Qbeta Replicase Amplification

Q-beta replicase is an RNA-dependent RNA polymerase derived from the bacteriophage Q-beta (Haruna and Spiegelman, 1965). It comprises four different subunits with only one polypeptide (i.e., subunit II) encoded in the Q-beta phage genome. The other subunits are generated by the host protein synthesizing apparatus (30S ribosomal protein S1, elongation factor EF-Tu and EF-Ts) (Blumenthal and Landers, 1976). Q-beta replicase has stringent specificity for its templates (Wu et al., 1992). Only a few naturally occurring RNAs can serve as Q-beta replicase templates, including plus and minus Q-beta RNAs and several smaller "variant" RNAs from in vitro replication reactions (Wu et al., 1992). Such replicase recognizes the specific structure within the template and initiates new strand synthesis from the 3' end of the template without the need of primers. Because the daughter strands also serve as templates for the enzyme, RNA production proceeds exponentially. A single probe molecule can yield a detectable amount of product RNA in a 30-min amplification reaction.

Midvariant-1 (MDV) RNA is a 220-nucleotide-long variant that can be recognized and replicated by Q-beta replicase (Wu et al., 1992). Within the RNA sequence, Kramer and his colleagues inserted a link sequence to which additional probe sequences can be inserted. These recombinant RNAs can then serve as vehicles for amplifying probe sequences to million-folds to allow easy detection of the products by conventional methods such as dot blot and fluorescence. To eliminate nonspecific amplification of the probe, two RNA fragments were made, each containing only half of the probe sequence, and none of them were amplifiable (Fig. 13.3). Upon hybridization to the target, two fragments of the probe sequence were brought together and were subsequently ligated to yield a fully replicable RNA (Tyagi et al., 1996). Some of the properties of Q-beta replicase amplification are summarized in Table 13.1.

Q-beta replicase-based assay has been successfully used to detect various microorganisms such as Chlamydia trachomatis, Mycobacterium tuberculosis, and HIV (Tyagi et al., 1996). Shah et al. described a "dual capture" method to detect C. trachomatis in urogenital samples (Shah et al., 1994). In this method, the hybrids between chlamydial-specific MDV RNAs and chlamydial rRNA targets were captured onto magnetic beads via a separate capture probe. After washing, these

MDV RNA

Figure 13.3. Schematic representation of the Q-beta replicase assay. The two fragments of a recombinant MDV RNA probe hybridize to a target, bringing the two ends in close proximity. After removal of unbound probes, the RNA probes are linked together by a T4 DNA ligase to form a fully replicable RNA, which is then amplified exponentially by Q-beta replicase.

hybrids were released and recaptured to eliminate nonspecific binding of the MDV RNAs to the beads. The chlamydial-specific MDV RNAs were then amplified by Q-beta replicase in the presence of propidium iodide, and detection was carried out in a real-time fashion using a kinetic fluorescence reader. The analytical sensitivity of the assay was 1000 molecules. In their study of 94 urogenital samples, the assay detected 5 of the 6 culture-positive samples and did not detect C. trachomatis target in 85 of the 88 culture-negative samples.

An automatic instrument (Galileo) was developed to process the samples and detect amplification products in a closed disposable test pack to reduce contamination (Smith et al., 1997). In a clinical trial, Smith et al. (1997) designed a recombinant MDV-1 RNA containing a probe sequence specific for 23S rRNA of Mycobacterium tuberculosis. Seven hundred eighty respiratory tract samples

(sputum or bronchoalveolar lavage specimens) were tested using this assay, and the results were compared with those of culture and microscopic examination of acid-fast staining bacillus. Seventy-one out of the 90 (78.9%) culture-positive samples were found positive when tested in the assay, while 7% of the culture-negative samples were assay positive, corresponding to a sensitivity of 79% and a specificity of 93%. After discrepancy analysis, the sensitivity and specificity for the assay were 84% and 97%, respectively. A total of 69.2% of smear-negative (culture-positive) samples were detected by the assay. Although relatively good sensitivity and specificity were demonstrated in this study, the assay and the instrument have not yet been implemented for routine use in clinical laboratory settings.

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