title: Evaluation of DNA extraction methods and their clinical application for direct detection of causative bacteria in continuous ambulatory peritoneal dialysis culture fluids from patients with peritonitis by using broad-range PCR.
abstract: BACKGROUND
  The aims of this study were to compare several DNA extraction methods and 16S rDNA primers and to evaluate the clinical utility of broad-range PCR in continuous ambulatory peritoneal dialysis (CAPD) culture fluids.
  
  
  METHODS
  Six type strains were used as model organisms in dilutions from 10(8) to 10(0) colony-forming units (CFU)/mL for the evaluation of 5 DNA extraction methods and 5 PCR primer pairs. Broad-range PCR was applied to 100 CAPD culture fluids, and the results were compared with conventional culture results.
  
  
  RESULTS
  There were some differences between the various DNA extraction methods and primer sets with regard to the detection limits. The InstaGene Matrix (Bio-Rad Laboratories, USA) and Exgene Clinic SV kits (GeneAll Biotechnology Co. Ltd, Korea) seem to have higher sensitivities than the others. The results of broad-range PCR were concordant with the results from culture in 97% of all cases (97/100). Two culture-positive cases that were broad-range PCR-negative were identified as Candida albicans, and 1 PCR-positive but culture-negative sample was identified as Bacillus circulans by sequencing. Two samples among 54 broad-range PCR-positive products could not be sequenced.
  
  
  CONCLUSIONS
  There were differences in the analytical sensitivity of various DNA extraction methods and primers for broad-range PCR. The broad-range PCR assay can be used to detect bacterial pathogens in CAPD culture fluid as a supplement to culture methods.
authors.affiliation: Department of Laboratory Medicine, Inje University College of Medicine, Busan, Korea.
authors.forename: Si Hyun
authors.initials: SH
authors.lastname: Kim
authors.forename: Haeng Soon
authors.initials: HS
authors.lastname: Jeong
authors.forename: Yeong Hoon
authors.initials: YH
authors.lastname: Kim
authors.forename: Sae Am
authors.initials: SA
authors.lastname: Song
authors.forename: Ja Young
authors.initials: JY
authors.lastname: Lee
authors.forename: Seung Hwan
authors.initials: SH
authors.lastname: Oh
authors.forename: Hye Ran
authors.initials: HR
authors.lastname: Kim
authors.forename: Jeong Nyeo
authors.initials: JN
authors.lastname: Lee
authors.forename: Weon-Gyu
authors.initials: WG
authors.lastname: Kho
authors.forename: Jeong Hwan
authors.initials: JH
authors.lastname: Shin
mesh_terms: D001407:Bacillus / Q000235:genetics / Q000302:isolation & purification; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D002176:Candida albicans / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D005821:Genetic Techniques / Q000592:standards; D006801:Humans; D010531:Peritoneal Dialysis, Continuous Ambulatory; D010538:Peritonitis / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic