title: [Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing].
abstract: OBJECTIVE
  To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene.
  
  
  METHODS
  RtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity.
  
  
  RESULTS
  The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, P<0.05). Nested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, P<0.01). The detection sensitivity of Sanger sequencing varied with the viral loads, especially in samples with low viral copies (HBV DNA ≤3log10 copies/ml), where the sensitivity was 78%, significantly lower than that of pyrosequencing (100%, P<0.01). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity.
  
  
  CONCLUSIONS
  Compared with nested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.
authors.affiliation: Department of Nosocomial Infection Control, Southern Medical University, Guangzhou, China. sunshumei99@126.com
authors.forename: Shumei
authors.initials: S
authors.lastname: Sun
authors.forename: Hao
authors.initials: H
authors.lastname: Zhou
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Ziyou
authors.initials: Z
authors.lastname: Hu
authors.forename: Jinlin
authors.initials: J
authors.lastname: Hou
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008875:Middle Aged; D010756:Phosphoric Acids; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D004279:DNA, Viral; D010756:Phosphoric Acids; D011933:Reagent Kits, Diagnostic