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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.

SUBMITTER: Jiang T

PROVIDER: S-EPMC8092547 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission.MethodsA novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples.ResultsThe SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63°C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed.ConclusionThe mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world.

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

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