Allele-Specific Recombinase Polymerase Amplification to Detect Sickle Cell Disease in Low-Resource Settings

Abstract

Sickle cell disease (SCD) is a group of common, life-threatening disorders caused by a point mutation in the β globin gene. Early diagnosis through newborn and early childhood screening, parental education, and preventive treatments are known to reduce mortality. However, the cost and complexity of conventional diagnostic methods limit the feasibility of early diagnosis for SCD in resource-limited areas worldwide. Although several point-of-care tests are commercially available, most are antibody-based tests, which cannot be used in patients who have recently received a blood transfusion. Here, we describe the development of a rapid, low-cost nucleic acid test that uses real-time fluorescence to detect the point mutation encoding hemoglobin S (HbS) in one round of isothermal recombinase polymerase amplification (RPA). When tested with a set of clinical samples from SCD patients and healthy volunteers, our assay demonstrated 100% sensitivity for both the β A globin and β S globin alleles and 94.7 and 97.1% specificities for the β A globin allele and β S globin allele, respectively ( n = 91). Finally, we demonstrate proof-of-concept sample-to-answer genotyping of genomic DNA from capillary blood using an alkaline lysis procedure and direct input of diluted lysate into RPA. The workflow is performed in <30 min at a cost of <$5 USD on a commercially available benchtop fluorimeter and an open-source miniature fluorimeter. This study demonstrates the potential utility of a rapid, sample-to-answer nucleic acid test for SCD that may be implemented near the point of care and could be adapted to other disease-causing point mutations in genomic DNA.

A Simple, Cost-Effective, and Extraction-Free Molecular Diagnostic Test for Sickle Cell Disease Using a Noninvasive Buccal Swab Specimen for a Limited-Resource Setting

“…Hence, the authors suggested further confirmatory validation. Another study by Natoli et al reported a strategy for allele-specific detection of the sickle cell mutation using blood samples [ 18 ]. In their assay, two separate reactions for each sample are required to detect AA and SS genotypes, which is the major limitation of the above study.…”

A simple, cost-effective and extraction-free molecular diagnostic test for sickle cell disease in noninvasive buccal swab specimen for a limited-resource setting

“…Another study by Natoli et al have reported a strategy for allele-specific detection of sickle cell mutation using blood samples. 18 In their assay, two separate reactions for each sample are required to detect AA and SS genotypes, which is the major limitation of the above study.…”

A genosensor for detecting single-point mutations in dendron chips after blocked recombinase polymerase amplification

“…For the detection of single-point mutations, some RPA-based approaches have been described, such as RPA-microarray hybridization, 8 real-time RPA, 9 and zip-RPA. 10 A relevant variant, called blocked-RPA, was developed for selective amplification of particular DNA variants based on the enrichment of minority alleles.…”