title: Development of a panel of seven duplex real-time PCR assays for detecting 13 streptococcal superantigens.
abstract: 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.
authors.affiliation: Institute 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.
authors.forename: Peng
authors.initials: P
authors.lastname: Yang
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Peng
authors.forename: Shujuan
authors.initials: S
authors.lastname: Cui
authors.forename: Junbin
authors.initials: J
authors.lastname: Shao
authors.forename: Xuping
authors.initials: X
authors.lastname: Zhu
authors.forename: Daitao
authors.initials: D
authors.lastname: Zhang
authors.forename: Huijie
authors.initials: H
authors.lastname: Liang
authors.forename: Quanyi
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D013297:Streptococcus pyogenes / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D018089:Superantigens / Q000032:analysis* / Q000235:genetics
chemical_list: D000942:Antigens, Bacterial; D017931:DNA Primers; D018089:Superantigens; C117321:streptococcal superantigen SSA
references.citation: Chem Immunol Allergy. 2007;93:1-23
references.pmid: 17369697
references.citation: J Clin Microbiol. 2010 Mar;48(3):842-51
references.pmid: 20042624
references.citation: J Clin Microbiol. 2010 Apr;48(4):1150-60
references.pmid: 20164275
references.citation: J Infect Dis. 2001 Oct 1;184(7):853-60
references.pmid: 11509997
references.citation: Lancet Infect Dis. 2003 Apr;3(4):191-200
references.pmid: 12679262
references.citation: Clin Microbiol Rev. 2000 Jul;13(3):470-511
references.pmid: 10885988
references.citation: Nucleic Acids Res. 2002 Mar 15;30(6):1292-305
references.pmid: 11884626
references.citation: Clin Exp Immunol. 2003 Sep;133(3):299-306
references.pmid: 12930353
references.citation: Epidemiol Infect. 2009 Oct;137(10):1414-9
references.pmid: 19243651