Establishment of Coamplification at Lower Denaturation Temperature PCR/Fluorescence Melting Curve Analysis for Quantitative Detection of Hepatitis B Virus DNA, Genotype, and Reverse Transcriptase Mutation and Its Application in Diagnosis of Chronic Hepatitis B

Publication date: Available online 10 October 2019Source: The Journal of Molecular DiagnosticsAuthor(s): Can Liu, Jinpiao Lin, Zhen Xun, Jinlan Huang, Er Huang, Tianbin Chen, Yujue He, Ni Lin, Bin Yang, Qishui OuDynamic and real-time hepatitis B virus (HBV) DNA, genotype, and reverse transcriptase mutation analysis plays an important role in diagnosing and monitoring chronic hepatitis B (CHB) and in assessing the therapeutic response. We established a highly sensitive coamplification at lower denaturation temperature PCR (COLD-PCR) coupled with probe-based fluorescence melting curve analysis (FMCA) for precision diagnosis of CHB patients. The imprecision with %CV and detection limit of HBV DNA detected by COLD-PCR/FMCA were 2.58% to 4.42% and 500 IU/mL, respectively. For mutation, the imprecision and detection limit were 3.35% to 6.49% and 1%, respectively. Compared with Sanger sequencing, the coincidence rates of genotype and mutation were 96.0% and 82.5%, respectively, whereas the inconsistent data resulted from a low proportion (<20%) of mixed genotypes or mixed mutations. The mutation ratio in HBV infection patients was as follows: hepatitis B e antigen (HBeAg)–positive infection (0/0.0%) < HBeAg-negative infection (16/4.5%) < HBeAg-positive hepatitis (30/5.5%) < HBeAg-negative hepatitis (36/6.5%). In patients with entecavir therapy, the proportion of mutation at baseline or week 4 in virologic response (VR) group was <4%, whereas in the partia...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research