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.
10.3343/alm.2012.32.2.119
- Author:
Si Hyun KIM
1
;
Haeng Soon JEONG
;
Yeong Hoon KIM
;
Sae Am SONG
;
Ja Young LEE
;
Seung Hwan OH
;
Hye Ran KIM
;
Jeong Nyeo LEE
;
Weon Gyu KHO
;
Jeong Hwan SHIN
Author Information
1. Department of Laboratory Medicine, Inje University College of Medicine, Busan, Korea. jhsmile@inje.ac.kr
- Publication Type:Original Article ; Evaluation Studies ; Research Support, Non-U.S. Gov't
- Keywords:
DNA extraction;
Broad-Range PCR;
CAPD culture fluid
- MeSH:
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
- From:Annals of Laboratory Medicine
2012;32(2):119-125
- CountryRepublic of Korea
- Language:English
-
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.
