title: Establishment of a novel two-probe real-time PCR for simultaneously quantification of hepatitis B virus DNA and distinguishing genotype B from non-B genotypes.
abstract: OBJECTIVE
  Establishment of a simple, rapid and economical method for quantification and genotyping of hepatitis B virus (HBV) is of great importance for clinical diagnosis and treatment of chronic hepatitis B patients. We hereby aim to develop a novel two-probe real-time PCR for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes.
  
  
  METHODS
  Conserved primers and TaqMan probes for genotype B and non-B genotypes were designed. The linear range, detection sensitivity, specificity and repeatability of the method were assessed. 539 serum samples from HBV-infected patients were assayed, and the results were compared with commercial HBV quantification and HBV genotyping kits.
  
  
  RESULTS
  The detection sensitivity of the two-probe real-time PCR was 500IU/ml; the linear range was 10(3)-10(9)IU/ml, and the intra-assay CVs and inter-assay CVs were between 0.84% and 2.80%. No cross-reaction was observed between genotypes B and non-B. Of the 539 detected samples, 509 samples were HBV DNA positive. The results showed that 54.0% (275/509) of the samples were genotype B, 39.5% (201/509) were genotype non-B and 6.5% (33/509) were mixed genotype. The coincidence rate between the method and a commercial HBV DNA genotyping kit was 95.9% (488/509, kappa=0.923, P<0.01). In addition, high correlation (R(2)=0.94, P<0.05) and good consistency between our assay and a commercial HBV DNA qPCR kit were achieved.
  
  
  CONCLUSIONS
  A novel two-probe real-time PCR method for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes was established. The assay was sensitive, specific and reproducible which can be applied to areas prevalent with HBV genotypes B and C, especially in China.
authors: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China; The Genetic Diagnosis Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors: Wei
authors: W
authors: Wang
authors: Department of Laboratory Medicine, Shanxi Provincial People's Hospital, Taiyuan, China.
authors: Hongpin
authors: H
authors: Liang
authors: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China; The Genetic Diagnosis Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors: Yongbin
authors: Y
authors: Zeng
authors: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China; The Genetic Diagnosis Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors: Jinpiao
authors: J
authors: Lin
authors: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China; The Genetic Diagnosis Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors: Can
authors: C
authors: Liu
authors: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China; The Genetic Diagnosis Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors: Ling
authors: L
authors: Jiang
authors: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China; The Genetic Diagnosis Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors: Bin
authors: B
authors: Yang
authors: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China; The Genetic Diagnosis Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China. Electronic address: ouqishui@mail.fjmu.edu.cn.
authors: Qishui
authors: Q
authors: Ou
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D015342:DNA Probes / Q000097:blood / Q000235:genetics*; D004279:DNA, Viral / Q000097:blood / Q000235:genetics*; D005260:Female; D005838:Genotype; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D019562:Viral Load / Q000379:methods / Q000592:standards*; D055815:Young Adult
chemical_list: D015342:DNA Probes; D004279:DNA, Viral
keywords: Genotyping; Hepatitis B virus; Real-time PCR; Viral concentration