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Multiplex-invasive reaction-assisted qPCR for quantitatively detecting the abundance of EGFR exon 19 deletions in cfDNA.
Analytical Methods ( IF 2.7 ) Pub Date : 2020-06-08 , DOI: 10.1039/d0ay00897d Yunmei Tang 1 , Bingjie Zou 2 , Runyuan Wang 1 , Nan Luo 1 , Xiemin Qi 2 , Guohua Zhou 2, 3 , Qinxin Song 1
Analytical Methods ( IF 2.7 ) Pub Date : 2020-06-08 , DOI: 10.1039/d0ay00897d Yunmei Tang 1 , Bingjie Zou 2 , Runyuan Wang 1 , Nan Luo 1 , Xiemin Qi 2 , Guohua Zhou 2, 3 , Qinxin Song 1
Affiliation
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Exon 19 deletions (19-Del) on the epidermal growth factor receptor (EGFR) gene are vital biomarkers for guiding tyrosine kinase inhibitor (TKI) treatment and the diagnosis of non-small cell lung cancer (NSCLC). However, it is difficult for conventional qPCR to quantitatively detect all 19-Del targets of EGFR, especially for cfDNA samples. Herein, a multiplex invasive reaction-assisted qPCR was proposed by employing a multiplex invasive reaction to distinguish 19-Del DNA targets from wild DNA targets and report them with different fluorescence signals in each PCR cycle. As all 19-Del targets have the same amplification efficiency and very similar invasive reaction efficiencies, the 19-Del abundance in a sample could be quantified by using the difference between the Ct values (ΔCt) of the deletion targets and the wild targets without the requirement of a standard calibration curve. Combining the high sensitivity of PCR and the high specificity of the invasive reaction, this method can detect 10 copies of the deletion targets and lower than 0.1% deletion abundance. The results were 100% consistent with ARMS-PCR for the 38 tumor tissues tested and were in good agreement with next-generation sequencing for quantifying the abundance of EGFR 19-Del in 15 cfDNA samples, showing the great potential of the method for liquid biopsies.
中文翻译：
多重入侵反应辅助qPCR用于定量检测cfDNA中EGFR外显子19缺失的丰度。
表皮生长因子受体（EGFR）基因上的第19外显子缺失（19-Del）是指导酪氨酸激酶抑制剂（TKI）治疗和诊断非小细胞肺癌（NSCLC）的重要生物标志物。但是，常规qPCR难以定量检测EGFR的所有19-Del靶标，特别是对于cfDNA样品。在本文中，通过采用多重侵入性反应来区分19-Del DNA靶与野生DNA靶并在每个PCR循环中报告不同的荧光信号，提出了多重侵入性反应辅助qPCR。由于所有的19德尔目标具有相同的扩增效率和非常相似的侵入性反应效率，19德尔丰度的样品中可以通过使用之间的差值被量化Ç吨（Δ值Çt）删除目标和野生目标，无需标准校准曲线。结合PCR的高灵敏度和侵袭性反应的高特异性，该方法可检测10个拷贝的缺失靶标，且缺失丰度低于0.1％。结果与用于38个肿瘤组织的ARMS-PCR的结果100％一致，并且与用于定量分析15个cfDNA样品中EGFR 19-Del丰度的下一代测序技术吻合良好，显示了液体活检方法的巨大潜力。
更新日期：2020-07-09
中文翻译：
多重入侵反应辅助qPCR用于定量检测cfDNA中EGFR外显子19缺失的丰度。
表皮生长因子受体（EGFR）基因上的第19外显子缺失（19-Del）是指导酪氨酸激酶抑制剂（TKI）治疗和诊断非小细胞肺癌（NSCLC）的重要生物标志物。但是，常规qPCR难以定量检测EGFR的所有19-Del靶标，特别是对于cfDNA样品。在本文中，通过采用多重侵入性反应来区分19-Del DNA靶与野生DNA靶并在每个PCR循环中报告不同的荧光信号，提出了多重侵入性反应辅助qPCR。由于所有的19德尔目标具有相同的扩增效率和非常相似的侵入性反应效率，19德尔丰度的样品中可以通过使用之间的差值被量化Ç吨（Δ值Çt）删除目标和野生目标，无需标准校准曲线。结合PCR的高灵敏度和侵袭性反应的高特异性，该方法可检测10个拷贝的缺失靶标，且缺失丰度低于0.1％。结果与用于38个肿瘤组织的ARMS-PCR的结果100％一致，并且与用于定量分析15个cfDNA样品中EGFR 19-Del丰度的下一代测序技术吻合良好，显示了液体活检方法的巨大潜力。
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