Abstract
BackgroundSickle cell anaemia is a common, life-threatening disorder caused by a point mutation in the β globin gene. The high cost and complexity of conventional diagnostic methods limit the scope and sustainability of newborn screening for sickle cell anaemia and other haemoglobin disorders in resource-limited areas. 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. Additionally, very low levels of sickle haemoglobin (HbS) in premature babies and some newborns may cause errors in immunoassays. DNA analysis provides the most reliable approach to diagnose haemoglobin disorders. However, DNA analysis is not currently available at the point of care due to its cost and complexity. Here, we describe a rapid, low-cost nucleic acid test that uses real-time fluorescence to detect the point mutation encoding HbS in one round of isothermal recombinase polymerase amplification. MethodsVenous and capillary blood samples were obtained from healthy volunteers who were recruited by word of mouth. Venous blood samples from patients with varying genotypes seen in the Sickle Cell Disease Program at Texas Children's Hematology Center were collected and deidentified under IRB-approved exemption. Genotypes, as determined by qualitative isoelectric focusing combined with quantitative high performance liquid chromatography (HPLC) to confirm a diagnosis, were provided with the blood samples. The assay was evaluated with genomic DNA extracted from a set of blood samples of varying genotypes using the Axxin T8-ISO, a low-cost, field-deployable benchtop fluorimeter. FindingsThe developed assay demonstrated 100% sensitivity for both the βA globin and βS globin alleles, and 94·7% and 97·1% specificity for the βA globin allele and βS globin allele, respectively (n=91). We also demonstrate a complete sample-to-answer workflow using a two-step lysis method and direct input of lysate into the allele-specific assay, and genotyping of blood samples in an open-source, custom built fluorimeter whose components cost less than US$850. InterpretationThis sample-to-answer nucleic acid test has the potential to be adapted to other disease-causing point mutations in genomic DNA. The technology described could streamline diagnosis in resource-limited settings of patients with sickle cell anaemia who have recently undergone a blood transfusion, with a comparable cost to protein-based tests. FundingNIH K23-HL148548-01A1.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.