Open AccessMultiple Single-Nucleotide Polymorphism Detection for Antimalarial Pyrimethamine Resistance via Allele-Specific PCR Coupled with Gold Nanoparticle-Based Lateral Flow Biosensor
Author(s) -
Tingting Jiang,
Yan Huang,
Weijia Cheng,
Yifei Sun,
Wei Wei,
Kai Wu,
Chen Shen,
Xiaolong Fu,
Haifeng Dong,
Jian Li
Publication year - 2021
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.01063-20
Subject(s) - single nucleotide polymorphism , pyrimethamine , biology , allele , polymerase chain reaction , polymorphism (computer science) , drug resistance , virology , microbiology and biotechnology , genetics , plasmodium falciparum , malaria , gene , genotype , immunology
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.