PCR-GoldMag Lateral Flow Assay And Its Clinical Applications For Genotyping Of ApoE And PLD3 Polymorphisms

Single-nucleotide polymorphisms (SNPs) are regarded as biomarkers for assessing disease susceptibility and predicting the patients’ response to medication. Apolipoprotein E (ApoE) is a major cholesterol carrier that supports lipid transport and injury repair in the human brain. Pharmacogenomic studies indicate that ApoE polymorphism informs pre-symptomatic risk for a wide variety of diseases, increases risk of cardiovascular disease and neurodegenerative disorders, including Alzheimer’s disease (AD). The phospholipases (phospholipase Al, A2, B, C and D) that can specifically hydrolyze the intramolecular ester bond to regulate lipids metabolism. Two rare coding variants in PLD3 (p.V232M and p.A442A) significantly associated with AD risk have been identified in western populations, in 2014. And carriers of these variants have a twofold increased risk for AD. With the intensified aging trend of Chinese population and increasing needs for detection of chronic disease, screening the susceptibility genes is imminent in a large population. This not only provides information about disease susceptibility genes, but also prevents the disease and saves significant health care resources.Thus far, a number of methods have been established for SNP detection. Such methods include direct sequencing, real-time PCR, DNA microarrays, and so on. However, most of these techniques require expensive and sophisticated instruments that may not be available in laboratories with limited resources. Therefore, an easy-to-use, affordable and high throughput technique for the genotyping with high efficiency is urgently needed.With the rapid advancing of nanotechnology in life science, nano-materials have become a focus of interest in biomedical application. Nanoparticles associated lateral flow method for nucleic acid analysis is a simple and rapid assay technique, which enables visual detection of the target DNA and eliminates complicated steps including pipetting, incubation, washing, and data analysis. Combination of PCR with lateral flow assay (LFA) for the detection of SNP has being quickly adopted for diagnosis and genetic analysis.In the present study, we established a PCR-GoldMag nanoparticle-based lateral flow assay (LFA) platform that enables rapid visual detection of genetic polymorphisms without instrumentation for genotyping. And then this assay was also employed to determine the ApoE and PLD3 variants (V232M and A442A), respectively, for molecular etiology and for clinical prevention and treatment. Herein, the following four aspects are highlighted:1. We developed a PCR-GoldMag Lateral Flow Assay for SNP analysis. The assay could be implemented via two steps:(1) The multiple AS-PCR amplification of DNA sequence containing SNP sites. The wild-type specific primer had a 3’-end nucleotide complementary to the wild-type allelic variant and was labelled at the 5’-end with digoxin, whereas the mutant-type specific primer was complementary to the mutant allele and labelled at the 5’-end with the fluorescein. A common primer was also included in the PCR reaction mixture, which was carried biotin at the 5’-end. (2) Detection. To visually detect PCR product, a LFA based on GoldMag nanoparticle is employed. The product was detected on the LFA through dual immunoreactions (anti-FITC/digoxin antibody on the GoldMag nanoparticle and FITC/digoxin on the duplex, streptavidin on the LFS test zone and biotin on the duplex). The LFA is disposable and allows visual detection and confirmation of the genotyping products within 10 min without the need for specialized instruments.2. Detection of ApoE genotypes based on PCR-GoldMag Lateral Flow Assay. Design the primers for the detection of ApoE genotypes and screen the allele-specific primers. And the conditions of PCR, LFS preparation and detecting conditions were optimized systematically. The elaborately designed PCR-GoldMag Lateral Flow Assay was examined with various quantities of DNA samples with known ApoE genotypes. The limit of detection for human genomic DNA is as low as 10 ng. The specificity was also confirmed by different ApoE genetypes, and no false positive result was produced.3. A case-control study was designed to examine the association between ApoE genotype and susceptibility to complex diseases, including AD and Ischemic stroke (IS). The study subjects were 53 patients with AD,130 patients with IS, and 105 population controls. The ApoE genotypes were detected by PCR-GoldMag Lateral Flow Assay. And the acuracy of LFA results were reconfinned with DNA sequencing. The results showed that the detectable rate of the novel method is much higher than the sequencing method. About AD, there were significant differences in the genotype (P<0.01) and allele (P<0.01) frequencies of the ApoE polymorphism between AD patients and controls. The E4 allele was significantly associated with an increased risk of AD (P=0.000; OR=5.412,95%CI. 2.603-11.254). About ischemic stroke (IS), it was also found that the presence of E4 allele was higher in IS patients compared to controls (P=0.024); Moreover, a significantly elevated risk of IS was also seen in E4 carriers,2.19 times higher than that of E3 carriers (OR=2.19,95%CI.1.092-4.390). Our data provide preliminary evidence that ApoE E4 allele is a susceptibility gene for AD and IS in Chinese Han populations.4. Detection of PLD3 rare coding variants based on PCR-GoldMag Lateral Flow Assay, including p.V232M and p.A442A. These single base mutations were detected by the developed method and direct sequencing which is an existing gold standard method, the concordance rate was 100%. In order to examine whether these variants are genetic risk factors in patients with AD from mainland China, we detected V232M and A442A in Chinese Han population including 53 patients with AD and 105 population controls, using PCR-GoldMag Lateral Flow Assay. As a result, only one A442A variant was identified in all subjects. These findings suggest the two rare coding variants in PLD3 identified in western population might not play an important role in AD risk in mainland China. A larger sample size should be further confirmed in future studies.In this report, we present a PCR-GoldMag lateral flow assay for rapid and sensitive detection of SNP from nucleic acid samples, which has been the use of our patented Gold magnetic nanoparticles (GoldMag) instead of colloidal gold. Primer screening ensures specificity and GoldMag nanoparticles provide high sensitivity. Our clinical data have demonstrated that PCR-LFA has a high concordance rate but outperforms with higher detectable rate compared with sequencing results. Due to its superior sensitivity and specificity with low cost and easy operation and no need of expensive instrument and time-consuming step, this technology shows great promise for screening of important SNPs related to diseases or medical conditions. It lays a foundation for the research of the susceptibility genes screening and prevention and management of chronic disease.