F. Bekkaoui et al., Rapid detection of the mecA gene in methicillin resistant staphylococci using a colorimetric Cycling Probe Technology, DIAG MICR I, 34(2), 1999, pp. 83-90
A Cycling Probe Technology (CPT) assay was developed for the defection of t
he 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 fu
rther cycling. CPT-EIA uses streptavidin-coated microplate wells to capture
uncut probe followed by detection with horseradish-peoxidase conjugated an
ti-fluorescein antibody. The assay was compared to PCR and shown to accurat
ely detect the presence or absence of the mecA gene in 159 staphylococcal c
linical isolates. The CPT-EIA assay takes two hours starting from cultured
cells compared with the 24-48 h required for detection of methicillin resis
tance by conventional susceptibility tests. (C) 1994 Elsevier Science Inc.