Abstract
Nucleic acid amplification technologies (NAATs) represent powerful tools in clinical microbiology, particularly in areas where traditional culture-based methods alone prove insufficient. A notable advantage is in reducing the time from taking samples to reporting results. This, and the specificity and sensitivity imparted by NAATs, can help to improve patient care. Both thermal and isothermal NAATs have been adapted to aid diagnosis in clinical laboratories. Current molecular diagnostic assays are generally high-tech, and are expensive to buy and perform. Easy-to-use NAATs are beginning to appear, not only facilitating acceptable throughput in clinical laboratories, but also allowing tests to move out of the laboratory, closer to the point of care. Demand for simpler, miniaturized equipment and assays, and the trend toward personalized medicine, is leading towards the development of fully integrated automation and home-use kits. The integration of diverse disciplines, such as genomics, molecular biology, microelectromechanical systems, microfluidics, microfabrication, and organic chemistry, is behind the emerging DNA microarray technology. Development of DNA microchips allows the simultaneous detection of potentially thousands of target sequences, not only favoring high throughput, but also the potential for genotyping patient subsets with respect to their response to particular drug types (pharmakogenomics). It is envisaged that the future of probe-based technologies will see the development of fully integrated assays and devices suitable for nonskilled users.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Tansuphasiri, U. (2001) Detection of Mycobacterium tuberculosis from sputum collected on filter paper and stored at room temperature for 5 days by PCR assay and culture. J. Med. Assoc. Thai. 84, 1183–1189.
Diagnostics Intelligence. (1996) Profiting from Gene-Based Diagnostics. CTB International Publishing, Mapelwood, USA.
Towner, K. J., Talbot, D. C. S., Curran, R., Webster, C. A., and Humphreys, H. (1998) Development and evaluation of a PCR-based immunoassay for the rapid detection of methicillin resistant Staphylococcus aureus. J. Med. Microbiol. 47, 607–613.
Hookey, J. V., Richardson, J. F., and Cookson, B. D. (1998) Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene. J. Clin. Microbiol. 36, 1083–1089.
Jonas, D., Grundmann H., Hartung D., Daschner F. D., and Towner K. J. (1999) Evaluation of the mecA femB duplex polymerase chain reaction for detection of methicillin resistant Staphylococcus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 18, 643–647.
Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996) Real time quantitative PCR. Genome Res. 6, 986–994.
Tyagi, S. and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nature Biotech. 14, 303–308.
Sixou, S., Szoka, F. C. Jr., Green, G. A., Giusti, B., Zon, G., and Chin, D. J. (1994) Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET). Nucleic Acids Res. 22, 662–668.
Whiley, D. M., LeCornec, G. M., Mackay, I. M., Siebert, D. J., and Sloots, T. P. (2002) A real-time PCR assay for the detection of Neisseria gonorrhoeae by LightCycler. Diagn. Microbiol. Infect. Dis. 42, 85–89.
Qi, Y., Patra, K., Liang, X., Williams, L. E., Rose, S., Redkar, R. J., et al. (2001) Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis. Appl. Environ. Microbiol. 67, 3720–3727.
Makino, S. I, Cheun, H. I., Watari, M., Uchida, I., and Takeshi, K. (2001) Detection of anthrax spores from the air by real-time PCR. Lett. Appl. Microbiol. 33, 237–240.
McDonald, R., Cao, T., and Borschel, R. (2001) Multiplexing for the detection of multiple biowarfare agents shows promise in the field. Mil. Med. 166, 237–239.
Kievits, T., van Gemen, R., van Strijp, D., Schukkink, R., Dircks, M., Adriaanse, H., et al. (1991) NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection. J. Virol. Methods 35, 273–286.
Min, J. and Baeumner, A. J. (2002) Highly sensitive and specific detection of viable Escherichia coli in drinking water. Anal. Biochem. 303, 186–193.
Foolen, H., Sillikens, P., van de Wiel-van de Meer, M., and Fox, J. D. (2001) Nuclisens Basic Kit NASBA including an internal control and “real-time” detection for diagnosis of enterovirus infections. J. Microbiol. Methods 47, 96.
Fox, J. D., Hibbitts, S., Rahman, A., and Westmorland, D. (2001) Nuclisens Basic Kit NASBA with “end point” and “real-time” detection for diagnosis of respiratory virus infections. J. Microbiol. Methods 47, 96.
Damen, M., Sillekens, P., Cuypers, H. T., Frantzen, I., and Melsert, R. (1999) Characterization of the quantitative HCV NASBA assay. J. Virol. Methods 82, 45–54.
Lizardi, P. M. and Kramer, F. R. (1991) Exponential amplification of nucleic acids: new diagnostics using DNA polymerases and RNA replicases. Trends Biotech. 9, 53–58.
Smith, J. H., Radcliffe, G, Rigby, S., Mahan, D., Lane, D. J., and Klinger, J. D. (1997) Performance of an automated Q-beta replicase amplification assay for Mycobacterium tuberculosis in a clinical trial. J. Clin. Microbiol. 35, 1484–1491.
Beggs, M. L., Cave, M. D., Marlowe, C., Cloney, L., Duck, P., and Eisenach, K. D. (1996) Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction. J. Clin. Microbiol. 34, 2985–2989.
Cloney, L., Marlowe, C., Wong, A., Chow, R., and Bryan R. (1999) Rapid detection of mecA in methicillin resistant Staphylococcus aureus using Cycling Probe Technology. Mol. Cell Probes 13, 191–197.
Cooksey, R. C., Holloway, B. P., Oldenburg, M. C., Listenbee, S., and Miller, C. W. (2000) Evaluation of the invader assay, a linear signal amplification method, for identification of mutations associated with resistance to rifampin and isoniazidin Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 44, 1296–1301.
Hessner, M. J., Budish, M. A., and Friedman, K. D. (2000) Genotyping of factor V G1691A (Leiden) without the use of PCR by invasive cleavage of oligonucleotide probes. Clin. Chem. 46, 1051–1056.
Wharam, S. D., Marsh, P., Lloyd, J. S., Ray, T. D., Mock, G. A., Assenberg, R., et al. (2001) Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure. Nucleic Acids Res. Methods Online 29, E54.
Hall, M. J., Wharam, S. D., Weston, A., Cardy, D. L. N., and Wilson, W. H. (2002) Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA. BioTechniques 32, 604–611.
Levi, K., Bailey, C., Marsh, P., Cardy, D. L. N., and Towner, K. J. (2001) Evaluation of a novel isothermal amplification assay (CytAMP™) for direct detection of methicillin resistant Staphylococcus aureus (MRSA). J. Microbiol. Methods [Abstract] 47, 100.
Levi, K., Bailey, C., Bennett, A., Marsh, P., Candy, D. L., and Towner, K. J. (2003) Evaluation of an isothermal signal amplification method for rapid detection of methicillin resistant Staphylococcus aureus from patient-screening swabs. J. Clin. Microbiol. 41, 3187–3191.
Crosby, L. (2002) IVDs for home-use testing: issues in diagnosis and monitoring. IVD Technology 8, 18–21.
Westin, L., Xu, X., Miler, C., Wang, L., Edman, C. F., and Nerenberg, M. (2000) Anchored multiplex amplification on a microelectronic chip array. Nature Biotech. 18, 199–204.
Sosnowski, R. G., Tu, E., Butler, W. F., O’Connell, J. P., and Heller, M. J. (1997) Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control. Proc. Natl. Acad. Sci. USA 94, 1119–1123.
Syvänen, A. C. and Söderlund, H. (2002) DNA sandwiches with silver and gold. Nature Biotech. 20, 349–350.
Donaldson, A. L., Hearps, A., and Alexandersen, S. (2001) Evaluation of a portable, “real-time” PCR machine for FMD diagnosis. Veterinary Record 149, 430.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Marsh, P., Cardy, D.L.N. (2004). Molecular Diagnostics. In: Woodford, N., Johnson, A.P. (eds) Genomics, Proteomics, and Clinical Bacteriology. Methods in Molecular Biology™, vol 266. Humana Press. https://doi.org/10.1385/1-59259-763-7:167
Download citation
DOI: https://doi.org/10.1385/1-59259-763-7:167
Publisher Name: Humana Press
Print ISBN: 978-1-58829-218-6
Online ISBN: 978-1-59259-763-5
eBook Packages: Springer Protocols