We developed a new method for ABO genotyping using a multiplex single-base primer extension reaction. The method allows for the simultaneous detection of six SNP sites in the ABO gene (nt 261, 297, 681, 703, 802, and 803) and the determination of ABO genotypes from their combinations. It enabled ABO genotyping of all samples of peripheral blood DNA extracted from 103 Japanese individuals, and had a highly satisfactory detection sensitivity being capable of genotyping 0.1 ng of genomic DNA. Using this method, we were able to determine ABO genotypes of minute stain samples, heated bloodstains, aged bloodstains and mixed samples. Experiments with samples from 26 animal species and bacterial samples to test the species-specificity of the method showed that genotyping was possible in the chimpanzee and gorilla, but their genotypes were extremely rare in humans. In addition, we applied this method to casework samples, and successfully determined ABO genotypes of bones, teeth, muscles, organs, nails, and semen-contaminated vaginal fluid in which ABO grouping by conventional serological techniques was not possible. This new method enables the sensitive, simultaneous detection of six SNP sites in the ABO gene by two specific reactions, i.e. PCR and a primer extension reaction. Therefore, it holds promise as an effective method of ABO genotyping particularly for forensic samples.

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The eŠect of pH on color development was examined in detail to detect higher sensitivity of ABO blood groups substances in saliva and semen by means of an indirect enzyme-linked immunosorbent assay (ELISA) using secondly antibody conjugated with horseradish peroxidase. The use of McIlvaine buŠer was the most eŠective for color development. Deep color was observed at pH 5.0, and intensity was about 32 times stronger than that obtained with the Tris HCl buŠer (pH 7.6) that was widely used in the Japanese Criminal Investigation Laboratories. Owing to the increased sensitivity, B-type and O-type blood of non-secretors semen, which could not be detected by ELISA, were successfully determined under the present conditions. Through this study, we found the interesting fact that commercially available monoclonal antibodies can be divided into two groups: One group reacts with secretor sample alone and the other reacts with both secretor and non-secretor samples. Using these two classiˆed antibodies, we were able to discriminate between secretor and non-secretor and to speculate the blood type of one sample from two mixed samples.