[Application of PCR for the detection of mutations in the genes coding for beta-lactamases].
BACKGROUND
The purpose of this study was to develop a satisfactory technique to detect punctual mutations in blaTEM genes.


METHODS
The strains [E. coli HB 101 pBR322 (TEM-1), E. coli J62 RP4 (TEM-2)] were submitted to PCR with primers PL1 and PL2 which amplify the genetic region susceptible of punctual mutations. Then, we developed Southern blot and hybridization with oligonucleotide probes (GIn 37, Lys 37 y Thr 261), corresponding to first and last mutations.


RESULTS
A region of 841 bp was amplified using the primers previously described. Hybridization experiments with the Thr 261 probe gave positive signal with both strains (both carry the mutation); GIn 37 only hybridized with TEM-1 and Lys 37 only with TEM-2.


CONCLUSIONS
The use of primers which amplify all the region susceptible of mutations in blaTEM and oligonucleotide probes allows the specific detection of point modifications in the original genes by the use of digoxigenin-labeled oligonucleotide probes.
Gallego
L
L
Departamento de Microbiología, Facultad de Medicina, Universidad del País Vasco, Bilbao.
Basaras
M
M
Alonso
R
R
Sarria
L
L
Cisterna
R
R
D000218:Adamantane / Q000031:analogs & derivatives; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D004076:Digoxigenin; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D001618:beta-Lactamases / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; C056342:beta-lactamase TEM-2; D004076:Digoxigenin; D000218:Adamantane
Aplicación de PCR para la detección de mutaciones en los genes codificantes de betalactamasas.