title: Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.
abstract: BACKGROUND
  In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner.
  
  
  OBJECTIVE
  Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS.
  
  
  METHODS
  60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells.
  
  
  RESULTS
  The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS.
  
  
  CONCLUSIONS
  Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay.
authors.affiliation: The George Washington University, School of Public Health and Health Services, Department of Epidemiology and Biostatistics, Washington, DC 20037, USA. jajordan@gwu.edu
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Jordan
authors.forename: Christine O
authors.initials: CO
authors.lastname: Ibe
authors.forename: Miranda S
authors.initials: MS
authors.lastname: Moore
authors.forename: Christel
authors.initials: C
authors.lastname: Host
authors.forename: Gary L
authors.initials: GL
authors.lastname: Simon
mesh_terms: D000328:Adult; D001769:Blood / Q000821:virology*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D003890:Desiccation; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D014773:Virology / Q000379:methods*
chemical_list: D004279:DNA, Viral