title: Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious agents.
abstract: BACKGROUND
  Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR). Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification.
  
  
  METHODS
  Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium solani were mixed with an internal control virus and treated with: 1) proteinase K; 2) lyticase and 3) lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid Herpesvirus. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous Fungi with HRM (real-time PCR followed by high-resolution melting analysis).
  
  
  RESULTS
  Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for Fungi and Proteinase K for Bacteria. The addition of lyticase and proteinase K did not improve results. For Fungi the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification.
  
  
  CONCLUSIONS
  Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis.
authors.affiliation: Laboratoire du Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France.
authors.forename: Pablo
authors.initials: P
authors.lastname: Goldschmidt
authors.affiliation: Laboratoire du Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Degorge
authors.affiliation: Laboratoire du Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France.
authors.forename: Lilia
authors.initials: L
authors.lastname: Merabet
authors.affiliation: Laboratoire du Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Chaumeil
mesh_terms: D001203:Ascomycota / Q000235:genetics / Q000302:isolation & purification*; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004271:DNA, Fungal / Q000032:analysis* / Q000302:isolation & purification; D019286:Endopeptidase K; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D006801:Humans; D009097:Multienzyme Complexes; D010447:Peptide Hydrolases; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D004271:DNA, Fungal; D009097:Multienzyme Complexes; C025310:lyticase; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D010447:Peptide Hydrolases; D019286:Endopeptidase K