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Revision as of 18:19, 23 October 2020
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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.
Since we wanted to differentiate between Flavobacterium species, instead of antibody-antigen interaction, our detection test is based on nucleic acid hybridization. We found out that for identification purposes, 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 marker genes, which allows us to identify an exact bacteria species. 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 highly specific, we made a multiple sequence alignment with 16S rRNA genes from other species within the same genus using Clustal Omega tool (1. 2. 4.). Unique target sequences for F. columnare and F. psychrophilum were selected based on the absence of matching alignments between sequences.
Picture 1
Also we found that rpoC gene can be used as marker from F. psychrophilum. We decided to create probes for this gene sequence as well.
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.
However, regarding the WHO guidelines for point-of-care testing, our detection tool should be not only sensitive, specific and user-friendly, but also it should be affordable for target customers1. Keeping in mind that HDA amplification, performed using a commercial kit, is still too expensive, we have decided to search for new alternatives to reduce the cost of the test as much as possible.
Our solution to this problem - helimerase. This protein complex contains two enzymes – Thermoanaerobacter tengcongensis UvrD helicase (TteUvrD) and Bacillus stearothermophilus DNA polymerase I large fragment (BstPol). TteUvrD is fused with one part of coiled-coil structure WinZip-A2 (WZA2) via linker L1 and possesses a maltose-binding protein (MBP) and 10xHis Tag in the N terminal end. BstPol is fused with the second coiled-coil part WinZip-B1 (WZB1) through linker L1 and possesses StrepII tag in the N terminal end2. It was presumed that this non-covalent fusion strategy through coiled-coil structures should improve HDA reaction by letting to amplify longer DNA fragments as well as it allows to perform amplification reaction without using additional proteins such as MutL or others SSB proteins, which are used in this type of reactions2,3.
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.
After detection of an exact flavobacterium species, farmers need to start an exact treatment process as soon as possible. Currently, fish infected with flavobacterial diseases are treated with different types of antibiotics13. Scientific data shows14 that the most abundant antibiotics used for salmon cultivation was quinolone, which consumption (by mass) in 2007 reached 821,997 tonnes. That is only in Norway! Other commonly used antibiotics in farms are oxolinic acid and florfenicol, which consumption reached 681 kg in 2008 and 166 kg in 2010 respectively. This enormous usage of wide variety antibiotics forces the evolving of antibiotic-resistant bacterial fish pathogens. These numbers raise huge concerns and questions on how we can reduce the usage of antibiotics?
Scientific data already shows that some F. psychrophilum isolates already have reduced susceptibility to quinolones, oxolinic acid, and enrofloxacin1. To reduce the amount of antibiotics used in aquaculture farms, our second goal is the development of two exogenous fish infection treatment systems, which are based on quorum sensing mechanisms.
Flavobacterial diseases are caused when Flavobacterium forms the same biofilm on fish fins or gills15. Our target bacteria - Flavobacterium uses signaling molecules for cell-cell communication. Quorum sensing is a bacterial communication process that leads to the regulation of genes and response to changes2–4. The quorum sensing has two distinguished systems - AHL and AI-2 for Gram-negative bacteria for now4–6. We used the circumstances in our advantage: build a genetic circuit that is enhanced by quorum sensing signaling molecules.
Usually, in order to prevent fish infections, vaccines are injected intraperitoneally. Because of manual handling, which is unavoidable in such a type of vaccination, fish experience a lot of stress and it paradoxically weakens their immune system. With the aim to avoid such consequences, we have set a third goal - to create a prevention system based on subunit vaccines against bacterial and viral fish infections.
R. Meškys et al. iGEM Vilnius-Lithuania 2020
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