Abstract
Single/double strand conformation polymorphism (SSCP/DSCP) analysis of polymerase chain reaction (PCR) amplified DNA fragments has emerged as a simple and sensitive screening method for the detection of small genomic variations. In this study we investigated the use of a four-color fluorescent-dye-labeled PCR-SSCP/DSCP technique as a screening method to enable the identification of microorganisms. PCR-SSCP/DSCP analysis was performed on 14 kDa fusion protein-encoding gene fragments, derived from strains belonging to different species of the genus Orthopoxvirus, amplified with fluorescent-dye-labeled primers. An automated gel analyzer was used for SSCP/DSCP analysis of the products. Preliminary results indicate that the PCR-SSCP/DSCP approach is a powerful method for the detection and identification of strain-specific nucleotide sequence polymorphism within the 14 kDa protein encoding gene. Therefore, it is concluded that this PCR-SSCP/DSCP technique represents a sensitive, reproducible and highly discriminatory procedure for molecular typing of pathogens.
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References
Czemy, C.-P., Johann, S., Hölzle, L. and Meyer, H. (1994) Epitope detection in the envelope of intracellular naked Orthopox viruses and identification of encoding genes, Virology 200, 764–777.
Fujimoto, S., Marshall, B. and Blaser, M.J. (1994) PCR-based restriction fragment length polymorphism typing of Helicobacter pylori. J. Clin. Microbiol. 32, 331–334.
Glava..., D. and Dean, M. (1993) Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations, Hum. Mutat. 2, 404–414.
Hayashi, K., Orita, M., Suzuki, Y. and Sekiya, T. (1989) Use of labeled primers in polymerase chain reaction (LP-PCR) for a rapid detection of the product, Nucleic Acids Res. 17, 3605.
Liu, Q. and Sommer, S.S. (1994) Parameters affecting the sensitivities of dideoxy fingerprinting and SSCP. PCR Methods Appl. 4, 97–108.
Makino, R., Yazyu, H. Kishimoto, Y., Sekiya, T. and Hayashi, K. (1992) F-SSCP: Fluorescence-based Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) Analysis. PCR Methods Applic. 2, 10–13.
Orfermans, M.T.C., Sonneveld, R.D., Bakker, E., Deutz-Terlouw, P.P., de Geus, B. and Rozing, J. (1995) Denaturing and non-denaturing gel electrophoresis as methods for the detection of junctional diversity in rearranged T cell receptor sequences, J. Immunol. Methods 181, 101–114.
Offermans, M.T.C., Struyk, L., de Geus, B., Breedveld, F.C., van den Elsen, P.J. and Rozing, J. (1996) Direct assessment of junctional diversity in rearranged T cell receptor b chain encoding genes by combined heteroduplex and single strand conformation polymorphism (SSCP) analysis. J. Immunol. Methods 191, 21–31.
Olsen, J.E. and Skov, M. (1994) Genomic lineage of Salmonella enterica serovar Dublin, Vet. Microbiol. 40, 271–282.
Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K. and Sekiya, T. (1989a) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA 86, 2766–2770.
Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K. (1989b) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction, Genomics 5, 874–879.
Preston, M.A. and Borczyk A.A. (1994) Genetic variability and molecular typing of Shigella sonnei strains isolated in Canada, J. Clin. Microbiol. 32, 1427–1430.
Telenti, A., Imboden, P., Marchesi, F., Schmidheini, T. and Bodmer, T. (1993) Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis, Antimicrob. Agents Chemother. 37, 2054–2058.
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Offermans, M.T.C., Meyer, H., Zegers, N.D. (2000). Identification of Pathogens Using Single/Double Strand Conformation Polymorphism (SSCP/DSCP) Analysis. In: Stopa, P.J., Bartoszcze, M.A. (eds) Rapid Methods for Analysis of Biological Materials in the Environment. NATO ASI Series, vol 30. Springer, Dordrecht. https://doi.org/10.1007/978-94-015-9534-6_20
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DOI: https://doi.org/10.1007/978-94-015-9534-6_20
Publisher Name: Springer, Dordrecht
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