Simultaneous quantitative detection of 12 pathogens using high-resolution CE-SSCP.
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.
Shin
Gi Won
GW
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea.
Hwang
Hee Sung
HS
Oh
Mi-Hwa
MH
Doh
Junsang
J
Jung
Gyoo Yeol
GY
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000175:diagnosis* / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D011092:Polyethylene Glycols; D018807:Polymorphism, Single-Stranded Conformational; D011409:Propylene Glycols; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D004269:DNA, Bacterial; C116176:PEO-PPO-PEO; D011409:Propylene Glycols; D012336:RNA, Ribosomal, 16S; D011092:Polyethylene Glycols