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Published on: September 25, 2018
Single-Drop Blood Detection of Common G6PD Mutations in Thailand Based on Allele-Specific Recombinase Polymerase
Punchalee Mungkalasut1,2, Pattaraporn Nimsamer3, Poonlarp Cheepsunthorn4
1Interdisciplinary Program of Biomedical Sciences, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand.
Insights
A new point-of-care test accurately detects Glucose 6-phosphate dehydrogenase (G6PD) deficiency, a common inherited condition. This rapid G6PD test is crucial for preventing drug-induced hemolytic anemia in malaria-endemic regions.
Area of Science:
- Molecular Biology
- Genetics
- Medical Diagnostics
Background:
- Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most prevalent inherited enzymopathy globally.
- Accurate identification of G6PD deficiency is critical for preventing hemolytic anemia triggered by antimalarial drugs, especially in malaria-prone rural areas.
- Phenotypic diagnosis of intermediate G6PD deficiency presents challenges due to overlapping activity levels, leading to potential misinterpretations.
Purpose of the Study:
- To develop a rapid, precise, and field-deployable point-of-care test (POCT) for diagnosing common G6PD mutations.
- To enable effective screening of heterozygous G6PD deficiency cases in resource-limited settings.
- To address the diagnostic challenges associated with G6PD deficiency and intermediate phenotypes.
Main Methods:
- Development of an allele-specific recombinase polymerase amplification (AS RPA) assay combined with CRISPR-Cas12a technology.
- Design of AS primers targeting wild-type and mutant alleles of G6PD Mahidol and G6PD Viangchan mutations.
- Visual detection of targeted RPA amplicons using CRISPR-Cas12a systems with specific protospacer adjacent motifs.
Main Results:
- The developed assays demonstrated high sensitivity and specificity for detecting G6PD Mahidol and G6PD Viangchan mutations.
- Sensitivity reached 93.62% for G6PD Mahidol and 98.15% for G6PD Viangchan.
- Specificities were 88.71% for G6PD Mahidol and 99.02% for G6PD Viangchan, with overall accuracies of 91.67% and 98.72%, respectively.
- The entire assay process, from DNA extraction to detection, was completed in approximately 52 minutes.
Conclusions:
- The AS RPA-CRISPR-Cas12a platform offers a high-performance solution for G6PD genotyping.
- This technology has significant potential for use as a point-of-care diagnostic tool for common G6PD mutations.
- The developed assay facilitates accurate G6PD deficiency screening, particularly in low-resource settings, aiding in the prevention of adverse drug reactions.
Abstract:
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 and G6PD Viangchan 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 and G6PD Viangchan assays reached 93.62 and 98.15% sensitivity, respectively. The specificity was 88.71% in Mahidol and 99.02% in G6PD Viangchan. The diagnosis accuracy of the G6PD Mahidol and G6PD Viangchan 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 and G6PD Viangchan detection assays and the potential use of G6PD genotyping as POCT.
