Abstract
Among the molecular diagnostic methods for bacteria-induced diseases, capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) combined with 16S rRNA gene-specific PCR has enormous potential because it can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S rRNA gene-specific markers into separate peaks. 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 report, a protocol for the detection of 12 pathogenic bacteria is provided. 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 the easy identification of the pathogens.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Briese T et al (2005) Diagnostic system for rapid and sensitive differential detection of pathogens. Emerg Infect Dis 11:310–313
Hall RH (2002) Biosensor technologies for detecting microbiological foodborne hazards. Microbes Infect 4:425–432
Hebart H et al (2000) Early detection of Aspergillus infection after allogeneic stem cell transplantation by polymerase chain reaction screening. J Infect Dis 181:1713–1719
Schlundt J (2002) New directions in foodborne disease prevention. Int J Food Microbiol 78:3–17
Lin LH et al (2011) Rectal swab sampling followed by an enrichment culture-based real-time PCR assay to detect Salmonella enterocolitis in children. Clin Microbiol Infect 17:1421–1425. doi:10.1111/j.1469-0691.2010.03450.x
Lampel KA, Orlandi PA, Kornegay L (2000) Improved template preparation for PCR-based assays for detection of food-borne bacterial pathogens. Appl Environ Microbiol 66:4539–4542
Grace MB et al (2003) Development and assessment of a quantitative reverse transcription-PCR assay for simultaneous measurement of four amplicons. Clin Chem 49:1467–1475
Andersen PS et al (2003) Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening. Hum Mutat 21:455–465
Bouallagui H et al (2004) Microbial monitoring by molecular tools of a two-phase anaerobic bioreactor treating fruit and vegetable wastes. Biotechnol Lett 26:857–862
Chachkhiani M et al (2004) 16S rDNA characterisation of bacterial and archaeal communities during start-up of anaerobic thermophilic digestion of cattle manure. Bioresour Technol 93:227–232
Leclerc M, Delgenes JP, Godon JJ (2004) Diversity of the archaeal community in 44 anaerobic digesters as determined by single strand conformation polymorphism analysis and 16S rDNA sequencing. Environ Microbiol 6:809–819
Dabert P, Delgenes JP, Godon JJ (2005) Monitoring the impact of bioaugmentation on the start up of biological phosphorus removal in a laboratory scale activated sludge ecosystem. Appl Microbiol Biotechnol 66:575–588
Khammar N et al (2005) Link between spatial structure of microbial communities and degradation of a complex mixture of volatile organic compounds in peat biofilters. J Appl Microbiol 98:476–490
King S, McCord BR, Riefler RG (2005) Capillary electrophoresis single-strand conformation polymorphism analysis for monitoring soil bacteria. J Microbiol Methods 60:83–92
Chung JH et al (2007) Parallel analysis of antimicrobial activities in microbial community by SSCP based on CE. Electrophoresis 28:2416–2423
Shin GW et al (2008) A two-step quantitative pathogen detection system based on capillary electrophoresis. Anal Biochem 383:31–37
Shin GW et al (2009) A new single-step quantitative pathogen detection system: template-tagging followed by multiplex asymmetric PCR using common primers and CE-SSCP. Electrophoresis 30:2728–2736
Kim SJ et al (2010) Triblock copolymer matrix-based capillary electrophoretic microdevice for high-resolution multiplex pathogen detection. Electrophoresis 31:1108–1115
Shin GW et al (2010) A novel pathogen detection system based on high-resolution CE-SSCP using a triblock copolymer matrix. J Sep Sci 33:1639–1643
Shin GW et al (2010) Simultaneous quantitative detection of 12 pathogens using high-resolution CE-SSCP. Electrophoresis 31:2405–2410
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hwang, H.S., Shin, G.W., Chung, B., Na, J., Jung, G.Y. (2013). Multiplex and Quantitative Pathogen Detection with High-Resolution Capillary Electrophoresis-Based Single-Strand Conformation Polymorphism. In: Phillips, T., Kalish, H. (eds) Clinical Applications of Capillary Electrophoresis. Methods in Molecular Biology, vol 919. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-029-8_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-029-8_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-028-1
Online ISBN: 978-1-62703-029-8
eBook Packages: Springer Protocols