Biochip for the Simultaneous Identification of Beta-Lactamase and Carbapenemase Genes Conferring Bacterial Resistance to Beta-Lactam Antibiotics

Bacterial beta-lactamases and carbapenemases confer resistance to beta-lactam antibiotics, including penicillins, cephalosporins, carbapenems, and monobactams. Their wide distribution among the bacteria that cause infectious diseases in humans and animals represent a global threat. We have developed a biochip with colorimetric detection based on horseradish peroxidase for the simultaneous identification of genes for all clinically relevant class A beta-lactamases and carbapenemases of classes A, B, and D, including 25 single substitutions in the nucleotide sequence encoding the key amino acid substitutions in class A beta-lactamases. The conditions for allele-specific hybridization of biotin-labeled target DNA with oligonucleotide probes immobilized on the surface of the biochip have been optimized. A method of multiplex amplification of all of the studied genes in one reaction with the simultaneous incorporation of biotin was developed to obtain the target DNA. The biochip was validated with mixtures of the beta-lactamase and carbapenemase genes, as well as 68 DNA samples isolated from clinical strains of gram-negative bacteria. The total DNA sample analysis time was ~4 h. A high specificity of the identification of genes in mixtures was demonstrated, which can be used in the study of multidrug-resistant bacteria.