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Genotyping of human platelet antigen‐15 by single closed‐tube T m ‐shift method
Author(s) -
ZHOU S. H.,
LIU M.,
AN W. X.,
LIANG X. H.,
YU W. J.,
GONG B. L.,
PIAO F. Y.
Publication year - 2012
Publication title -
international journal of laboratory hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.705
H-Index - 55
eISSN - 1751-553X
pISSN - 1751-5521
DOI - 10.1111/j.1751-553x.2011.01344.x
Subject(s) - genotyping , antigen , platelet , tube (container) , microbiology and biotechnology , immunology , medicine , chemistry , biology , materials science , biochemistry , genotype , gene , composite material
Summary Introduction: 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. Conclusion: 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.