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CV002 Rapid microbial antibiotic resistance detection using DNA-microarrays: Genotyping of beta-lactamases
Wissenschaftliches Programm 55. DGHM-Tagung 29. September-l. Oktober 2003 in Dresden Abstracts - Kurzvortr~ge
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species would have an impact on clinical decisions. To enhance an immediate clinical applicability and to reduce costs, the chip contains multiple separated identical hybridisation areas. This enables interrogation of up to six specimens on one chip. The total process from the specimen, including sample preparation, amplification, hybridisation and scanning, takes less than 4 h manually. For the analysis of complex mixed samples, such as patient specimens, a set of primers with group-specificity, multiplexed or assayed singly, was also developed. This new parallel testing format opens new perspectives in terms of patient management for bacterial diseases by allowing a number of genetic tests to be simultaneously run.
Rapid microbial antibiotic resistance detection using DNA-microarrays: Genotyping of betalactamases Bachmann, T.T.1; Jung, V.1; Susa, M.2; Knabbe, C.2; Schmid, R.D. 1
1University of Stuttgart; Institute of Technical Biochemistry 2Robert Bosch Hospital; Center of Diagn. Medicine Clinical use of antibiotics has increased the selective pressure for resistant bacteria. A major class of microbial resistance stems from the occurrence of betalactamases. These enzymes are ubiquitously distributed in microorganisms and are responsible for microbial antibiotic resistance directed towards different kinds of beta-lactam antibiotics. Today over 340 different betalactamase sequences have been described. TEM betalactamases are one group of serine beta-lactamases (others are OXA, SHV, etc.) in which some members show the Extended Spectrum Beta-Lactamase phenotype conferring resistance to extended spectrum cephalosporins and monobactams. In addition inhibitor resistant TEM beta lactamases (IRT) mutants occur that display decreased susceptibility to beta-lactamase inhibitors like clavulanic acid, tazobactam and sulbactam. The clinical standard procedures of pathogen and resistance identification are tedious and can require up to three days before the resistance can be determined unequivocally. DNA microarrays offer a promising alternative as they allow rapid and accurate genotyping of biological samples. An oligonucleotide microarray for the identification of single nucleotide polymorphisms (SNPs) of 119 untill today described TEM beta-lactamases was developed with a consensus PCR amplification and a fluorescence labeling procedure. The DNA microarray was able to identify ESBL and/or IRT TEM beta-lactamases within less than 4 hours and could be validated to standard clinical proced u res.
http://www.dghm.org
106
3 ~&~"~'~vo~ °~
species would have an impact on clinical decisions. To enhance an immediate clinical applicability and to reduce costs, the chip contains multiple separated identical hybridisation areas. This enables interrogation of up to six specimens on one chip. The total process from the specimen, including sample preparation, amplification, hybridisation and scanning, takes less than 4 h manually. For the analysis of complex mixed samples, such as patient specimens, a set of primers with group-specificity, multiplexed or assayed singly, was also developed. This new parallel testing format opens new perspectives in terms of patient management for bacterial diseases by allowing a number of genetic tests to be simultaneously run.
Rapid microbial antibiotic resistance detection using DNA-microarrays: Genotyping of betalactamases Bachmann, T.T.1; Jung, V.1; Susa, M.2; Knabbe, C.2; Schmid, R.D. 1
1University of Stuttgart; Institute of Technical Biochemistry 2Robert Bosch Hospital; Center of Diagn. Medicine Clinical use of antibiotics has increased the selective pressure for resistant bacteria. A major class of microbial resistance stems from the occurrence of betalactamases. These enzymes are ubiquitously distributed in microorganisms and are responsible for microbial antibiotic resistance directed towards different kinds of beta-lactam antibiotics. Today over 340 different betalactamase sequences have been described. TEM betalactamases are one group of serine beta-lactamases (others are OXA, SHV, etc.) in which some members show the Extended Spectrum Beta-Lactamase phenotype conferring resistance to extended spectrum cephalosporins and monobactams. In addition inhibitor resistant TEM beta lactamases (IRT) mutants occur that display decreased susceptibility to beta-lactamase inhibitors like clavulanic acid, tazobactam and sulbactam. The clinical standard procedures of pathogen and resistance identification are tedious and can require up to three days before the resistance can be determined unequivocally. DNA microarrays offer a promising alternative as they allow rapid and accurate genotyping of biological samples. An oligonucleotide microarray for the identification of single nucleotide polymorphisms (SNPs) of 119 untill today described TEM beta-lactamases was developed with a consensus PCR amplification and a fluorescence labeling procedure. The DNA microarray was able to identify ESBL and/or IRT TEM beta-lactamases within less than 4 hours and could be validated to standard clinical proced u res.
http://www.dghm.org
106