Abstract
Background: The loop-mediated isothermal amplification (LAMP) technique is widely used in research and diagnostics, yet its underlying mechanisms remain poorly understood. Methods: The experiment utilized 31 synthetic gene templates (500-560 base pairs) from the 16S rRNA V8 region of bacterial phyla, paired with specific primer sets (F3, FIP, BIP, B3) individually designed for template amplification. Wet lab amplification experiments were conducted at 68°C for 120 minutes using 105 template molecules per reaction, and were validated using 1.2% agarose gel electrophoresis. Dry lab amplification was predicted using our model data and analysis for LAMP-process pipeline. Predictions centered on assessing dumbbell and ladder formation near the 3′ end of primer sequences. SeqKit software facilitated 1512 condition explorations, with evaluations of primer sensitivity, specificity, accuracy, and F1 values. Results: Predictive LAMP amplification experiment yielded mixed results, suggesting some primer issues. Six primer sets displayed consistent amplification across templates. We identified two models with optimal conditions based on F1 scores exceeding 0.64, highlighting the balance between sensitivity and specificity. Parameters like oligomer length and mismatch tolerance have significantly influenced predictive accuracy. Conclusion: This novel LAMP prediction model can enhance primer design and promote more LAMP applications.
Keywords
Isothermal gene amplification, predictive LAMP, primer and template sequences, synthetic gene
Philippine Journal of Veterinary Medicine 