Solid-Phase Microextraction Enables Isolation of BRAF V600E Circulating Tumor DNA from Human Plasma for Detection with a Molecular Beacon Loop-Mediated Isothermal Amplification Assay

摘要

Circulating tumor DNA (ctDNA) is a promising biomarker that can provide a wealth of information regarding the genetic makeup of cancer as well as provide a guide for monitoring treatment. Methods for rapid and accurate profiling of ctDNA are highly desirable in order to obtain the necessary information from this biomarker. However, isolation of ctDNA and its subsequent analysis remains a challenge due to the dependence on expensive and specialized equipment. In order to enable widespread implementation of ctDNA analysis, there is a need for low-cost and highly accurate methods that can be performed by nonexpert users. In this study, an assay is developed that exploits the high specificity of molecular beacon (MB) probes with the speed and simplicity of loop-mediated isothermal amplification (LAMP) for the detection of the BRAF V600E single-nucleotide polymorphism (SNP). Furthermore, solid-phase microextraction (SPME) is applied for the successful isolation of clinically relevant concentrations (73.26 fM) of ctDNA from human plasma. In addition, the individual effects of plasma salts and protein on the extraction of ctDNA with SPME are explored. The performed work expands the use of MB-LAMP for SNP detection as well as demonstrates SPME as a sample preparation tool for nucleic acid analysis in plasma.