Research Article: Ultrasensitive detection of serum hepatitis B virus by coupling ultrafiltration DNA extraction with real-time PCR

Date Published: February 9, 2017

Publisher: Public Library of Science

Author(s): Bin Wu, Feng Xiao, Peiwen Li, Yan Du, Jinqiong Lin, Kaihua Ming, Bin Chen, Xiuxia Lei, Banglao Xu, Dayu Liu, Isabelle A. Chemin.

http://doi.org/10.1371/journal.pone.0170290

Abstract

A 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.

Serum 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.

The 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). The UF-qPCR assay is reliable, highly sensitive, affordable and time-saving, and the method can be used for ultrasensitive detection of serum HBV.

Partial Text

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]. Over 2 billion people throughout the world have been infected with HBV and more than 350 million of them are chronically infected carriers [2]. 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]. The presence of HBV DNA in peripheral blood is a reliable marker of active HBV replication. In comparison with immunoassays, HBV DNA detection and quantification are more useful in the diagnosis of infection, therapeutic decision-making, and assessment of the response to therapy [4]. In addition, 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. To date, a series of HBV DNA detection assays have been developed, including spot hybridization technique [5], quantitative real-time PCR (qPCR) [6], DNA biosensor [7], loop-mediated isothermal amplification assay (LAMP) [8], rolling circle amplification (RCA) [9], et al. Among these assay systems, qPCR is strongly recommended by current consensus guidelines due to its sensitivity, specificity, accuracy and broad dynamic range [10]. 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].

Since the viral load of HBV patients varies greatly, qPCR assays with a wide detection range are highly desirable. The ultrasensitive HBV DNA detection assays emerged as this study required. The research has demonstrated that the detection of low level of HBV DNA was essential in order to determine the necessity of antiviral treatment [29]. The improved detection sensitivity of ultrasensitive HBV DNA detection assays is essentially dependent upon an increased sample input volume [30]. Therefore, a large volume of sample is necessary for the preparation of increased amounts of DNA template. The majority of the commercial ultrasensitive assays employ the MBs method [17–20]. Despite the high recovery obtained, these methods are dependent upon costly and specialized equipment. In contrast, UF is a simple and low-cost sample preparation tool. With its advantage in handling large volumes of sample, UF has the potential to be employed as a nucleic acids preparation facility for the ultrasensitive DNA detection assays.

 

Source:

http://doi.org/10.1371/journal.pone.0170290

 

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