Multiplex detection of ctDNA mutations in plasma of colorectal cancer patients by PCR/SERS assay

Abstract

Molecular diagnostic testing of KRAS and BRAF mutations has become critical in the management of colorectal cancer (CRC) patients. Some progress has been made in liquid biopsy detection of mutations in circulating tumor DNA (ctDNA), which is a fraction of circulating cell-free DNA (cfDNA), but slow analysis for DNA sequencing methods has limited rapid diagnostics. Other methods such as quantitative PCR and more recently, droplet digital PCR (ddPCR), have limitations in multiplexed capacity and the need for expensive specialized equipment. Hence, a robust, rapid and facile strategy is needed for detecting multiple ctDNA mutations to improve the management of CRC patients. To address this significant problem, herein, we propose a new application of multiplex PCR/SERS (surface-enhanced Raman scattering) assay for the detection of ctDNA in CRC, in a fast and non-invasive manner to diagnose and stratify patients for effective treatment. Methods: To discriminate ctDNA mutations from wild-type cfDNA, allele-specific primers were designed for the amplification of three clinically important DNA point mutations in CRC including KRAS G12V, KRAS G13D and BRAF V600E. Surface-enhanced Raman scattering (SERS) nanotags were labelled with a short and specific sequence of oligonucleotide, which can hybridize with the corresponding PCR amplicons. The PCR/SERS assay was implemented by firstly amplifying the multiple mutations, followed by binding with multicolor SERS nanotags specific to each mutation, and subsequent enrichment with magnetic beads. The mutation status was evaluated using a portable Raman spectrometer where the fingerprint spectral peaks of the corresponding SERS nanotags indicate the presence of the mutant targets. The method was then applied to detect ctDNA from CRC patients under a blinded test, the results were further validated by ddPCR. Results: The PCR/SERS strategy showed high specificity and sensitivity for genotyping CRC cell lines and plasma ctDNA, where as few as 0.1% mutant alleles could be detected from a background of abundant wild-type cfDNA. The blinded test using 9 samples from advanced CRC patients by PCR/SERS assay was validated with ddPCR and showed good consistency with pathology testing results. Conclusions: With ddPCR-like sensitivity yet at the convenience of standard PCR, the proposed assay shows great potential in sensitive detection of multiple ctDNA mutations for clinical decision-making.

Keywords: CRC; PCR/SERS assay; ctDNA; ddPCR; multiplex detection.

Figure 1

Scheme of the multiplex PCR/SERS assay. Multiplex mutation-specific primers were used to amplify mutant targets (the wild-type dsDNA is shown in black). Amplicons were then labelled with mutation-specific nanotags and enriched with streptavidin magnetic beads. The status of mutations was then analysed with SERS spectrum where unique spectral peaks demonstrated the presence of targeting mutations.

Figure 2

Specific amplification of the mutant targets by PCR and specific identification of PCR amplicons by SERS nanotags. (A, B, C) Left: Gel electrophoresis verified the specific amplification of the mutant gDNA with the corresponding individual primers, where no amplification of the wild-type gDNA was observed. Right: Specific detection of individual mutant PCR products with the corresponding single SERS nanotags; (D) Multiplex detection of the mutant targets with 3-plex primers and 3-plex SERS nanotags. NTC is the no template control.

Figure 3

Detection of low levels of KRAS G12V mutation load. (A) Gel electrophoresis image, (B) typical raw Raman spectra and (C) bar graph of average SERS intensities at 1376 cm^-1 over a range of mutation loads for 10,000 input copies. NTC is the no template control. Error bar represents standard deviation (SD) of 3 independent experiments.

Figure 4

Detection of serially diluted BRAF V600E mutation. (A) Typical raw Raman spectra, (B) Bar graph of average SERS intensities at 1342 cm^-1 over a range of 0% to 10% of BRAF mutant allele frequency (MAF). Error bar represents SD of 3 independent experiments. (C) ddPCR results for serially diluted samples (# copy number per sample, 4 ng of template was used in each case).

Figure 5

Detection of BRAF V600E mutation using cfDNA from CRC patients. (A) Bar graph of average SERS intensities at 1342 cm^-1 for patients' samples, where 61T represents the patient #61 after 4 months of chemotherapy. (B) Results for ddPCR assay (# copy number per sample).