TaqManマルチプレックスリアルタイムPCRによるABO遺伝子型検査法の確立とその法科学的検証

抄録

 In forensic investigations, ABO genotyping via detection of single nucleotide polymorphisms (SNPs) in the ABO gene is often performed to estimate the ABO phenotype of case samples. This study involved the developmental validation of the ABO genotyping method performed by multiplex real-time PCR using TaqMan probes targeting major O or B allele-specific SNPs. We first evaluated the genotyping accuracy of this method by using 2 ng of blood DNA and confirmed that the ABO genotypes could be correctly determined from all samples. To validate the effect of unbalanced amplification of heterozygous samples on the genotyping accuracy, we analyzed various amounts (0.03125-1.0 ng) of two blood DNA samples of the BO genotype, which were heterozygous for both O and B allele-specific SNPs. No false genotypes were observed, even in the analysis of low-template DNA, although 1 ng of template DNA was required for stable detection. We also analyzed BO blood DNA mixed with humic acid or hematin. Although inhibition of PCR by using high concentrations of both inhibitors caused “no amplification” results, no false genotypes were observed under any condition. We confirmed that the correct genotypes could be obtained by using 1 ng of DNA from blood stains stored at room temperature for 2-36 years. Finally, we analyzed the DNA from five animal species (chimpanzee, Japanese macaque, dog, cat, rat) and confirmed that several probes reacted strongly to the primate DNA and weakly to the feline DNA, indicating that careful attention should be paid in the analysis of samples that may be derived from these species. Because the genotypes of multiple samples can be simultaneously determined within about 30 min after DNA quantification, this method is expected to be useful for forensic ABO genotyping.