文章基本信息

标题：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
本地全文：下载
作者：渡邊賢 ; 豊間根耕地 ; 山岸孝幸等
期刊名称：日本法科学技術学会誌
印刷版ISSN：1880-1323
电子版ISSN：1881-4689
出版年度：2022
卷号：27
期号：2
页码：185-194
DOI：10.3408/jafst.829
语种：Japanese
出版社：Japanese Association of Forensic Science and Technology
摘要：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.
关键词：ABO blood group system;ABO genotyping;single nucleotide polymorphism;multiplex real-time PCR