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.
J Mol Diagn
; 21(6): 1106-1116, 2019 11.
Article
em En
| MEDLINE
| ID: mdl-31607557
ABSTRACT
Dynamic 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 partial VR group, it was mostly ≥4%. COLD-PCR/FMCA provides a novel tool with high sensitivity, convenience, and practicability for the simultaneous quantification of HBV DNA, genotype, and mutation. It might be used for distinguishing the different phases of HBV infection and predicting VR of CHB patients.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Vírus da Hepatite B
/
Reação em Cadeia da Polimerase
/
Hepatite B Crônica
/
Mutação
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
J Mol Diagn
Ano de publicação:
2019
Tipo de documento:
Article