monitoring established lamivudine (Lamivudine, LAM) is widely recognized as the most effective and safe antiviral nucleoside similar drugs, but the long-term use can be induced by the the HBV virus mutates, causing viral resistance. Mainly due to the tyrosine - methionine - Asp - aspartic acid (YMDD) Ribbon variation of HBV lamivudine resistance. ,Widespread view of the medical institutions in China laboratory to carry out real-time PCR detection, these labs have quantitative PCR analyzer, therefore this article for such instruments, the use of selective primers and TaqMan probe technology, the establishment of a new adaptation real-time PCR method for class equipment. We detected 187 cases of clinical hepatitis B patients with serum samples using this method, the observation of HBV YMDD mutation by sequencing as the gold standard verification. The results show that this method has a rapid, sensitive and specific characteristics in the clinical diagnosis of YMDD mutation. The second part of the real-time PCR using TaqMan probe method used for the detection of hepatitis B virus genotypes using TaqMan probe real-time fluorescence quantitative PCR method for quantitative detection and genotyping of HBV DNA of 150 patients with HBV samples then containing poison more than 5000IU/ml 113 specimens and one case sequencing genotyping. For the DNA sequence of the samples obtained through PCR and sequencing of the 114 patients, all using the the NCBI online parting tool (www.ncbi.nlm.nih.gov/projects/genotyping/ formpage.cgi) DNA sequence alignment analysis. Conducted HBV genotyping. Verify the fast detection of hepatitis B virus genotype B, C mixed genotype B, C genotype, with good specificity and high compliance rate with greater clinical significance. Summary In summary, quantitative PCR with specific, rapid, sensitive, accurate, wide linear range, safe and easy to operate and many other advantages. It has great potential in the field of chemical and biochemical analysis, especially in the field of clinical examination, as a new, fast and effective detection method will be rapid application development.

Establishment and Application of a Real-time PCR Method for Monitoring YMDD Motif Mutations Associated with Lamivudine Resistance and Hepatitic B Virus Genotypes

Abstract

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