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KMID : 1100720120320020119
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Annals of Laboratory Medicine
2012 Volume.32 No. 2 p.119 ~ p.125
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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
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Kim Si-Hyun
Ryu Min-Hee
Kim Yeong-Hoon
Song Sae-Am
Lee Ja-Young
Oh Seung-Hwan
Kim Hye-Ran
Lee Jeong-Nyeo
Kho Weon-Gyu
Shin Jeong-Hwan
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Abstract
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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 108 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.
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KEYWORD
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DNA extraction, Broad-Range PCR, CAPD culture fluid
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