title: [Development and application of a new hepatitis C virus genotyping method with polymerase chain reaction-reverse blot dot technique].
abstract: OBJECTIVE
  Using polymerase chain reaction-reverse blot dot (PCR-RDB) technique to establish a new method for hepatitis C virus (HCV) genotyping and to study the distribution of HCV genotypes in Foshan area.
  
  
  METHODS
  HCV primers and probes were designed in 5'-untranslated region (nt-1-nt-299) of HCV. HCV RNA in serum was isolated and purified, and its cDNA was obtained by reversed transcription. Nested PCR using biotin-labelled primers, was done. PCR products were hybridized with immobilized specific probes (genotype 1a to 3b) on Biodyne C membrane to genotype HCV by color development while adding POD and TMB. A certain judgment could be made according to the position of color reaction. The reliability of this new method was verified by sequencing. HCV RNA levels in serum were determined by real time fluorescent quantitative (FQ)-PCR. 60 FQ-PCR-positive HCV sera from Foshan area were genotyped using this assay.
  
  
  RESULTS
  All 60 sera could be successfully genotyped by PCR-RBD. 50 (83.3%) cases were found to be genotype 1b, 2 (3.3%) as genotype 1a and 2 (3.3%) as genotype 2a while 5 (8.0%) to be mixture of genotype 1a and 1b, and 1 (1.7%) to be mixture of genotypes 1b and 2a. No genotypes 2b, 3a and 3b were found. The results of PCR-RDB genotyping methods coincided with sequence analysis.
  
  
  CONCLUSIONS
  Newly established HCV genotyping system was proved to be sensitive, specific, precise and economic, thus suitable for clinical and epidemiologic studies. The results of HCV genotyping showed that genotype 1b was the predominant genotype in Foshan area.
authors.affiliation: Institute of Medical Researches, the First People's Hospital of Foshan, Foshan 528000, China.
authors.forename: Guang
authors.initials: G
authors.lastname: Yang
authors.forename: Shu
authors.initials: S
authors.lastname: Chen
authors.forename: Jin-huan
authors.initials: JH
authors.lastname: Cui
authors.forename: Jian-hua
authors.initials: JH
authors.lastname: Si
authors.forename: Jia-ju
authors.initials: JJ
authors.lastname: Tan
mesh_terms: D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity