The Establishment And Application Of A Mutation Specific Polymerase Chain Reaction For Detecting Hepatitis B Virus Genome Mutations At Nt551

This study is to establish a method to investigate the distribution of the HBsAg mutant at nt551 among hepatitis B patients in China. A mutation specific polymerase chain reaction (msPCR) was established for amplifying HBV DNA with a mutation at nt551. Four sets of primer pairs, P551A-PPS, P551G-PPS, P551C-PPS and P551T-PPS, with the same sequences except for one base at 3' terminus were designed and synthesized according to the known HBV genome sequences and the popular HBV subtypes, adr and adw, in China. At the basis of regular PCR method, the specific conditions for amplifying HBV DNAs with a mutation at nt551 were optimized by adjusting annealing temperature and the primer concentration. When the annealing temperature was raised to 71癈 and primer concentration was 800nmol/L, HBV DNAs with nt551A and nt551G were amplified specifically by P551A-PPS and P551G-PPS, respectively; At 72癈 and primer concentration of 200nmol/L, HBV DNAs with nt551C and nt551T were amplified specifically by P551C-PPS and P551T-PPS, respectively. To test the specificity and sensitivity of the msPCR, 16 of HBV S DNA samples from hepatitis B patients were detected. It was shown that 14 of them were positive for nt551A, one was positive for nt551G, and the other one was positive for nt551T. The results were confirmed by nucleotide sequencing. To further investigate the application potential of the msPCR, 101 serum samples taken from Hepatitis B patients was detected. Three of nt551G mutants were examined out from the 101 serum samples which were positive for S gene. Totally, the distributing probability of mutants with a G at nt551 among 117 samples is 3.42%, that with a C at nt551 is 0%, that with a T at nt551 is 0.85%, and that with an A at nt551 is 95.73%.