多重实时PCR和测序技术用于RHC基因分型的研究

Genotyping of RHC through multiplex real-time PCR and DNA sequencing

目的探讨多重实时PCR和测序技术用于RHC基因分型的可行性。方法随机收集2019年11月2日—11月6日期间242名健康献血者血样。使用血清学方法检测标本的Rh CcEe血型表型。使用检测RHCE基因第1外显子48C>G多态性及第2内含子109 bp插入序列的多重实时PCR方法对血液标本进行RHC基因分型。使用基因测序方法对以上2个多态性位点检测结果不一致的标本进行基因分型。结果经多重实时PCR基因分型,2个多态性位点分型结果一致者共235例(97.10%),这些标本的分型结果和血清学方法是完全一致的。2个多态性位点分型结果不一致者共7例(2.90%),其中第1外显子48C>G多态性位点分型结果与血清学方法不符者共4例,假阳性率12.5%(4/32),而第2内含子的109 bp插入序列分型结果与血清学方法不符者共3例,假阴性率1.43%(3/210)。对于多重实时PCR2个多态性位点分型不一致的标本,进行第2外显子测序,测序结果和血清学完全一致。结论使用检测RHCE基因第1外显子48C>G多态性及第2内含子109 bp插入片段的多重实时PCR进行RHC基因分型,若二者结果一致,则基因分型结果可靠,若二者检测结果不一致,则可能存在假阴性或假阳性,需要进一步利用基因测序确定。

Objective To determine the feasibility of RHC genotyping through multiplex real-time PCR and DNA sequencing. Methods A total of 242 participants during November 2 to 6, 2019 were randomly recruited from healthy blood donors. Rh CcEe phenotypes of the samples were tested by serological test. RHC of the samples were genotyped by targeting exon 1 48 C > G mutation and intron 2 109 bp insert of the RHCE gene through multiplex real-time PCR. The samples presenting discrepant typing results on the above-mentioned two mutations were genotyped by DNA sequencing. Results Both of the 48 C>G mutation and 109 bp insert were presented in 235(97.10%) cases, which were in full concordance with serotypes. Exon 1 48 C > G mutation with absent intron 2 109 bp insert was detected in 7(2.90%) cases, while false-positive prediction of C by 48 C>G mutation in 4 cases(4/32, 12.5%) and false-negative prediction of C by intron 2 109 bp insert in 3 cases(3/210, 1.43%). The samples presenting discrepant PCR results were subjected to exon2 sequencing, and the sequencing results were matched with the serological test. Conclusion Reliable RHC genotyping is possible with the exon 1 48 C > G/intron 2 109 bp insert multiplex PCR approach, however, sole present of 48 C > G mutation or intron 2 109 bp insert may predict false positive C or false negative C, and further DNA sequencing on exon2 is needed.