Ann Lab Med.  2012 Mar;32(2):119-125. 10.3343/alm.2012.32.2.119.

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

• Kim SH
• Jeong HS
• Kim YH
• Song SA
• Lee JY
• Oh SH
• Kim HR
• Lee JN
• Kho WG
• Shin JH

• KMID: 1245251
• DOI: http://doi.org/10.3343/alm.2012.32.2.119

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 100 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.

Keyword

DNA extraction; Broad-Range PCR; CAPD culture fluid

MeSH Terms

Bacillus/genetics/isolation & purification
Bacteria/genetics/*isolation & purification
Candida albicans/genetics/isolation & purification
DNA Primers/genetics
DNA, Bacterial/*analysis/isolation & purification
*Genetic Techniques/standards
Humans
Peritoneal Dialysis, Continuous Ambulatory
Peritonitis/*microbiology
Polymerase Chain Reaction
Reagent Kits, Diagnostic
Sequence Analysis, DNA

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