Malaria journal · 2019
Bead-based assays to simultaneously detect multiple human inherited blood disorders associated with malaria.
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Grignard Lynn, Mair Catherine, Curry Jonathan, Mahey Laleta, Bastiaens Guide J H, Tiono Alfred B, Okebe Joseph, Coulibaly Sam A, Gonçalves Bronner P, Affara Muna, Ouédraogo Alphonse, Bougouma Edith C, Sanou Guillaume S, Nébié Issa, Lanke Kjerstin H W, Sirima Sodiomon B, d'Alessandro Umberto, Clark Taane G, Campino Susana, Bousema Teun, Drakeley Chris
Аннотация
BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (G6PDd), haemoglobin C (HbC) and S (HbS) are inherited blood disorders (IBD) common in populations in malaria endemic areas. All are associated to some degree with protection against clinical malaria whilst additionally G6PDd is associated with haemolysis following treatment with 8-aminoquinolines. Measuring the prevalence of these inherited blood disorders in affected populations can improve understanding of disease epidemiology. Current methodologies in epidemiological studies commonly rely on individual target amplification and visualization; here a method is presented to simultaneously detect the polymorphisms and that can be expanded to include other single nucleotide polymorphisms (SNPs) of interest.
METHODS: Human DNA from whole blood samples was amplified in a novel, multiplex PCR reaction and extended with SNP-specific probes in an allele specific primer extension (ASPE) to simultaneously detect four epidemiologically important human markers including G6PD SNPs (G202A and A376G) and common haemoglobin mutations (HbS and HbC). The products were hybridized to magnetic beads and the median fluorescence intensity (MFI) was read on MAGPIX® (Luminex corp.). Genotyping data was compared to phenotypical data generated by flow cytometry and to established genotyping methods.
RESULTS: Seventy-five samples from Burkina Faso (n = 75/78, 96.2%) and 58 samples from The Gambia (n = 58/61, 95.1%) had a G6PD and a HBB genotype successfully assigned by the bead-based assay. Flow cytometry data available for n = 61 samples further supported the concordance between % G6PD normal/deficient cells and genotype.
CONCLUSIONS: The bead based assay compares well to alternative measures of genotyping and phenotyping for G6PD. The screening is high throughput, adaptable to inclusion of multiple targets of interest and easily standardized.
METHODS: Human DNA from whole blood samples was amplified in a novel, multiplex PCR reaction and extended with SNP-specific probes in an allele specific primer extension (ASPE) to simultaneously detect four epidemiologically important human markers including G6PD SNPs (G202A and A376G) and common haemoglobin mutations (HbS and HbC). The products were hybridized to magnetic beads and the median fluorescence intensity (MFI) was read on MAGPIX® (Luminex corp.). Genotyping data was compared to phenotypical data generated by flow cytometry and to established genotyping methods.
RESULTS: Seventy-five samples from Burkina Faso (n = 75/78, 96.2%) and 58 samples from The Gambia (n = 58/61, 95.1%) had a G6PD and a HBB genotype successfully assigned by the bead-based assay. Flow cytometry data available for n = 61 samples further supported the concordance between % G6PD normal/deficient cells and genotype.
CONCLUSIONS: The bead based assay compares well to alternative measures of genotyping and phenotyping for G6PD. The screening is high throughput, adaptable to inclusion of multiple targets of interest and easily standardized.