Development of an optimized RPA-PfAgo detection system for MTHFR C677T polymorphism genotyping

Lianghui Chen; Cheng Wei; Yaqun Liu; Meihua Liao; Jialin Wang; Jiaqi Chen; Peikui Yang; Liejun Li; Chengsong Xie; Min Lin; Zhenxia Zhang; Yuzhong Zheng

doi:10.1016/j.gene.2024.148544

Development of an optimized RPA-PfAgo detection system for MTHFR C677T polymorphism genotyping

Gene. 2024 May 9:922:148544. doi: 10.1016/j.gene.2024.148544. Online ahead of print.

Authors

Lianghui Chen¹, Cheng Wei¹, Yaqun Liu², Meihua Liao¹, Jialin Wang³, Jiaqi Chen³, Peikui Yang³, Liejun Li⁴, Chengsong Xie⁵, Min Lin⁶, Zhenxia Zhang³, Yuzhong Zheng⁷

Affiliations

¹ Reproductive Medicine, Guangxi Medical and Health Key Discipline Construction Project, The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise 533000, China; Industrial College of Biomedicine and Health Industry, Youjiang Medical University for Nationalities, Baise 533000, China.
² School of Life Sciences and Food Technology, Hanshan Normal University, Chaozhou 521041, China; Guangdong Hybribio Biotech Co., Ltd., Chaozhou 521041, China; Guangdong Taiantang Pharmaceutical Co., Ltd., Shantou 515000, China. Electronic address: 20200048@hstc.edu.cn.
³ School of Life Sciences and Food Technology, Hanshan Normal University, Chaozhou 521041, China.
⁴ Guangdong Hybribio Biotech Co., Ltd., Chaozhou 521041, China.
⁵ Guangdong Taiantang Pharmaceutical Co., Ltd., Shantou 515000, China.
⁶ Industrial College of Biomedicine and Health Industry, Youjiang Medical University for Nationalities, Baise 533000, China; School of Life Sciences and Food Technology, Hanshan Normal University, Chaozhou 521041, China.
⁷ Industrial College of Biomedicine and Health Industry, Youjiang Medical University for Nationalities, Baise 533000, China; School of Life Sciences and Food Technology, Hanshan Normal University, Chaozhou 521041, China. Electronic address: zhengyuzhon@hstc.edu.cn.

PMID: 38734187
DOI: 10.1016/j.gene.2024.148544

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.

Keywords: Genotyping; MTHFR C677T polymorphism; Point-of-care testing; Pyrococcus furiosus Argonaute (PfAgo); Recombinase polymerase amplification (RPA).