Apolipoprotein E genotyping using PCR-GoldMag lateral flow assay and its clinical applications

Abstract

A polymerase chain reaction-gold magnetic nanoparticles lateral flow assay (PCR-GoldMag LFA) has been developed via integrating multiplex amplification refractory mutation system PCR (multi‑ARMS‑PCR) with GoldMag‑based LFA for the visual detection of single‑nucleotide polymorphisms (SNPs). This assay was applied to genotype Apolipoprotein E (ApoE). ApoE genotyping is important due to the predictive value for the development of coronary artery disease and Alzheimer's disease. The method requires two steps: i) Simultaneous amplifications of the two polymorphic codons (ApoE 158 and 112), performed in separated reactions using multi‑ARMS‑PCR; and ii) detection of the wild‑type and mutant PCR products via dual immunoreactions, which can be performed in ~5 min. Within two LFAs, anti‑digoxin antibody‑conjugated GoldMag probes bind digoxin‑labeled wild‑type PCR products, and anti‑fluorescein isothiocyanate (FITC) antibody-conjugated GoldMag probes bind FITC‑labeled mutant PCR products. All PCR products are biotin labeled and are detected by streptavidin-coated regions on the LFA strip, resulting in a red color. The current approach is capable of detecting the SNPs of ApoE in ~1.5 h, with a broad detection range from 10‑1,000 ng of genomic DNA. Thus, the present protocol may facilitate simple, fast and cost‑effective screening for important SNPs, as demonstrated by the evaluation of the prevalence of ApoE variants in a Han Chinese cohort.