Difference between revisions of "Team:NYU Abu Dhabi/Description"

Revision as of 14:07, 24 October 2017

Issue
Shiga toxin-producing Escherichia coli (STEC) causes over 70,000 infections per year in the United States alone. A portion of these individuals will experience kidney failure after 6 days, 50% of which will require renal replacement therapy.[1] Conventional pathogen detection requires a laboratory setting using PCR, which requires multiple annealing and extension steps that can take over 3 hours and involves expensive equipment.

Gold Medal Requirement: Improving an previous iGEM Project
Shiga toxin is an exotoxin which consists of a toxin A subunit and cell-binding B subunit. The B subunit binds to a globotriasylceramide Gb3 receptor, which is expressed on the surface of target cells, an interaction which is responsible for the toxin’s entry into the host cell.[2] 2016 Team NYU Abu Dhabi exploited the binding of Gb3 to subunit B to detect for the presence of STEC. Their prototype compared the migration pattern of a bound Gb3-subunit B complex to a non-bound subunit B using a PAGE gel. Their device was estimated to take 45 minutes and their prototyping process ran into several factors that affected affordability and accessibility.

Project
Due to these difficulties in validating their prototype, and based on responses from food vendors in Pakistan and Indonesia, we have produced a rapid, affordable, portable device that allows for the detection of STEC using loop-mediated isothermal amplification (LAMP). This is a highly specific, efficient and rapid DNA amplification technique that uses 4-6 primers that bind to 6-8 distinct regions of target DNA. The selectivity of our system was tested using the rfbE gene, a non-toxic coding sequence required for O157-antigen synthesis. Reagents were lyophilized into distinct microfluidic channels, and sample DNA from inoculated broth was introduced into the system. Heating was supplied in the form of commercial, disposable hand warmers that were found to sustain the required temperature for up to 9 hours with insulation. The reaction was visualized using a handheld fluorescence microscope that can easily be substituted with a smartphone.

Results
The LAMP technique was shown to be more sensitive than conventional PCR techniques without the need for a heat lysis or centrifugation steps. The reaction generated fluorescent products after excitation with blue LEDs after 20 minutes. Our system achieves detection limits to add here without the need for laboratory equipment. The device is estimated to cost approximately $70 USD. This device offers a power, rapid method for pathogen detection for future point-of-care diagnostic applications.

[1] Borgatta, B.; Kmet-Lunaček, N.; Rello, J., E. coli O104:H4 outbreak and haemolytic–uraemic syndrome. Medicina Intensiva (English Edition) 2012, 36 (8), 576-583.
[2] Pacheco, A. R., Sperandio, V., Shiga toxin in enterohermorrhagic E. coli: regulation and novel anti-virulence strategies. Front. Cell. Infect. Microbiol. 2012, 2 (81), 1-12.
[3] New England BioLabs. Isothermal Amplification. Accessed October 19, 2017.

@@ Line 99: / Line 99: @@
   <p class="section-content">
-         The most common type of bacterial infection stems from contact with <i>Escherichia coli </i>, which when ingested can cause a variety of symptoms ranging from nausea to diarrhea. Shiga toxin-producing  <i>E. coli </i> (STECs) are responsible for the majority of foodborne  <i>E. coli </i> infections because the shiga toxin produced inhibits protein synthesis in all cells. While most countries now have stringent food safety regulations in place to prevent the sale of contaminated foods, small scale manufacturers, particularly street food vendors, often do not have access, time or pressure to consult laboratories about the safety of their food. Therefore, STEC-illnesses are still a major problem in countries that revolve around street food.
+        <b> Issue </b> </br>
+Shiga toxin-producing <i>Escherichia coli </i>(STEC) causes over 70,000 infections per year in the United States alone. A portion of these individuals will experience kidney failure after 6 days, 50% of which will require renal replacement therapy.[1] Conventional pathogen detection requires a laboratory setting using PCR, which requires multiple annealing and extension steps that can take over 3 hours and involves expensive equipment.
 </p>
     <p class="section-content">
-Our project aims to produce a portable device that allows for the detection of STEC through the use of loop-mediated isothermal amplification (LAMP), a technique that is similar to, but more sensitive than, polymerase chain reaction (PCR). Using the LAMP technique, we are targeting the genes that have been identified in shiga-toxin producing <i>E. coli</i>, namely stx1B, stx2B, rfbE, and eae. For each gene, we designed a set of 4-6 primers, which includes the forward and backward outer primers, the forward and backward inner primers, and the forward and backward loop primers if applicable. The forward inner primer initiates the amplification, followed by the strand-displacing DNA polymerase which separates the target DNA duplex. Synthesis initiated by the forward outer primer at an upstream target region subsequently displaces the first product, causing a stem-loop structure to form at the end of the first product due to the inner primer sequence complementarity. The annealing and strand-displacing processes continue from the opposite direction, yielding a dumbbell-shaped structure that contains more annealing sites at the inner primer sequences for further amplification.
+<b>Gold Medal Requirement: Improving an previous iGEM Project</b> </br>
+Shiga toxin is an exotoxin which consists of a toxin A subunit and cell-binding B subunit. The B subunit binds to a globotriasylceramide Gb3 receptor, which is expressed on the surface of target cells, an interaction which is responsible for the toxin’s entry into the host cell.[2] 2016 Team NYU Abu Dhabi exploited the binding of Gb3 to subunit B to detect for the presence of STEC. Their prototype compared the migration pattern of a bound Gb3-subunit B complex to a non-bound subunit B using a PAGE gel. Their device was estimated to take 45 minutes and their prototyping process ran into several factors that affected affordability and accessibility.
 </p>
   <p class="section-content">
-In order to ease the use of this technique for food vendors, we are building a device that will consist of two main components: the heater and a custom-made cartridge. The cartridge will contain three different chambers connected by pipes and valves. The first chamber is a cooking chamber where the samples will be heated to 95℃ to trigger the lysis process. The second chamber is where the LAMP technique will occur, with a chamber temperature of approximately 65℃ for the amplification reaction to occur. The result of the amplification can be visualized by a colorimetric assay using a color-changing dye in the third chamber. The reaction tubes will be prepared in powder form and premixed, ensuring that the user only needs to insert their food sample for testing. The device is also designed such that the cartridge is easily disposable and a new cartridge can be inserted every time the customer want to check a food sample. This ensures that the whole setup will be clear from contamination.
+        <b> Project </b> </br>
+Due to these difficulties in validating their prototype, and based on responses from food vendors in Pakistan and Indonesia, we have produced a rapid, affordable, portable device that allows for the detection of STEC using loop-mediated isothermal amplification (LAMP). This is a highly specific, efficient and rapid DNA amplification technique that uses 4-6 primers that bind to 6-8 distinct regions of target DNA. The selectivity of our system was tested using the rfbE gene, a non-toxic coding sequence required for O157-antigen synthesis. Reagents were lyophilized into distinct microfluidic channels, and sample DNA from inoculated broth was introduced into the system. Heating was supplied in the form of commercial, disposable hand warmers that were found to sustain the required temperature for up to 9 hours with insulation. The reaction was visualized using a handheld fluorescence microscope that can easily be substituted with a smartphone.
 </p>
 <p class="section-content">
-The end goal of our project is to provide food vendors an opportunity to easily and quickly detect for the presence of STEC in their products to ensure that they are complying with government standards efficiently and conveniently. The results of each test will eventually be uploaded into a database that provides consumers with the date, location and result of each STEC test. This will ensure that both vendor and consumer are safe, leading to a decrease in the incidence of foodborne  <i>E.coli </i> infections.
+        <b> Results </b> </br>
+The LAMP technique was shown to be more sensitive than conventional PCR techniques without the need for a heat lysis or centrifugation steps. The reaction generated fluorescent products after excitation with blue LEDs after 20 minutes. Our system achieves detection limits to add here without the need for laboratory equipment. The device is estimated to cost approximately $70 USD. This device offers a power, rapid method for pathogen detection for future point-of-care diagnostic applications.
 </p>
+<p class="section-content">
+[1] Borgatta, B.; Kmet-Lunaček, N.; Rello, J., E. coli O104:H4 outbreak and haemolytic–uraemic syndrome. <i>Medicina Intensiva (English Edition)</i> 2012, 36 (8), 576-583.</br>
+[2] Pacheco, A. R., Sperandio, V., Shiga toxin in enterohermorrhagic E. coli: regulation and novel anti-virulence strategies. Front. Cell. Infect. Microbiol. 2012, 2 (81), 1-12. </br>
+[3] New England BioLabs. Isothermal Amplification. Accessed October 19, 2017.</br>
+</p>
 </div>