Rapid, Highly Sensitive, and Label-Free Pathogen Assay System Using a Solid-Phase Self-Interference Recombinase Polymerase Amplification Chip and Hyperspectral Interferometry

• Published in: Analytical chemistry (Washington) 2022-02, Vol.94 (6), p.2926-2933
• Main Authors: Jin, Xiangyu, Fu, Rongxin, Du, Wenli, Shan, Xiaohui, Mao, Zeyin, Deng, Anni, Lin, Xue, Su, Ya, Yang, Han, Lv, Wenqi, Zhong, Hao, Huang, Guoliang
• Format: Article
• Language: English
• Subjects: Amplification; Assaying; Chemistry; Deoxyribonucleic acid; Dihydrofolate reductase; Dimers; DNA; Fluorescence; Gene polymorphism; Genotyping; Identification methods; Infectious diseases; Interference; Interferometry; Nucleic Acid Amplification Techniques - methods; Nucleotides; Nucleotidyltransferases; Pathogens; Plasmodium falciparum; Plasmodium falciparum - genetics; Polymorphism; Recombinase; Recombinases; rRNA 18S; Sensitivity; Sensitivity and Specificity; Single-nucleotide polymorphism; Solid phases
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.1c04858

Recombinase polymerase amplification (RPA) is a useful pathogen identification method. Several label-free detection methods for RPA amplicons have been developed in recent years. However, these methods still lack sensitivity, specificity, efficiency, or simplicity. In this study, we propose a rapid, highly sensitive, and label-free pathogen assay system based on a solid-phase self-interference RPA chip (SiSA-chip) and hyperspectral interferometry. The SiSA-chips amplify and capture RPA amplicons on the chips, rather than irrelevant amplicons such as primer dimers, and the SiSA-chips are then analysed by hyperspectral interferometry. Optical length increases of SiSA-chips are used to demonstrate RPA detection results, with a limit of detection of 1.90 nm. This assay system can detect as few as six copies of the target 18S rRNA gene of Plasmodium falciparum within 20 min, with a good linear relationship between the detection results and the concentration of target genes (R 2 = 0.9903). Single nucleotide polymorphism (SNP) genotyping of the dhfr gene of Plasmodium falciparum is also possible using the SiSA-chip, with as little as 1% of mutant gene distinguished from wild-type loci (m/wt). This system offers a high-efficiency (20 min), high-sensitivity (6 copies/reaction), high-specificity (1% m/wt), and low-cost (∼1/50 of fluorescence assays for RPA) diagnosis method for pathogen DNA identification. Therefore, this system is promising for fast identification of pathogens to help diagnose infectious diseases, including SNP genotyping.