Detection of Antimalarial Resistance-Associated Mutations in Plasmodium falciparum via a Platform of Allele-Specific PCR Combined with a Gold Nanoparticle-Based Lateral Flow Assay.

Abstract

Since single nucleotide polymorphisms (SNPs) have attracted attention, there have been many explorations and improvements in screening and detection methods for SNPs. Traditional methods are complex and time-consuming and rely on expensive instruments. Therefore, there is an urgent need for a low-cost, simple, and accurate method that is convenient for use in resource-poor areas. Thus, a platform based on allele-specific PCR (AS-PCR) and a gold nanoparticle-based lateral flow assay (LFA) was developed, optimized, and used to detect the SNPs of the drug resistance gene pfmdr1. Subsequently, the system was assessed on clinical isolates and compared with nested PCR followed by Sanger sequencing. The sensitivity and specificity of the AS-PCR-LFA platform were up to 99.43% and 100%, respectively, based on the clinical isolates. The limit of detection is approximately 150 fg/μL for plasmid DNA as the template and 50 parasites/μL for dried filter blood spots from clinical isolates. The established and optimized AS-PCR-LFA system is more adaptable and rapidly translated to SNP analysis of other drug resistance genes and genetic diseases. In addition, while actively responding to the point-of-care testing policy, it also contributes to the Global Malaria Eradication Program. IMPORTANCE Rapid detection of single nucleotide polymorphisms (SNPs) is essential for malaria treatment. Based on the techniques of allele-specific PCR (AS-PCR) and lateral flow assay (LFA), an accurate and powerful platform for SNP detection of pfmdr1 was developed and evaluated with plasmid and clinical isolates. It offers a useful tool to identify antimalarial drug resistance and can support the effort to eliminate malaria globally.