Jump to main content
Jump to site search
PLANNED MAINTENANCE Close the message box

Scheduled maintenance work on Wednesday 27th March 2019 from 11:00 AM to 1:00 PM (GMT).

During this time our website performance may be temporarily affected. We apologise for any inconvenience this might cause and thank you for your patience.


Issue 5, 2019
Previous Article Next Article

Ultrasensitive and quantitative detection of EGFR mutations in plasma samples from patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-PNA PCR clamp

Author affiliations

Abstract

Circulating tumour DNA (ctDNA) is a potential proxy for tumour tissues. However, the analysis of mutations and mutational abundance using ctDNA remains challenging because ctDNA is present at low levels. In addition, the concordance between plasma and tumour tissues requires further investigation by high-sensitivity techniques. Here, we established an ultrasensitive, quantitative method for detecting rare mutations in plasma samples based on a dual PNA clamping-mediated LNA-PNA PCR clamp (LNA-dPNA PCR clamp). The novelty of our method is the coupling of PNA clamping with one-tube nested PCR to dually block wild-type DNA amplification and efficiently amplify mutant DNA. Then, four hotspot EGFR mutations (EGFR L858R, EGFR Exon 19 deletion, EGFR T790M, and EGFR C797S) were detected by our proposed method. Finally, we evaluated the concordance between plasma and tumour tissues by simultaneously detecting EGFR L858R by ddPCR and LNA-dPNA PCR clamp in 132 tissues and matched plasma samples from patients with NSCLC. For the four EGFR mutations, the amplification sensitivity of the LNA-dPNA PCR clamp was 100 copies per reaction, and the linearity was from 100 to 106–107 copies per reaction. The limit of detection for the LNA-dPNA PCR clamp was 0.01%–0.1%. The LNA-dPNA PCR clamp was similarly consistent with ddPCR in quantifying mutational abundance (R2 = 0.9568) and exhibited similar limit of detection (0.01%–0.1% vs. 0.01%), sensitivity (19.6 vs. 21.7), specificity (94.2 vs. 91.9), and concordance (68.2 vs. 67.4) to those of ddPCR for ctDNA detection. In conclusion, the LNA-dPNA PCR clamp will provide a labour-saving, cost-saving, ultrasensitive tool for detecting and quantifying plasma EGFR mutations.

Graphical abstract: Ultrasensitive and quantitative detection of EGFR mutations in plasma samples from patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-PNA PCR clamp

Back to tab navigation

Supplementary files

Publication details

The article was received on 17 Dec 2018, accepted on 19 Dec 2018 and first published on 21 Jan 2019


Article type: Paper
DOI: 10.1039/C8AN02446D
Citation: Analyst, 2019,144, 1718-1724

  •   Request permissions

    Ultrasensitive and quantitative detection of EGFR mutations in plasma samples from patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-PNA PCR clamp

    S. Zhang, Z. Chen, C. Huang, C. Ding, C. Li, J. Chen, J. Zhao and L. Miao, Analyst, 2019, 144, 1718
    DOI: 10.1039/C8AN02446D

Search articles by author

Spotlight

Advertisements