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

• Published in: Talanta (Oxford) 2024-08, Vol.276, p.126224, Article 126224
• Main Authors: Mirlohi, Maryam Sadat, Pishbin, Esmail, Dezhkam, Rasool, Kiani, Mohammad Javad, Shamloo, Amir, Salami, Siamak
• Format: Article
• Language: English
• Subjects: Allele-specific LAMP; Alleles; Humans; KRAS mutation; Lab-On-A-Chip Devices; Lab-on-Disc; Molecular diagnosis; Molecular Diagnostic Techniques - methods; Mutation; Nucleic Acid Amplification Techniques - methods; Peptide nucleic acids; Peptide Nucleic Acids - chemistry; Peptide Nucleic Acids - genetics; Proto-Oncogene Proteins p21(ras) - genetics
• ISSN: 0039-9140; 1873-3573; 1873-3573
• DOI: 10.1016/j.talanta.2024.126224

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 (Tf) values. Preclinical specificity and sensitivity, assessed using different cutoffs for Eva-Green fluorescence Tf 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. [Display omitted] •New Lab-on-a-Disc system uses PNA/LNA in LAMP for precise, accurate detection of frequent G12D KRAS mutation.•Increased sensitivity (80%) and specificity (90%) via varied cut-off values for LNA-PNA LAMP reactions.•Multiple endpoint result detection methods: naked-eye observation, RGB image analysis, and fluorescence Tf values.•Crafting a reliable microfluidic disc with rotary control and advanced induction heating ensures high reproducibility (CV% < 3.5% and 5.5%).•Suitable for point-of-care diagnostics and compact lab devices, detecting multiple mutations, personalized medicine.