title: Multiplex polymerase chain reaction with DNA pooling: a cost-effective strategy of genotyping rare blood types.
abstract: OBJECTIVE
  This work aims to develop a multiplex polymerase chain reaction combined with DNA pooling for mass screening for rare blood types.
  
  
  BACKGROUND
  The differences in most blood group antigens are associated with single-nucleotide polymorphisms (SNPs), which are used in detecting blood antigen expression at the molecular level. However, all existing sequence-specific primers polymerase chain reaction (PCR-SSP) assays for blood typing genotype one or several SNPs individually. DNA pooling is a way that reduces the amount of genotyping required.
  
  
  METHODS
  A sensitive multiplex PCR-SSP assay testing pooled DNA was established to detect the rare Fy(b)  and S alleles. It was applied to screen a total of 4490 donor samples via testing 898 DNA pools. The samples in the positive pools were further tested individually. Then the positive samples, including Fy(a-b+)/Fy(a+b+) and S+s-/S+s+ genotypes, were tested via two PCR-SSP assays for alleles Fy(a)  and s. The rare genotypes Fy(a-b+) and S+s- were verified using serologic tests and sequencing analysis.
  
  
  RESULTS
  Two hundred and fifty-four donors were tested positive for the Fy(b)  allele, whereas 101 donors were positive for the S allele. Among the 254 Fy(b+) donors, 5 were Fy(a-b+) and 249 were Fy(a+b+). Among the 101 S+ donors, 3 were S+s- and 98 were S+s+. The rare Fy(b)  and S alleles comprised 2·28 and 1·16%, respectively. The PCR-SSP assays were confirmed by sequencing analysis and serological test.
  
  
  CONCLUSIONS
  A multiplex PCR assay was combined with DNA pooling to reduce the number of tests required, making large-scale screening feasible.
authors.affiliation: School of Life Science, East China Normal University, Shanghai, China.
authors.forename: Y-L
authors.initials: YL
authors.lastname: He
authors.affiliation: Blood Group Reference Laboratory, Shanghai Blood Center.
authors.forename: H-H
authors.initials: HH
authors.lastname: Gao
authors.affiliation: Blood Group Reference Laboratory, Shanghai Blood Center.
authors.forename: L-Y
authors.initials: LY
authors.lastname: Ye
authors.affiliation: Blood Group Reference Laboratory, Shanghai Blood Center.
authors.forename: Z-H
authors.initials: ZH
authors.lastname: Guo
authors.affiliation: School of Life Science, East China Normal University, Shanghai, China.
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.affiliation: Blood Group Reference Laboratory, Shanghai Blood Center.
authors.forename: Z-Y
authors.initials: ZY
authors.lastname: Zhu
mesh_terms: D000483:Alleles; D001782:Blood Donors; D001787:Blood Group Incompatibility / Q000517:prevention & control; D001803:Blood Transfusion; D003362:Cost-Benefit Analysis; D017931:DNA Primers; D004375:Duffy Blood-Group System / Q000235:genetics*; D005820:Genetic Testing / Q000191:economics / Q000379:methods*; D060005:Genotyping Techniques / Q000191:economics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011956:Receptors, Cell Surface / Q000235:genetics*; D013048:Specimen Handling
chemical_list: C473745:ACKR1 protein, human; D017931:DNA Primers; D004375:Duffy Blood-Group System; D011956:Receptors, Cell Surface