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Towards detection of methicillin resistance using LAMP-CRISPR diagnostics

Over a century, we have gone from the breakthrough discovery of antibiotics to witnessing an alarming progressive decline of their effectiveness, as antimicrobial resistance (AMR) continues to rise. Some strains of bacteria have become “superbugs”, developing resistance to multiple forms of treatment. Methicillin-resistant Staphylococcus aureus (MRSA) affects all countries, however sub-Saharan Africa is bearing a particularly heavy burden. AMR directly caused 1.27 million deaths globally in 2019 and contributed to an additional 4.95 million deaths. This makes it a bigger killer than HIV or malaria. Predicted mortality annually by 2050 could be as high as 10 million. In the case of sepsis, rapid diagnostic testing is of paramount importance. To discriminate at POC between MRSA and MSSA (methicillin susceptible), simultaneous testing of staph specific genes (nuc, femA) and the methicillin resistance variants (mecA, mecB, mecC) is required. Such diagnostic testing will be feasible using DNAiTECHs low-cost method PLACID: paper-based LAMP-CRISPR integrated diagnostics.

Loop-mediated isothermal amplification (LAMP) is a rapid method for molecular amplification; however, it is prone to false positive detection when using non-specific indicators such as dsDNA binding dyes. Using CRISPR combined with LAMP, we confer high specificity and sensitivity with intense visual fluorescence coming up within a minute. The effectiveness of combining LAMP with CRISPR is demonstrated below with amplification of mecA.

Left LAMP reaction alone showing mecA positive sample and a delayed negative control (minus mecA DNA) reaction which tests positive. This is an example of a LAMP reaction generating false positives.

Middle When the same LAMP products were tested for Cas12a activity in tubes, only the mecA positive reaction generated fluorescence, the signal reached full intensity within 3 minutes. The PAM site which targets the amplicon ensures only real positive reactions are detected. The insert shows the smartphone camera image of the tube fluorescence at the end of the reaction on the DNAiTECH Gen2 instrument. 1 negative control, 2 positive control.

Right: Testing the LAMP-CRISPR on DNAiTECH’s PLACID chips. Only the +mecA was positive, and the visibly intense fluorescence signal was detected on the chip within a minute. DNAiTECH's PLACID technology, due for release in 2024 will be a powerful tool for delivering low cost WHO ASSURED diagnostics.

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