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Updated: May 9, 2026
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
Published on: August 6, 2011
Development of a panel of seven duplex real-time PCR assays for detecting 13 streptococcal superantigens
Peng Yang1, Xiaomin Peng, Shujuan Cui
1Institute for Infectious Disease and Endemic Disease Control, Beijing Center for Disease Prevention and Control (CDC), Beijing Research Center for Preventive Medicine, Capital Medical University School of Public Health, No,16 He Pingli Middle Street, Dongcheng District, Beijing 100013, China.
Background:
Streptococcal 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.
Methods:
Primer 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.
Results:
Using 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.
Conclusions:
The 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.
