title: Genotyping of human platelet antigen-15 by single closed-tube Tm-shift method.
abstract: BACKGROUND
  Genotyping of human platelet antigens (HPA) is useful for the diagnosis and prevention of platelet alloimmune syndromes. HPA-15 might play an important role in the development of platelet alloimmune syndromes. There are several disadvantages in the conventional methods for HPA-15 genotyping. The aim of this study was to develop a new method for HPA-15 genotyping by using single closed-tube melting temperature (T(m))-shift genotyping.
  
  
  METHODS
  Two GC-rich tails of different lengths were attached to 5'-end of HPA-15 allele-specific PCR primers, such that HPA-15 alleles can be discriminated by the T(m)s of the PCR products. One hundred blood samples were genotyped for HPA-15 by the T(m)-shift and conventional polymerase chain reaction with sequence-specific primers (PCR-SSP).
  
  
  RESULTS
  The comparison of the PCR-SSP and the T(m)-shift method showed four discordant results in one hundred samples tested. Confirmatory results demonstrated that the PCR-SSP produced several errors, whereas HPA-15 genotyping by T(m)-shift is correct. The retesting results of T(m)-shift method were consistent with those of the initial testing.
  
  
  CONCLUSIONS
  The single closed-tube T(m)-shift method for HPA-15 genotyping is high-throughput, rapid, reliable, reproducible and cost-effective and it is superior to conventional PCR-SSP used in routine genotyping of HPA-15.
authors.affiliation: Dalian Blood Center, Dalian, China.
authors.forename: S H
authors.initials: SH
authors.lastname: Zhou
authors.forename: M
authors.initials: M
authors.lastname: Liu
authors.forename: W X
authors.initials: WX
authors.lastname: An
authors.forename: X H
authors.initials: XH
authors.lastname: Liang
authors.forename: W J
authors.initials: WJ
authors.lastname: Yu
authors.forename: B L
authors.initials: BL
authors.lastname: Gong
authors.forename: F Y
authors.initials: FY
authors.lastname: Piao
mesh_terms: D000483:Alleles; D015703:Antigens, CD / Q000235:genetics*; D016824:Antigens, Human Platelet / Q000235:genetics*; D058851:GPI-Linked Proteins / Q000235:genetics; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D044366:Transition Temperature
chemical_list: D015703:Antigens, CD; D016824:Antigens, Human Platelet; C120523:CD109 protein, human; D058851:GPI-Linked Proteins; D009363:Neoplasm Proteins