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

• Published in: Annals of laboratory medicine 2012, 32(2), , pp.119-125
• Main Authors: Kim, Si Hyun, Jeong, Haeng Soon, Kim, Yeong Hoon, Song, Sae Am, Lee, Ja Young, Oh, Seung Hwan, Kim, Hye Ran, Lee, Jeong Nyeo, Kho, Weon-Gyu, Shin, Jeong Hwan
• Format: Article
• Language: English
• Subjects: Bacillus - genetics; Bacillus - isolation & purification; Bacteria - genetics; Bacteria - isolation & purification; Candida albicans - genetics; Candida albicans - isolation & purification; DNA Primers - genetics; DNA, Bacterial - analysis; DNA, Bacterial - isolation & purification; Genetic Techniques - standards; Humans; Original; Peritoneal Dialysis, Continuous Ambulatory; Peritonitis - microbiology; Polymerase Chain Reaction; Reagent Kits, Diagnostic; Sequence Analysis, DNA; 병리학
• ISSN: 2234-3806; 2234-3814
• DOI: 10.3343/alm.2012.32.2.119

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. 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. 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. 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.