Single‐molecule detection of cancer mutations using a novel PCR‐LDR‐qPCR assay

Ruiz, Cristian; Huang, Jianmin; Giardina, Sarah F.; Feinberg, Philip B.; Mirza, Aashiq H.; Bacolod, Manny D.; Soper, Steven A.; Barany, Francis

doi:10.1002/humu.23987

Cited by 9 publications

(7 citation statements)

References 68 publications

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“…Moreover, the test involves many washing and incubation steps with a total analysis time of about 2 h [ 48 ]. Compared to the previously reported biosensors for ctDNA detection in real samples ( Table S3 ) [ 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 ], the advantages of the proposed multi-plex test are related mostly to the cost, run time, and simplicity, as well as the universality and advanced multiplexing potential.…”

Section: Discussionmentioning

confidence: 92%

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

et al. 2022

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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.

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Section: Discussionmentioning

confidence: 92%

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

et al. 2022

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“…Many researchers combined PCR with LDR for gene identification. For example, Ruiz et al applied this method to establish a specific, rapid, and practical liquid biopsy strategy [ 22 ]. Zhang Xinya et al used this method to detect the D614G mutation in the fragmented coronavirus S gene down to a 40-nt fragment length, which was a shorter template length than that required for the probe method of fluorescent quantitative PCR and had an advantage in detecting fragmented templates [ 23 ].…”

Section: Ligase-related Nucleic Acid Amplification Techniquesmentioning

confidence: 99%

Advances in ligase-based nucleic acid amplification technology for detecting gene mutations: a review

Liu

Wang

et al. 2022

Mol Cell Biochem

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Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore methods to detect gene mutations, which may help in disease diagnosis, medication guidance, and so on. Currently, the detection methods, such as whole-genome sequencing and polymerase chain reaction, have some limitations in terms of cost and sensitivity. Ligase (an enzyme) can recognize base mismatch as a commonly used tool in genetic engineering. Therefore, the ligase-related nucleic acid amplification technology for detecting gene mutations has become a research hotspot. In this study, the main techniques explored for detecting gene mutations included the ligase detection reaction, ligase chain reaction, rolling circle amplification reaction, enzyme-assisted polymerase chain reaction, and loop-mediated isothermal amplification reaction. This review aimed to analyze the aforementioned techniques and mainly present their advantages and disadvantages, sensitivity, specificity, cost, detection time, applications, and so on. The findings may help develop sufficient grounds for further studies on detecting gene mutations.

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“…In a separate article, a similar approach was employed to demonstrate the sensitivity of our PCR-LDR-qPCR assay, designed to interrogate mutations in cfDNA samples. 54 5' CG CA C CG CT CG AT GC GT G GC GA GC TA…”

Section: Single-molecule Detection Of Vim Methylation By Pixel Pcr-ldr-qpcrmentioning

confidence: 99%

Application of Multiplex Bisulfite PCR–Ligase Detection Reaction–Real-Time Quantitative PCR Assay in Interrogating Bioinformatically Identified, Blood-Based Methylation Markers for Colorectal Cancer

Bacolod

Mirza

Huang

et al. 2020

The Journal of Molecular Diagnostics

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The analysis of CpG methylation in circulating tumor DNA fragments has emerged as a promising approach for the noninvasive early detection of solid tumors, including colorectal cancer (CRC). The most commonly employed assay involves bisulfite conversion of circulating tumor DNA, followed by targeted PCR, then real-time quantitative PCR (alias methylation-specific PCR). This report demonstrates the ability of a multiplex bisulfite PCReligase detection reactionereal-time quantitative PCR assay to detect seven methylated CpG markers (CRC or colon specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with approximately 3000 copies of fragmented peripheral blood DNA) and CRC patientederived cell-free DNAs. This scalable assay is designed for multiplexing and incorporates steps for improved sensitivity and specificity, including the enrichment of methylated CpG fragments, ligase detection reaction, the incorporation of ribose bases in primers, and use of uracil DNA glycosylase. Six of the seven CpG markers (located in promoter regions of PPP1R16B, KCNA3, CLIP4, GDF6, SEPT9, and GSG1L) were identified through integrated analyses of genome-wide methylation data sets for 31 different types of cancer. These markers were mapped to CpG sites at the promoter region of VIM; VIM and SEPT9 are established epigenetic markers of CRC. Additional bioinformatics analyses show that the methylation at these CpG sites negatively correlates with the transcription of their corresponding genes.

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Single‐molecule detection of cancer mutations using a novel PCR‐LDR‐qPCR assay

Cited by 9 publications

References 68 publications

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

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

Advances in ligase-based nucleic acid amplification technology for detecting gene mutations: a review

Application of Multiplex Bisulfite PCR–Ligase Detection Reaction–Real-Time Quantitative PCR Assay in Interrogating Bioinformatically Identified, Blood-Based Methylation Markers for Colorectal Cancer

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