Applications and Variations of Isothermal RNA Amplifications

The transcription-based ITA techniques have been given various names especially during the early stages of development. As described above, names that have appeared in the literature include NASBA, TMA, TAS, and 3SR (Table 12.1) (Kwoh et al., 1989; Fahy et al., 1991; Guilfoyle et al., 1997; Deiman et al., 2002). The major differences between these procedures are found in the detection systems, and this flexibility is achieved by the modification of the 5' end of the P2 primer to bind a detection probe (Fig. 12.1). Kwoh et al. used oligo-coated Sephacryl beads to bind the amplicon (Kwoh et al., 1989), but a number of modifications have emerged. Electrochemiluminescent (ECL)-labeled probes (van Gemen et al., 1994) have been incorporated into a basic system design that allows extensive flexibility in assay development. Enzyme-linking technologies have been incorporated into a gel assay (ELGA) that uses a horseradish peroxidase-labeled probe to detect the amplicon. In this case, bound and free probe are separated on a poly-acrylamide gel, and the products are visualized using the peroxidase substrate

Table 12.1. Selected applications of commercially available non-PCR-mediated target amplification techniques for detection of microorganisms.

Test name

Type

Use

Manufacturer

URL

NucliSens

NASBA

HIV-1 viral

bioMerieux, Inc.

http://www.biomerieux.com

HIV-1 QT

load

Durham, NC, USA

HIV-1/HCV

TMA

Plasma donor

Gen-Probe

http://www.gen-probe.com

Assay (Procleix)

screen

San Diego, CA, USA

NucliSens

NASBA

CMV pp67 mRNA

bioMerieux, Inc. Durham, NC, USA

http://www.biomerieux.com

BDProbeTecET

SDA

C. trachomatis,

Becton

http://www.bd.com

DNA Amplified

N. gonorrhoeae

Dickinson

Assay

Sparks, MD USA

Amplified MTD

TMA

Mycobacterium tuberculosis

Gen-Probe San Diego, CA, USA

http://www.gen-probe.com

BD ProbeTec ET

SDA

Legionella

Becton Dickinson

http://www.bd.com

DNA Amplified

pneumophila

Sparks, MD,

Assay

USA

(van der Vliet et al., 1993). Samuelson et al. designed a capture probe to bind amplified products to a streptavidin-coated plate that is followed by the application of a digoxigenin-labeled detection probe (Samuelson et al., 1998). The TMA reaction uses an acridinium ester-labeled DNA probe to detect the amplicon via luminescence (also called a hybridization protection assay; HPA) (Arnold et al., 1989).

Initial studies using transcription-based ITA methods focused upon detection of HIV RNA as an important example of RNA targeting (Guatelli et al., 1990; Bush et al., 1992; van Gemen et al., 1993a; 1993b; Sherefa et al., 1998). Recently, it has been incorporated into a real-time format (de Baar et al., 2001), and the use of molecular beacons and other fluorescent detection systems are enabling development of high-throughput and quantitative assays (Arens, 1993; Romano et al., 1997; Kamisango et al., 1999; Greijer et al., 2001; Yates et al., 2001). Targeting RNA has also been used as an indicator of cell viability (Simpkins et al., 2000; Keer and Birch, 2003) and to assess antimicrobial treatment regimens where problems may be caused by the presence of nonviable organisms (Morre et al., 1998). Because RNA is relatively unstable compared with DNA, detection of RNA is abetter indicator of viability as in the case of cytomegalovirus infection (Amorim et al., 2001) and helps to facilitate differentiation of reactivation or acute infection from latent presence of the virus (Hodinka, 1998; Preiser et al., 2001; Caliendo et al., 2002; Hebart et al., 2002).

The transcription-based ITA technology is also well suited to distinguish between viral and proviral sequences. This technology has been applied to detection of HIV after anti-retroviral therapy (Bruisten et al., 1993), as well as after therapies of CMV (Greijer et al., 2001; Goossens et al., 2004) human herpes virus 8 (Polstra et al., 2003), and Epstein-Barr virus (Brink et al., 1998), respectively. The direct detection of RNA has been applied to cancer diagnostics, for instance, in transcription detection from bcr3-abl2 and bcr2-abl2 junctions to diagnose chronic myeloid leukemia (Sooknanan et al., 1993; Langabeer, 2002). The detection of telomerase activity is expected to be a new diagnostic and prognostic marker of human cancer (Hirose et al., 1998). A sensitive TMA assay coupled with the hybrid protection assay was developed that could measure the addition of telomeric repeats with a sensitivity and reproducibility equal to or greater than that of PCR-based telomeric repeat amplification assay (Hirose et al., 1998). With the recent discoveries of small regulatory RNA molecules (Novina and Sharp, 2004), perhaps an entirely new field of direct RNA detection will develop using self-sustained transcription-based ITA principles.

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