A Rapid and Specific Genotyping Platform for Plasmodium falciparum Chloroquine Resistance via Allele-Specific PCR with a Lateral Flow Assay.

Abstract

ABSTRACTSingle-nucleotide polymorphisms and genotyping related to genetic detection are several of the focuses of contemporary biotechnology development. Traditional methods are complex, take a long time, and rely on expensive instruments. Therefore, there is an urgent need for a rapid, simple, and accurate method convenient for use in resource-poor areas. Thus, a platform based on allele-specific PCR (AS-PCR) combined with a lateral flow assay (LFA) was developed, optimized, and used to detect the genotype of the Plasmodium falciparum chloroquine transporter gene (pfcrt). Subsequently, the system was assessed by clinical isolates and compared with Sanger sequencing. The sensitivity and specificity of the AS-PCR-LFA platform were 95.83% (115/120) and 100% (120/120), respectively, based on the clinical isolates. The detection limit of plasmid DNA was approximately 3.38 × 105 copies/μL. In addition, 100 parasites/μL were used for the dried filter blood spots from clinical isolates. The established rapid genotyping technique is not limited to antimalarial drug resistance genes but can also be applied to genetic diseases and other infectious diseases. Thus, it has realized the leap and transformation from scientific research theory to practical application and actively responds to the point-of-care testing policy.IMPORTANCE Accurate recognition of the mutation and genotype of genes are essential for the treatment of infectious diseases and genetic diseases. Based on the techniques of allele-specific PCR (AS-PCR) and a lateral flow assay (LFA), a rapid and useful platform for mutation detection was developed and assessed with clinical samples. It offers a powerful tool to identify antimalarial drug resistance and can support malaria control and elimination globally.