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KMID : 0387720210320010028
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Korean Journal of Blood Transfusion
2021 Volume.32 No. 1 p.28 ~ p.34
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Detection of RHD 1227G>A and 1222T>C Using PCR-Restriction Fragment Length Polymorphism
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Kim Woo-Shin
Park Geon
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Abstract
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Background: DEL is an RhD variant that cannot be detected by routine serologic tests because of the extremely low expression of the RhD antigen. Detecting the common genotypes of RHD 1227G＞A and 1222T＞C in Korean DEL is important for safe and efficient blood transfusions. Therefore, in this study, a PCR-restriction enzyme fragment polymorphism (RFLP) method was applied to detect RHD 1227G＞A and 1222T＞C.
Methods: DNA extracted from the blood of each segment of 56 units of RhD-negative red blood cell were used.
The promoter, exon 7 and exon 9 of RHD, and exon 9 of RHCE were amplified. The PCR products of RHD exon 7, RHD exon 9, and RHCE exon 9 were treated with the restriction enzymes HpyAV and MspI, and the RFLP patterns were observed by electrophoresis. The results of PCR-RFLP of RHD exon 9 were confirmed by PCR-direct sequencing.
Results: RHCE exon 9 was amplified in all 56 DNAs. RHD promoters, exon 7, and exon 9 were all amplified in 10 samples, RHD promoter, exon 7, and exon 9 were not amplified in 38 samples, and RHD promoter only was amplified in eight samples. As a result of the RHD exon 9 PCR-RFLP performed on 10 samples with all targets amplified, 10 samples were determined to be 9 samples with 1227G＞A and 1 sample with 1222T＞C.
The PCR-RFLP result and the sequencing result were 100% identical.
Conclusion: PCR-RFLP using HpyAV and MspI is a reliable and applicable method for detecting RHD 1227G>A and 1222T＞C in serologically RhD negative samples.
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KEYWORD
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RHD, DEL, Genotyping, RFLP
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