Development of an optimized RPA-PfAgo detection system for MTHFR C677T polymorphism genotyping.
Gene
; 922: 148544, 2024 Sep 05.
Article
in En
| MEDLINE
| ID: mdl-38734187
ABSTRACT
This study introduces an efficient RPA-PfAgo detection system for the MTHFR C677T polymorphism, proposing a potential strategy to simplify the genotyping process. By optimizing recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) nucleases, we achieved DNA amplification at a constant temperature. The assay was fine-tuned through meticulous primer and guide DNA selection, with optimal conditions established at 2.0 µL of MgAc, a reaction temperature of 42 °C, and a 10-minute reaction time for RPA. Further optimization of the PfAgo cleavage assay revealed the ideal concentrations of MnCl2, guide DNA, molecular beacon probes, the PfAgo enzyme, and the RPA product to maximize sensitivity and specificity. Clinical validation of 20 samples showed 100% concordance with Sanger sequencing, confirming the method's precision. The RPA-PfAgo system is a promising tool for on-site genotyping, with broad applications in personalized medicine and disease prevention.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Methylenetetrahydrofolate Reductase (NADPH2)
/
Genotyping Techniques
Limits:
Humans
Language:
En
Journal:
Gene
Year:
2024
Document type:
Article
Affiliation country:
Suiza