Quantitative Determination of Beta-Lactamase mRNA in the RNA Transcripts of Antibiotic-Resistant Bacteria Using Colorimetric Biochips

A method for the quantitative determination of mRNA of TEM-type serine beta-lactamases in bacteria resistant to beta-lactam antibiotics has been developed. The method includes several stages: the isolation of a total RNA fraction from a bacterial culture and the production of the beta-lactamase target DNA in sequential reverse transcription and polymerase chain reaction with the incorporation of a biotin label, and the hybridization of the target DNA on colorimetric biochips. Sample preparation conditions were optimized to increase the yield of the analyzed target DNA. A calibration curve for determining the amount of mRNA was constructed using a standard sample of beta-lactamase TEM-1 mRNA obtained by transcription in vitro. The advantage of using a standard sample corresponding to the full-length blaTEM-1 gene is that it passes through all the stages of analysis in parallel with the test samples with the same efficiency. The detection limit for TEM-1 beta-lactamase mRNA was 0.40 ± 0.05 amol/mL; the range of determined concentrations was from 1.0 amol/mL to 2000 fmol/mL, and the relative standard deviation did not exceed 12%. The duration of analysis after the production of a bacterial culture was about 7 h. The developed method can be used to study the conditions for the expression of beta-lactamase genes in bacteria resistant to antimicrobial drugs.