Methodology

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A thorough literature research about microfluidics, antibiotic resistance, loop-mediated isothermal amplification (LAMP) molecular biology technique, labeled primers, and electrical materials was conducted. Particularly, the pressing need detect bacteria with resistance to beta-lactam antibiotics in places such as hospitals or residual water was identified. Thereafter, the CTX-M-15 allele was selected as a molecular marker of interest that could be used to identify a wide collection of resistant bacteria via LAMP-mediated amplification. This technique is able to produce large amounts of the selected sequence in isothermal conditions, reducing costs and complexity, gaining advantage over PCR (Foo, et al., 2020; Salinas & Little, 2012).

To make this test even more efficient, it was proposed that the reaction would be done inside a paper-based portable microfluidic device since its physical properties would permit an easy compound separation and transportation. Meanwhile, the constant temperature needed to carry out the amplification was determined to be provided by a recyclable battery-powered resistor, which would allow its use on-field. Subsequently, the signaling method that the device would produce if the amplification of the CTX-M-15 gene was carried out was proposed. For this, the method of TaqMan hairpin primers was chosen. This labeled oligonucleotide would emit a green fluorescence thanks to the detachment of the 6-FAM fluorophore in case of annealing to the sequence of interest. Finally, the use of a UV light camera was proposed that would allow fluorescence to be visualized in the case of a positive test, or the lack of it in the case of a negative test.

All the technical details such as the design programs, material specifications, reagents, primer designs, expected molecular mechanisms, and device accommodation were determined based on scientific literature to finally present a complete protocol that spans from the design of the device, experimental steps, and  result interpretation.