[Digoxigenin-labelled probes for detection of TEM-type beta-lactamases using the PCR technique].
BACKGROUND
Production of DNA probes is time-consuming and inefficient. We have developed a method for the obtention of digoxigenin-labeled probes to detect TEM-type betalactamases using the polymerase chain reaction. The amplification product was a 516 bp fragment internal to bla-TEM-1 from pBR 322.


METHODS
The techniques developed included extraction of plasmid DNA by lisis by alkali, electroelution, electrophoresis in agarose gels, polymerase chain reaction and hydridization with a DNA probe digoxigenin labeled.


RESULTS
We obtained by polymerase chain reaction 1500 ng of probe using 1 ng of target DNA. Developing classic methods the amount of probe was 75 ng from 1 microgram of target DNA. The time to obtain the probe was 3 hours, instead of a week with other methods.


CONCLUSIONS
We conclude that polymerase chain reaction is a good alternative to classic methods to obtain digoxigenin-labeled DNA probes.
Gallego
L
L
Departamento de Microbiología, Facultad de Medicina y Odontología, Universidad del País Vasco, Bilbao.
Aranguiz
A
A
Sarriá
L
L
Alonso
R
R
Colom
K
K
Cisterna
R
R
D001483:Base Sequence; D015342:DNA Probes / Q000302:isolation & purification*; D004076:Digoxigenin; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D001618:beta-Lactamases / Q000032:analysis*
D015342:DNA Probes; D001618:beta-Lactamases; D004076:Digoxigenin
Obtención mediante la técnica de PCR de sonda marcada con digoxigenina para detectar betalactamasas tipo TEM.