Genotyping of human platelet antigen-15 by single closed-tube Tm-shift method.

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.