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.

Sensitive genotyping of mutations in the EGFR gene from NSCLC patients using PCR-GoldMag lateral flow device

“…One novelty of this method is that the PCR-GoldMag LFA using our patented GoldMag as a carrier for the first time detected small deletions, especially in tumor. Our method has been successfully applied to detect some single nucleotide polymorphisms, such as C677T on MTHFR and ApoE genotyping 22 . Herein, we also detected the small insertions/deletions in EGFR gene by our method.…”

“…GoldMag lateral flow device combined with ARMS-PCR was set up as a simple and rapid genotyping method for methylenetetrahydrofolate reductase ( MTHFR ) C677T 21 . The new PCR-GoldMag lateral flow device for SNP detection, including MTHFR C677T and Apolipoprotein E polymorphisms, has been validated by sequencing for more than 2,000 genomic DNAs in 6 hospitals in China, and showed a high specificity and sensitivity 21 , 22 . This method allows for rare signals to be detected with greater sensitivity, tends to be faster and cheaper, and hence can be used as “targeted method” for genotyping of EGFR gene.…”

Apolipoprotein E Genotyping Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism with General-Purpose Agarose and Lithium Borate Buffer

“…APOE 유전형 검사를 위한 DNA을 기반으로 한 기법에는 염기 서열분석법 [6], polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) [7], single-strand conformation polymorphism [8], allele-specific PCR [9,10], fluorescence resonance energy transfer-based melting curve analysis [11], real-time Taqman PCR [12], real-time SYBR Green PCR [13], high-resolution melting curve analysis [14], denaturing high-performance liquid chromatography [15], SNaP-shot mini-sequencing [16], MALDI-TOP mass spectrometry [17], PCR-GoldMag lateral flow assay [18] Ladder (Takara)를 이용하였다.…”

Enhanced Stability of Gold Magnetic Nanoparticles with Poly(4-styrenesulfonic acid-co-maleic acid): Tailored Optical Properties for Protein Detection

“…Customizing GoldMag for detecting target molecules based on coating with, polymers has a high utility. It has been reported that the dispersity of GoldMag can improved through surface modification with various macromolecular organic compounds, such as 11-mercaptoundecanoic acid (MUA) [ 7 ], polystyrenesulfonate (PSS) [ 8 ], and polyethylenimine (PEI) [ 9 ]. MUA was brought on the surface of GoldMag using the ligand-exchange strategy.…”