A multiplexed, allele-specific recombinase polymerase amplification assay with lateral flow readout for sickle cell disease detection.
Lab Chip
; 24(17): 4115-4127, 2024 08 20.
Article
en En
| MEDLINE
| ID: mdl-39051493
ABSTRACT
Isothermal nucleic acid amplification tests have the potential to improve disease diagnosis at the point of care, but it remains challenging to develop multiplexed tests that can detect ≥3 targets or to detect point mutations that may cause disease. These capabilities are critical to enabling informed clinical decision-making for many applications, such as sickle cell disease (SCD). To address this, we describe the development of a multiplexed allele-specific recombinase polymerase amplification (RPA) assay with lateral flow readout. We first characterize the specificity of RPA using primer design strategies employed in PCR to achieve point mutation detection, and demonstrate the utility of these strategies in achieving selective isothermal amplification and detection of genomic DNA encoding for the healthy ßA globin allele, or genomic DNA containing point mutations encoding for pathologic ßS and ßC globin alleles, which are responsible for most sickle cell disorders. We then optimize reaction conditions to achieve multiplexed amplification and identification of the three alleles in a single reaction. Finally, we perform a small pilot study with 20 extracted genomic DNA samples from SCD patients and healthy volunteers - of the 13 samples with valid results, the assay demonstrated 100% sensitivity and 100% specificity for detecting pathologic alleles, and an overall accuracy of 92.3% for genotype prediction. This multiplexed assay is rapid, minimally instrumented, and when combined with point-of-care sample preparation, could enable DNA-based diagnosis of SCD in low-resource settings. The strategies reported here could be applied to other challenges, such as detection of mutations that confer drug resistance.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Técnicas de Amplificación de Ácido Nucleico
/
Recombinasas
/
Alelos
/
Anemia de Células Falciformes
Límite:
Humans
Idioma:
En
Revista:
Lab Chip
Asunto de la revista:
BIOTECNOLOGIA
/
QUIMICA
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Reino Unido