Real-time fluorogenic PCR assays for the detection of entA, the gene encoding staphylococcal enterotoxin A.
Staphylococcal enterotoxin A (SEA) is among the most potent of the growing list of known enterotoxins produced by Staphylococcus aureus. SEA, a 27 kDa monomeric protein, is encoded by the entA gene. We have developed two real-time fluorogenic PCR assays for the detection of nucleic acid sequences in entA. The assays are useful in detecting and identifying strains of S. aureus that produce SEA and can serve a confirmatory role in determining the presence of SEA in food samples. The assays were tested in two real-time PCR formats, using either dye-labeled DNA probes corresponding to each primer set that are degraded by the 5' exonuclease activity of Taq polymerase, or a PCR master mix that contains the DNA-binding dye SYBR Green. In both formats the assays have a limit of detection of between 1 and 13 copies of a S. aureus genome that contains a copy of entA. Neither assay cross-reacted with genomic DNA isolated from other strains of S. aureus or other species.
Horsmon
Jennifer R
JR
U.S. Army Edgewood Chemical Biological Center, AMSRD-ECB-RT-BM, Aberdeen Proving Ground, 5183, Edgewood, MD 21010, USA.
Cao
Cheng J
CJ
Khan
Akbar S
AS
Gostomski
Mark V
MV
Valdes
James J
JJ
O'Connell
Kevin P
KP
D001431:Bacteriological Techniques / Q000379:methods*; D004768:Enterotoxins / Q000235:genetics* / Q000302:isolation & purification; D005456:Fluorescent Dyes; D005516:Food Microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics*
D004768:Enterotoxins; D005456:Fluorescent Dyes; C026066:enterotoxin A, Staphylococcal