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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초록
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.
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 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.
참고문헌 (23)
- Petti CA, "The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods", J Clin Microbiol, 43, pp.6123~6125 (2005)
- Shin JH, "Clinical significance and species identification of rapidly growing mycobacteria isolated from routine blood cultures", Korean J Lab Med, 26 (2005)
- Rantakokko-Jalava K, "Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR", J Clin Microbiol, 40, pp.4211~4217 (2002)
- Yoo TH, "Usefulness of 23S rRNA amplification by PCR in the detection of bacteria in CAPD peritonitis", Am J Nephrol, 26, pp.115~120 (2006)
- Sontakke S, "Use of broad range16S rDNA PCR in clinical microbiology", J Microbiol Methods, 76, pp.217~225 (2009)
- Fenollar F, "Usefulness of broad-range PCR for the diagnosis of osteoarticular infections", Curr Opin Rheumatol, 28, pp.463~470 (2008)
- Drancourt M, "16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates", J Clin Microbiol, 38, pp.3623~3630 (2000)
- Fuursted K, "Broad-range PCR as a supplement to culture for detection of bacterial pathogens in patients with a clinically diagnosed spinal infection", Scand J Infect Dis, 40, pp.772~777 (2008)
- Zucol F, "Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification", J Clin Microbiol, 44, pp.2750~2759 (2006)
- Maiwald M, "Broad-range PCR for detection and identification of bacteria", pp.379~390 (2004), ASM Press
- Piraino B., "Peritonitis as a complication of peritoneal dialysis", J Am Soc Nephrol, 9, pp.1956~1964 (1998)
- Kim DK, "Changes in causative organisms and their antimicrobial susceptibilities in CAPD peritonitis: a single center’s experience over one decade", Perit Dial Int, 24, pp.424~432 (2004)
- Zelenitsky S, "Analysis of microbiological trends in peritoneal dialysis-related peritonitis from 1991 to 1998", Am J Kidney Dis, 36, pp.1009~1013 (2000)
- Lee JY, "Two Cases of Peritonitis Caused by Kocuria marina in Patients Undergoing Continuous Ambulatory Peritoneal Dialysis", JOURNAL OF CLINICAL MICROBIOLOGY, 47, pp.3376~3378 (2009), AMER SOC MICROBIOLOGY
- Goldenberger D, "Molecular diagnosis of bacterial endocarditis by broad-range PCR amplification and direct sequencing", J Clin Microbiol, 35, pp.2733~2739 (1997)
- Mignard S, "16S rRNA sequencing in routine bacterial identification: a 30-month experiment", J Microbiol Methods, 65, pp.574~581 (2006)
- Edwards U, "Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA", Nucleic Acids Res, 17, pp.7843~7853 (1989)
- Lane DJ, "Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses", Proc Natl Acad Sci USA, 82, pp.6955~6959 (1985)
- Rovery C, "PCR detection of bacteria on cardiac valves of patients with treated bacterial endocarditis", J Clin Microbiol, 43, pp.163~167 (2005)
- Eden PA, "Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reactionamplified 16S rRNA-specific DNA", Int J Syst Bacteriol, 41, pp.324~325 (1991)
- Piraino B, "Peritoneal dialysis-related infections recommendations: 2005 update", Perit Dial Int, 25, pp.107~131 (2005)
- Chomczynski P, "Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction", Anal Biochem, 162, pp.156~159 (1987)
- Kemp M, "Demonstration by PCR and DNA sequencing of Corynebacterium pseudodiphtheriticum as a cause of joint infection and isolation of the same organism from a surface swab specimen from the patient", J Med Microbiol, 54, pp.689~691 (2005)
