title: A comparison of different pre-lysis methods and extraction kits for recovery of Streptococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood.
abstract: Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidase pre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6-85.1, p<0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2-8.9, p<0.001), compared with no pre-lysis. Differences in yield due to pre-lysis were 2-3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture.
authors.affiliation: Centre for Infection and Immunity, Queens University Belfast, Northern Ireland, United Kingdom; Regional Virus Laboratory/Department of Microbiology, Belfast Health and Social Care Trust, Northern Ireland, United Kingdom.
authors.forename: Rachael M
authors.initials: RM
authors.lastname: Burke
authors.affiliation: Regional Virus Laboratory/Department of Microbiology, Belfast Health and Social Care Trust, Northern Ireland, United Kingdom.
authors.forename: James P
authors.initials: JP
authors.lastname: McKenna
authors.affiliation: Regional Virus Laboratory/Department of Microbiology, Belfast Health and Social Care Trust, Northern Ireland, United Kingdom.
authors.forename: Ciara
authors.initials: C
authors.lastname: Cox
authors.affiliation: Regional Virus Laboratory/Department of Microbiology, Belfast Health and Social Care Trust, Northern Ireland, United Kingdom.
authors.forename: Peter V
authors.initials: PV
authors.lastname: Coyle
authors.affiliation: Centre for Infection and Immunity, Queens University Belfast, Northern Ireland, United Kingdom; Royal Belfast Hospital for Sick Children, Northern Ireland, United Kingdom.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Shields
authors.affiliation: Centre for Infection and Immunity, Queens University Belfast, Northern Ireland, United Kingdom; Regional Virus Laboratory/Department of Microbiology, Belfast Health and Social Care Trust, Northern Ireland, United Kingdom. Electronic address: derek.fairley@belfasttrust.hscni.net.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Fairley
mesh_terms: D001419:Bacteria / Q000302:isolation & purification; D001431:Bacteriological Techniques; D001433:Bacteriolysis; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology; D064349:Genome, Microbial; D005898:Glass; D006801:Humans; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods; D011933:Reagent Kits, Diagnostic; D012697:Serine Endopeptidases / Q000378:metabolism; D013048:Specimen Handling / Q000379:methods*; D013290:Streptococcal Infections / Q000382:microbiology*; D013292:Streptococcus agalactiae / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase
keywords: Achromopeptidase; Extraction; Group B Streptococcus