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.
Nucleic acid detection is widely used in the amplification and quantitation of nucleic acids from biological samples. While polymerase chain reaction (PCR) enjoys great popularity, expensive thermal cyclers are required for precise temperature control. Loop-mediated isothermal amplification (LAMP) enables highly sensitive, rapid, and low-cost amplification of nucleic acids at constant temperatures. LAMP detection often relies on double-stranded DNA-binding dyes or metal indicators that lack sequence selectivity. Molecular beacons (MBs) are hairpin-shaped oligonucleotide probes whose sequence specificity in LAMP provides the capability of differentiating between singlenucleotide polymorphisms (SNPs). Digital droplet LAMP (ddLAMP) enables a large number of independent LAMP reactions to be performed and provides quantification of target DNA sequences. However, a major challenge with ddLAMP is the requirement of expensive droplet generators to form homogeneous microdroplets. In this study, we demonstrate for the first time that a three-dimensional (3D) printed droplet generation platform can be coupled to a LAMP assay featuring MBs as sequencespecific probes. The low-cost 3D printed droplet generator system was designed, and its customizability was demonstrated in the formation of monodisperse ddLAMP assay-in-oil microdroplets. Additionally, a smartphone-based imaging system is demonstrated to increase accessibility for point-of-care applications. The MB-ddLAMP assay is shown to discriminate between two SNPs at various amplification temperatures to afford a useful platform for sequence-specific, sensitive, and accurate DNA quantification. This work expands the utility of MBs to ddLAMP for quantitative studies in the detection of SNPs and exploits the customizability of 3D printing technologies to optimize the homogeneity, size, and volume of oil-in-water microdroplets.
Loop-mediated
isothermal amplification (LAMP) holds great potential
for point-of-care (POC) diagnostics due to its speed and sensitivity.
However, differentiation between spurious amplification and amplification
of the target sequence is a challenge. Herein, we develop the use
of molecular beacon (MB) probes for the sequence-specific detection
of LAMP on commercially available lateral flow immunoassay (LFIA)
strips. The detection of three unique DNA sequences, including ORF1a
from SARS-CoV-2, is demonstrated. In addition, the method is capable
of detecting clinically relevant single-nucleotide polymorphisms (BRAF
V600E). For all sequences tested, the LFIA method offers similar sensitivity
to fluorescence detection using a qPCR instrument. We also demonstrate
the coupling of the method with solid-phase microextraction to enable
isolation and detection of the target sequences from human plasma,
pond water, and artificial saliva. Lastly, a 3D printed device is
designed and implemented to prevent contamination caused by opening
the reaction containers after LAMP.
Nucleic
acid analysis has been at the forefront of the COVID-19
global health crisis where millions of diagnostic tests have been
used to determine disease status as well as sequencing techniques
that monitor the evolving genome of SARS-CoV-2. In this study, we
report the development of a sample preparation method that decreases
the time required for DNA isolation while significantly increasing
the sensitivity of downstream analysis. Functionalized planar supports
are modified with a polymeric ionic liquid sorbent coating to form
thin film microextraction (TFME) devices. The extraction devices are
shown to have a high affinity for DNA while also exhibiting high reproducibility
and reusability. Using quantitative polymerase chain reaction (qPCR)
analysis, the TFME devices are shown to require low equilibration
times while achieving higher preconcentration factors than solid-phase
microextraction (SPME) by extracting larger masses of DNA. Rapid desorption
kinetics enable higher DNA recoveries using desorption solutions that
are less inhibitory to qPCR and loop-mediated isothermal amplification
(LAMP). To demonstrate the advantageous features of the TFME platform,
a customized leuco crystal violet LAMP assay is used for visual detection
of the ORF1ab DNA sequence from SARS-CoV-2 spiked into artificial
oral fluid samples. When coupled to the TFME platform, 100% of LAMP
reactions were positive for SARS-CoV-2 compared to 66.7% obtained
by SPME for a clinically relevant concentration of 4.80 × 106 DNA copies/mL.
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