A new PCR method for direct determination of RHD zygosity

Abstract

To establish a direct method for the determination of RHD zygosity. Two pairs of primers were designed specific for hybrid Rhesus box and exon 1 of RHD, respectively. Combined with a pair of internal control primers, a new dual-tube PCR method was established. One hundred and fifty-two DNA samples, of which the sequence of RHD and RH genotypes were known, and samples from 359 donors or patients, of which the Rh phenotypes were determined, were evaluated by taking a recent restriction fragment length polymorphism (RFLP) method as reference. The RHD zygosities of 152 samples, including 92 Rh-negative, 28 D(el), 3 partial D, 5 weak D and 24 Rh-positive, were determined in concordance with the known genotypes. It showed that the PCR method could detect RHD(+)/RHD(+), RHD(+)/RHD(-) and RHD(-)/RHD(-) zygosities in one test. The PCR results of 359 known phenotype samples including 12 husbands, 18 family members, 48 Rh-negative and 281 Rh-positive, were identical with RFLP except for one Rh-positive sample, which was tested as RHD(+)/RHD(-) heterozygote by PCR, whereas RHD(-)/RHD(-) homozygote by RFLP. Apparently, RFLP revealed a false negative result detecting downstream Rhesus box. The dual-tube PCR is a less-labored and more rapid method for the determination of RHD zygosity comparing RFLP. It can be used routinely in the laboratories in clinics and blood banks.