Bacteria invade and grow in the urinary tract (the kidneys, ureters, bladder, and urethra). Most urinary tract infections occur in the bladder or urethra.They are mainly caused by bacteria, although fungi and some viruses have also been implicated. E. coli is the dominant causative agent in all patient groups, causing 80–90% of all UTIs and hence serves as a model pathogen for studying UTIs.It is the 2nd most commonly diagnosed infectious disease worldwide. It is the most common bacterial infection in children. We use the aptamer based detection.Aptamers are single-stranded oligonucleotides,fold into defined architectures and bind to targets such as proteins, inhibit protein–protein interactions,elicit therapeutic effects such as antagonism,discovered using SELEX. (Systematic Evolution of Ligands byEXponential enrichment)The target protein used is Fimh. FimH adhesin confers a higher tropism on E. coli for extraintestinal sites and urinary bladder. FimH is selected as target for aptamer designing. N-terminal for FimH being highly conservative is considered for aptamer binding and tagged with 6 Histidine tags since we target cell membrane protein.Selex is further carried out and aptamers are obtained, which is further used in the designing of the diagnostic kit.
The method we will be adapting to design kit is known as Aptamer-based lateral flow assay. A two-colored line result indicates negativity and a control-only line indicates positivity.Typically, antibodies comprise both the specific recognition molecules in the conjugate pad and the target-binding biosensors and capture molecules on the membrane. Because nucleotide acid aptamers can recognize targets of interest specifically, antibodies for LFA can be replaced with aptamers. Aptamer-based LFAs (ALFA) offers a more flexible design and the ability to detect a wider range of targets.
Our main goal is to create a kit which will detect the presence of uropathogens through color change due to disintegration of the gold nanoparticle aggregation which is tagged to the aptamers. And to optimize the signal to noise ratio we are planning to do different assays and also are trying to find the optimum sample to aptamer ratio which can be use.
We intend to make the APT4UTI kit a rapid detection kit which would provide the results in 20 minutes or less with absolutely no expertise in the field. The usual cost of antibody based detection kits available in the market need to have expert invigilation and a laboratory setup and maintenance. With this kit there will be no need for such a highly maintained infrastructure and expert presence which reduces the cost to build the setup and also reduces the burden on the healthcare professionals. The taboo of not speaking in public about urinary tract infection can also be tackled because an infected person can test in a private space on their own. With high reduction in the cost of infrastructure, manforce payment and daily visit fee of the individual, this kit will prove to be highly cost effective.
On the industry level, we as a team have pitched the idea for a startup and it has been appreciated by the state government. We are in the process of taking it to the small scale manufacturing level, where the cost of manufacturing a kit as a hardware will be estimated.
The aim of this project is to develop a novel aptamer based low cost UTI detection kit at the POC. Efforts are made to design the kit that will follow lateral flow assay with aptamer binding principle. Aptamers are better than antibodies in this regard for detection of uropathogens and hence aptamer based strategy is used. The aptamers for the kit are designed and screened and the kit is designed. Physically when the urine sample is loaded, the kit will show colorimetric changes depending on the presence of the uropathogen.