Single-Tube Multiplex PCR-SSP for the Detection of RHD Variant Alleles Commonly Found in Serologically D- Phenotype Individuals in a Thai Population.

INTRODUCTION

The molecular basis of the gene found in serologically D- phenotype individuals differs with race/ethnicity. Therefore, we aimed to develop a single-tube multiplex PCR-sequence specific primer (multiplex PCR-SSP) to detect variant alleles commonly found in serologically D- phenotype individuals in a Thai population.

METHODS

In total, 205 blood samples with a serologically D- phenotype were tested using a single-tube multiplex PCR-SSP targeted exons 1, 4, 7, 10, and c.1227G>A in exon 9 in combination with a hybrid , and results were confirmed by direct DNA sequencing.

RESULTS

In a single-tube multiplex PCR-SSP, three patterns of amplified exons were observed: total deletion of the gene, Asian-type DEL, and hybrid. The allele frequencies of , , and hybrid were 83.4%, 12.9%, and 2%, respectively. All of the Asian-type DEL samples present the allele (predicted RhCE phenotype: C/E+).

CONCLUSION

This study successfully established a simple and reliable molecular diagnostic platform for analyzing variant alleles commonly found in serologically D- phenotype individuals in a Thai population. This technique could enable broader (Asian-type DEL) analyses in high-prevalence areas such as Thailand and other countries in East and Southeast Asia, serving as an example for blood bank routine settings.