Development of a new duplex real-time polymerase chain reaction assay for hepatitis B viral DNA detection

Shipeng Sun,Kuo Zhang,Jinming Li,Lunan Wang,Shuang Meng,Rui Zhang

doi:10.1186/1743-422x-8-227

Abstract

BackgroundQuantification of hepatitis B virus (HBV) DNA can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy. However, probably because of mismatches between the template and primer/probe, HBV DNA in some HBV infections could not be detected using currently available commercial assays with single primer/probe. By aligning the HBV sequences, we developed a duplex real-time polymerase chain reaction (PCR) assay using two sets of primers/probes and a specific armored DNA as internal control (IC).ResultsThe limit of the duplex real-time PCR assay was 29.5 IU/ml, whereas the specificity was 100%. The within-run precision coefficient of variation (CV) ranged from 1.02% to 2.73%, while the between-run CV ranged from 0.83% to 1.25%. The optimal concentration of armored DNA IC in the HBV DNA duplex real-time PCR assay was 1 000 copies/ml. Data from 69 serum samples with HBV infection showed that the performance of the duplex real-time PCR assay was comparable to that of the COBAS Ampliprep/Cobas Taqman (CAP/CTM) HBV assay and was superior to those of the domestic commercial HBV assays.ConclusionsThe duplex real-time PCR assay is sufficiently sensitive, specific, accurate, reproducible and cost-effective for the detection of HBV DNA. It is suitable for high throughput screening and frequent HBV DNA level monitoring.

Highlights

Quantification of hepatitis B virus (HBV) DNA can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy
Optimal concentration of IC To establish the optimum copy number of internal control to be added to the tubes for HBV DNA duplex real-time polymerase chain reaction (PCR) assay, a chequerboard assay was performed in which serially diluted HBV standards (5 × 105 to 0 IU/ml) were spiked with four different copy numbers (105 to 0) of the internal control armored DNA (Table 2)
The IC copies more than 1 000 changed the threshold cycles (Ct) for almost all the standards which resulted in the underestimation of the copy number

Summary

Introduction

Quantification of hepatitis B virus (HBV) DNA can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy. Probably because of mismatches between the template and primer/probe, HBV DNA in some HBV infections could not be detected using currently available commercial assays with single primer/probe. An estimated 600,000 persons worldwide die each year due to the acute or chronic consequences of hepatitis B caused by the hepatitis B virus (HBV) infection [1]. HBV infection is a leading cause of death in China [2]. Serologic tests were used routinely for the diagnosis of HBV infection. During the window period of hepatitis B virus infection, early diagnosis and follow-up of infection cannot be achieved by serologic tests. Some studies indicate that HBV may be transmitted by individuals with occult HBV infection (OHB), that is, persons who have no serologic evidences of ongoing HBV replication [4,5]

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Discussion

Conclusion