Grants and collaborations
The standard protocol for virus detection in patient samples is nucleic extraction followed by amplification by Quantitative Polymerase Chain Reaction (qPCR) or Loop Mediated Isothermal Amplification (LAMP). For RNA viruses a reverse transcription step precedes qPCR (RT-qPCR) and LAMP (RT-LAMP).
The nucleic acid extraction step is critical to remove components of the viral transport media (VTM) and of the saliva/buccal swab which inhibit the qPCR/RT-qPCR and LAMP/RT-LAMP. In the RVDR Project, the project partners will develop a series of qPCR and LAMP reagents, which are not inhibited by saliva, removing the cost and time of nucleic acid extraction steps in clinical diagnostics.
TargetEx Ltd. aims to develop new, innovative diagnostic products. One product group aims to reduce the time to perform PCR and RT-PCR reactions by increasing the rate of the polymerase enzyme and enhancing the stability of the reverse transcriptase enzyme. The other product group to be developed will be applicable to next generation sequencing (NGS), particularly for the amplification of long DNA fragments.
In both cases, we design and perform the necessary protein modifications and then produce small amounts of protein prototypes that can be compared with existing diagnostic methods. Realizing such approaches, we hope that we can develop better products than our competitors.
Successfully completed grants:
Diagnostic procedures based on quantitative polymerase chain reaction (qPCR) are complex, slow, require high equipment, use reagents, and require extensive practice. Isothermal chain reaction (LAMP) is a DNA synthesis method in which the DNA strand is copied at a constant temperature (60-65˚C). Applying LAMP in diagnostic procedures, no cyclic change of temperature is required as in case of a “normal” PCR reaction test. The LAMP reaction can even be performed in a water bath. The resulting product can be detected by a color reaction: if there is a product, a color change is obtained, or in the case of a more sensitive method, a fluorescent dye can be used, which can be excited by blue light. Using one pathogen-specific oligonucleotide probe at a time, a ’yes / no’ response can be obtained in as little as 40-60 minutes. Such a measurement method has already been developed for the detection of several pathogens, such as coronavirus. During the execution of the project, we developed reagents that could reliably identify a virus or bacterium in less than 20 minutes, even in a district physician’s office, using the specificity of the polymerase reaction.
In connection with the fight against the coronavirus epidemic, we have developed a new type of protein-based vaccine that elicits an innate immune response and an acquired immunity, thus making our body resistant to the attack of the real virus. The idea was to combine two proteins in the vaccine: one is the part of the flagellin protein, the main component of the locomotion organelle of the salmonella bacterium, which had been shown to be a useful carrier or excipient to increase the effectiveness of vaccines. The other component of the fusion protein was the cell surface-binding portion of the „spike” protein responsible for recognizing the target cell on the surface of selected animal coronaviruses. It was proved that by incorporating the receptor-binding domain of the coronavirus, which directed entry into infected cells, inside the flagellin, provided the way for the production of an effective vaccine.
The goal of the project was to develop and make manufacture-ready a novel, innovative, market-ready, ISO certified RNase inhibitor that is often used in research and human diagnostics. For this, we produced a protein that was more stable than the marketed inhibitors due to particular changes in the amino acid composition. Besides, we developed a buffer system that increased the stability of the protein. The project continued until the end of 2020.
The aim of this collaboration was to develop novel ON-type gene expression systems based on the currently available, OFF-type E-Rex and PEACE systems, where the term “ON-type” means that gene expression is switched on in the presence, and off in the absence of the relevant drugs (erythromycin, phloretin). Turning these OFF-type systems into ON-type (i.e. achieving reverse molecules) also included and required the development of a novel computational modelling methodology for such systems. Further aim was to improve the recently developed, already ON-type RuX system (regulated by RU486). With the international SwitchItOn consortium, a combination of three SMEs and an academia, we combined the needed expertise and knowledge to bring novel switches for the 2nd generation of models of human disease to the market. The project had a website (http://switchiton.eu/) and ended in Q2 of 2021.