Discrimination of single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis using a surface plasmon resonance biosensor

摘要

Single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis, the mutations of which cause drug resistance of the infectious agent, were detected and discriminated using a surface plasmon resonance biosensor system. Thiol-modified oligonucleotides of the selected sequence (the probe) and 1-mercapto-6-hexanol were immobilized on a gold sensor surface. Hybridization between immobilized probe P2 and perfectly matched target T2 as well as a single base mismatched target TN was investigated in buffer solutions of various stringencies. Discrimination of perfectly matched and single base mismatched targets is achieved due to a difference in the level of their hybridization with the immobilized probe depending on stringency of the buffer solution. In 0.5×SSC buffer solution (7.5 mM sodium citrate, pH 7, containing 75 mM NaCl), sensor response at T2 injection into the measuring sensor cell was 16 times that at TN injection. The experimental results on surface hybridization between the studied oligonucleotides demonstrated a good correlation with theoretical calculations of thermodynamic parameters of these interactions in the solution. The described approach could be proposed as a basis for creating a biosensor for real-time label-free diagnostics of drug-resistant tuberculosis.