TECHNOLOGY

HOW IT WORKS

Drug-resistant tuberculosis often arises from subtle variations in bacterial genome (mutations). Currently, the conventional  approach for detecting antibiotic resistance requires prolonged cultivation of bacteria under strictly controlled conditions. This is a well-established yet laborious procedure that often impairs early treatment. Therefore, the need for rapid molecular tests is constantly increasing, especially in  high TB burden­ countries worldwide.

INNOVATIVE – UNIQUE – RAPID

EMPE Diagnostics’ tests combine molecular tools called padlock probes with lateral flow biosensor to identify antibiotic resistance in a fast and accurate manner. Such a novel combination enables highly specific identification of multiple segments and/or positions in TB DNA and provide the results in less than 2h, in a convenient and easy to interpret PRESENCE/ABSENCE format by developing visual signals.

SPECIFIC – ADAPTABLE – ROBUST

Mutation identification principal is based on proprietary technology of padlock probes [1] and circle-to-circle isothermal DNA amplification (C2CA) [2]. Padlocks are highly selective DNA probes that become circularized when bound to a specific region of DNA. This technology allows for interrogation of thousands of independent DNA positions (DNA sequences) in a single assay, without complications of artefacts observed in traditional multiplexed PCR assays. EMPE Diagnostics’ probe panel is engineered to detect the specific sequences in TB DNA, coding resistance or susceptibility to various antibiotics. Robust DNA identification with padlock probes is created with strong scientific foundation reflected in hundreds of peer-reviewed publications in high impact journals such as Science, Nature Genetics, Nature Biotechnology, Nature Methods and PNAS among others.

ISOTHERMAL – SENSITIVE

To magnify successful DNA detection event, circularized padlock probes become replicated (amplified, polymerized) through a two-step process called Circle-to-Circle Amplification, C2CA. First, Phi29 DNA polymerase copies circles into long DNA polymers containing hundreds of identical repeats of the original padlock probe. The polymer is cleaved into individual units that are in turn re-circularized. This re-circularization and consequent second amplification followed by another monomerization yield vast amounts of polymer units that are finally detected [3] on the EMPE Diagnostics’ test cassette. Depending on an antibiotic resistance profile of the bacteria, distinctive types of polymer units are generated.

USER FRIENDLY – ACCURATE

Our biosensing devices are engineered around two principal objectives: clear-cut and user friendliness. Cassette casing encapsulates a nitrocellulose membrane strip with multiple engraved detection zones. When sample is applied into the application window, the polymer units flow along the membrane dragged by simple capillary forces. Units selectively attach to their respective zone areas, producing a visible pattern reflecting bacteria’s antibiotic resistance profile. All our cassettes contain convenient labels along the sample preview window to facilitate simple interpretation of the test result.

We at EMPE Diagnostics continuously develop empowering technologies in house as well as through collaborations with leading researchers at Uppsala Science Park/hub, Stockholm University, Karolinska Institute and other international consortia.

REFERENCES

[1] Nilsson, M. et al. (1994). Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265 (5181), 2085–2088.

[2] Dahl, F. et al. (2004). Circle-to-circle amplification for precise and sensitive DNA analysis. PNAS, 101 (13), 4548–4553.

[3] Pavankumar AR. et al. (2016). Proficient Detection of Multi-Drug-Resistant Mycobacterium tuberculosis by Padlock Probes and Lateral Flow Nucleic Acid Biosensors. ACS Anal. Chem, 88(8):4277-84.