Optimization of Nested Polymerase Chain Reaction Hepatitis B Virus X Gene and Detection of Mutation Related to Liver Carcinoma

• Main Author: Romlah, Sitti
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45745
• Institution: Institut Teknologi Bandung

Hepatitis B virus (HBV) infects more than two billions people, 350 millions among them are chronically infected. Develepment of liver carcinogenesis became chronic HBV infection was related with HBV’s genotype and subgenotype, x gene mutations, and HBV DNA titer. To determine mutation of the x gene, gene amplification prior to analysis is required. One of method that could be used is nested PCR. Previous research had been able to amplify the x gene of several HBV DNA clinic isolates with a titer range between 10 5 -10 8 IU DNA/ml. However, the condition of that nested PCR was not applicable to DNA isolates with unknown titer. The purpose of this study was to optimize nested PCR conditions for gen x HBV clinic sample with unknown titer, to determine the genotype and subgenotype of HBV DNA isolate, and mutation on the gene. Methods: Nested PCR to amplify x gene was performed with two phases. DNA fragment produced in the first phase has theoretical length of 707 bp, while the second phase yielded two DNA fragments with the theoretical size of 469 bp (F1) and 395 bp (F2), respectively. Optimization was conducted by increasing DNA concentration using alcohol precipitation, varying primer concentrations, and diluting first phase PCR product. The sequence of nested PCR products were determined by sequencing and analyzed further by BLAST program to obtain the genotype and subgenotype. The presence of mutation of the x gene was analyzed by ClustalW program. Results: Succesfull optimization is obtained by concentrated DNA samples up to 1000µg/ml, decreasing primer concentration from 40 µM to 20 µM, and diluting first phase PCR product from 100x to 25x and 10x. A total of 14 clinic samples out of 28 samples were successfully amplified using the optimized conditions. Ten sequenced samples gave 5 isolates with B genotype (B2 and B3 subgenotypes) and 5 samples with C genotype (C1 and C5 subgenotypes). DNA alignment for analyzing x gene mutation showed one liver carcinoma-associated mutation (G1653T) which caused change in the codon encoding histidin to tyrosin. Several new mutations were also observed. Conclusion: Optimization of nested PCR for HBV clinic isolate with unknown DNA titer has been done and increased the percentage of amplification to 50%. Of ten determined clinic samples, five isolates have B genotype (B2 and B3 genotypes) and 5 isolates have C genotype (C1 and C5 subgenotypes). One liver carcinoma-associated mutation was detected.