Detection and differentiation of in vitro-spiked bacteria by real-time PCR and melting-curve analysis.
We introduce a consensus real-time PCR protocol for the detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and plasma. This prototype detection system enables an exact Gram stain classification and, in particular, screening for specific species of 17 intensive care unit-relevant bacteria by means of fluorescence hybridization probes and melting-curve analysis in a one-run experiment. One strain of every species was tested at a final density of 10(6) CFU/ml. All bacteria examined except Staphylococcus aureus and Staphylococcus epidermidis could be differentiated successfully; S. aureus and S. epidermidis could only be classified as "Staphylococcus species." The hands-on time for preparation of the DNA, performance of the PCR, and evaluation of the PCR results was less than 4 h. Nevertheless, this prototype detection system requires more clinical validation.
Klaschik
S
S
Department of Anesthesiology and Intensive Care Medicine. Institute for Hygiene and Public Health, University of Bonn, Bonn, Germany. klaschik@ukb.uni-bonn.de
Lehmann
L E
LE
Raadts
A
A
Book
M
M
Gebel
J
J
Hoeft
A
A
Stuber
F
F
D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000254:growth & development; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D017404:In Situ Hybridization, Fluorescence; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods
D004269:DNA, Bacterial