Colorimetric Nucleic Acid Research Articles

Preamplification-free viral RNA diagnostics with single-nucleotide resolution using MARVE, an origami paper-based colorimetric nucleic acid test.

The evolution and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgent concerns as they pose the risk of vaccine failure and increased viral transmission. However, affordable and scalable tools allowing rapid identification of SARS-CoV-2 variants are not readily available, which impedes diagnosis and epidemiological surveillance. Here we present a colorimetric nucleic acid assay named MARVE (multiplexed, preamplification-free, single-nucleotide-resolved viral evolution) that is convenient to perform and yields single-nucleotide resolution. The assay integrates nucleic acid strand displacement reactions with enzymatic amplification to colorimetrically sense viral RNA using a metal ion-incorporated DNA probe (TEprobe). We provide detailed guidelines to design TEprobes for discriminating single-nucleotide variations in viral RNAs, and to fabricate a test paper for the detection of SARS-CoV-2 variants of concern. Compared with other nucleic acid assays, our assay is preamplification-free, single-nucleotide-resolvable and results are visible via a color change. Besides, it is smartphone readable, multiplexed, quick and cheap ($0.30 per test). The protocol takes ~2 h to complete, from the design and preparation of the DNA probes and test papers (~1 h) to the detection of SARS-CoV-2 or its variants (30-45 min). The design of the TEprobes requires basic knowledge of molecular biology and familiarity with NUPACK or the Python programming language. The fabrication of the origami papers requires access to a wax printer using the CAD and PDF files provided or requires users to be familiar with AutoCAD to design new origami papers. The protocol is also applicable for designing assays to detect other pathogens and their variants.

Janus Fe3O4-Au@Pt nanozymes based lateral flow assay for enhanced-sensitive colorimetric detection of influenza A virus

The traditional colloidal gold-based lateral flow assay (LFA) is widely applied in point-of-care testing (POCT) for pathogens. However, its disadvantage of low sensitivity greatly limits its effectiveness in early detection. In this work, we developed an enhanced-sensitive colorimetric nucleic acid lateral flow assay (NALFA) based on Janus Fe3O4-Au@Pt nanoparticles (Fe3O4-Au@Pt NPs) for H1N1 influenza virus detection. Dumbbell-like heterodimer Fe3O4-Au@Pt NPs were prepared by a seed-mediated method, and their unique Janus structure endowed them with both good plasmonic property and high peroxidase-like activity. With the Fe3O4-Au@Pt NPs as NALFA signal labels, qualitative detection was achieved by observing the color change with the naked eyes, and quantitative results were obtained by measuring the gray values on test strips. Due to the strong peroxidase-like activity of Fe3O4-Au@Pt NPs, they could quickly catalyze 3,3′,5,5′-tetramethylbenzidine (TMB) to oxidize to the dark blue chelate (oxTMB), thus resulting in greatly enhanced color on the strips. The limit of detection (LOD) of NALFA reached as low as 50 pM, which was 20-fold lower than that of before catalysis. Therefore, the constructed Janus Fe3O4-Au@Pt NPs could effectively improve the performance of traditional colloidal gold LFA and provide a potential tool for the development of sensitive sensing platforms.

Signal amplified colorimetric nucleic acid detection based on autocatalytic hairpin assembly.

Herein, a nucleic acid assay based on autocatalytic hairpin assembly (ACHA) was proposed. In this system, two split G-quadruplex sequences were integrated into H1 and H2, respectively. And a DNA strand with the same sequence to target DNA was integrated into the assistant hairpin H3. In the presence of target DNA, the hairpin structure of H1 was opened and catalytic hairpin assembly (CHA) was activated, and then a series of DNA assembly steps based on the toehold-mediated DNA strand displacement were triggered and the product H1-H2 with sticky ends on both sides was formed. On the one side of H1-H2, the split two G-quadruplex sequences were close enough to form the intact G-quadruplex for the signal readout. At the same time, two sticky ends on the other side of H1-H2 hybridized with H3 and a new sticky end with the sequence same to the target DNA was exposed, which can immediately trigger the autocatalytic hairpin assembly reaction, and then the reaction rate of CHA was effectively accelerated and the colorimetric signal was significantly amplified. This ACHA signal amplified strategy has been successfully applied for the rapid and colorimetric nucleic acid detection.

AI-driven colorimetric nucleic acid test for tilapia lake virus: A large-scale, point-of-care diagnostic model for future emerging diseases

It is indisputable that the world is currently facing several health challenges, which include outbreaks of diseases. One way to limit the spread of such diseases is to have simple yet efficient bioassays that work well in large-scale operation. To address this issue, a new artificial intelligence (AI) based colorimetric DNA/RNA detection platform was developed using tilapia lake virus as a model. The assay uses a newly formulated indicator dye and colorimetric nucleic acid amplification done in a thermos or a simple heating block to produce visible results that can be observed by the naked eye. To ensure high-throughput screening, an AI-based analysis web application was developed that can be used on computers, tablets, and smartphones to accurately and quickly determine the colorimetric results. With a genetic material extraction procedure that takes only 5 min and requires no equipment, our assay can be completed within an hour from sampling to readout, with 96% accuracy and a sensitivity approaching a single copy of the target virus. The entire assay incubation can be done in a simple thermos, as a substitute for thermal cyclers, rendering its potential use in remote areas where access to electricity is restricted. To demonstrate the versatility of our platform, we modified our testing solution by changing pathogen-specific primers and tested it on various target pathogens that infect humans, animals, and plants. The results were accurate, indicating that the platform has the potential to be used for point-of-care disease diagnosis in the future.

Microfluidic-based colorimetric nucleic acid detection of pathogens

In this review, we highlight the most recent advances in the nucleic acid detection of viruses, bacteria, fungi, and protozoa in a portable fashion using colorimetric-based microfluidic devices.

CRISPR-Based Colorimetric Nucleic Acid Tests for Visual Readout of DNA Barcode for Food Authenticity.

Food authenticity is a critical issue associated with the economy, religion, and food safety. Herein, we report a label-free and colorimetric nucleic acid assay for detecting DNA barcodes, enabling the determination of food authenticity with the naked eye. This method, termed the CRISPR-based colorimetric DNA barcoding (Cricba) assay, utilizes CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR associated protein) to specifically recognize the polymerase chain reaction (PCR) products for further trans-cleavaging the peroxidase-mimicking G-quadruplex DNAzyme. Based on this principle, the presence of the cytochrome oxidase subunit I gene could be directly observed with the naked eye via the color change of 3,3',5,5'-tetramethylbenzidine sulfate (TMB). The whole detection process, including PCR amplification and TMB colorimetric analysis, can be completed within 90 min. The proposed assay can detect pufferfish concentrations diluted to 0.1% (w/w) in a raw pufferfish mixture, making it one of the most sensitive methods for food authenticity. The robustness of the assay was verified by testing four common species of pufferfish, including Lagocephalus inermis, Lagocephalus spadiceus, Takifugu bimaculatus, and Takifugu alboplumbeus. The assay is advantageous in easy signal readout, high sensitivity, and general applicability and thus could be a competitive candidate for food authenticity.

Colorimetric Approach for Nucleic Acid Salmonella spp. Detection: A Systematic Review.

Water- and food-related health issues have received a lot of attention recently because food-poisoning bacteria, in particular, are becoming serious threats to human health. Currently, techniques used to detect these bacteria are time-consuming and laborious. To overcome these challenges, the colorimetric strategy is attractive because it provides simple, rapid and accurate sensing for the detection of Salmonella spp. bacteria. The aim of this study is to review the progress regarding the colorimetric method of nucleic acid for Salmonella detection. A literature search was conducted using three databases (PubMed, Scopus and ScienceDirect). Of the 88 studies identified in our search, 15 were included for further analysis. Salmonella bacteria from different species, such as S. Typhimurium, S. Enteritidis, S. Typhi and S. Paratyphi A, were identified using the colorimetric method. The limit of detection (LoD) was evaluated in two types of concentrations, which were colony-forming unit (CFU) and CFU per mL. The majority of the studies used spiked samples (53%) rather than real samples (33%) to determine the LoDs. More research is needed to assess the sensitivity and specificity of colorimetric nucleic acid in bacterial detection, as well as its potential use in routine diagnosis.

CASCADE: Naked eye-detection of SARS-CoV-2 using Cas13a and gold nanoparticles

The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials.Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs.In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases.

Ultrasensitive colorimetric miRNA detection based on magnetic 3D DNA walker and unmodified AuNPs

Abstract Gold nanoparticles (AuNPs) based colorimetric assay is a promising platform for simple and low-cost point-of-care (POC) testing, but tends to have low sensitivity, which limits their clinical applications for trace nucleic acid assays. Herein, a simple colorimetric assay was developed by using unmodified AuNPs and magnetic 3D DNA walker, which could detect as few as 16.7 fM of miRNA-155, thus easily solves the main technical challenge of AuNPs-based colorimetric nucleic acid assays. In this platform, hundreds of substrate sing-stranded DNA (DNA tracks) and dozens of blocked walking strands were attached on an Au-Fe3O4 nanocomposite, applied as a magnetic 3D DNA walker. The target could activate this DNA walker powered by DNAzyme cleavage mechanism, generating a great deal of single-stranded product (displaced ssDNA). After magnetic separation, the generated displaced ssDNA (supernatant) was incubated with AuNPs to protect the unmodified nanoparticles from aggregation in high-salt solutions. Based on this, the concentration of miRNA-155 could be visualized through a color change from grayish black to purple and quantitatively determined by UV–vis spectroscopy. Furthermore, we demonstrated that our approach could be used to detect target miRNA-155 in real human serum samples without complicated sample pretreatment. Thus, the magnetic 3D DNA walker-AuNPs colorimetric method is a facile, sensitive and specific detection platform that shows great potential for development of next-generation POC nucleic acids diagnostics.

Colorimetric Nucleic Acid Detection Based on Gold Nanoparticles with Branched DNA

Nucleic acid detection is becoming increasingly important in the diagnostics of genetic diseases for biological analysis. We herein propose gold nanoparticles as probe for colorimetric detection of nucleic acids with branched DNA nanostructures, which enables a novel and simple colorimetric biosensor. In our system, the target DNA specifically triggered two short-chain ssDNA probes to generate branched DNA nanostructures (Y-shape DNA), which prevent AuNPs from aggregation in aqueous NaCl solution. On the contrary, when the target DNA did not exist, gold nanoparticles were unstable and aggregated easily because there is no anti-aggregation function from Y-shape DNA. Sensor response was found to be proportional to the target DNA concentration from 5 to 100[Formula: see text]nM, with detection limits determined as 5[Formula: see text]nM. The developed platform is for colorimetric nucleic acid detection without enzymes, label and modification holds great promise for practical applications.

Colorimetric Polymerase Chain Reaction Enabled by a Fast Light-Activated Substrate Chromogenic Detection Platform

Miniaturization of nucleic acid tests (NATs) into portable, inexpensive detection platforms may aid disease diagnosis in point-of-care (POC) settings. Colorimetric signals are ideal readouts for portable NATs, and it remains of high demand to develop color readouts that are simple, quantitative, and versatile. Thus motivated, we report a fast light-activated substrate chromogenic polymerase chain reaction (FLASH PCR) that uses DNA intercalating dyes (DIDs) to enable colorimetric nucleic acid detection and quantification. The FLASH system is established on our finding that DID-DNA intercalation can promote the rapid photooxidation of chromogenic substrates through light-induced production of singlet oxygen. Using this principle, we have successfully converted DID-based fluorescent PCR assays into colorimetric FLASH PCR. To demonstrate the practical applicability of FLASH PCR to POC diagnosis, we also fabricated two readout platforms, including a portable electronic FLASH reader and a paper-based FLASH strip. Using the FLASH reader, we were able to detect as low as 60 copies of DNA standards, a limit of detection (LOD) comparable with commercial quantitative PCR. The FLASH strip further enables the reader-free detection of PCR amplicons by converting the colorimetric signal into the visual measurement of distance as a readout. Finally, the practical applicability of the FLASH PCR was demonstrated by the detection and/or quantification of nucleic acid markers in diverse clinical and biological samples.

Pixelated colorimetric nucleic acid assay

Conjugated polyelectrolytes (CPEs) have been widely used as reporters in colorimetric assays targeting nucleic acids. CPEs provide naked eye detection possibility by their superior optical properties however, as concentration of target analytes decrease, trace amounts of nucleic acid typically yield colorimetric responses that are not readily perceivable by naked eye. Herein, we report a pixelated analysis approach for correlating colorimetric responses of CPE with nucleic acid concentrations down to 1 nM, in plasma samples, utilizing a smart phone with an algorithm that can perform analytical testing and data processing. The detection strategy employed relies on conformational transitions between single stranded nucleic acid-cationic CPE duplexes and double stranded nucleic acid-CPE triplexes that yield distinct colorimetric responses for enabling naked eye detection of nucleic acids. Cationic poly[N,N,N-triethyl-3-((4-methylthiophen-3-yl)oxy)propan-1-aminium bromide] is utilized as the CPE reporter deposited on a polyvinylidene fluoride (PVDF) membrane for nucleic acid assay. A smart phone application is developed to capture and digitize the colorimetric response of the individual pixels of the digital images of CPE on the PVDF membrane, followed by an analysis using the algorithm. The proposed pixelated approach enables precise quantification of nucleic acid assay concentrations, thereby eliminating the margin of error involved in conventional methodologies adopted for interpretation of colorimetric responses, for instance, RGB analysis. The obtained results illustrate that a ubiquitous smart phone could be utilized for point of care colorimetric nucleic acids assays in complex matrices without requiring sophisticated software or instrumentation.

Label-free pathogen detection by a deoxyribozyme cascade with visual signal readout

A colorimetric nucleic acid based test for label-free pathogen detection has been developed and used for the detection of the Zika virus. The test relies on nucleic acid sequence-based amplification (NASBA) of a viral RNA followed by interrogation of the amplicon by a cascade of deoxyribozymes constituting a visual split deoxyribozyme (vsDz) probe. The probe consists of a split phosphodiesterase deoxyribozyme, which forms its catalytic core upon binding to a specific amplicon fragment. The catalytically active complex recognizes and cleaves an inhibited peroxidase-like deoxyribozyme (PDz), thereby activating it. Active PDz catalyzes hydrogen peroxide-mediated oxidation of a colorless substrate into a colored product, thereby generating a visible signal. Viral RNA (106 copies/mL or higher) triggers intense color within 2 h. The test selectively differentiates between Zika and closely related dengue and West Nile viruses. The reported technology combines isothermal amplification and visual detection and therefore represents a basis for the future development of a cost-efficient and instrument-free method for point-of-care nucleic acid analysis.

Colorimetric ultrasensitive detection of DNA based on the intensity of gold nanoparticles with dark-field microscopy.

We present an ultrasensitive colorimetric nucleic acid assay based on the intensity of gold nanoparticles (Au NPs) using dark field microscopy. In the absence of target DNA, two hairpin-like DNA strands with protruding single-stranded DNA (ssDNA) can be absorbed onto the Au NP surface via non-covalent interactions between the exposed nitrogen bases of ssDNA and Au NPs, which inhibits Au NPs from aggregating in a high concentration of salt media, while in the presence of target DNA, two hairpin DNA strands hybridize with target DNA to form double-stranded DNA (dsDNA). After hybridization chain reaction (HCR) amplification, rigid dsDNA polymers are formed, which results in serious Au NP aggregation in the salt environment. By measuring the intensity change of yellow and red dots on a dark-field image, the concentration of target DNA can be accurately quantified with a limit of detection (LOD) of 66 fM.

Colorimetric Nucleic Acid Detection on Paper Microchip Using Loop Mediated Isothermal Amplification and Crystal Violet Dye.

Nucleic acid detection is of paramount importance in monitoring of microbial pathogens in food safety and infectious disease diagnostic applications. To address these challenges, a rapid, cost-effective label-free technique for nucleic acid detection with minimal instrumentations is highly desired. Here, we present paper microchip to detect and quantify nucleic acid using colorimetric sensing modality. The extracted DNA from food samples of meat as well as microbial pathogens was amplified utilizing loop-mediated isothermal amplification (LAMP). LAMP amplicon was then detected and quantified on a paper microchip fabricated in a cellulose paper and a small wax chamber utilizing crystal violet dye. The affinity of crystal violet dye toward dsDNA and positive signal were identified by changing the color from colorless to purple. Using this method, detection of Sus scrofa (porcine) and Bacillus subtilis (bacteria) DNA was possible at concentrations as low as 1 pg/μL (3.43 × 10 -1 copies/μL) and 10 pg/μL (2.2 × 103 copies/μL), respectively. This strategy can be adapted for detection of other DNA samples, with potential for development of a new breed of simple and inexpensive paper microchip at the point-of-need.

Target-triggered autonomous assembly of DNA polymer chains and its application in colorimetric nucleic acid detection

A novel target-triggered DNA autonomous assembly pathway for the formation of one-dimensional DNA polymer chains was proposed based on the catalyzed hairpin assembly and sticky end self-assembly, which led to an enzyme-free and signal amplified gold nanoparticle (AuNP) based colorimetric nucleic acid assay with picomolar sensitivity and specificity.

Colorimetric Nucleic Acid Testing Assay for RNA Virus Detection Based on Circle-to-Circle Amplification of Padlock Probes

We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 10(3) viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.

Rapid detection of human rotavirus using colorimetric nucleic acid sequence-based amplification (NASBA)–enzyme-linked immunosorbent assay in sewage treatment effluent

A colorimetric nucleic acid sequence-based amplification–enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for rapid detection and identification of human rotavirus. Oligonucleotide primers targeting gene 9 encoding a serotype-specific antigen VP7 were selected and used for the amplification of viral RNA by the isothermal NASBA process, resulting in the accumulation of biotinylated RNA amplicons. Amplicons were hybridized with a specific amino-linked oligonucleotide probe covalently immobilized on microtiter plates. The DNA–RNA hybrids were colorimetrically detected by the addition of streptavidin–peroxidase conjugate and tetramethylbenzidine substrate. Using the NASBA-ELISA system, as little as 0.2 PFU (4×10 1 PFU ml −1) and 15 PFU (3×10 3 PFU ml −1) of rotavirus were detected within 6 h in spiked MQ water and sewage treatment effluent respectively. No interference was encountered in the amplification and detection of rotavirus in the presence of non-target RNA or DNA. Moreover, the presence of non-target bacteria and virus does not generate any non-specific signal, confirming the specificity of the developed NASBA-ELISA system and its effectiveness in specifically detecting rotavirus. The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

Rapid detection of human rotavirus using colorimetric nucleic acid sequence-based amplification (NASBA)-enzyme-linked immunosorbent assay in sewage treatment effluent.

A colorimetric nucleic acid sequence-based amplification-enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for rapid detection and identification of human rotavirus. Oligonucleotide primers targeting gene 9 encoding a serotype-specific antigen VP7 were selected and used for the amplification of viral RNA by the isothermal NASBA process, resulting in the accumulation of biotinylated RNA amplicons. Amplicons were hybridized with a specific amino-linked oligonucleotide probe covalently immobilized on microtiter plates. The DNA-RNA hybrids were colorimetrically detected by the addition of streptavidin-peroxidase conjugate and tetramethylbenzidine substrate. Using the NASBA-ELISA system, as little as 0.2 PFU (4 x 10(1) PFU ml(-1)) and 15 PFU (3 x 10(3) PFU ml(-1)) of rotavirus were detected within 6 h in spiked MQ water and sewage treatment effluent respectively. No interference was encountered in the amplification and detection of rotavirus in the presence of non-target RNA or DNA. Moreover, the presence of non-target bacteria and virus does not generate any non-specific signal, confirming the specificity of the developed NASBA-ELISA system and its effectiveness in specifically detecting rotavirus. The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

Determination of Listeria in Dairy and Environmental Samples: Comparison of a Cultural Method and a Colorimetric Nucleic Acid Hybridization Assay

The efficiency of the International Dairy Federation (IDF) Listeria detection method (IDF Standard 143:1990) was compared to that of a colorimetric nucleic acid hybridization assay. Out of 250 naturally contaminated cheese and environmental samples tested, the IDF method revealed 153 and the Gene-Trak assay 144 positive samples, respectively. Hence, the Gene-Trak assay gave a false-negative rate of 5.9%. False-positive samples could not be detected. When Oxford and LPM agar were compared for suitability as the streaking medium for the Gene-Trak assay, more satisfactory results were obtained with the Oxford agar. Both the overall number of positive samples and the mean optical density values of the Gene-Trak-positive samples were higher with Oxford than with LPM agar. The extension of sample enrichment time from 1 d (Gene-Trak-assay), and 2 d (IDF method), respectively, to 7 d resulted in a small increase in the number IDF method-positive samples but led to a drastic decrease in the number of Gene-Trak-positive samples.