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Multiplex polymerase chain reaction with DNA pooling: a cost-effective strategy of genotyping rare blood types


Correspondence: Prof. Zi-Yan Zhu, Blood Group Reference Laboratory, Shanghai Blood Center, Hongqiao Road 1191, Shanghai 200051, China.

Tel.: +86 (0)21 62758027x1115; fax: +86 (0)21 62700547;




This work aims to develop a multiplex polymerase chain reaction combined with DNA pooling for mass screening for rare blood types.


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.


A sensitive multiplex PCR-SSP assay testing pooled DNA was established to detect the rare Fyb 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 Fya and s. The rare genotypes Fy(a−b+) and S+s− were verified using serologic tests and sequencing analysis.


Two hundred and fifty-four donors were tested positive for the Fyb 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 Fyb and S alleles comprised 2·28 and 1·16%, respectively. The PCR-SSP assays were confirmed by sequencing analysis and serological test.


A multiplex PCR assay was combined with DNA pooling to reduce the number of tests required, making large-scale screening feasible.

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