AccuCopy quantification combined with pre-amplification of long-distance PCR for fast analysis of intron 22 inversion in haemophilia A
AccuCopy quantification combined with pre-amplification of long-distance PCR for fast analysis of intron 22 inversion in haemophilia A
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DOI：
10.1016/j.cca.2016.04.015
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发表时间：
2016-07-01
影响因子：
5
通讯作者：
Wang, Xuefeng
中科院分区：
文献类型：
--
作者：
Ding, Qianlan;Wu, Xi;Wang, Xuefeng
背景资料：目的：建立一种检测重型血友病A患者内含子22倒位（Inv 22）的方法。方法：设计了两种检测方法：一种是采用LD-PCR（PLP）的多重预扩增技术，分别扩增野生型和嵌合型int 22 h等位基因;载体嵌合性试验与基因分型试验相似，除了通过从两种组合中的每一种中除去一种引物而仅扩增嵌合INT 22 H等位基因。AccuCopy检测用于定量PLP products.Results：PLP产品模式的基因分型测试允许识别所有已知的Inv 22。定量模式准确地代表了产品模式。164份样本的基因分型结果与LD-PCR检测结果一致。载体嵌合试验的检出限（LOD）为至少2%的Inv 22杂合细胞。对两个散发家系中2名Inv 22阴性的母亲进行检测，家系1母亲的血液和发根嵌合率分别为83%和> 20%，家系2的结果均为阴性。结论：AccuCopy定量结合PLP（AQ-PLP）法是一种快速、可靠的Inv 22基因分型方法，对检测Inv 22携带者嵌合体具有较高的敏感性。(C)© 2016 Elsevier B. V.版权所有。
Background: To develop a digitalized intron 22 inversion (Inv22) detection in patients with severe haemophilia A.Methods: The design included two tests: A genotyping test included two multiplex pre-amplification of LD-PCR (PLP) with two combinations of five primers to amplify wild-type and chimeric int22h alleles; a carrier mosaicism test was similar to the genotyping test except only amplification of chimeric int22h alleles by removing one primer from each of two combinations. AccuCopy detection was used to quantify PLP products.Results: PLP product patterns in the genotyping test allowed identifying all known Inv22. Quantitative patterns accurately represented the product patterns. The results of 164 samples detected by the genotyping test were consistent with those obtained by LD-PCR detection. Limit of detection (LOD) of the carrier mosaicism test was at least 2% of heterozygous cells with Inv22. Performing the test in two obligate mothers with negative Inv22 from two sporadic pedigrees mosaic rates of blood and hair root of the mother from pedigree 1 were 83% and >20%, respectively and negative results were obtained in pedigree 2.Conclusions: AccuCopy quantification combined with PLP (AQ-PLP) method was confirmed to be rapid and reliable for genotyping Inv22 and highly sensitive to carrier mosaicism detection. (C) 2016 Elsevier B.V. All rights reserved.