Innovative PNA-LB mediated allele-specific LAMP for KRAS mutation profiling on a compact lab-on-a-disc device.

Tailored healthcare, an approach focused on individual patients, requires integrating emerging interdisciplinary technologies to develop accurate and user-friendly diagnostic tools. KRAS mutations, prevalent in various common cancers, are crucial determinants in selecting patients for novel KRAS inhibitor therapies. This study presents a novel state-of-the-art Lab-on-a-Disc system utilizing peptide nucleic acids-loop backward (PNA-LB) mediated allele-specific loop-mediated isothermal amplification (LAMP) for detecting the frequent G12D KRAS mutation, signifying its superiority over alternative mutation detection approaches. The designed Lab-on-a-Disc system demonstrated exceptional preclinical and technical precision, accuracy, and versatility. By applying varying cutoff values to PNA- LB LAMP reactions, the assay's sensitivity and specificity were increased by 80 % and 90 %, respectively. The device's key advantages include a robust microfluidic Lab-on-a-Disc design, precise rotary control, and a cutting-edge induction heating module. These features enable multiplexing of LAMP reactions with high reproducibility and repeatability, with CV% values less than 3.5 % and 5.5 %, respectively. The device offers several methods for accurate endpoint result detection, including naked-eye observation, RGB image analysis using Python code, and time of fluorescence (T _f ) values. Preclinical specificity and sensitivity, assessed using different cutoffs for Eva-Green fluorescence T _f values and pH-sensitive dyes, demonstrated comparable performance to the best standard methods. Overall, this study represents a significant step towards tailoring treatment strategies for cancer patients through precise and efficient mutation detection technologies.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Siamak Salami reports financial support was provided by Iran National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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