Multiple SNPs Detection Based on Lateral Flow Assay for Phenylketonuria Diagnostic.

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Genetics

Background:

• Current single nucleotide polymorphism (SNP) detection methods are often complex, costly, and time-consuming, hindering rapid clinical application, especially for multiple SNPs associated with genetic diseases.
• The need for efficient, accessible diagnostic tools for genetic disorders is critical for timely intervention and management.

Purpose of the Study:

• To develop and validate a sensitive, low-cost, and user-friendly point-of-care testing (POCT) system for simultaneous detection of multiple SNPs.
• To evaluate the performance of the developed system for genotyping pathogenic SNPs in the phenylalanine hydroxylase (PAH) gene, the cause of phenylketonuria.

Main Methods:

• Development of an Amplification Refractory Mutation System (ARMS) combined with Gold Magnetic Nanoparticles (GMNPs) and Lateral Flow Assay (LFA), termed the ARMS-LFA system.
• Utilized uniform reaction conditions for simultaneous multiplex SNP detection, with results interpretable via a magnetic reader or visual inspection.
• Validated the system by genotyping seven pathogenic SNPs in the PAH gene and assessing its DNA quantification capabilities.

Main Results:

• The ARMS-LFA system achieved high sensitivity (0.04 pg/μL detection limit for plasmid DNA) and specificity.
• Demonstrated a linear dynamic range for DNA quantification from 0.02 to 2 pg/μL of plasmid DNA.
• Clinical trials involving 23 families (69 individuals) showed a high concordance rate of 97.8% compared to DNA sequencing for PAH gene SNPs.

Conclusions:

• The developed ARMS-LFA system is a sensitive, cost-effective, and easy-to-use POCT method for simultaneous detection of multiple SNPs.
• This technology holds significant promise for the rapid screening and diagnosis of genetic diseases caused by multiple SNPs in clinical settings.
• The system's high accuracy and applicability in clinical trials underscore its potential as a valuable diagnostic tool.