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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
Annals of Laboratory Medicine ; : 119-125, 2012.
Article in English | WPRIM | ID: wpr-89103
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.
Subject(s)

Full text: Available Index: WPRIM (Western Pacific) Main subject: Peritonitis / Reagent Kits, Diagnostic / Bacillus / Bacteria / Candida albicans / DNA, Bacterial / Polymerase Chain Reaction / Genetic Techniques / Peritoneal Dialysis, Continuous Ambulatory / Sequence Analysis, DNA Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Annals of Laboratory Medicine Year: 2012 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Peritonitis / Reagent Kits, Diagnostic / Bacillus / Bacteria / Candida albicans / DNA, Bacterial / Polymerase Chain Reaction / Genetic Techniques / Peritoneal Dialysis, Continuous Ambulatory / Sequence Analysis, DNA Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Annals of Laboratory Medicine Year: 2012 Type: Article