Difference between revisions of "Team:Vilnius-Lithuania/Design"

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<div class="grandHeading" style="margin-top:32px" id="section-text">DETECTION</div>
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<div class="scrollBlock noanim">
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<div class="content noanim">
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<div class="h2 larger">OVERVIEW</div>
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<p style="margin-bottom: 8px;">
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Lorem ipsum dolor sit amet, consectetur adipiscing elit. Morbi quis ante odio. Sed eleifend, eros at bibendum ullamcorper, nisl nisl mattis ligula, ut euismod lorem elit sit amet ipsum. Suspendisse scelerisque, odio vitae malesuada mattis, sem augue mattis velit, sit amet ornare lacus velit nec nibh. Curabitur sed lectus sapien. Suspendisse finibus urna volutpat mi consequat lacinia. In ut felis quis purus tempus gravida. Vestibulum eget gravida risus. Vivamus porta dui et nulla fringilla, nec fermentum felis placerat. Pellentesque dictum risus quis aliquam lacinia. Duis mauris arcu, rhoncus eu felis a, laoreet volutpat ipsum. Ut sed aliquam libero. Sed placerat sem nec hendrerit pellentesque.
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</p>
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</div>
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</div>
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</main>
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<div class="grandHeading" style="margin-top:32px">SEE OUR DESIGN FLOW</div>
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<div class="overviewIndex">
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<div>
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<a href="#headingDetection" class="title">DETECTION</a>
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<a href="#headingHDA" class="item"><div></div><span>Helicase-Dependent Amplification</span></a>
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<a href="#headingBioinf" class="item"><div></div><span>Bioinformatic Analysis</span></a>
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<a href="#headingHelimerase" class="item"><div></div><span>Helimerase</span></a>
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<a href="#headingLFA" class="item"><div></div><span>Lateral Flow Assay</span></a>
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</div>
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<div>
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<a href="#headingTreatment" class="title">TREATMENT</a>
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<a href="#headingLysin" class="item"><div></div><span>Endolysin & Exolysin System</span></a>
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<a href="#headingToxin" class="item"><div></div><span>Toxin & Antitoxin System</span></a>
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</div>
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<div>
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<a href="#headingPrevention" class="title">PREVENTION</a>
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<a href="#headingSubunit" class="item"><div></div><span>Subunit Vaccines</span></a>
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</div>
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</div>
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<div class="grandHeading" style="margin-top:32px">DETECTION</div>
 
<main>
 
<main>
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<div class="headingForIndex notvisible">Detection</div>
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<div id="headingDetection" class="headingForIndex notvisible" id="headingDetection">Detection</div>
 
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<div class="scrollBlock noanim">
 
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<div class="content noanim">
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</main>
 
</main>
 
<main>
 
<main>
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<div class="headingForIndex notvisible">Bioinformatic Analysis</div>
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<div class="headingForIndex notvisible" id="headingBioinf">Bioinformatic Analysis</div>
 
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<div class="scrollBlock noanim">
 
<div class="content noanim">
 
<div class="content noanim">
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<main>
 
<main>
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<div class="headingForIndex notvisible">Helicase-dependent amplification</div>
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<div class="headingForIndex notvisible" id="headingHDA">Helicase-dependent amplification</div>
 
<div class="scrollBlock noanim">
 
<div class="scrollBlock noanim">
 
<div class="content noanim">
 
<div class="content noanim">
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</main>
 
</main>
 
<main>
 
<main>
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<div class="headingForIndex notvisible">Helimerase</div>
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<div class="headingForIndex notvisible" id="headingHelimerase">Helimerase</div>
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<div class="scrollBlock">
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<div class="scrollBlock" style="height:unset;grid-template-rows:40vh auto">
 
<div class="scrollItem">
 
<div class="scrollItem">
 
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{ length: 0.03, title: "Conjugate Pad", text: "Fluid absorbed into sample pad flows to the subsequent membrane called a <b>conjugate pad</b>. On this membrane, labelled biorecognition molecules - <b>gold nanoparticles conjugated with detection probes</b> are dispersed. Upon contact with a moving liquid sample, the pad easily releases biorecognition molecules. According to literature research, <b>13 nm gold nanoparticles</b> are the most suitable for LFA based on nucleic acid hybridization<sup>1</sup>. For this reason, we chose to synthesize 13 nm gold nanoparticles using sodium citrate reduction method otherwise called Turkevich method." },
 
{ length: 0.03, title: "Conjugate Pad", text: "Fluid absorbed into sample pad flows to the subsequent membrane called a <b>conjugate pad</b>. On this membrane, labelled biorecognition molecules - <b>gold nanoparticles conjugated with detection probes</b> are dispersed. Upon contact with a moving liquid sample, the pad easily releases biorecognition molecules. According to literature research, <b>13 nm gold nanoparticles</b> are the most suitable for LFA based on nucleic acid hybridization<sup>1</sup>. For this reason, we chose to synthesize 13 nm gold nanoparticles using sodium citrate reduction method otherwise called Turkevich method." },
 
{ length: 0.065, title: "Gold Nanoparticles", text: "Gold nanoparticles can be bought premade or synthesized in the laboratory. Usually, the Turkevich method with slight modifications is used for synthesis in which HAuCl<sub>4</sub> is reduced by trisodium citrate under reflux conditions. In this technique, citrate ions act as both - stabilizing and reducing agents. The size of gold nanoparticles is determined by the amount of sodium citrate added. With a lower concentration of sodium citrate, larger gold nanoparticles are produced. For LFA test development we also decided to use tannic acid in the gold nanoparticles synthesis process since it helps to maintain the uniformity of gold nanoparticles size." },
 
{ length: 0.065, title: "Gold Nanoparticles", text: "Gold nanoparticles can be bought premade or synthesized in the laboratory. Usually, the Turkevich method with slight modifications is used for synthesis in which HAuCl<sub>4</sub> is reduced by trisodium citrate under reflux conditions. In this technique, citrate ions act as both - stabilizing and reducing agents. The size of gold nanoparticles is determined by the amount of sodium citrate added. With a lower concentration of sodium citrate, larger gold nanoparticles are produced. For LFA test development we also decided to use tannic acid in the gold nanoparticles synthesis process since it helps to maintain the uniformity of gold nanoparticles size." },
−
{ length: 0.065, title: "Detection Probe", text: "Synthesized gold nanoparticles must be functionalized with <b>detection probes</b>. Detection probe is a ssDNA molecule with <b>5’ thiol group modification</b> created to hybridize to HDA produced specific to species ssDNA amplicon. To functionalize gold nanoparticles a method, which includes the step of “aging” with NaCl is used. Salt helps to overcome the long-ranged electrostatic repulsion between the DNA and gold nanoparticles since they are both negatively charged<sup>2</sup>. Our team created probes with <b>poly-A</b> spacer, so an alternative and quicker, low pH assisted method can be used for functionalization<sup>3</sup>. In pH 3, adenine is protonated, and this reduces the repulsion between DNA and gold nanoparticle." },
+
{ length: 0.095, title: "Detection Probe", text: "Synthesized gold nanoparticles must be functionalized with <b>detection probes</b>. Detection probe is a ssDNA molecule with <b>5’ thiol group modification</b> created to hybridize to HDA produced specific to species ssDNA amplicon. To functionalize gold nanoparticles a method, which includes the step of “aging” with NaCl is used. Salt helps to overcome the long-ranged electrostatic repulsion between the DNA and gold nanoparticles since they are both negatively charged<sup>2</sup>. Our team created probes with <b>poly-A</b> spacer, so an alternative and quicker, low pH assisted method can be used for functionalization<sup>3</sup>. In pH 3, adenine is protonated, and this reduces the repulsion between DNA and gold nanoparticle.<br>We also used tannic acid in the synthesis process since it helps to maintain the uniformity of gold nanoparticles size. It is crucial to remember that HAuCl<sub>4</sub> is corrosive so a glass or plastic spatula must be used also all glassware has to be cleaned with aqua regia to avoid gold nanoparticle aggregation during synthesis<sup>2</sup>." },
−
{ length: 0.03, text: "We also used tannic acid in the synthesis process since it helps to maintain the uniformity of gold nanoparticles size. It is crucial to remember that HAuCl<sub>4</sub> is corrosive so a glass or plastic spatula must be used also all glassware has to be cleaned with aqua regia to avoid gold nanoparticle aggregation during synthesis<sup>2</sup>." },
+
 
{ length: 0.10, title: "Hybridization", text: "<b>Conjugate pad</b> filled with biorecognition molecules is essential for nucleic acid hybridization.<br>If specific ssDNA from the HDA reaction is present in the sample, then the first hybridization reaction occurs between the <b>detection probe and ssDNA amplicon</b>. Newly formed complex immediately flows to the subsequent nitrocellulose membrane.<br>If the added sample does not have a ssDNA amplicon, then no hybridization occurs in the conjugate pad. Further flows labelled gold nanoparticles unhybridized to ssDNA." },
 
{ length: 0.10, title: "Hybridization", text: "<b>Conjugate pad</b> filled with biorecognition molecules is essential for nucleic acid hybridization.<br>If specific ssDNA from the HDA reaction is present in the sample, then the first hybridization reaction occurs between the <b>detection probe and ssDNA amplicon</b>. Newly formed complex immediately flows to the subsequent nitrocellulose membrane.<br>If the added sample does not have a ssDNA amplicon, then no hybridization occurs in the conjugate pad. Further flows labelled gold nanoparticles unhybridized to ssDNA." },
−
{ length: 0.05, title: "Test Line", text: "Mentioned nitrocellulose membrane has two lines with dispersed DNA probes. The first line is called <b>the test line</b>, and the further line is <b>the control line</b>. On the test line, capture probes are dispersed. The capture probe is a ssDNA molecule with 3’ biotin modification which is necessary so that the probe could be immobilized <b>via a biotin-streptavidin non-covalent bond<b> on the membrane." },
+
{ length: 0.11, title: "Test Line", text: "Mentioned nitrocellulose membrane has two lines with dispersed DNA probes. The first line is called <b>the test line</b>, and the further line is <b>the control line</b>. On the test line, capture probes are dispersed. The capture probe is a ssDNA molecule with 3’ biotin modification which is necessary so that the probe could be immobilized <b>via a biotin-streptavidin non-covalent bond</b> on the membrane.<br>When the fluid reaches the test line, another hybridization reaction occurs between the <b>capture probe and ssDNA amplicon-gold nanoparticle complex</b>. Since capture probes are immobilized on the membrane, the resulting complex does not move any further and stays on the test line. Eventually, due to hybridization gold nanoparticles start to accumulate and the red line becomes visible to the naked eye. If there is no specific ssDNA amplicon in the sample, then no hybridization takes place and labeled gold nanoparticles move further the membrane." },
−
{ length: 0.06, text: "When the fluid reaches the test line, another hybridization reaction occurs between the <b>capture probe and ssDNA amplicon-gold nanoparticle complex</b>. Since capture probes are immobilized on the membrane, the resulting complex does not move any further and stays on the test line. Eventually, due to hybridization gold nanoparticles start to accumulate and the red line becomes visible to the naked eye. If there is no specific ssDNA amplicon in the sample, then no hybridization takes place and labeled gold nanoparticles move further the membrane." },
+
{ length: 0.15, title: "Control Line", text: "In the conjugate pad, not all labelled gold nanoparticles hybridize to ssDNA amplicon, meaning that there are gold nanoparticles with free detection probes. This is important for <b>the control line</b> where control probes are immobilized via the same biotin-streptavidin bond. The only difference is that the control probe has a 5’ biotin modification and is complementary to the detection probe. For this reason, hybridization between <b>free detection probe on gold nanoparticle and control probe</b> occurs resulting in the red line formation. Mentioned hybridization reaction has to occur during each test because it shows that the test operated correctly.<br>We chose to use Hi-Flow plus HF180 nitrocellulose membrane, which has a nominal capillary <b>flow time of 180 seconds/4cm</b>, which is relatively slow. The migration speed of the fluid is critical because it affects the response of the test. Slower flow membrane results in a longer time assay, but response signal is higher, and lines are better defined<sup>1</sup>. To get the best results optimization is needed, for this reason we decided to create a mathematical model which helps to find out the best place for capture and control probes." },
−
{ length: 0.05, title: "Control Line", text: "In the conjugate pad, not all labelled gold nanoparticles hybridize to ssDNA amplicon, meaning that there are gold nanoparticles with free detection probes. This is important for <b>the control line</b> where control probes are immobilized via the same biotin-streptavidin bond. The only difference is that the control probe has a 5’ biotin modification and is complementary to the detection probe. For this reason, hybridization between <b>free detection probe on gold nanoparticle and control probe</b> occurs resulting in the red line formation. Mentioned hybridization reaction has to occur during each test because it shows that the test operated correctly." },
+
−
{ length: 0.10, text: "We chose to use Hi-Flow plus HF180 nitrocellulose membrane, which has a nominal capillary <b>flow time of 180 seconds/4cm</b>, which is relatively slow. The migration speed of the fluid is critical because it affects the response of the test. Slower flow membrane results in a longer time assay, but response signal is higher, and lines are better defined<sup>1</sup>. To get the best results optimization is needed, for this reason we decided to create a mathematical model which helps to find out the best place for capture and control probes." },
+
 
{ length: 0.12, title: "Absorbent Pad", text: "Finally, the last membrane is <b>the absorbent pad</b> which helps in maintaining the flow rate of the liquid over the membrane and stops backflow of the sample. Also, it absorbs excess fluid. Different kinds of membranes are assembled with overlapping ends (2mm) to ensure that continuous flow is achieved and mounted over a backing card which helps as support and makes it easy to handle the test." },
 
{ length: 0.12, title: "Absorbent Pad", text: "Finally, the last membrane is <b>the absorbent pad</b> which helps in maintaining the flow rate of the liquid over the membrane and stops backflow of the sample. Also, it absorbs excess fluid. Different kinds of membranes are assembled with overlapping ends (2mm) to ensure that continuous flow is achieved and mounted over a backing card which helps as support and makes it easy to handle the test." },
 
],
 
],
Line 234: Line 261:
 
sections: [
 
sections: [
 
{ length: 0.15, title: "Sample Processing", text: "The specimens obtained from the fish gills are lysed mechanically or using ionic liquids. For the exact species identification, we have selected <b><i>F. columnare</i></b>, and <b><i>F. psychrophilum</i> 16S rRNA</b> DNA fragments and rpoC gene, obtained from <i>F. psychrophilum</i>." },
 
{ length: 0.15, title: "Sample Processing", text: "The specimens obtained from the fish gills are lysed mechanically or using ionic liquids. For the exact species identification, we have selected <b><i>F. columnare</i></b>, and <b><i>F. psychrophilum</i> 16S rRNA</b> DNA fragments and rpoC gene, obtained from <i>F. psychrophilum</i>." },
−
{ length: 0.15, title: "Asymmetric HDA", text: "These genomic DNA marker sequences for further analysis are being amplified during <b>asymmetric helicase dependent amplification</b>. This system is based on a natural mechanism where two complementary DNA strands are separated by thermostable <b>DNA helicase</b> and coated with single-stranded DNA-binding proteins (<b class=\"semi\">SSB</b>). After generation of ssDNA two sequence-specific primers hybridise to each border of ssDNA." },
+
{ length: 0.30, title: "Asymmetric HDA", text: "These genomic DNA marker sequences for further analysis are being amplified during <b>asymmetric helicase dependent amplification</b>. This system is based on a natural mechanism where two complementary DNA strands are separated by thermostable <b>DNA helicase</b> and coated with single-stranded DNA-binding proteins (<b class=\"semi\">SSB</b>). After generation of ssDNA two sequence-specific primers hybridise to each border of ssDNA.<br>As it is an asymmetric HDA assay, the limiting primer is used up with the purpose to obtain ssDNA templates. After hybridization, DNA polymerase extends these annealed to the template primers. Newly synthesised fragments are then used as substrates for DNA helicase, which enters the next round of isothermal amplification." },
−
{ length: 0.15, text: "As it is an asymmetric HDA assay, the limiting primer is used up with the purpose to obtain ssDNA templates. After hybridization, DNA polymerase extends these annealed to the template primers. Newly synthesised fragments are then used as substrates for DNA helicase, which enters the next round of isothermal amplification." },
+
 
{ length: 0.42, title: "HDA Products", text: "Finally, after asymmetric HDA assay, due to excess reverse primer concentration in the assay, ssDNA templates are prepared for the lateral flow assay test." },
 
{ length: 0.42, title: "HDA Products", text: "Finally, after asymmetric HDA assay, due to excess reverse primer concentration in the assay, ssDNA templates are prepared for the lateral flow assay test." },
 
],
 
],

Revision as of 14:54, 22 October 2020

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OVERVIEW

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SEE OUR DESIGN FLOW
DETECTION
Detection
Overview

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.

  1. A bioinformatic analysis of the marker gene sequences which does not match between Flavo species. Creation of LFA DNA probes and HDA primers.
  2. Helicase-dependent asymmetric DNA amplification (HDA) of the marker gene fragments.
  3. Lateral flow assay membrane test that just in a few minutes identifies an exact pathogen.

Bioinformatic Analysis
Bioinformatic Analysis

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.

Helicase-dependent amplification
Helicase-dependent amplification

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.

Helimerase
WZB1WZA2TteUvrDBstPolStrepII Tag10xHis TagMBP TagL1L1
Helimerase

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
Lateral Flow Assay

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.

TREATMENT
Treatment

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.

Choose treatment system
Endolysin & Exolysin System
Toxin & Antitoxin System
Endolysin & Exolysin System
Toxin & Antitoxin System
PREVENTION
Prevention

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.

Subunit vaccines
References
Big Mega Super Duper List of References

R. Meškys et al. iGEM Vilnius-Lithuania 2020