title: Multiplex PCR for joint amplification of carbapenemase genes of molecular classes A, B, and D.
abstract: Here we present a method for joint amplification of genes of carbapenemases of molecular classes A, B, and D for hybridization analysis on DNA microarrays. Using new-generation DNA polymerase KAPA2G Fast (KAPA Biosystems, USA) together with optimization of the conditions for the multiplex PCR with 20 primer pairs allowed us to carry out joint amplification of full-length genes of seven different types of carbapenemases (KPC, VIM, IMP, SPM, SIM, GIM, and OXA) with simultaneous inclusion of biotin as a label. Yield of the labeled PCR product sufficient for further analysis by microarray hybridization was achieved 40 min after the start of the reaction. This reduced the total duration of DNA identification techniques, including sample preparation stage, to 4 h. The method for gene identification by DNA microarrays with the improved stage of amplification of specific carbapenemase genes was tested with clinical strains of gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae spp. with different sensitivity towards carbapenems according to phenotyping tests. All clinical strains of A. baumannii resistant to carbapenems were found to have genes of OXA-type carbapenemases (subtypes OXA-51, OXA-23, OXA-40, and OXA-58), and clinical strains of P. aeruginosa resistant to carbapenems were found to possess the gene of VIM-type metallo-beta-lactamase (subtype VIM-2). When testing clinical strains sensitive to carbapenems, carbapenemase genes were not detected. Thus, the method of identifying carbapenemase genes on DNA microarrays is characterized by high accuracy and can be used in clinical microbiology laboratories for express diagnostics of resistance to carbapenems.
authors.affiliation: Lomonosov Moscow State University, Chemical Faculty, Moscow, 119991, Russia. mrubtsova@gmail.com.
authors.forename: Yu I
authors.initials: YI
authors.lastname: Pobolelova
authors.forename: M M
authors.initials: MM
authors.lastname: Ulyashova
authors.forename: M Yu
authors.initials: MY
authors.lastname: Rubtsova
authors.forename: A M
authors.initials: AM
authors.lastname: Egorov
mesh_terms: D040981:Acinetobacter baumannii / Q000201:enzymology; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004755:Enterobacteriaceae / Q000201:enzymology; D008274:Magnesium / Q000737:chemistry; D060885:Multiplex Polymerase Chain Reaction; D020411:Oligonucleotide Array Sequence Analysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D001618:beta-Lactamases / Q000145:classification / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001618:beta-Lactamases; C063912:carbapenemase; D008274:Magnesium; D004121:Dimethyl Sulfoxide