Signal Mediated Amplification of RNA Technology

Signal-mediated amplification of RNA technology (SMART) is a novel isothermal amplification technology that uses a three-way junction (3WJ) structure to facilitate target-dependent production of multiple copies of a RNA product (Wharam et al., 2001). The 3WJ structure is composed of two target-specific single-stranded DNA probes (the "template" probe and the "extension" probe) and a target sequence. Both probes have a longer region that hybridizes to the target at adjacent sites and a shorter region that only hybridizes to each other in the presence of the target, thus forming the three-way junction (3WJ) structure (Fig. 13.4A). In addition, the template probe also contains a nonfunctional single-stranded T7 RNA polymerase promoter sequence. After 3WJ formation and addition of Bst DNA polymerase, the polymerase extends the short probe (extension probe) along the single-stranded template probe to form a functional double-stranded promoter for T7 RNA polymerase. In the presence of T7 RNA polymerase, multiple copies of RNA can be synthesized (Fig. 13.4B). Both Bst DNA polymerase and T7 RNA polymerase can function under the same reaction condition, hence the reaction can be performed in a single tube. In order to further improve the signal, a second template oligonucleotide (probe for RNA amplification) containing a second T7 promoter sequence can be added to the reaction to allow the RNAs generated from 3WJ to bind, which, in turn, allows its extension by Bst DNA polymerase and generation of secondary RNAs by T7 RNA polymerase, ultimately leading to a further increase in RNA yield (Fig. 13.4B). The RNA product can be measured by an enzyme-linked oligosorbent assay. This assay is capable of generating a detectable signal from 50 nmol single-stranded synthetic target, 10 ng bacterial genomic DNA, or 0.1 ng total bacterial RNA (or 104 bacteria) (Wharam et al., 2001). Some of the properties of SMART are summarized in Table 13.1.

Levi et al. evaluated the SMART assay (CytAMP assay kit, Cytocell Ltd., Adderbury, Oxford, UK) for the rapid detection of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA) (Levi et al., 2003). Two sets of probes were designed against the coa (coagulase) and mecA (methicillin resistance) genes, respectively, hence, simultaneous identification of S. aureus and methicillin

Figure 13.4. Schematic representation of the SMART assay. (A) Formation of a 3WJ. Extension and template probes anneal to the target, and only then can they hybridize with each other. The short extension probe has a free 3'-OH to allow extension. The template probe includes a single-stranded (nonfunctional) T7 RNA polymerase promoter (Pr) and sequences to allow the capture and detection of the RNA signal. The 3' end of the template probe is blocked (x) by phosphorylation to prevent extension. (B) Extension and transcription generate an RNA signal. Bst DNA polymerase extension of the extension probe generates a double-stranded (ds), hence functional, T7 RNA polymerase promoter (Pr), allowing transcription of multiple copies of an RNA signal (RNA1) by T7 RNA polymerase. If required, RNA CD anneals to a second template (probe for RNA amplification), leading to further extension and transcription by the DNA and RNA polymerases to generate increased amounts of a second RNA signal (RNA (2)).

Figure 13.4. Schematic representation of the SMART assay. (A) Formation of a 3WJ. Extension and template probes anneal to the target, and only then can they hybridize with each other. The short extension probe has a free 3'-OH to allow extension. The template probe includes a single-stranded (nonfunctional) T7 RNA polymerase promoter (Pr) and sequences to allow the capture and detection of the RNA signal. The 3' end of the template probe is blocked (x) by phosphorylation to prevent extension. (B) Extension and transcription generate an RNA signal. Bst DNA polymerase extension of the extension probe generates a double-stranded (ds), hence functional, T7 RNA polymerase promoter (Pr), allowing transcription of multiple copies of an RNA signal (RNA1) by T7 RNA polymerase. If required, RNA CD anneals to a second template (probe for RNA amplification), leading to further extension and transcription by the DNA and RNA polymerases to generate increased amounts of a second RNA signal (RNA (2)).

(oxacillin) resistance is possible. The detection limit of the assay was 2 x 105 and 106 CFU/assay for mecA and coa, respectively. When tested with S. aureus isolates, the assay detected 113 MRSA among 396 S. aureus with 100% sensitivity and specificity, compared with a mecA-femB PCR assay. When 100 enrichment broths containing sets of screening swabs from individual patients were tested, the presence of MRSA was detected in 19, 24, and 31 enrichment broths by SMART assay, conventional culture, and mecA-femB PCR, respectively. Six enrichment broths were found negative by SMART assay but positive by both PCR and culture. Five of these contained an equivalence of 102 to 105 CFU/assay (below the predicted detection limit of 2 x 105 CFU/assay for SMART assay), and the sixth contained an equivalence of 106 CFU/assay. Overall, culture and SMART had similar sensitivities and specificities relative to those of PCR.

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