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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.
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.
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. 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 visualised using a handheld fluorescence microscope that can easily be substituted with a smartphone.
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.
[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.
