A novel pyrosequencing strategy for RHD zygosity for predicting risk of hemolytic disease of the fetus and newborn.

Laboratory medicine Pub Date : 2024-03-07 DOI:10.1093/labmed/lmad051

Piao Lv, Jixin Li, Yuan Yao, Xinxin Fan, Chixiang Liu, Hui Li, Huayou Zhou

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Abstract

Objective: The aim of this study was the development of an accurate and quantitative pyrosequence (PSQ) method for paternal RHD zygosity detection to help risk management of hemolytic disease of the fetus and newborn (HDFN).

Methods: Blood samples from 96 individuals were genotyped for RHD zygosity using pyrosequencing assay. To validate the accuracy of pyrosequencing results, all the samples were then detected by the mismatch polymerase chain reaction with sequence-specific primers (PCR-SSP) method and Sanger DNA sequencing. Serological tests were performed to assess RhD phenotypes.

Results: Serological results revealed that 36 cases were RhD-positive and 60 cases were RhD-negative. The concordance rate between pyrosequencing assay and mismatch PCR-SSP assay was 94.8% (91/96). There were 5 discordant results between pyrosequencing and the mismatch PCR-SSP assay. Sanger sequencing confirmed that the pyrosequencing assay correctly assigned zygosity for the 5 samples.

Conclusion: This DNA pyrosequencing method accurately detect RHD zygosity and will help risk management of pregnancies that are at risk of HDFN.

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用于预测胎儿和新生儿溶血性疾病风险的新型 RHD 基因型热测序策略。

研究目的本研究旨在开发一种准确、定量的热释光测序（PSQ）方法，用于检测父系RHD基因型，以帮助进行胎儿和新生儿溶血病（HDFN）的风险管理：方法：使用热释光测序法对96人的血样进行RHD基因分型。为验证热释光测序结果的准确性，所有样本均采用序列特异引物错配聚合酶链反应（PCR-SSP）法和桑格DNA测序法进行检测。血清学检测用于评估 RhD 表型：血清学结果显示，36 例为 RhD 阳性，60 例为 RhD 阴性。热释光测序法与错配 PCR-SSP 法的吻合率为 94.8%（91/96）。有 5 例热释光测序与错配 PCR-SSP 检测结果不一致。桑格测序证实，热释光测序法正确地确定了这 5 个样本的合子数：这种 DNA 热释光测序方法能准确检测出 RHD 基因型，有助于对有 HDFN 风险的孕妇进行风险管理。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Laboratory medicine

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