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

Why is STEC a major concern?

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.

Mode of Action

Symptoms of STEC infections

What are we doing?

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 sample wells in a PDMS chip, and sample DNA from broth inoculated with rfbE+ DH5alpha was introduced into the system. The 65 ºC temperature was achieved using a Peltier Modular Cooling system with a 6 Volts, 1.5 Amperes external power supply adapter. The reaction was visualized under UV and blue light. It is envisioned that a smartphone will suffice for capturing the output of the reaction.

How are we doing it?

Why E.coLAMP?

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.

Our changes

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.

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 $50 USD. This device offers a power, rapid method for pathogen detection for future point-of-care diagnostic applications. After running the gels, it was noticed that larger volume (25μl) gave better sensitivity, however, due to low availability of reagents 12.5μl instead of 25μl was used. Testing with 12.5μl showed it achieved limited detection of 10 ⁶ cells/ml, which is the FDA minimum infections dose for healthy adult human. Therefore, even with a smaller amount of reagents the detection was sensitive enough to detect harmful dose of the toxin.