Target Amplification Systems

Target amplification systems are defined as nucleic acid amplification procedures in which many copies of the nucleic acid targets are made, which include PCR, nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), or strand displacement amplification (SDA). Among them, PCR and PCR-derived techniques are the best-developed and most widely used methods of nucleic acid amplification (Saiki et al., 1988; Eisenstein, 1990; Mullis, 1990). Commercial products as well as user-developed PCR-based NAA techniques are available for the detection of microbial pathogens, identification of clinical isolates, and strain subtyping (Tang et al., 1999; Hayden, 2004). PCR-derived techniques, such as reverse transcription PCR, nested PCR, multiplex PCR, arbitrary primed PCR, and broad-range PCR, have collectively expanded the flexibility and power

Table 10.1. Nucleic acid amplification methods.

Amplification

Manufacturer/license

Temperature

Nucleic acid

Amplification method

category

(trade mark)

Enzymes used

requirement

target

Main references

Polymerase chain

Target

Roche Molecular

Taq DNA polymerase

Thermal cycler

DNA or RNA

(Saiki et al., 1988; Mullis,

reaction (PCR)

System, Inc., Branchburg, NJ, USA (Amplicor)

1990)

Transcription-mediated

Target

Gen-Probe, Inc., San

Reverse transcriptase,

Isothermal

RNA or DNA

(Kwohetal., 1989; La

amplification (TMA)

Diego, CA, USA (APTIMA)

RNA polymerase, RNase H

Rocco et al., 1994)

Nucleic acid

Target

Organon-Teknika, Corp.,

Reverse transcriptase,

Isothermal

RNA or DNA

(Compton et al., 1991;

sequence-based

Durham, NC, USA

RNA polymerase,

Revets et al., 1996)

amplification

(Nuclisens)

RNase H

(NASBA)

Strand displacement

Target

Becton-Dickinson,

Restrictive

Isothermal

DNA or RNA

(Walker et al., 1992;

amplification (SDA)

Sparks, MD, USA (ProbTec)

endonucleonase, DNA polymerase

Hellyer et al., 1996)

Invader technology

Probe

Third Wave, Madison, WI, USA

Cleavase

Isothermal

DNA or RNA

(Brow et al., 1996; Rossetti et al., 1997)

Cycling probe

Probe

ID Biomedical Corp.,

Rnase H

Isothermal

DNA

(Duck et al., 1990; Cloney

technology (CPT)

Vancouver, Canada

et al., 1999)

Ligase chain reaction

Probe

Abbott Laboratories,

DNA ligase

Thermal cycler

DNA or RNA

(Wu and Wallace, 1989;

(LCR)

Abbott Park, IL, USA (LCx)

Cecil et al., 2001)

Hybrid capture system

Signal

Digene Diagnostics, Inc., Silver Spring, MD, USA

None

Isothermal

DNA

(Brown et al., 1993; Mazzulli et al., 1999)

Branched DNA (bDNA)

Emeryville, CA, USA

None

Isothermal

DNA or RNA

(Urdea et al., 1991; Lau et al., 1993)

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 bioMerieux 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 etal., 1999).

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