Team:Vilnius-Lithuania/test/Design
Since it's inception iGEM has worked to ensure that excellence in synthetic biology goes beyond what happens in the lab. Decisions in science and engineering shape, and are shaped by, the societies we create.
Our detection system is based on identification of an exact Flavobacterium species marker gene fragments. This detection design is made up of these three main steps.
- A bioinformatic analysis of the marker gene sequences which does not match between Flavo species. Creation of LFA DNA probes and HDA primers.
- Helicase dependent asymmetric DNA amplification (HDA) of the marker gene fragments.
- Lateral-flow assay membrane test that just in a few minutes identifies an exact pathogen.
Our test is based on nucleic acid hybridization instead of antibody-antigen recognition since we wanted to differentiate between Flavobacterium species. We found out that for this purpose, nucleic acids are a more reliable and specific source than antibodies SALTINIS. The first step in developing a lateral flow assay test based on nucleic acid hybridization is choosing genes for species identification. According to our literature research 16S rRNA gene is a suitable candidate for this purpose because it is present in almost all bacteria and its function did not change over time2. To make sure that flavotest is specific we made a multiple sequence alignment with 16S rRNA genes from other species within the same Flavobacterium columnare, F. branchiophilum and F. psychrophilum species using Clustal Omega tool. Unique target sequences for F. columnare and F. psychrophilum were selected based on the absence of matching alignments between them (pic. 1).
We used Flavobacterium columnare and Flavobacterium psychrophilum 16S rRNA gene as a unique marker to each of the species. After bioinformatics analysis, a specific region in the sequences where no matches between the bases were found was chosen. For the short fragment, we created detection and capture probes.
With the aim to create a rapid, specific and cost-effective point-of-care detection system, at first, we needed to find the most suitable isothermal DNA amplification method. This method should be usable for farmers who have no scientific background. This factor pinpoints a huge need to be able to perform these isothermal reactions with as minimal pipetting steps as possible by means of avoiding errors and false-positive results. Although, amplification of marker sequences should be done in constant temperature by the needs of cheap and fully-portable equipment. By leading these main requirements, we have separated some isothermal amplification methods such as helicase dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), strand-displacement amplification (SDA) and rolling circle amplification (RCA)1.
However, LAMP, SDA or RCA amplification methods have their own limitations such as complicated reaction schemes or multiplex sets of primers. Also, it should be mentioned that each of these methods are incapable of amplifying DNA targets of sufficient length required for lateral flow assay test2. After further analysis, we found out that in order to fulfil these goals, helicase dependent amplification would be a perfect solution. This method allows us to make our detection test as specific as possible by using an exact length of target sequences. Thus, it provides a simple reaction scheme and enables the generation of single-stranded DNA fragments, which are essential for lateral flow assay test development3.
Since it's inception iGEM has worked to ensure that excellence in synthetic biology goes beyond what happens in the lab. Decisions in science and engineering shape, and are shaped by, the societies we create.
Lateral flow assay (LFA) is a simple method that can be used for isothermal amplification results visualisation. The use of the test is very intuitive and requires no prior training. Also, this LFA based test method is cost-effective and portable. Because of this, LFA is commonly used in remote locations where access to scientific laboratories is limited. For these reasons, we have decided that the best strategy for rapid flavobacterium caused infections detection tool development is the combination of HDA and LFA methods. since the users of this detection kit would be people without a scientific background.
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Since it's inception iGEM has worked to ensure that excellence in synthetic biology goes beyond what happens in the lab. Decisions in science and engineering shape, and are shaped by, the societies we create.
Since it's inception iGEM has worked to ensure that excellence in synthetic biology goes beyond what happens in the lab. Decisions in science and engineering shape, and are shaped by, the societies we create. Social,
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