Team:NYU Abu Dhabi/Description

Why is STEC a major concern?

Globally, 1 in every 10 people is affected by foodborne diseases each year. According to 2010 data, 600 million were affected by such diseases and 420,000 died as a result. Countries of low- and middle-income are the most affected due to unsafe practices of food production and storage. Enteropathogenic E. coli is one of the main causes of death due to foodborne disease.
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

What is the mode of action?

Shiga toxin is an exotoxin comprised of a toxic A subunit and a cell-binding B subunit. The B subunit binds to a globotriaosylceramide Gb3 receptor. This receptor is expressed on the surface of cells. Binding of the B subunit to the Gb3 receptor causes the shiga toxin to be endocytosed into the cell. Once inside, shiga toxin inhibits protein synthesis and induces apoptosis.⁴

What are the symptoms of STEC infections?

Shiga toxin-producing E. coli (STEC) is one of the most prevalent enteropathogenic E. coli strains. In the US alone, this strain causes 70,000 infections a year. The most common symptom of such infections is diarrhea. Even more worrying is the fact that 6-9% of those cases derived in life-threatening hemolytic uremic syndrome (HUS). 5-7% of the individuals affected by HUS did not survive.⁵

What are we doing?

Due to the geographical location of NYUAD, which is in close proximity to countries where food safety is a daily, life-threatening concern, we were inspired to design and deliver an efficient solution to reduce the severity of this issue. We have produced a rapid, affordable and portable device that allows for the detection of Shiga toxin-producing Escherichia coli using loop-mediated isothermal amplification (LAMP).

How are we doing it?

LAMP 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. This technique was shown to be more specific than colony PCR without the need for heat lysis or centrifugation steps. The selectivity of our system was tested using the rfbE gene, a non-toxic coding sequence required for O157-antigen synthesis. The presence of the O-antigen confers resistance against phagocyte killing.

The samples are contained in a PDMS chip that contains wells for positive control, negative control, and three samples. Heating is supplied by a Peltier Modular Cooling. The reaction temperature of 65˚C was achieved using the Peltier system supplied with a 6V, 1.5A external power supply. The reaction was visualized under UV and blue light. It is envisioned that a smartphone will suffice for capturing the output of the reaction.⁵

Why E.coLAMP?

After surveying potential device users hailing from various parts of the world, we discovered that there was a shortage of quick and reliable methods to detect pathogenic E. coli. Most available methods are time- and resource-consuming, as they require the expertise of laboratory-trained technicians. More than 70% of the survey participants indicated the need of a method to detect pathogenic E. coli in less than 30 minutes. In response to this, we tailored our device to complete a diagnostic reaction within 20 minutes from the the beginning of sample submission to the attainment of the fluorescence signal.

There is a lack of stringent government regulations regarding food safety in many parts of the world. As most food vendor stalls are not subjected to government procedures and regulations about food safety, it is our priority to protect potentials consumer from the possibility of contracting harmful diseases or toxins. Our device aims to address this issue by allowing consumers to own a cost-effective, easy-to-use handheld device that can assess the safety of a product that they wish to buy. The results of our device can be easily recorded, exported to an Excel file, and uploaded to a public database, which can be accessed by anyone.

STEC are often detected in undercooked meat, raw milk, and raw produce. In the food industry, it is important to prioritize food hygiene and prevent the sale of contaminated food to potential buyers. Our device is well-suited as a surveillance method for food vendors, ranging from small-scale individual sellers at traditional markets to large-scale supermarkets selling meat and produce as well as cooked food. Its rapid and easy method allows users from all kinds of background to interpret its results and discard the contaminated food accordingly. This device offers a simple solution to easily prevent and stem outbreaks.

As a proof of concept, we have acquired specific amplification of the target gene rfbE specific to STEC O157:H7 by using four LAMP primers rather than the usual pair of primers in PCR. In the future, the LAMP primers can be designed to target the shiga toxin stx1 and stx2 gene as well, which we deliberately chose to exclude in this project due to our consideration of the iGEM safety standards. The device is not just limited to detecting the presence of shiga toxin. In this project, we also demonstrated our device’s ability to detect malaria using primers specifically targeted for Plasmodium falciparum DNA. By simply changing the LAMP primers, we will be able to detect all known pathogens and viruses. The versatility of the technique and the design of the device allows for a broad range of diagnostic uses.

By using a 12.5µL reaction volume of primers and LAMP Master Mix, we have shown that our lower limit of detection is 10⁶ cells/mL. This is the FDA minimum infectious dose for healthy adult humans. Improved limit of detection can also be attained by using a 25µL reaction volume. Additionally, we only needed a swab of the food sample to attain such sensitivity.

Another major concern for most diagnostic methods is the high cost per use. Problems arise when a device is accurate but expensive to use or inversely cost-effective but not reliable. We aim to solve both problems by bringing the price of the device as low as possible while still maintaining high sensitivity and accuracy. About 44% of the survey participants indicated that they would pay between USD 40-150 for this device. By optimising the materials and size of the device, we have currently reduced the price to as low as 70 USD per chip.

iGEM NYU Abu Dhabi 2016 Project

Award: Silver medal

In many developing countries, people depend on reasonably priced and conveniently available street food. However, lack of action taken by governments to regulate street food vendors has led to the prevalence of severe street food-related illnesses. One of the primary microbial contaminants in street food is E. coli O157:H7, which acts by secreting the Shiga-like toxin (SLT). Currently, there is no detection method for SLT outside of a lab setting, thus putting the consumers of foods at risk. Our project aims to develop a device that would be used by street vendors and restaurant owners to verify the safety of their products. Through our device, we exploit the binding of Gb3 to subunit B of the Shiga toxin, and compare the migration pattern of the bound Gb3-subunit B complex to a non bound subunit B. A shift in the migration pattern on a PAGE gel will occur when Gb3 is bound, indicating the presence of the toxin in the food sample. If no shift occurs in the SLT migration pattern, this implies the absence of the toxin within the sample, and reflects the safety status of the food.⁶

Our improvements

Shiga toxin is an exotoxin that consists of two subunits. Subunit B binds to Gb3 receptor expressed in the surface of target cells and permits the entry of subunit A, which inhibits protein synthesis.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 that of free subunit B using a PAGE gel. Their device was estimated to take 45 minutes and their prototyping process ran into several issues that negatively impacted the specificity, affordability, and accessibility of the product. However, based on feedback from food vendors, we discovered that very few individuals would wait so long for to obtain results. Since they also had issues with expressing their protein of interest, we decided to target a DNA sequence for our device.

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. 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.
4. 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.
5. "Virulence factors of entertoxigenic E. coli" from Epidemiology of Infectious Diseases. Available at: http://ocw.jhsph.edu. Copyright © Johns Hopkins Bloomberg School of Public Health. Creative Commons BY-NC-SA.
6. Team NYU Abu Dhabi - 2016 (2017) 2016igemorg. Available at: https://2016.igem.org/Team:NYU-AD [Accessed October 28, 2017].