ViennaLab Diagnostics GmbH, Gaudenzdorfer Gürtel 43-45,Vienna, Austria.
J Clin Pathol. 2011 Mar;64(3):252-6. doi: 10.1136/jcp.2010.081414. Epub 2011 Jan 22.
To evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched PCR followed by reverse-hybridisation of biotinylated amplification products to oligonucleotide probes immobilised as an array of parallel lines on nitrocellulose test strips.
The strip assay was used to analyse genomic DNA isolated from 120 formalin-fixed paraffin-embedded (FFPE) ovarian tissue samples. The samples were analysed in parallel using a biochip-based protocol (biochip assay) covering the same mutation spectrum, and results were compared with respect to sensitivity, specificity and operational input.
The strip assay identified 19 (16%) of 120 FFPE samples to carry a KRAS mutation; results were in agreement with those obtained by biochip hybridisation. Both assays had an analytical sensitivity of 1% when performed on FFPE-extracted DNA with approximately the same operational input needed for post-PCR processing. In contrast to the biochip assay, strip assay hybridisation may be automated to a large extent.
The strip assay is an accurate and sensitive tool for the low to medium throughput detection of KRAS mutation in genomic DNA isolated from FFPE tissue.
评估一种设计用于灵敏检测 KRAS 基因密码子 12 和 13 中 10 种突变的反向杂交检测（条带检测）。该条带检测依赖于突变富集 PCR，然后将生物素标记的扩增产物反向杂交到固定在硝酸纤维素测试条上的寡核苷酸探针上，探针呈平行线条阵列排列。
该条带检测用于分析从 120 个福尔马林固定石蜡包埋（FFPE）卵巢组织样本中分离的基因组 DNA。使用基于生物芯片的协议（生物芯片检测）对样本进行平行分析，该协议涵盖相同的突变谱，并比较了敏感性、特异性和操作输入。
条带检测鉴定出 120 个 FFPE 样本中的 19 个（16%）携带 KRAS 突变；结果与生物芯片杂交检测结果一致。当对 FFPE 提取的 DNA 进行检测时，两种检测方法的分析灵敏度均为 1%，且在后 PCR 处理过程中需要的操作输入大致相同。与生物芯片检测不同，条带检测杂交可以在很大程度上实现自动化。
该条带检测是一种用于从 FFPE 组织中分离的基因组 DNA 中低至中通量检测 KRAS 突变的准确、敏感工具。
