373 Vox Sanguinis (2007) 92, 373–380 ORIGINAL PAPER © 2007 The Author(s) Journal compilation © 2007 Blackwell Publishing Ltd. DOI: 10.1111/j.1423-0410.2007.00902.x Blackwell Publishing Ltd Real-time multiplex allele-specific polymerase chain reaction for genotyping of the Duffy antigen, the Plasmodium vivax invasion receptor T. N. Sousa, B. A. M. Sanchez, I. P. Cerávolo, L. H. Carvalho & C. F. A. Brito Laboratory of Malaria, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil Background and Objectives Duffy blood group is of major interest in clinical medicine as it is not only involved in blood-transfusion risks and occasionally in neonatal haemolytic disease, but it is also the receptor for the human malaria parasite Plasmodium vivax in the erythrocyte invasion. The aim of this study was to develop a rapid and inexpensive approach for high-throughput Duffy genotyping. Materials and methods This paper reported the development of a Duffy genotyping assay based on multiplex real-time polymerase chain reaction (PCR) using SYBR Green I fluorescent dye. Results By using this approach for Duffy genotyping we obtained the same results as that for the conventional allele-specific PCR, however, in a high-throughput assay. The Duffy genotyping of field samples demonstrated that P. vivax-infected individuals showed a significantly higher prevalence of two functional alleles than Plasmodium falciparum-infected and non-infected individuals. This finding corroborates the hypothesis that the presence of two functional alleles increases the risk of P. vivax infection. Conclusion This methodology may be suitable for epidemiological studies, particularly for exploring the relationship between Duffy alleles and malaria susceptibility, and also for identification of transfusional incompatibility in blood banks. Key words: DARC, Duffy blood group, malaria, Plasmodium vivax, real-time PCR, SYBR green fluorescent dye. Received: 14 September 2006, revised 28 December 2006, accepted 8 January 2007, published online 15 March 2007 Introduction The Duffy blood group, also known as Duffy antigen receptor for chemokines (DARC), is of biological and clinical import- ance, as antibodies against Duffy antigens are responsible for some cases of transfusion incompatibility and haemolytic disease of the newborn [1,2]. Furthermore, besides being a receptor for various chemokines, the Duffy antigen is the obligatory receptor for invasion of Plasmodium vivax malaria parasite [3,4]. The Duffy antigen was first reported in a polytransfused haemophiliac patient, who had an alloantibody against an antigen denoted as Fy a [5]. The antithetical antigen, Fy b , was described 1 year later [6]. Fy a and Fy b antigens are the products of two codominant alleles FY *A and FY *B on the chromosome 1 FY locus [7]. The single copy FY gene is com- posed of two exons, which encodes for a major product of 336 amino acids [8]. The two common alleles in Caucasians, FY *A and FY *B, differ by a single base substitution (125G>A) resulting in the replacement of a glycine with an aspartic acid at residue 42 in the extracellular domain of Duffy antigen [9,10]. T. N. Souza and B. A. M. Sanchez contributed equally to this work. Correspondence: Cristiana Ferreira Alves de Brito, Laboratory of Malaria, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Avenida Augusto de Lima, 1715, Belo Horizonte, Brazil, 30190-002 E-mail: cristiana@cpqrr.fiocruz.br