Multiple Single-Nucleotide Polymorphism Detection for Antimalarial Pyrimethamine Resistance via Allele-Specific PCR Coupled with Gold Nanoparticle-Based Lateral Flow Biosensor

Abstract

Molecular genotyping holds tremendous potential to detect antimalarial drug resistance (ADR) related to single nucleotide polymorphisms (SNPs). However, it relies on the use of complicated procedures and expensive instruments. Thus, rapid point-of-care testing (POCT) molecular tools are urgently needed for field survey and clinical use. Herein, a POCT platform consisting of multiple-allele-specific PCR (AS-PCR) and a gold nanoparticle (AuNP)-based lateral flow biosensor was designed and developed for SNP detection of the Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene related to pyrimethamine resistance. The multiple-AS-PCR utilized 3' terminal artificial antepenultimate mismatch and double phosphorothioate-modified allele-specific primers. The duplex PCR amplicons with 5' terminal labeled with biotin and digoxin are recognized by streptavidin (SA)-AuNPs on the conjugate pad and then captured by anti-digoxin antibody through immunoreactions on the test line to produce a golden red line for detection. The system was applied to analyze SNPs in Pfdhfr N51I, C59R, and S108N of 98 clinical isolates from uncomplicated P. falciparum malaria patients. Compared with the results from nested PCR followed by Sanger DNA sequencing, the sensitivity was 97.96% (96/98) for N51I, C59R, and S108N. For specificity, the values were 100% (98/98), 95.92% (94/98), and 100% (98/98) for N51I, C59R, and S108N, respectively. The limit of detection is approximately 200 fg/μl for plasmid DNA as the template and 100 parasites/μl for blood filter paper. The established platform not only offers a powerful tool for molecular surveillance of ADR but also is easily extended to interrelated SNP profiles for infectious diseases and genetic diseases.

Keywords: allele-specific PCR; antimalarial drug resistance; lateral flow biosensor; point-of-care testing (POCT); single nucleotide polymorphisms (SNPs).

FIG 1

Schematic illustration of the allele-specific PCR (AS-PCR) combined with lateral flow assay (LFA) system. (A) Principle of AS-PCR for every single nuclear polymorphism. (B) Sample preparation and target amplification. (C) Structure of labeled lateral flow device. (D) Results were analyzed based on the signal read-out by visual interpretation in LFA, agarose gel electrophoresis, and DNA sequencing.

FIG 2

Sensitivity and specificity evaluation of selected primers for pfdhfr gene with AS-PCR-LFA system through visualized interpretation. The final concentrations of serial dilutions of plasmid pEASY-T1/Pfdhfr in the PCR system (20 μl) were from 200 pg/μl to 2 fg/μl. W and M on the sample lanes represent the wild-type and mutation primer, respectively; C and T represent the control line and test line, respectively.

FIG 3

Genotyping result (take partial samples as an example) provided by the AS-PCR-LFA system through visualized interpretation (A), agarose gel electrophoresis (B), and DNA sequencing (reverse sequencing) (C).