Rapid detection of the mecA gene in methicillin resistant staphylococci using a colorimetric cycling probe technology.
A Cycling Probe Technology (CPT) assay was developed for the detection of the mecA gene from methicillin resistant staphylococcal cultures. The assay is based on a colorimetric enzyme-immuno-assay (EIA) and uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5'-terminus and biotin at the 3'-terminus. The reaction occurs at a constant temperature that allows the target DNA to anneal to the probe. RNase H cuts the RNA portion, allowing the cut fragments to dissociate from the target, making it available for further cycling. CPT-EIA uses streptavidin-coated microplate wells to capture uncut probe followed by detection with horseradish-peroxidase conjugated anti-fluorescein antibody. The assay was compared to PCR and shown to accurately detect the presence or absence of the mecA gene in 159 staphylococcal clinical isolates. The CPT-EIA assay takes two hours starting from cultured cells compared with the 24-48 h required for detection of methicillin resistance by conventional susceptibility tests.
Bekkaoui
F
F
ID Biomedical Corporation, Burnaby, B.C., Canada.
McNevin
J P
JP
Leung
C H
CH
Peterson
G J
GJ
Patel
A
A
Bhatt
R S
RS
Bryan
R N
RN
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics*; D003124:Colorimetry; D005798:Genes, Bacterial; D006602:Hexosyltransferases; D006801:Humans; D007124:Immunoenzyme Techniques; D008712:Methicillin / Q000494:pharmacology; D016106:Methicillin Resistance / Q000235:genetics*; D015336:Molecular Probe Techniques; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics*; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000494:pharmacology; D010458:Peptidyl Transferases; D016133:Polymerase Chain Reaction / Q000379:methods; D016914:Ribonuclease H / Q000378:metabolism; D013203:Staphylococcal Infections / Q000382:microbiology; D013210:Staphylococcus / Q000187:drug effects / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D016914:Ribonuclease H; D002267:Muramoylpentapeptide Carboxypeptidase; D008712:Methicillin