Division of Pathology, School of Molecular Medical Sciences, University of Nottingham, Nottingham, UK.
J Clin Pathol. 2010 Feb;63(2):134-40. doi: 10.1136/jcp.2009.070508.
Mutation detection in tumours will become increasingly important in pathological diagnosis as 'predictive' mutations are identified. A cheap and reliable test that works on formalin-fixed paraffin-embedded (FFPE) tissue is required.
The quick-multiplex-consensus (QMC)-PCR protocol was developed to be used with high-resolution melting (HRM) analysis. The assay was compared with Sanger sequencing. Robustness of the assay was tested in DNA from FFPE tissue.
QMC-PCR with HRM could detect a minimum of 2.5% of mutant alleles (compared with 20% detectable for Sanger sequencing). Ten mutation hotspots in KRAS, BRAF, PIK3CA and CDC4 were screened in 29 cell lines with 100% sensitivity and specificity. Forty-three FFPE colorectal tumours were sequenced for hotspots in KRAS and PIK3CA and then screened by QMC-PCR. There was 100% sensitivity, although, of 21 mutations detected by QMC-PCR, 16 were confirmed by sequencing (71% specificity, positive predictive value 76%). All 43 samples were then screened for mutations in all 10 hotspots. Of 430 tests, 43 (10%) showed aberrant melting and 36 were confirmed mutant (positive predictive value 84%). As our technique is more sensitive than direct sequencing, the remaining seven tests are probably sequencing false-negatives. Precision tests showed that there was little intra-assay and interassay variation.
QMC-PCR with HRM is a simple, robust and inexpensive technique which had greater sensitivity than Sanger sequencing. It allows multiple mutation hotspots to be rapidly screened and is thus highly suited to mutation detection in DNA derived from FFPE tissues.
随着“预测性”突变的确定，肿瘤中的突变检测在病理诊断中将变得越来越重要。需要一种廉价、可靠且适用于福尔马林固定石蜡包埋（FFPE）组织的测试。
开发了快速多重共识（QMC）-PCR 方案，用于高分辨率熔解（HRM）分析。该测定法与 Sanger 测序进行了比较。在 FFPE 组织的 DNA 中测试了测定法的稳健性。
QMC-PCR 与 HRM 相结合可检测到低至 2.5%的突变等位基因（而 Sanger 测序可检测到 20%的突变等位基因）。用 100%的灵敏度和特异性对 29 个细胞系中的 KRAS、BRAF、PIK3CA 和 CDC4 的 10 个突变热点进行了筛选。对 43 个 FFPE 结直肠癌肿瘤进行了 KRAS 和 PIK3CA 热点的测序，然后用 QMC-PCR 进行筛选。QMC-PCR 检测到的 21 个突变中有 100%的敏感性，但是，通过 QMC-PCR 检测到的 16 个突变中有 16 个通过测序证实（特异性 71%，阳性预测值 76%）。然后对所有 43 个样本进行了所有 10 个热点的突变筛查。在 430 次测试中，有 43 次（10%）出现异常熔解，其中 36 次被证实为突变（阳性预测值 84%）。由于我们的技术比直接测序更敏感，因此其余 7 次测试可能是测序假阴性。精密测试表明，内试验和间试验的变异很小。
QMC-PCR 与 HRM 是一种简单、稳健且廉价的技术，其灵敏度高于 Sanger 测序。它允许快速筛选多个突变热点，因此非常适合从 FFPE 组织中提取的 DNA 中的突变检测。
