title: Evaluation of five DNA extraction methods for purification of DNA from atherosclerotic tissue and estimation of prevalence of Chlamydia pneumoniae in tissue from a Danish population undergoing vascular repair.
abstract: BACKGROUND
  To date PCR detection of Chlamydia pneumoniae DNA in atherosclerotic lesions from Danish patients has been unsuccessful. To establish whether non-detection was caused by a suboptimal DNA extraction method, we tested five different DNA extraction methods for purification of DNA from atherosclerotic tissue.
  
  
  RESULTS
  The five different DNA extraction methods were tested on homogenate of atherosclerotic tissue spiked with C. pneumoniae DNA or EB, on pure C. pneumoniae DNA samples and on whole C. pneumoniae EB. Recovery of DNA was measured with a C. pneumoniae-specific quantitative real-time PCR. A DNA extraction method based on DNA-binding to spin columns with a silica-gel membrane (DNeasy Tissue kit) showed the highest recovery rate for the tissue samples and pure DNA samples. However, an automated extraction method based on magnetic glass particles (MagNA Pure) performed best on intact EB and atherosclerotic tissue spiked with EB. The DNeasy Tissue kit and MagNA Pure methods and the highly sensitive real-time PCR were subsequently used on 78 atherosclerotic tissue samples from Danish patients undergoing vascular repair. None of the samples were positive for C. pneumoniae DNA. The atherosclerotic samples were tested for inhibition by spiking with two different, known amounts of C. pneumoniae DNA and no samples showed inhibition.
  
  
  CONCLUSIONS
  As a highly sensitive PCR method and an optimised DNA extraction method were used, non-detection in atherosclerotic tissue from the Danish population was probably not caused by use of inappropriate methods. However, more samples may need to be analysed per patient to be completely certain on this. Possible methodological and epidemiological reasons for non-detection of C. pneumoniae DNA in atherosclerotic tissue from the Danish population are discussed. Further testing of DNA extraction methods is needed as this study has shown considerable intra- and inter-method variation in DNA recovery.
authors.affiliation: Department of Medical Microbiology and Immunology, Wilhelm Meyers Allé, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark. mygind@biobase.dk
authors.forename: Tina
authors.initials: T
authors.lastname: Mygind
authors.forename: Lars
authors.initials: L
authors.lastname: Østergaard
authors.forename: Svend
authors.initials: S
authors.lastname: Birkelund
authors.forename: Jes S
authors.initials: JS
authors.lastname: Lindholt
authors.forename: Gunna
authors.initials: G
authors.lastname: Christiansen
mesh_terms: D001161:Arteriosclerosis / Q000150:complications / Q000382:microbiology*; D002690:Chlamydia Infections / Q000150:complications / Q000453:epidemiology; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D003718:Denmark; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial