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1.
Anal Methods ; 15(21): 2641-2649, 2023 06 01.
Article in English | MEDLINE | ID: covidwho-2323864

ABSTRACT

Rapid detection of nucleic acids is integral for clinical diagnostics, especially if a major public-health emergency occurs. However, such detection cannot be carried out efficiently in remote areas limited by medical resources. Herein, a dual-labeled fluorescence resonance energy transfer (FRET) lateral flow assay (LFA) based on one-pot enzyme-free cascade amplification was developed for rapid, convenient, and sensitive detection of open reading frame (ORF)1ab of severe acute respiratory syndrome-coronavirus-2. The catalyzed hairpin assembly (CHA) reaction of two well-designed hairpin probes was initiated by a target sequence and generated a hybridization chain reaction (HCR) initiator. Then, HCR probes modified with biotin were initiated to produce long DNA nanowires. After two-level amplification, the cascade-amplified product was detected by dual-labeled lateral flow strips. Gold nanoparticles (AuNPs)-streptavidin combined with the product and then ran along a nitrocellulose membrane under the action of capillary force. After binding with fluorescent microsphere-labeled-specific probes on the T line, a positive signal (red color) could be observed. Meanwhile, AuNPs could quench the fluorescence of the T line, and an inverse relationship between fluorescence intensity and the concentration of the CHA-HCR-amplified product was formed. The proposed strategy achieved a satisfactory limit of detection of 2.46 pM for colorimetric detection and 174 fM for fluorescent detection, respectively. Benefitting from the features of being one-pot, enzyme-free, low background, high sensitivity, and selectivity, this strategy shows great potential in bioanalysis and clinical diagnostics upon further development.


Subject(s)
COVID-19 , Metal Nanoparticles , Humans , Gold , COVID-19/diagnosis , DNA/analysis , Nucleic Acid Hybridization
2.
Anal Methods ; 15(19): 2382-2390, 2023 05 18.
Article in English | MEDLINE | ID: covidwho-2315737

ABSTRACT

Rapid and accurate detection of a variety of pathogens is very important for the prevention, control, and diagnosis of infectious diseases. Herein, an ultrasensitive nucleic acid isothermal cascade amplification technique based on rolling circle amplification (RCA) coupled with hybridization chain reaction (HCR) was developed for ORF1ab (opening reading frame 1a/b) for SARS-CoV-2 detection. In this scheme, the ORF1ab sequence hybridized with a padlock probe to trigger RCA reaction. Specifically, the recognition site for a unique nicking enzyme was incorporated into the padlock probe to cut the RCA products into short intermediate amplicons, which contain dual HCR initiation sites and can be directly used as primers for HCR. HCR probes, H1 and H2, labeled with FAM (FAM-H1 and FAM-H2) spontaneously participated in the HCR and formed a long nicked dsDNA. Additional probes were quenched by graphene oxide (GO) via π-stacking to decrease the background signal. Meanwhile, the fluorescence signal can be strongly amplified by the synergistic effect of FAM and SYBR green I. The proposed RCA-HCR method can be used to detect ORF1ab at concentrations as low as 7.65 fM. Moreover, the reliability of the RCA-HCR method in serum samples has also been validated. Satisfactory recoveries ranging from 85% to 113% for ORF1ab can be obtained. Therefore, this facile and ultrasensitive RCA-HCR assay provides a new promising tool for ORF1ab analysis and can be extended to the detection of various kinds of pathogens and genetic biomarkers.


Subject(s)
COVID-19 , Humans , Reproducibility of Results , Limit of Detection , COVID-19/diagnosis , SARS-CoV-2/genetics , Nucleic Acid Hybridization
3.
Sci Rep ; 13(1): 6461, 2023 04 20.
Article in English | MEDLINE | ID: covidwho-2298135

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) pandemic has led to extensive virological monitoring by whole genome sequencing (WGS). Investigating the advantages and limitations of different protocols is key when conducting population-level WGS. SARS-CoV-2 positive samples with Ct values of 14-30 were run using three different protocols: the Twist Bioscience SARS­CoV­2 protocol with bait hybridization enrichment sequenced with Illumina, and two tiled amplicon enrichment protocols, ARTIC V3 and Midnight, sequenced with Illumina and Oxford Nanopore Technologies, respectively. Twist resulted in better coverage uniformity and coverage of the entire genome, but has several drawbacks: high human contamination, laborious workflow, high cost, and variation between batches. The ARTIC and Midnight protocol produced an even coverage across samples, and almost all reads were mapped to the SARS-CoV-2 reference. ARTIC and Midnight represent robust, cost-effective, and highly scalable methods that are appropriate in a clinical environment. Lineage designations were uniform across methods, representing the dominant lineages in Sweden during the period of collection. This study provides insights into methodological differences in SARS­CoV­2 sequencing and guidance in selecting suitable methods for various purposes.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Whole Genome Sequencing/methods , Sequence Analysis , Nucleic Acid Hybridization , Genome, Viral/genetics
4.
J Microbiol Methods ; 204: 106647, 2023 01.
Article in English | MEDLINE | ID: covidwho-2211151

ABSTRACT

The PCR technique is indispensable in biology and medicine, but some difficulties are associated with its use, including false positive or false negative amplifications. To avoid these issues, a non-amplification nucleic acid detection protocol is needed. In the present study, we propose a method in which nucleic-acid probe hybridization is combined with thio-NAD cycling to detect nucleic acids without amplification. We report our application of this method for the detection of the gene of MPT64 in Mycobacterium tuberculosis. Two different cDNA probes targeted the mpt64 gene: the first probe was used to immobilize the mpt64 gene, and the second probe, linked with alkaline phosphatase (ALP), was hybridized to a target sequence in the mpt64 gene. A substrate was then hydrolyzed by ALP, and a cycling reaction was conducted by a dehydrogenase with its co-factors (thio-NAD and NADH). The single-stranded DNA, double-stranded DNA, plasmid DNA for the mpt64 gene, and whole genome of M. tuberculosis var. BCG were detected at the level of 105-106 copies/assay, whereas the non-tuberculosis mycobacteria (e.g., M. avium, M. intracellulare, M. kansasii, and M. abscessus) were below the limits of detection. The present method enables us to avoid the errors inherent in nucleic acid amplification methods.


Subject(s)
Mycobacterium tuberculosis , NAD , Mycobacterium tuberculosis/genetics , Nontuberculous Mycobacteria/genetics , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization , DNA, Bacterial/genetics , DNA, Bacterial/analysis
5.
ACS Sens ; 7(12): 3692-3699, 2022 12 23.
Article in English | MEDLINE | ID: covidwho-2160150

ABSTRACT

We present the development of electrochemical impedance spectroscopy (EIS)-based biosensors for sensitive detection of SARS-CoV-2 RNA using multi-valent binding. By increasing the number of probe-target binding events per target molecule, multi-valent binding is a viable strategy for improving the biosensor performance. As EIS can provide sensitive and label-free measurements of nucleic acid targets during probe-target hybridization, we used multi-valent binding to build EIS biosensors for targeting SARS-CoV-2 RNA. For developing the biosensor, we explored two different approaches including probe combinations that individually bind in a single-valent fashion and the probes that bind in a multi-valent manner on their own. While we found excellent biosensor performance using probe combinations, we also discovered unexpected signal suppression. We explained the signal suppression theoretically using inter- and intra-probe hybridizations which confirmed our experimental findings. With our best probe combination, we achieved a LOD of 182 copies/µL (303 aM) of SARS-CoV-2 RNA and used these for successful evaluation of patient samples for COVID-19 diagnostics. We were also able to show the concept of multi-valent binding with shorter probes in the second approach. Here, a 13-nt-long probe has shown the best performance during SARS-CoV-2 RNA binding. Therefore, multi-valent binding approaches using EIS have high utility for direct detection of nucleic acid targets and for point-of-care diagnostics.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , RNA, Viral/genetics , Nucleic Acid Hybridization
6.
Analyst ; 147(23): 5306-5313, 2022 Nov 21.
Article in English | MEDLINE | ID: covidwho-2096841

ABSTRACT

We report the development of a portable absorption (PortAbs)-based pathogen nucleic acid detection system using peptide nucleic acid (PNA) and a cyanine dye, DiSc2(5). When the dye binds to the PNA-DNA hybrid, it results in a characteristic ∼110 nm shift in the dye absorbance, which we measure using PortAbs. The protocol involves amplification of the target DNA, PNA-DNA hybridization and dye complexing steps followed by absorption measurement. The system is built using a broad-spectrum photodiode whose output is amplified and then measured by a high resolution (24 or 32 bit) analog-to-digital converter. The excitation pulses of light are delivered by a color-changing LED. The sequence of excitation, measurement and display of results are all controlled by an embedded Raspberry-Pi board (or alternatively a laptop). At higher concentrations of the target amplicon (∼200 ng), the color change can be detected visually. At lower concentrations, PortAbs outperforms a plate reader and can detect target DNA as low as 30 ng or approximately 10 nM which is at least 10 fold better than previously reported studies. We validate the methodology using SARS-CoV-2 clinical samples containing about 1000 copies of the viral RNA and show that the entire workflow takes about 90 min. The cost of the complete standalone system is less than INR 40 000 (approx. 500 USD).


Subject(s)
COVID-19 , Nucleic Acids , Peptide Nucleic Acids , Humans , Peptide Nucleic Acids/genetics , SARS-CoV-2 , Nucleic Acid Hybridization , DNA/genetics
7.
Talanta ; 247: 123542, 2022 Sep 01.
Article in English | MEDLINE | ID: covidwho-1852117

ABSTRACT

In this work we present a powerful, affordable, and portable biosensor to develop Point of care (POC) SARS-CoV-2 virus detection. It is constructed from a fast, low cost, portable and electronically automatized potentiostat that controls the potential applied to a disposable screen-printed electrochemical platform and the current response. The potentiostat was designed to get the best signal-to-noise ratio, a very simple user interface offering the possibility to be used by any device (computer, mobile phone or tablet), to have a small and portable size, and a cheap manufacturing cost. Furthermore, the device includes as main components, a data acquisition board, a controller board and a hybridization chamber with a final size of 10 × 8 × 4 cm. The device has been tested by detecting specific SARS-CoV-2 virus sequences, reaching a detection limit of 22.1 fM. Results agree well with those obtained using a conventional potentiostat, which validate the device and pave the way to the development of POC biosensors. In this sense, the device has finally applied to directly detect the presence of the virus in nasopharyngeal samples of COVID-19 patients and results confirm its utility for the rapid detection infected samples avoiding any amplification process.


Subject(s)
Biosensing Techniques , COVID-19 , Biosensing Techniques/methods , COVID-19/diagnosis , Humans , Nucleic Acid Hybridization , Point-of-Care Systems , SARS-CoV-2
8.
Mikrochim Acta ; 189(4): 171, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1777732

ABSTRACT

Gold nanotriangles (AuNTs) functionalized with dithiolated oligonucleotides have been employed to develop an amplification-free electrochemical biosensor for SARS-CoV-2 in patient samples. Gold nanotriangles, prepared through a seed-mediated growth method and exhaustively characterized by different techniques, serve as an improved electrochemical platform and for DNA probe immobilization. Azure A is used as an electrochemical indicator of the hybridization event. The biosensor detects either single stranded DNA or RNA sequences of SARS-CoV-2 of different lengths, with a low detection limit of 22.2 fM. In addition, it allows to detect point mutations in SARS-CoV-2 genome with the aim to detect more infective SARS-CoV-2 variants such as Alpha, Beta, Gamma, Delta, and Omicron. Results obtained with the biosensor in nasopharyngeal swab samples from COVID-19 patients show the possibility to clearly discriminate between non-infected and infected patient samples as well as patient samples with different viral load. Furthermore, the results correlate well with those obtained by the gold standard technique RT-qPCR, with the advantage of avoiding the amplification process and the need of sophisticated equipment.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Nucleic Acid Hybridization , Oligonucleotides , SARS-CoV-2/genetics
9.
Talanta ; 243: 123352, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1730116

ABSTRACT

Accurate identification of mutant pathogens derived from genetic polymorphisms is highly desired in clinical diagnosis. However, current detection methods based on Watson-Crick hybridization suffers from false positives due to the cross-reactivity of wild-type sequences. In this study, we developed an accurate identification of mutant pathogens by combining programmable DNAzyme and target nucleic acid sequence-triggered transcription. Single nucleotide variants (SNVs) are the most plentiful type of mutations in the genome. High specificity to discriminate SNV was first achieved by rational design of dual-hairpin DNA structure and DNAzyme's capability of site-specific cleavage. T7 RNA polymerase-mediated transcription amplification was introduced to exponentially increase the sensitivity by encompassing T7 promoter sequence into the dual-hairpin DNA structure. The design of this biosensor is fast and straightforward without many computational steps, and the highly sensitive biosensor can detect not only SNVs but also occasional insertions and large deletions in the genome. We showed that the assay could rapidly detect COVID-19 variant and methicillin-resistant Staphylococcus aureus (MRSA), and the limit of detection is 0.96 copy/µL. The modular design of functional DNA enables this biosensor be easily reconfigured and is useful diagnosis of emerging infectious diseases caused by mutant pathogens.


Subject(s)
Biosensing Techniques , DNA, Catalytic , Biosensing Techniques/methods , COVID-19/diagnosis , DNA, Catalytic/chemistry , DNA, Catalytic/genetics , Humans , Limit of Detection , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Hybridization , SARS-CoV-2/isolation & purification
10.
Mikrochim Acta ; 189(1): 34, 2021 12 23.
Article in English | MEDLINE | ID: covidwho-1633639

ABSTRACT

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel-based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel-based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings.


Subject(s)
Chemistry Techniques, Analytical/methods , DNA, Catalytic/chemistry , DNA, Complementary/analysis , Hydrogels/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , Capillary Action , Chemistry Techniques, Analytical/instrumentation , DNA, Catalytic/genetics , DNA, Complementary/genetics , Hydrogen-Ion Concentration , Inverted Repeat Sequences , Limit of Detection , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization
11.
Talanta ; 240: 123207, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1612034

ABSTRACT

In this work, we designed an ECL ratiometric biosensor with a three-stranded Y-type DNA (Y-DNA) probe and induced a hybridization chain reaction (HCR) for the highly sensitive detection of SARS-CoV-2 nucleic acid. The important component of this system is the self-assembled Y-Shaped probe based on three nucleic acids. Y1, Y2, and Y3 can be linked by complementary base pairing to Hairpin1 (H1), Hairpin2 (H2), and Ru modified DNA (Ru1), respectively. H1 and H2 can trigger the HCR reaction when activated by the SARS-CoV-2 RdRp gene and the 5' end of Ru1. The 5' end of Ru1 is modified with the Ru complex, which can produce a strong electrochemiluminescence luminescence signal at 620 nm under an applied voltage. Through the amplification of Y-DNA-induced HCR reaction, Ru1 on the electrode surface gradually increased, the ECL signal at 460 nm was gradually quenched, and the signal at 620 nm was steadily generated. The SARS-CoV-2 RdRp gene can be quantified according to the degree of decrease of ECL signal at 460 nm and the increase of ECL signal at 620 nm. Combining the two signal amplification strategies, this ratiometric ECL biosensor can accurately and efficiently detect the target gene with a detection limit of 59 aM.


Subject(s)
Biosensing Techniques , COVID-19 , Electrochemical Techniques , Humans , Luminescent Measurements , Nucleic Acid Hybridization , RNA-Dependent RNA Polymerase , SARS-CoV-2
12.
Biosensors (Basel) ; 11(7)2021 Jul 14.
Article in English | MEDLINE | ID: covidwho-1323111

ABSTRACT

Enteroviruses are ubiquitous mammalian pathogens that can produce mild to life-threatening disease. We developed a multimodal, rapid, accurate and economical point-of-care biosensor that can detect nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and oligonucleotides to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral nucleic acid sequence (23 bases), which was identified through in silico screening. Oligonucleotides were designed to demonstrate specific complementarity towards the target enteroviral nucleic acid to produce aggregated gold-oligonucleotide nanoconstructs. The conserved target enteroviral nucleic acid sequence (≥1 × 10-7 M, ≥1.4 × 10-14 g/mL) initiates gold-oligonucleotide nanoconstruct disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow assays that utilise gold-oligonucleotide nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (<60 s), and could be interpreted with a bespoke software and hardware electronic interface. We anticipate that our methodology will translate in silico screening of nucleic acid databases to a tangible enteroviral desktop detector, which could be readily translated to related organisms. This will pave the way forward in the clinical evaluation of disease and complement existing strategies to overcome antimicrobial resistance.


Subject(s)
Biosensing Techniques , Metal Nanoparticles , Nucleic Acids , Gold/chemistry , Humans , Metal Nanoparticles/chemistry , Nucleic Acid Hybridization , Oligonucleotides
13.
Sensors (Basel) ; 21(13)2021 Jun 29.
Article in English | MEDLINE | ID: covidwho-1288981

ABSTRACT

Coronavirus (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as a deadly pandemic. The genomic analysis of SARS-CoV-2 is performed using a reverse transcription-polymerase chain reaction (RT-PCR) technique for identifying viral ribonucleic acid (RNA) in infected patients. However, the RT-PCR diagnostic technique is manually laborious and expensive; therefore, it is not readily accessible in every laboratory. Methodological simplification is crucial to combat the ongoing pandemic by introducing quick, efficient, and affordable diagnostic methods. Here, we report how microcantilever sensors offer promising opportunities for rapid COVID-19 detection. Our first attempt was to capture the single-stranded complementary DNA of SARS-CoV-2 through DNA hybridization. Therefore, the microcantilever surface was immobilized with an oligonucleotide probe and detected using complementary target DNA hybridization by a shift in microcantilever resonance frequency. Our results show that microcantilever sensors can discriminate between complementary and noncomplementary target DNA on a micro to nanoscale. Additionally, the microcantilever sensors' aptitude toward partial complementary DNA determines their potential to identify new variants of coronavirus. Therefore, microcantilever sensing could be a vital tool in the effort to extinguish the spreading COVID-19 pandemic.


Subject(s)
COVID-19 , SARS-CoV-2 , DNA, Complementary , Humans , Nucleic Acid Hybridization , Pandemics , RNA, Viral
14.
Analyst ; 146(13): 4340-4347, 2021 Jun 28.
Article in English | MEDLINE | ID: covidwho-1262015

ABSTRACT

Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs) such as PCR and isothermal amplification methods provide excellent analytical performance and significantly faster turnaround times than conventional culture-based methods. However, the inherent cost and complexity of NAATs limit their application in resource-limited settings and the developing world. To help address this urgent need, we have developed a sensitive method for nucleic acid analysis based on padlock probe rolling circle amplification (PLRCA), nuclease protection (NP) and lateral flow detection (LFA), referred to as PLAN-LFA, that can be used in resource-limited settings. The assay involves solution-phase hybridization of a padlock probe to target, sequence-specific ligation of the probe to form a circular template that undergoes isothermal rolling circle amplification in the presence of a polymerase and a labeled probe DNA. The RCA product is a long, linear concatenated single-stranded DNA that contains binding sites for the labeled probe. The sample is then exposed to a nuclease which selectively cleaves single-stranded DNA, the double-stranded labeled probe is protected from nuclease digestion and detected in a lateral flow immunoassay format to provide a visual, colorimetric readout of results. We have developed specific assays targeting beta-lactamase resistance gene for monitoring of antimicrobial resistance and Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2, the novel coronavirus discovered in 2019) using the PLAN-LFA platform. The assay provides a limit of detection of 1.1 pM target DNA (or 1.3 × 106 copies per reaction). We also demonstrate the versatility and robustness of the method by performing analysis on DNA and RNA targets, and perform analysis in complex sample matrices like saliva, plant tissue extract and bacterial culture without any sample pretreatment steps.


Subject(s)
COVID-19 , SARS-CoV-2 , DNA Probes , Humans , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization
15.
Science ; 372(6545): 941-948, 2021 05 28.
Article in English | MEDLINE | ID: covidwho-1205995

ABSTRACT

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp614→Gly) variant with single-base resolution in patient samples.


Subject(s)
CRISPR-Associated Protein 9/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , RNA, Guide, Kinetoplastida/genetics , RNA, Viral/analysis , RNA/analysis , RNA/genetics , SARS-CoV-2/genetics , Base Sequence , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing , CRISPR-Cas Systems , Campylobacter jejuni , Humans , Nucleic Acid Hybridization , RNA, Bacterial/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , Spike Glycoprotein, Coronavirus/genetics
16.
Biosensors (Basel) ; 11(3)2021 Mar 06.
Article in English | MEDLINE | ID: covidwho-1143457

ABSTRACT

Surface Plasmon Resonance (SPR) is widely used in biological and chemical sensing with fascinating properties. However, the application of SPR to detect trace targets is hampered by non-specific binding and poor signal. A variety of approaches for amplification have been explored to overcome this deficiency including DNA aptamers as versatile target detection tools. Hybridization chain reaction (HCR) is a high-efficiency enzyme-free DNA amplification method operated at room temperature, in which two stable species of DNA hairpins coexist in solution until the introduction of the initiator strand triggers a cascade of hybridization events. At an optimal salt condition, as the concentrations of H1 and H2 increased, the HCR signals were enhanced, leading to signal amplification reaching up to 6.5-fold of the detection measure at 30 min. This feature enables DNA to act as an amplifying transducer for biosensing applications to provide an enzyme-free alternative that can easily detect complex DNA sequences. Improvement of more diverse recognition events can be achieved by integrating HCR with a phase-sensitive SPR (pSPR)-tested aptamer stimulus. This work seeks to establish pSPR aptamer system for highly informative sensing by means of an amplification HCR. Thus, combining pSPR and HCR technologies provide an expandable platform for sensitive biosensing.


Subject(s)
Biosensing Techniques , Nucleic Acid Hybridization , Surface Plasmon Resonance , Aptamers, Nucleotide/chemistry , DNA/chemistry , Gold/chemistry , Limit of Detection , Nucleic Acid Amplification Techniques
17.
Nat Commun ; 12(1): 1467, 2021 03 05.
Article in English | MEDLINE | ID: covidwho-1118805

ABSTRACT

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/virology , Colorimetry/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Hybridization/methods , Point-of-Care Testing , SARS-CoV-2/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , Humans , Phosphoproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
18.
Mol Cell Probes ; 56: 101707, 2021 04.
Article in English | MEDLINE | ID: covidwho-1087137

ABSTRACT

BACKGROUND: DNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primer or probe regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed. METHODS: We calculate the hybridization temperatures of primer/probe sets after aligning to SARS-CoV-2, SARS-CoV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5 ∘C to the fully matched reference temperature. RESULTS: We obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes. CONCLUSIONS: Some primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.


Subject(s)
Base Pair Mismatch , DNA Primers/metabolism , DNA Probes/metabolism , DNA, Viral/metabolism , Nucleic Acid Hybridization , Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Genome, Viral , Sequence Alignment , Temperature
19.
CRISPR J ; 3(6): 487-502, 2020 12.
Article in English | MEDLINE | ID: covidwho-990516

ABSTRACT

Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present coronavirus disease 2019 pandemic. Simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, current nucleic acid detection techniques are still limited by traditional amplification and hybridization. To overcome this limitation, here we developed CRISPR-Cas9-assisted DNA detection (CADD). In this detection, a DNA sample is incubated with a pair of capture single guide RNAs (sgRNAs; sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature (RT) for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa, and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally, the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cells, and viruses. In particular, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples was successfully detected by the method. All detections can be finished in 30 min at RT. This detection holds promise for rapid on-the-spot detection or point-of-care testing.


Subject(s)
CRISPR-Associated Protein 9/metabolism , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Hybridization/methods , Animals , CRISPR-Cas Systems , DNA, Viral/genetics , Genetic Engineering/methods , Humans , Limit of Detection , Papillomavirus Infections/genetics , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism
20.
Biosens Bioelectron ; 166: 112436, 2020 Oct 15.
Article in English | MEDLINE | ID: covidwho-665846

ABSTRACT

Our recent experience of the COVID-19 pandemic has highlighted the importance of easy-to-use, quick, cheap, sensitive and selective detection of virus pathogens for the efficient monitoring and treatment of virus diseases. Early detection of viruses provides essential information about possible efficient and targeted treatments, prolongs the therapeutic window and hence reduces morbidity. Graphene is a lightweight, chemically stable and conductive material that can be successfully utilized for the detection of various virus strains. The sensitivity and selectivity of graphene can be enhanced by its functionalization or combination with other materials. Introducing suitable functional groups and/or counterparts in the hybrid structure enables tuning of the optical and electrical properties, which is particularly attractive for rapid and easy-to-use virus detection. In this review, we cover all the different types of graphene-based sensors available for virus detection, including, e.g., photoluminescence and colorimetric sensors, and surface plasmon resonance biosensors. Various strategies of electrochemical detection of viruses based on, e.g., DNA hybridization or antigen-antibody interactions, are also discussed. We summarize the current state-of-the-art applications of graphene-based systems for sensing a variety of viruses, e.g., SARS-CoV-2, influenza, dengue fever, hepatitis C virus, HIV, rotavirus and Zika virus. General principles, mechanisms of action, advantages and drawbacks are presented to provide useful information for the further development and construction of advanced virus biosensors. We highlight that the unique and tunable physicochemical properties of graphene-based nanomaterials make them ideal candidates for engineering and miniaturization of biosensors.


Subject(s)
Betacoronavirus/isolation & purification , Biosensing Techniques , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Graphite , Pneumonia, Viral/diagnosis , Viruses/isolation & purification , Antigen-Antibody Reactions , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Biosensing Techniques/trends , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/instrumentation , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/statistics & numerical data , Colorimetry , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , DNA, Viral/analysis , DNA, Viral/genetics , Electrochemical Techniques , Equipment Design , Graphite/chemistry , Humans , Luminescence , Nanostructures/chemistry , Nucleic Acid Hybridization , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Quantum Dots/chemistry , SARS-CoV-2 , Spectrum Analysis, Raman , Surface Plasmon Resonance , Virology/methods , Viruses/genetics , Viruses/pathogenicity
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