title: A comparison of molecular methods for hepatitis B virus (HBV) DNA detection from oral fluid samples.
abstract: The objective of the present study was to evaluate four commercial DNA extraction methods and three PCR protocols for hepatitis B virus (HBV) detection in artificially contaminated oral fluid samples. The extraction protocols were selected based on ease of use and cost, and were also compared with respect to sensitivity and cost. Prior PCR optimization was conducted, in which the sample volume for DNA extraction and the concentrations of DNA and Taq DNA polymerase in the PCR were adjusted. One-round PCR, used to amplify the core region of the HBV genome, achieved high levels of sensitivity in comparison with nested and semi-nested PCR experiments that were designed for the amplification of HBV surface protein genes. Of the four extraction protocols evaluated, the RTP DNA/RNA Virus Mini kit and the QIAamp DNA Mini kit gave the highest recovery rates, presenting 20 copies of HBV DNA ml(-1) as the limit of detection. These results suggest that HBV DNA can be detected from oral fluid samples but that the optimization of the PCR assays and the choice of extraction methods must be determined by laboratories before the implementation of this method in routine diagnostics.
authors.affiliation: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Moyra Machado
authors.initials: MM
authors.lastname: Portilho
authors.affiliation: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Patrícia Pais
authors.initials: PP
authors.lastname: Martins
authors.affiliation: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Lampe
authors.affiliation: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Livia Melo
authors.initials: LM
authors.lastname: Villar
mesh_terms: D000328:Adult; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D006509:Hepatitis B / Q000175:diagnosis* / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D009055:Mouth / Q000821:virology*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000191:economics / Q000379:methods; D014773:Virology / Q000191:economics / Q000379:methods*
chemical_list: D004279:DNA, Viral