Single-Drop Blood Detection of Common G6PD Mutations in Thailand Based on Allele-Specific Recombinase Polymerase Amplification with CRISPR-Cas12a

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited enzymopathy. Identification of the G6PD deficiency through screening is crucial to preventing adverse effects associated with hemolytic anemia following antimalarial drug exposure. Therefore, a rapid and precise field-based G6PD deficiency diagnosis is required, particularly in rural regions where malaria is prevalent. The phenotypic diagnosis of the G6PD intermediate has also been a challenging issue due to the overlapping of G6PD activity levels between deficient and normal individuals, leading to a misinterpretation. The availability of an accurate point-of-care testing (POCT) for G6PD genotype diagnosis will therefore increase the opportunity for screening heterozygous cases in a low-resource setting. In this study, an allele-specific recombinase polymerase amplification (AS RPA) with clustered regularly interspaced short palindromic repeats-Cas12a (CRISPR-Cas12a) was developed as a POCT for accurate diagnosis of common G6PD mutations in Thailand. The AS primers for the wild type and mutant alleles of G6PD Mahidol(G487A) and G6PD Viangchan(G871A) were designed and used in RPA reactions. Following application of CRISPR-Cas12a systems containing specific protospacer adjacent motif, the targeted RPA amplicons were visualized with the naked eye. Results demonstrated that the G6PD Mahidol(G487A) and G6PD Viangchan(G871A) assays reached 93.62 and 98.15% sensitivity, respectively. The specificity was 88.71% in Mahidol(G487A) and 99.02% in G6PD Viangchan(G871A). The diagnosis accuracy of the G6PD Mahidol(G487A) and G6PD Viangchan(G871A) assays was 91.67 and 98.72%, respectively. From DNA extraction to detection, the assay required approximately 52 min. In conclusion, this study demonstrated the high performance of an AS RPA with the CRISPR-Cas12a platform for G6PD Mahidol(G487A) and G6PD Viangchan(G871A) detection assays and the potential use of G6PD genotyping as POCT.