Detection of blood RhCE genotype based on PCR-SSP technique
Objective To establish a rapid genotyping method for RhCE blood group based on PCR-sequence specific primer(PCR-SSP)technique.Methods A total of 152 EDTA-anticoagulant blood samples were collected from the First Affiliated Hospital of Ningbo University from September to December 2023.RhD and RhCE blood types were detected by tube method,indirect anti-human sphere method and microcolumn gel card method,respectively.DNA was extracted from the samples using silico matrix column method.Sanger sequencing was used to select the RhCE gene standard sequence.The PCR-SSP technique was used to determine four sequence-specific primers for the detection two pairs of allele of RhC and Rhc,RhE and Rhe genes.TA cloning technique was used to obtain recombinant plasmids,and the sensitivity and anti-interference performance of sequence-specific primers were evaluated.The genes in all blood sampes were detected wity PCR-SSP technique,and the consistency between PCR-SSP and serological results was evaluated.Results The standard sequences of different RhCE genotypes were successfully determined by using three specimens with phenotypes of CCee,CcEe and ccEE,respectively.Four sequence-specific primer pairs were effective in distinguishing among RhC and Rhc,RhE and Rhe alleles.Sequence-specific primers were detected with the interference of the corresponding allele 1∶128.The sensitivity ranged from 104to 105 copies/μL.The consistence rate between RhE,Rhe and Rhc genotype detection and phenotype results was 100.00%in clinical samples,while the cosistence rate for RhC genotype was 92.76%,that was 98.39%and 88.89%in RhD positive and negative background population,respectively.Conclusion PCR-SSP technique is feasible in rapid identification of RhCE blood group genotype in clinical samples.The results of RhE,Rhe and Rhc genotypes are all consistent with the phenotype results.However,RhD blood group background will affect RhC genotyping results,so two or more methods are needed for the acurate detection.
RhCE blood typePCR-sequence specific primerGenotypingSingle nucleotide polymorphism