A more sensitive platform for the detection of low-abundance BRAFV600E mutations
A more sensitive platform for the detection of low-abundance BRAFV600E mutations
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DOI：
10.1007/s11010-012-1282-2
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发表时间：
2012-03
影响因子：
4.3
通讯作者：
Weiqin Jiang;Weibin Wang;Fangfang Fu;X. Teng;Haohao Wang;Haiyong Wang;L. Teng
中科院分区：
文献类型：
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作者：
Weiqin Jiang;Weibin Wang;Fangfang Fu;X. Teng;Haohao Wang;Haiyong Wang;L. Teng
识别低丰度突变对于癌症的治疗和诊断非常重要。由于肿瘤异质性的可能性，癌症相关突变的有效检测在很大程度上取决于所用方法的灵敏度。为了确认突变检测平台是否影响 BRAFV600E 的认知患病率及其与甲状腺乳头状癌 (PTC) 临床病理特征的相关性，我们比较了桑格测序 (SS)、焦磷酸测序 (PS) 和新建的等位基因特异性实时 PCR (AS-qPCR) 装置，用于在中国传统变异 PTC 队列中检测 BRAFV600E。建立了准确的质粒标准品来评估三个平台的检测限。在这项研究中，AS-qPCR 被发现在检测突变方面最灵敏、最可靠。 AS-qPCR 检测到的突变由于丰度低而无法通过 SS 或 PS 检测到，通过突变富集平台 COLD-PCR 和 SS 进行确认。通过 AS-qPCR 分析时，BRAFV600E 与更具攻击性的表型相关。我们的结果表明，报道的 PTC 中 BRAFV600E 突变的患病率被低估，临床环境中应应用更灵敏的方法，例如 AS-qPCR。
Identifying low-abundance mutations is important for the therapy and diagnose of cancer. Since the potential for tumor heterogeneity, the efficient detection of cancer-relevant mutations largely depends on the sensitivity of the methods employed. To confirm whether the mutation detection platforms affect the perceived prevalence of the BRAFV600Eand its correlation with clinicopathologic features in papillary thyroid carcinomas (PTC), we compared Sanger Sequencing (SS), Pyrosequencing (PS), and a newly built allele-specific real-time PCR (AS-qPCR) apparatus for the detection of BRAFV600Ein a Chinese cohort of conventional variant PTC. Accurate plasmid standards were built to assess the limit of detection of the three platforms. In this research, AS-qPCR has been found both the most sensitive and reliable at detecting mutation. The mutations detected by AS-qPCR which were not detected by SS or PS due to low abundance were confirmed by mutation enrichment platform COLD-PCR followed by SS. When analyzed by AS-qPCR, BRAFV600Ewas associated with a more aggressive phenotype. Our results indicate that the reported prevalence of the BRAFV600Emutations in PTC has been underestimated and more sensitive methods such as AS-qPCR should be applied in clinical settings.