A new DNA sensor system that can quantitatively detect the tuberculosis-causing bacteria, is introduced. The detection method relies on measuring the enzymatic activity of a specific mycobacterial enzyme called topoisomerase IA (TOP1A).
This research is the first to demonstrate how the catalytic properties of a TOP1A can be exploited for the precise detection of bacteria. The detection principle is based on a solid support-anchored DNA substrate which is converted into a closed DNA circle upon interaction with the specific enzyme target, extracted from the mycobacteria. The DNA circles then serve as a template for rolling circle amplification, generating multiple tandem copies that can be visualized in a fluorescence microscope.
By using purified mycobacterial TOP1A and extracts from various non-mycobacterial and mycobacterial species, it was demonstrated that the assay is specific, sensitive, and quantitative.
Moreover, to allow for the detection in small biological samples, the assay has been combined with the use of mycobacteriophages specific for the model bacteria Mycobacterium smegmatis which can lyse the bacteria releasing the active enzyme. In this way it was possible to detect M. smegmatis added to human saliva and reach a detection of 0.6 or 0.9 million colony-forming units (CFU) per milliliter of mycobacteria, which falls within the clinically relevant range of infection numbers.
This adaptable assay, suitable for resource-limited settings, is a promising step towards developing a new point-of-care diagnostic test for tuberculosis.