title: Development of a new duplex real-time polymerase chain reaction assay for hepatitis B viral DNA detection.
abstract: BACKGROUND
  Quantification 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).
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, People's Republic of China.
authors.forename: Shipeng
authors.initials: S
authors.lastname: Sun
authors.forename: Shuang
authors.initials: S
authors.lastname: Meng
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Kuo
authors.initials: K
authors.lastname: Zhang
authors.forename: Lunan
authors.initials: L
authors.lastname: Wang
authors.forename: Jinming
authors.initials: J
authors.lastname: Li
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D016903:Drug Monitoring / Q000379:methods; D006509:Hepatitis B / Q000175:diagnosis* / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral