Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications

Abstract

In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.

Design and Validation of a Three-Dimensional Printer-Based System Enabling Rapid, Low-Cost Construction of the Biosensing Areas of Lateral Flow Devices for Immunoassays and Nucleic Acid Assays

“…DNA was isolated from a tissue biopsy sample of a patient with colorectal cancer, which had a known KRAS mutation (G12 V mutation) analyzed by next-generation sequencing (NGS). The isolated DNA was subjected to PCR and primer extension reaction (PEXT) for the simultaneous discrimination of three major KRAS mutations from the normal KRAS gene, as previously described . The multiplex PEXT product was analyzed with the LFA.…”

A Molecular Lateral Flow Assay for SARS-CoV-2 Quantitative Detection

“…The first set was compatible with the DNA sequence for SARS-CoV-2, and the second one corresponded to the internal standard DNA sequence. The coupling reaction was performed as described previously [ 38 ].…”

Gold Nanoparticles as a Biosensor for Cancer Biomarker Determination

“…Combining MutS-AuNPs with sequence hybridiza-tion can change the resonance frequency detected by a microcantilever resonator [122]. It can also be applied to liquid biopsy-related incidents, including the detection of circulating tumor cells and the detection of ctDNA for the diagnosis of cancer [123][124][125].…”