Simultaneous quantitative detection of 12 pathogens using high-resolution CE-SSCP
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- Title
- Simultaneous quantitative detection of 12 pathogens using high-resolution CE-SSCP
- Authors
- Gi Won Shin; Hee Sung Hwang; Mi-Hwa Oh; Doh, J; Gyoo Yeol Jung
- Date Issued
- 2010-07
- Publisher
- WILEY (Inter Science)
- Abstract
- Several methods based on screening for a 16S ribosomal RNA gene marker have been developed for rapid and sensitive detection of pathogenic microorganisms. One such method, CE-based SSCP (CE-SSCP), has enormous potential because the technique can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S ribosomal RNA gene-specific markers unless combined with additional modification steps. A high-resolution CE-SSCP system that uses a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer matrix was recently developed and shown to effectively separate highly similar PCR products. In this study, we developed a method based on a high-resolution CE-SSCP system using a poly(ethyleneoxide)-poly(propyleneoxide)poly(ethyleneoxide) triblock copolymer that is capable of simultaneous and quantitative detection of 12 clinically important pathogens. Pathogen markers were amplified by PCR using universal primers and separated by CE-SSCP; each marker peak was well separated at baseline and showed a characteristic mobility, allowing easy identification of pathogens. A series of experiments using different amounts of genomic pathogen DNA showed that the method had a limit of detection of 0.31-1.56 pg and a dynamic range of approximately 10(2). These results indicate that high-resolution CE-SSCP systems have considerable potential in the clinical diagnosis of bacteria-induced diseases.
- Keywords
- 16S rRNA gene; CE-SSCP; High-resolution; Pathogen detection; Polymer matrix; CONFORMATION POLYMORPHISM ANALYSIS; REAL-TIME PCR; CAPILLARY-ELECTROPHORESIS; DNA MICROARRAY; SYSTEM; DIAGNOSTICS; COPOLYMER; IDENTIFICATION; COMMUNITIES; SEPARATION
- URI
- https://oasis.postech.ac.kr/handle/2014.oak/17585
- DOI
- 10.1002/ELPS.201000091
- ISSN
- 0173-0835
- Article Type
- Article
- Citation
- ELECTROPHORESIS, vol. 31, no. 14, page. 2405 - 2410, 2010-07
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