Development of a LAMP simulation and selection pipeline to predict primer success

Issue Date

1-2024

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.

Source or Periodical Title

Philippine Journal of Veterinary Medicine

ISSN

0031-7705

Volume

61

Issue

1

Page

26-38

Document Type

Article

Frequency

semi-annually

Physical Description

illustrations; tables, graphs

Language

English

En – AGROVOC descriptors

DNA SEQUENCES; NUCLEIC ACID AMPLIFICATION TECHNIQUES; PREDICTION THEORY; LAMPS

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