Abstract
The polymerase chain reaction (PCR) is a fundamental part of modern molecular biology. Fluorescence-based PCR methods also are now available, which enable rapid, specific, and sensitive assays for the amplification and analysis of deoxyribonucleic acid (DNA). These methods are performed in closed-tube format, thereby reducing the risk of contamination between stages. In addition, post-PCR processing, such as clean-up steps and gel electrophoresis, are eliminated as the results are read via an integrated fluorimeter. An example of this methodology is fluorescence-based PCR using dual-labeled probes, termed dual-labeled end-point fluorescence PCR. This method uses oligonucleotide probes that are dual-labeled with a reporter dye and quencher dye. The method has the advantage that DNA extraction, liquid handling, PCR, and analysis also can be fully automated. In this chapter, the simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples is described.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Saiki, R. K., Gelfand, D. H., Stoffel, S., et al. (1988) Primer-directed enzymatic amplification of, DNA with a thermostable, DNA polymerase. Science 239, 487–491.
Clarke, S. C. (2002) Nucleotide sequence-based typing of bacteria and the impact of automation. Bioessays 24, 858–862.
Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W., and, Deetz, K. (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting, PCR product and nucleic acid hybridization. PCR Methods, Appl. 4, 357–362.
Tyagi, S. and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308.
Wright, P. A. and Wynford-Thomas, D. (1990) The polymerase chain reaction: miracle or mirage? A critical review of its uses and limitations in diagnosis and research. J. Pathol. 162, 99–117.
Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237–238.
Guiver, M., Levi, K., and Oppenheim, B. A. (2001) Rapid identification of candida species by TaqMan PCR. J. Clin. Pathol. 54, 362–366.
Heid, C. A., Stevens, J., Livak, K. J., and, Williams, P. M. (1996) Real time quantitative PCR. Genome, Res. 6, 986–994.
Stryer, L. (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47, 819–846.
Cardullo, R. A., Agrawal, S., Flores, C., Zamecnik, P. C., and, Wolf, D. E. (1988) Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 85, 8790–8794.
Higuchi, R., Fockler, C., Dollinger, G., and, Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (NY) 11, 1026–1030.
Guiver, M., Borrow, R., Marsh, J., et al. (2000) Evaluation of the applied biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA. FEMS Immunol. Med. Microbiol. 28, 173–179.
Diggle, M. A., Edwards, G. F., and, Clarke, S. C. (2001) Automation of fluorescence-based PCR for confirmation of meningococcal disease. J. Clin. Microbiol. 39, 4518–4519.
Diggle, M. A. and Clarke, S. C. (2003) Detection and genotyping of meningococci using a nested PCR approach. J. Med. Microbiol. 52, 51–57.
Smith, K., Diggle, M. A., and, Clarke, S. C. (2004) Automation of a fluorescence-based multiplex PCR for the laboratory confirmation of common bacterial pathogens. J. Med. Microbiol. 53, 115–117.
Taha, M. K., Alonso, J. M., Cafferkey, M., et al. (2005) Interlaboratory comparison of PCR-based identification and genogrouping of Neisseria meningitidis. J. Clin. Microbiol. 43, 144–149.
Diggle, M. A., Smith, K., Girvan, E. K., and, Clarke, S. C. (2003) Evaluation of a fluorescence-based PCR method for identification of serogroup a meningococci. J. Clin. Microbiol. 41, 1766–1768.
Guiver, M., Fox, A. J., Mutton, K., Mogulkoc, N., and, Egan, J. (2001) Evaluation of CMV viral load using TaqMan CMV quantitative PCR and comparison with CMV antigenemia in heart and lung transplant recipients. Transplantation 71, 1609–1615.
Smith, K., Diggle, M. A., and, Clarke, S. C. (2003) Comparison of commercial, DNA extraction kits for extraction of bacterial genomic DNA from whole-blood samples. J. Clin. Microbiol. 41, 2440–2443.
McChlery, S. M. and Clarke, S. C. (2003) The use of hydrolysis and hairpin probes in real-time PCR. Mol. Biotechnol. 25, 267–274.
Duke, T., Curtis, N., and, Fuller, D. G. (2003) The management of bacterial meningitis in children. Expert Opin. Pharmacother. 4, 1227–1240.
Peltola, H. (1983) Meningococcal disease: still with us. Rev. Infect. Dis. 5, 71–91.
van, Deuren, M., Meis, J. F. (2001) Meningococcal disease. N. Engl. J. Med. 345, 699–703.
Hart, C. A., Cuevas, L. E., Marzouk, O., Thomson, A. P., Sills, J. (1993) Management of bacterial meningitis. J. Antimicrob. Chemother. 32, 49–59.
O’Donovan, D., Iversen, A., Trounce, J., Curtis, S. (2000) Outbreak of group, C meningococcal infection affecting two preschool nurseries. Commun. Dis. Public Health 3, 177–180.
Gilmore, A., Jones, G., Barker, M., Soltanpoor, N., Stuart, J. M. (1999) Meningococcal disease at the University of Southampton: outbreak investigation. Epidemiol. Infect. 123, 185–192.
Rosenstein, N. E., Fischer, M., Tappero, J. W. (2001) Meningococcal vaccines. Infect. Dis. Clin. North Am. 15, 155–169.
Corless, C. E., Guiver, M., Borrow, R., Edwards-Jones, V., Fox, A. J., Kaczmarski, E. B. (2001) Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J. Clin. Microbiol. 39, 1553–1558.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Clarke, S.C. (2006). Detection of Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae in Blood and Cerebrospinal Fluid Using Fluorescence-Based PCR. In: O’Connor, L. (eds) Diagnostic Bacteriology Protocols. Methods in Molecular Biology™, vol 345. Humana Press. https://doi.org/10.1385/1-59745-143-6:69
Download citation
DOI: https://doi.org/10.1385/1-59745-143-6:69
Publisher Name: Humana Press
Print ISBN: 978-1-58829-594-1
Online ISBN: 978-1-59745-143-7
eBook Packages: Springer Protocols