Abstract
BackgroundStreptococcal superantigens (SAgs) are the major virulence factors of infection in humans for group A Streptococcus (GAS) bacteria. A panel consisting of seven duplex real-time PCR assays was developed to simultaneously detect 13 streptococcal SAgs and one internal control which may be important in the control of GAS-mediated diseases.MethodsPrimer and probe sequences were selected based on the highly conserved region from an alignment of nucleotide sequences of the 13 streptococcal SAgs. The reaction conditions of the duplex real-time PCR were optimized and the specificity of the duplex assays was evaluated using SAg positive strains. The limit of detection of the duplex assays was determined by using 10-fold serial dilutions of the DNA of 13 streptococcal SAgs and compared to a conventional polymerase chain reaction (PCR) method for evaluating the duplex assays sensitivity.ResultsUsing the duplex assays, we were able to differentiate between 13 SAgs from Streptococcus strains and other non-Streptococcus bacteria without cross-reaction. On the other hand, the limit of detection of the duplex assays was at least one or two log dilutions lower than that of the conventional PCR.ConclusionsThe panel was highly specific (100%) and the limit of detection of these duplex groups was at least ten times lower than that obtained by using a conventional PCR method.
Highlights
Streptococcal superantigens (SAgs) are the major virulence factors of infection in humans for group A Streptococcus (GAS) bacteria
13 streptococcal SAgs have been identified in GAS to date, including SpeA, SpeC, SpeG, SpeH, SpeI, SpeJ, SpeL, SpeK, SpeM, Ssa, SmeZ and SpeB, SpeF exhibiting the SAg activity [4,5,6,7]. 13 conventional polymerase chain reaction (PCR) using 1 primer pair for each SAgs performed in the same volume of reaction mixture and using the standard number of cycles used in previous studies of streptococcal SAgs detection [4]
Specificity of the duplex real-time PCR assay To assess the specificity, the Sag positive GAS strains and other closely related non-GAS strains were selected for amplification using the real-time PCR assay
Summary
Streptococcal superantigens (SAgs) are the major virulence factors of infection in humans for group A Streptococcus (GAS) bacteria. 13 conventional polymerase chain reaction (PCR) using 1 primer pair for each SAgs performed in the same volume of reaction mixture and using the standard number of cycles used in previous studies of streptococcal SAgs detection [4]. These tests are not suitable for effective, fast, high-throughput routine diagnostic screening because they are labor intensive, the samples are prone to contamination and have relatively lower sensitivity. A rapid and efficient method for surveillance of streptococcal SAgs in clinical samples is required
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