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首页> 外文期刊>Transfusion: The Journal of the American Association of Blood Banks >A convenient qualitative and quantitative method to investigate RHD-RHCE hybrid genes
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A convenient qualitative and quantitative method to investigate RHD-RHCE hybrid genes

机译：一种方便的定性和定量方法来研究RHD-RHCE杂交基因

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摘要

BACKGROUND: Molecular biology techniques, such as single specific-primer polymerase chain reaction (PCR), denaturing-high performance liquid chromatography, direct sequencing, next-generation sequencing, and microarray platforms, contribute to the efficient genotyp-ing of the human blood group RHD gene. However, some alleles remain undetermined in rare cases in DNA samples carrying two copies of the RHD gene, which challenge the identification of D-CE hybrid genes. STUDY DESIGN AND METHODS: We set up, in a single-tube format, a qualitative and quantitative assay based on multiplex PCR of short fluorescent fragments (QMPSF) to simultaneously amplify all 10 RHD exons on the one hand and all 10 RHCE exons on the other hand. RESULTS: The test proved to be useful to rapidly identify hybrid genes in hemizygous RHD samples carrying a hybrid D-CE gene and to resolve unknown genotypes by quantifying individual exons in compound heterozygous samples, but also unexpectedly helped to redefine the RHDV haplotype. While validating the test, two novel single-point variants, c.648G>C (p.L216F) and c.1048G>C (p.D350H), were found. CONCLUSION: For the first time, a QMPSF-based method is reliable to individually quantify the exons of both RH genes, including hybrid D-CE genes in compound heterozygous samples and may help to investigate samples with unknown RHD and/or RHCE status.
机译：背景技术分子生物学技术，如单一特异性底漆聚合酶链反应（PCR），变性 - 高效液相色谱，直接测序，下一代测序和微阵列平台，有助于人类血液组的有效基因型RHD基因。然而，一些等位基因在携带两份RHD基因的DNA样品中的罕见情况下仍未确定，这攻击D-Ce杂交基因的鉴定。研究设计和方法：以单管形式，基于短荧光片段（QMPSF）的多重PCR的定性和定量测定进行设置，同时放大所有10个RHD外显子，以及所有10个RHCE外显子另一方面。结果：证明是有用的，可用于快速鉴定携带杂交D-Ce基因的血液rHD样品中的杂合基因，并通过量化化合物杂合样品中的个体外显子来解决未知基因型，但也意外地帮助重新定义了Rhdv单倍型。在验证测试的同时，发现了两种新型单点变体，C.648G> C（P.L216F）和C.1048G> C（P.D350H）。结论：首次，基于QMPSF的方法可靠地是单独量化rh基因的外显子，包括化合物杂合子样品中的杂合D-Ce基因，并有助于研究具有未知RHD和/或RHCE状态的样品。

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