Molecular Detection of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Abstract

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2-3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.

Identification ofEscherichia coliO157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

“…To implement the use of improved detection methods with DNA microarrays, the present study evaluated photopolymerization, a novel colorimetric detection method (Kuck and Taylor, 2008;Sikes et al, 2008;Dawson et al, 2009), for genotyping E. coli O157 strains. To provide a proof of concept for testing photopolymerization, a low-density 30-mer DNA oligonucleotide array was constructed to target the E. coli O157 virulence genes eae, per, stx1, and stx2 (Table 1).…”

“…For photopolymerization to occur, a streptavidinconjugated photoinitiator is used to specifically label the microarrays that have been hybridized with the biotin-labeled, single-stranded DNA targets (Kuck and Taylor, 2008). After irradiating at a wavelength absorbed by the photoinitiator (l 404 nm), polymer forms exclusively in only a few minutes where the probe sequences were spotted on the microarray (Kuck and Taylor, 2008;Dawson et al, 2009); polymer formation can be observed after staining (Kuck and Taylor, 2008).…”

“…The specific colorimetric detection of signals on the microarray were detected with the ampliPHOXÔ Detection System (InDevR, Inc.), a light-initiated signal amplification through polymerization (Kuck and Taylor, 2008;Sikes et al, 2008;Dawson et al, 2009). The hybridized microarrays were labeled subsequently with ampliTAGÔ labeling solution (InDevR, Inc.) for 5 min at room temperature in a dark humidity chamber, and immediately after labeling, microarrays were rinsed briefly with Wash Buffer D, Wash Buffer C for 5 min, and then dried.…”

“…Improved procedures for pathogen surveillance are thus needed with sufficient sensitivity, cost effectiveness, and suitability for routine testing. To implement the use of better detection methods for categorizing E. coli O157 strains, the present study evaluated ampliPHOX, a novel and innovative colorimetric procedure that is based on light-initiated signal amplification through polymerization (Kuck and Taylor, 2008;Sikes et al, 2008;Dawson et al, 2009). Our findings demonstrate that photopolymerization is a simple and rapid method for the accurate genotyping of E. coli O157 strains with DNA microarrays.…”

“…Given the limitations of current procedures, the present study evaluated photopolymerization for identification of pathogenic E. coli, using low-cost reagents and instrumentation that are amenable to high-throughput sampling and routine pathogen surveillance (Kuck and Taylor, 2008). Previously, photopolymerization was employed successfully for molecular diagnostic assays detecting Streptococcus species (Dawson et al, 2009) and for genotyping influenza viruses (Kuck and Taylor, 2008). Real-time PCR methods have been developed for the identification of virulent strains of E. coli O157 (Kawasaki et al, 2010;Suo et al, 2010); however, the cost for the reagents and instrumentation required for these analyses with real-time PCR impede the use of this molecular method for routine strain characterization in smaller research facilities.…”