Team:NYU Abu Dhabi/Description
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. After running the gels, it was noticed that higher concentrations (25l) gave better sensitivity, however, due to the limitations of reagents 12.5l instead of 25l was used. Testing with 12.5l showed it achieved limited detection of 106 cells/ml, which is the FTA minimum infections dose for healthy adult human. Therefore, even with a smaller amount of reagents the detection was sensitive enough to detect harmful dosage of the toxin.
[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.
