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Multiplex polymerase chain reaction with DNA pooling: a cost‐effective strategy of genotyping rare blood types
Author(s) -
He Y.L.,
Gao H.H.,
Ye L.Y.,
Guo Z.H.,
Wang P.,
Zhu Z.Y.
Publication year - 2013
Publication title -
transfusion medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.471
H-Index - 59
eISSN - 1365-3148
pISSN - 0958-7578
DOI - 10.1111/j.1365-3148.2012.01198.x
Subject(s) - genotyping , polymerase chain reaction , genotype , multiplex polymerase chain reaction , multiplex , typing , biology , microbiology and biotechnology , serology , allele , single nucleotide polymorphism , genetics , gene , antibody
SUMMARY Objectives/Aims 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. Conclusion A multiplex PCR assay was combined with DNA pooling to reduce the number of tests required, making large‐scale screening feasible.