【Objective】 To explore the application of reverse dot blotting (RDB) assay in the simultaneous detection of the most common six Cantonese G6PD gene mutations. 【Methods】 Glucose 6 phosphate dehydrogenase (G6PD) deficiency was screened using the methemoglobin reduction test and G6PD/6-PGD ratio method. Multiplex PCR was used to amplify the objective fragments of G6PD gene. The specific oligonucleotide probes were designed and optimized using the Premier Primer 6.0 program. Nylon membrane was treated by (ethyl-3-(dimethylaminopropyl) carbodiimide

EDC) and then was dotted with the specific hybridization probes. The genotypes were judged by colorimetric reaction after the RDB. Moreover

the RDB assay was evaluated by using the sequencing result as a gold standard. 【Results】 Among 26 severe G6PD deficient patients

five individuals was found carrying 95 A→G mutation

three with 1024 C→T

eight with 1376 G→T

seven with 1388G→A

one with 392 G→T. Moreover

two G392T homozygote identified by DNA sequencing could not be correctly detected by the RDB assay. 【Conclusions】 The RDB assay could be used to simultaneously detect the most common six Cantonese G6PD gene mutations after optimizing probe design and hybridization condition.