A new method for simultaneous quantitative determination of hepatitis B virus pregenomic RNA and DNA
10.16781/j.0258-879x.2020.05.0564
- Author:
Bo-Wen XU
1
Author Information
1. Institute of Immunology and National Key Laboratory of Medical Immunology, College of Basic Medical Sciences, Naval Medical University, Second Military Medical University)
- Publication Type:Journal Article
- Keywords:
Chronic hepatitis B;
Covalently closed circular DNA;
DNA;
Duplex polymerase chain reaction;
Hepatitis B virus;
Pregenomic RNA;
Quantitative polymerase chain reaction
- From:
Academic Journal of Second Military Medical University
2020;41(5):564-569
- CountryChina
- Language:Chinese
-
Abstract:
Objective To design and establish a new method for simultaneous determination of hepatitis B virus (HBV) pregenomic RNA (pgRNA) and DNA from nucleic acid extracts. Methods We established a duplex fluorescence quantitative PCR system to determine HBV pgRNA and DNA. DNA gel electrophoresis and quantitative PCR were used to test the specificity and sensitivity. We tested the feasibility and accuracy by determining the HBV pgRNA and DNA in HepG2.2.15 cells and the culture supernatants. Results The established duplex fluorescence quantitative PCR system has a good specificity and sensitivity. When it was used to determine cell culture supernatants with different dilution ratios, the dilution ratios and results were well correlated. However, this method was more suitable for the determination of HBV pgRNA and DNA in cell culture supernatants, rather than cell samples. Conclusion Our method can avoid inaccuracy of HBV RNA determination caused by HBV DNA contaminant in nucleic acid extracts, and realize simultaneous detection of HBV pgRNA and DNA in one PCR reaction, which greatly improves the determination efficiency and has potential clinical application value..
