title: Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags.
abstract: Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research.
authors.affiliation: 1. Center for Personalized Nanomedicine. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Queensland, Australia.
authors.forename: Eugene J H
authors.initials: EJ
authors.lastname: Wee
authors.affiliation: 1. Center for Personalized Nanomedicine. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Queensland, Australia.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 3. Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia; 4. Department of Surgery, University of Melbourne, Austin Health. Heidelberg, Victoria, Australia.
authors.forename: Simon Chang-Hao
authors.initials: SC
authors.lastname: Tsao
authors.affiliation: 1. Center for Personalized Nanomedicine. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Queensland, Australia; 2. School of Chemistry and Molecular Biosciences, The University of Queensland, Queensland, Australia.
authors.forename: Matt
authors.initials: M
authors.lastname: Trau
mesh_terms: D004247:DNA / Q000097:blood* / Q000235:genetics*; D006801:Humans; D008545:Melanoma / Q000175:diagnosis* / Q000473:pathology*; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation; D012680:Sensitivity and Specificity; D013059:Spectrum Analysis, Raman / Q000379:methods*
chemical_list: D004247:DNA
keywords: BRAF; NRAS; SERS; cKIT; ctDNA; melanoma; multiplex PCR