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Visual Detection of Multiple Nucleic Acids in a Capillary Array

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Introducing Triplex Forming Oligonucleotide into Loop-Mediated Isothermal Amplification for Developing a Lateral Flow

Wei Chang¹, Po-Hao Chou^2,3, Cai-Tong Wu¹

¹Master and PhD Program in Biotechnology Industry, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.

|May 24, 2024

Summary

This study introduces a novel triplex-forming oligonucleotide (TFO) probe integrated into loop-mediated isothermal amplification (LAMP) to improve infectious disease detection specificity. The TFO-LAMP method enhances sequence specificity for detecting Group B Streptococcus (GBS).

Keywords:

Group B Streptococcus (GBS)Streptococcus agalactiae lateral flow assay (LFA)loop-mediated isothermal amplification (LAMP)triplex-forming oligonucleotide (TFO)

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Area of Science:

Molecular Biology
Biotechnology
Infectious Disease Diagnostics

Background:

Loop-mediated isothermal amplification (LAMP) is a sensitive method for infectious disease detection.
Conventional LAMP assays often lack precise sequence specificity.
Enhancing specificity is crucial for reliable molecular diagnostics.

Purpose of the Study:

To develop a more sequence-specific LAMP assay using triplex-forming oligonucleotide (TFO) probes.
To apply this TFO-integrated LAMP (TFO-LAMP) for the detection of Group B Streptococcus (GBS).
To create a nucleic acid testing biosensor based on the TFO-LAMP technology.

Main Methods:

Integrated a TFO probe into the LAMP process to target a specific DNA sequence (TFO targeting sequence, TTS).
Developed a screening method to identify high-affinity TFO sequences for LAMP integration.
Designed a TFO-LAMP assay utilizing a FAM-labeled TFO for detection via lateral flow biosensors.

Main Results:

The TFO-LAMP assay demonstrated high sequence specificity for GBS detection.
Sensitivity reached 85% at 300 copies and 100% at 30,000 copies of GBS.
The TFO-LAMP assay was successfully adapted for use with lateral flow test strips.

Conclusions:

Successfully developed a TFO-LAMP technology for enhanced sequence specificity in nucleic acid detection.
The TFO-LAMP assay is applicable for developing sensitive and specific biosensors, including lateral flow devices.
This technology holds potential for improved infectious disease diagnostics.