Diagnostic Guidelines for High-Resolution Melting Curve (HRM) Analysis: An Interlaboratory Validation of BRCA1 Mutation Scanning Using the 96-Well LightScanner™
Diagnostic Guidelines for High-Resolution Melting Curve (HRM) Analysis: An Interlaboratory Validation of BRCA1 Mutation Scanning Using the 96-Well LightScanner™
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DOI：
10.1002/humu.21004
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发表时间：
2009-06-01
期刊：
影响因子：
3.9
通讯作者：
Bakker, Egbert
中科院分区：
文献类型：
--
作者：
van der Stoep, Nienke;van Paridon, Chantal D. M.;Bakker, Egbert
通过测序对 BRCA1 进行遗传分析之前，通常会先进行扫描方法，例如变性梯度凝胶电泳 (DGGE)、蛋白质截短测试 (PTT) 或 DHPLC。高分辨率熔解曲线（HRM）分析是一种有前途且经济的高通量突变扫描方法。 EuroGentest 网络 (www.eurogentest.org) 旨在协助在诊断环境中引入新技术。因此，我们与 LightScanner (TM) (LS) 制造商 Idaho Technology 合作，对 HRM 进行了彻底、高标准的实验室间评估和验证。通过对 170 个变体的详细研究，我们制定了轻松设置和实施 HRM 作为新基因扫描技术的指南，该技术适用于单个诊断实验室的质量体系。该验证研究包括使用 96 孔 LS 进行 BRCA1 特异性突变筛查测试的描述。该测定包含 40 个扩增子，并使用具有统计学意义的精心设计的变体组和对照 DNA 样本进行评估。检测到所有杂合变异。此外，对九种常见多态性的基因型分析为这些频繁出现的非致病性变异创建了快速筛选和检测方法。一项使用总共 28 个患者来源 DNA 样本的盲法研究也实现了 100% 检测，平均特异性为 98%，表明假阳性 (FP) 发生率较低。 Hum Mutat 30, 899-909, 2009。(C) 2009 Wiley-Liss, Inc.
Genetic analysis of BRCA1 by sequencing is often preceded by a scanning method like denaturing gradient gel electrophoresis (DGGE), protein truncation test (PTT) or DHPLC. High-resolution melting curve (HRM) analysis is a promising and economical method for high-throughput Mutation scanning. The EuroGentest network (www.eurogentest.org) aims to assist with the introduction of novel technologies in the diagnostic setting. Therefore, we have performed a thorough and high, standard interlaboratory evaluation and validation of HRM, in collaboration with Idaho Technology, the manufacturer of the LightScanner (TM) (LS). Through this detailed study of 170 variants, we have generated guidelines for easy setup and implementation of HRM as a scanning technique for new genes, which are adaptable to the quality system of an individual diagnostic laboratory. This validation study includes the description of a BRCA1-specific mutation screening test using the 96-well LS. This assay comprises 40 amplicons and was evaluated using a statistically significant elaborate panel of variants and control DNA samples. All heterozygous variants were detected. Moreover, genotype analysis for nine common polymorphisms created a fast screening and detection method for these frequently occurring nonpathogenic variants. A blind study using a total of 28 patient-derived DNA samples resulted also in 100% detection and showed an average specificity of 98%, indicating a low incidence of false positives (FPs). Hum Mutat 30, 899-909, 2009. (C) 2009 Wiley-Liss, Inc.