title: Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.
abstract: BACKGROUND
  ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells.
  
  
  METHODS
  We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification.
  
  
  RESULTS
  Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing.
  
  
  CONCLUSIONS
  The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.
authors.affiliation: Laboratory of Clinical Chemistry and Immunology, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Taira
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Kazuyuki
authors.identifier: http://orcid.org/0000-0002-4056-7242
authors.initials: K
authors.lastname: Matsuda
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Takeichi
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Furukawa
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Sugano
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uehara
authors.affiliation: Laboratory of Clinical Chemistry and Immunology, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Okumura
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Honda
mesh_terms: D000017:ABO Blood-Group System / Q000032:analysis / Q000737:chemistry / Q000145:classification*; D004247:DNA / Q000032:analysis* / Q000235:genetics; D059788:Dried Blood Spot Testing; D060005:Genotyping Techniques; D006801:Humans; D057230:Limit of Detection; D009061:Mouth Mucosa / Q000166:cytology*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D013997:Time Factors
chemical_list: D000017:ABO Blood-Group System; D004247:DNA
keywords: ABO genotype; buccal cells; dried blood spot samples; droplet allele specific PCR; single nucleotide polymorphism