Recombinase polymerase amplification for single nucleotide polymorphism-specific detection of βC variant in sickle cell disease

Abstract

Sickle cell disease (SCD) comprises a group of inherited blood disorders caused by point mutations in the β-globin gene. SCD is characterized by at least one β^S globin allele and a second pathologic globin variant that results in predominant formation of hemoglobin S (HbS). Early diagnosis in low-and middle-income countries is limited by the high cost and complexity of DNA-based tests. Recombinase Polymerase Amplification (RPA) is an isothermal nucleic acid amplification technique that facilitates rapid and low-cost detection of genetic mutations. While RPA primers have been developed to detect wild-type β^A and β^S alleles, they can also amplify the β^C allele, the next most common hemoglobin variant causing SCD which requires distinct clinical management. We developed a novel allele-specific RPA fluorescent assay for selective detection of the β^C allele using primers incorporating Amplification Refractory Mutation System (ARMS) and Locked Nucleic Acid (LNA) modifications. Twelve forward primers with different modifications were screened to achieve β^C-specific amplification. The best-performing primer combined a single mismatch near the 3′ end with an LNA at the terminal base, enabling specific detection of β^C with a limit of detection of 100 copies per reaction. Key design insights include avoiding mismatches immediately before the LNA for consistent target amplification and positioning the LNA closer to the 3′ end to achieve less sensitive amplification. This study establishes an isothermal assay for SCD diagnosis and offers systemic design strategies for SNP-specific RPA assays. These findings have important implications for expanding affordable, rapid genetic testing for hemoglobinopathies in low-resource settings.