Molecular diagnostic of toxigenic Corynebacterium diphtheriae strain by DNA sensor potentially suitable for electrochemical point-of-care diagnostic.

ABSTRACT

The presented study is focused on the development of electrochemical genosensor for detection of tox gene fragment of toxigenic Corynebacterium diphtheriae strain. Together with our previous studies it fulfils the whole procedure for fast and accurate diagnostic of diphtheria at its early stage of infection with the use of electrochemical methods. The developed DNA sensor potentially can be used in more sophisticated portable device. After the electrochemical stem-loop probe structure optimization the conditions for real asymmetric PCR (aPCR) product detection were selected. As was shown it was crucial to optimize the magnesium and organic solvent concentrations in detection buffer. Under optimal conditions it was possible to selectively detect as low as 20.8 nM of complementary stand in 5 min or 0.5 nM in 30 min with sensitivity of 12.81 and 0.24 1⋅µM-1 respectively. The unspecific biosensor response was elucidated with the use of new electrode blocking agent, diethyldithiocarbamate. Its application in electrochemical genosensors lead to significant higher current values and the biosensor response even in conditions with magnesium ion depletion. The developed biosensor selectivity was examined using samples containing genetic material originated from a number of non-target bacterial species which potentially can be present in the human upper respiratory tract.