Advanced Techniques in Diagnostic Microbiology

160 H. Li and Y-W. Tang
of these methods in diagnostic laboratories across the world. Roche Molecular
System, the current holder of the PCR patents, has several PCR-based diagnostic
products available for infectious disease pathogen detection and quantitation (Tang
et al., 1999; Jungkind et al., 2002).
Given the patent restrictions on PCR and the expanding interest in nucleic
acid–based diagnosis, alternative amplification methods have been sought. Another
target amplification system, transcription-mediated amplification or nucleic
acid sequence–based amplification, involves several enzymes and a complex series
of reactions that all take place simultaneously at the same temperature and
in the same buffer (Kwoh et al., 1989; Compton, 1991). The advantages include
very rapid kinetics and the lack of requirement for a thermocycler. Isothermal
conditions in a single tube with a rapidly degradable product (RNA) help minimize
(but may not eliminate) contamination risks. Amplification of RNA not
only makes it possible to detect RNA viruses but also increases the sensitivity
of detecting bacterial and fungal pathogens by targeting high copy number RNA
templates. A TMA-based system manufactured by GenProbe Inc. has been used to
detect Mycobacterium tuberculosis in smear-positive sputum specimens, to confirm
Chlamydia trachomatis and Neisseria gonorrhoeae infection, as well as to
screen human immunodeficiency virus (HIV)-1 RNA in donor blood specimens
(La Rocco et al., 1994; Revets et al., 1996; Gaydos et al., 2003). NASBA system–
based products are commercially available from bioMérieux and have been used
for the detection of enteroviruses in cerebrospinal fluid and for the quantitation of
hepatitis C virus (HCV) levels in serum (Hollingsworth et al., 1996; Landry et al.,
2003).
Another isothermal, non-PCR target amplification technique is SDA, which
uses specific primers, a DNA polymerase, and restriction endonuclease to achieve
exponential amplification of the target (Walker et al., 1992). The key technology
behind SDA is the generation of site-specific nicks by the restriction endonuclease.
Since its initial description, it has evolved into a highly versatile tool that is technically
simple to perform but conceptually complex. Commercial kits have been
available from Becton Dickinson for diagnosis and monitoring of C. trachomatis,
N. gonorrhoeae, and M. tuberculosis infections (Hellyer et al., 1996; Spears et al.,
1997). The ProbeTec ET system combines amplification of nucleic acids by SDA
and real-time identification by using fluorescence resonance energy transfer (Little
et al., 1999).
Probe Amplification Systems
In probe amplification systems, many copies of the probe that hybridizes the target
nucleic acid are made (Birkenmeyer and Mushahwar, 1991). The ligase chain
reaction (LCR), cleavase-invader assay, and cycling probe technology (CPT) have
been successfully applied in diagnostic microbiology. A gapped LCR procedure,
which is designed following a target amplification method, such as PCR, can be
sensitive and useful for the detection of point mutations (Osioway, 2002). Although