Development of In-house SYBR Green I qPCR with Melting Curve Analysis for Detection of Basal Core Promoter A1762T/G1764A Mutation of Hepatitis B Virus.

Abstract

A1762T/G1764A basal core promoter (BCP) gene mutations  of hepatitis B virus (HBV) are associated  with disease progression in patients with chronic  HBV infection. Analysis of this mutation  requires  the use of molecular  technique whereas  the commercial  kits commonly  avail­ able in Thailand  are expensive  thus making  the patients  lack  the opportunity for  proper  care, This study aimed  to develop a molecular  diagnostic  method for  the detection  of BCP mutation at position A1762T/G1764A ofHBV using SYBR Green  I qPCR with melting curve analysis and consequent comparison of the diagnostic  performance with nested PCR with restriction analysis in 30 plasma DNA of chronic  hepatitis  B virus  patient  samples.  The results  of melting  curves showed that the average Tm  ±variance of A1762T/G1764A mutation  and wild type were 77.19

± 0.32  °C and 80.70  ± 0.37  °C respectively. By using the plasmid control vectors,  the realtime PCR  efficiency  and  the coefficient  of correlation of this method  were in acceptable  range and the minimum  detection limit of this method  was at 6,460 copies/mL.  Moreover,  the accuracy of this method was better than that of the nested PCR-RFLP when reinvestigated  discordant results with  direct  sequencing. In conclusion, SYBR Green  I qPCR  with  melting  curve  analysis  is a sensitive  and  reliable  technique for  detecting  and quantifying the A1762T/G1764A mutation. Moreover, the  cost  of  these  molecular   analyses  is  relatively  inexpensive  and  the  developed method  is suitable for applying  to HBV A1762T/G1764A mutation  detection  in Thailand.