Optimization of the Real-Time Loop-Mediated Isothermal Amplification (LAMP) Technique for Detecting the Fat Mass and Obesity-Associated (FTO) Gene
Keywords:
LAMP, FTO Gene, PrimersAbstract
The loop-mediated isothermal amplification (LAMP) technique enables rapid, highly sensitive, and specific nucleic acid amplification. In the field of genetic detection and amplification, polymerase chain reaction (PCR) has long been considered as a gold standard, though it has several limitations. This study aimed to optimize real-time LAMP (RT-LAMP) method for detecting the fat mass and obesity-associated (FTO) gene using blood samples. Six primers were used in the synthesis of the deoxyribonucleic acid (DNA) sequence by DNA polymerase, facilitating auto-cycling strand displacement. The primers included outer and inner primers, specifically F3 and B3, as well as forward inner primer (FIP that consists of F1c and F2) and backward inner primer (BIP that consists of B1c and B2). These primers were designed using Primer Explorer V5.0, and their sequences were checked for specificity using the BLAST program. A total of 2.5 mL of whole peripheral blood was extracted for DNA, which was then assessed for concentration, purity, and integrity. Twelve sets of primer concentrations were tested, and the most appropriate and optimal concentrations for amplifying and detecting the FTO gene were found to be 1.6 for FIP and BIP, and 0.8 for F3 and B3. The detection threshold value (Df) and threshold time (Tt) were 0.178 and 34:18 minutes, respectively, at a reaction temperature of 65°C. Validation of RT-LAMP optimization was performed using 2.0% agarose gel electrophoresis, which demonstrated as ladder-like pattern recognition, indicating the effectiveness of the RT-LAMP technique. The results showed that real-time LAMP can detect the FTO gene in facilitating rapid diagnosis. Therefore, real-time LAMP is a promising method that can be quantitatively studied to improve PCR performance in amplifying target DNA sequences.
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