a16SrRNA primers are included to serve as reaction control to ensure the presence of amplifiable template DNA.

a16SrRNA primers are included to serve as reaction control to ensure the presence of amplifiable template DNA.

temperature that is more suited to all the primer pairs, or to change the size of the amplicons to enable better visualization/detection. Multiplex PCR assays are very useful for characterizations of culture isolates, but when used for testing samples, which most likely contains mixed cultures, the amplicon patterns obtained may not necessarily have been derived from a single bacterial strain.

Selected EHEC multiplex PCR assays and the targets that were successfully combined, optimized, and used to specifically detect O157:H7 or other EHEC serotypes are listed in Table 2. In the following sections, a few of these multiplex PCR assays are presented.

Paton and Paton[23] designed an agarose gel-based, two-reaction system to identify EHEC strains. The first reaction selectively targets stx1, stx2, eae, and ehxA, while the second reaction differentiates O111 and O157 strains based on their rfb gene. Analysis of 52 previously characterized STEC strains isolated from food, human, or animal sources showed that the assays correctly identified all of the isolates and the determinants carried by each strain with no cross-reactivity to closely related strains. The stx2 primers also amplified all stx2 variants, except for stx2f, but the eae primers were designed for the conserved region of eae, and therefore did not identify the specific eae alleles carried by the strains.

Another gel-based multiplex PCR described by Feng and Monday[16] used a single PCR reaction to detect stx1, stx2 (including stx2c, stx2d, and stx2e), and both ehxA alleles to identify STEC. However, unlike other assays, this multiplex also incorporated primers to the g-intimin (eae) gene, the only allelic variant expressed by the O157:H7 group, and the +92 uidA mutation that has, thus far, only been observed in the O157:H7 serotype. This assay bears the advantage of being able to detect STEC and simultaneously identify EHEC of the O157:H7 serotype. Analysis of 38 strains showed that the assay correctly identified the determinants carried by EHEC,

STEC, and EPEC, and also identified all the O157:H7 strains tested. This assay will not identify other EHEC serotypes, which do not possess the +92 uidA mutation and may express intimins other than the g-allele.

Gel-based PCR assays can detect many genetic determinants in one reaction; however, it is time-consuming and provides data that are difficult to quantitate. Furthermore, gel-based results are solely based on amplicon sizes with no means to confirm that the product was derived from specific target amplification or was a consequence of a rare mispriming event. These limitations can be overcome by using real-time PCR (rt-PCR), which enables rapid amplification and real-time monitoring of results, and—when coupled with sequence-specific internal probes—guarantees the amplification of specific targets, which can also be quantified to estimate bacterial load. Reischl et al.[24] used rt-PCR and Fluorescence Resonance Energy Transfer (FRET) technology to develop two assays that amplified and detected sequence-specific EHEC virulence determinants in approximately 60 min. The first assay used a single primer pair to amplify stx genes, which were then differentiated into stx1 (at 640 nm) and/or the stx2 (at 705 nm) genes by toxin-specific, fluorophore-tagged hybridization probes. Melting temperature analysis of probe-hybridized amplicons also allowed the determination of some stx allelic variants, except for stx2f, which is genetically distinct and found in STEC isolated from pigeons. The second assay is similar, but uses two separate primer sets and target-specific, fluorophore-tagged hybridization probes to detect the eae (at 640 nm) and ehxA (at 705 nm) genes. The eae primers and probes appeared to be specific to the conserved regions, as the assay did not differentiate eae allelic variants. In the analysis of 431 STEC strains, 73 Stx-negative E. coli, and 118 other bacterial species, rt-PCR was found to be 100% sensitive and specific for stx1, eae, and ehxA, and had 96% and 100% sensitivity and specificity, respectively, for stx2.

Although rt-PCR offers many advantages, reaction kinetics and the limited number of distinct fluorophores presently available limit the number of targets that can be combined in a multiplex format. Furthermore, the cost of the equipment and special order reagents often precludes its current use in routine testing.

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Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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