Abstract
BackgroundA simple and reliable DNA extraction of hepatitis B virus (HBV) is critical in developing an ultrasensitive detection method for HBV infection. Current commercially available serum Hepatitis B Virus (HBV) DNA extraction methods are time-consuming, expensive and/or require specialized equipment, which hinders wide adoption of clinical laboratories. This study offers a report on an ultrasensitive HBV DNA detection method by coupling serum HBV DNA extraction by ultrafiltration (UF) with real-time PCR (qPCR) detection.MethodsSerum proteins were precipitated by phenol to release HBV DNA in the supernatant which was then transferred to the UF devices. The resultant DNA concentrate was eluted and released into qPCR pre-mixture. The UF-qPCR assay performance, including recovery rate, linearity, detection sensitivity, precision and diagnostic accuracy that compared to the CAP-CTM V2.0 assay by analyzing batched low viral load clinical samples was evaluated.ResultsThe recovery rate of the UF-based HBV DNA extraction method was above 80%. The assay linearity was demonstrated with a slope of 0.95 and R2 values of 0.99. Limit-of-detection (LOD) of the UF-qPCR assay was determined to be 12.1IU/ml. The coefficient of variation (CV) of HBV quantitation for high, low and limit titer samples was 2.28%, 5.77% and 25.59%, respectively. Accuracy of the UF-qPCR assay was confirmed with the reference panel, and there was a strong correlation between these two methods (R2 = 0.55, p < 0.01).ConclusionsThe UF-qPCR assay is reliable, highly sensitive, affordable and time-saving, and the method can be used for ultrasensitive detection of serum HBV.
Highlights
Hepatitis B Virus (HBV) infection is one of the most common causes of liver diseases ranging from acute hepatitis to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) [1]
This study offers a report on an ultrasensitive HBV DNA detection method by coupling serum HBV DNA extraction by ultrafiltration (UF) with real-time PCR detection
Accuracy of the UF-quantitative real-time PCR (qPCR) assay was confirmed with the reference panel, and there was a strong correlation between these two methods (R2 = 0.55, p < 0.01)
Summary
Hepatitis B Virus (HBV) infection is one of the most common causes of liver diseases ranging from acute hepatitis to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) [1]. The virological diagnosis and monitoring of the HBV infection are based on immunoassays detecting viral antigens and specific anti-HBV antibodies as well as nucleic acid detection assays targeting genomic material of the virus [3]. HBV DNA quantitation can be used to monitor viral replication kinetics in order to better understand the mechanisms of infection and the virologic response to antiviral therapy. The serum HBV DNA detection system using sensitive and accurate qPCR assays is crucial to predict the response to therapy, to determine therapy initiation, to monitor resistance to therapy, to establish treatment success, and to evaluate the risk factors for cirrhosis and the progression of HCC [11,12]
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