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
| Autor: | Ni Lin, Er Huang, Yujue He, Bin Yang, Zhen Xun, Jinpiao Lin, Qishui Ou, Tianbin Chen, Can Liu, Jinlan Huang |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Hepatitis B virus Guanine Biology medicine.disease_cause Polymerase Chain Reaction Sensitivity and Specificity Fluorescence Melting curve analysis Pathology and Forensic Medicine Viral Proteins 03 medical and health sciences chemistry.chemical_compound symbols.namesake Hepatitis B Chronic 0302 clinical medicine Limit of Detection Genotype medicine Humans Sanger sequencing Temperature High-Throughput Nucleotide Sequencing RNA-Directed DNA Polymerase Hepatitis B medicine.disease Molecular biology Reverse transcriptase Treatment Outcome 030104 developmental biology chemistry 030220 oncology & carcinogenesis DNA Viral Mutation symbols Mutation testing Molecular Medicine DNA |
| Zdroj: | The Journal of Molecular Diagnostics. 21:1106-1116 |
| ISSN: | 1525-1578 |
| DOI: | 10.1016/j.jmoldx.2019.08.001 |
| Popis: | 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 was4%, 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. |
| Databáze: | OpenAIRE |
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