Target Nucleic Acid Sequences Research Articles

Point-of-Care Molecular Testing with tinto rangTM: A Food Grade Safe Fluorophore for Colorimetric LAMP Assays at Low Resource Settings

LAMP (loop-mediated isothermal amplification) has proven to be a highly robust, sensitive, and cost-effective method for detecting specific target nucleic acid sequences in a single-tube reaction without the need for sophisticated instruments. Traditionally, pH-sensitive dyes, DNA-binding dyes like Ethidium Bromide(EtBr), and SYBR have been employed to detect amplified products. However, these dyes present several drawbacks, limiting the widespread adoption of LAMP in diagnostic applications. To address this limitation, a novel dye called tinto rangTM has been introduced with promising applications in nucleic acid analysis. In this study, we present an evaluation of tinto rangTM's performance in comparison to commercially available dyes commonly used in LAMP assays. One of the unique advantages of tinto rangTM is its capability to facilitate the analysis of amplified nucleic acid products through three distinct methods: visual color change, changes in relative fluorescence, and turbidity. In addition to assessing tinto rangTM's performance, we also investigated various approaches to tackle the issue of non-specific amplification, which has been a common challenge in LAMP reactions. The experimental results unequivocally demonstrate that tinto rangTM outperforms pH-indicating and other DNA-binding dyes used in LAMP assays, making it a superior alternative. This study paves the way for the broader adoption of tinto rangTM in various applications, including point-of-care diagnostics, disease monitoring, and molecular biology research.

Point-of-Care Molecular Testing with tinto rangTM: A Food Grade Safe Fluorophore for Colorimetric LAMP Assays at Low Resource Settings

LAMP (loop-mediated isothermal amplification) has proven to be a highly robust, sensitive, and cost-effective method for detecting specific target nucleic acid sequences in a single-tube reaction without the need for sophisticated instruments. Traditionally, pH-sensitive dyes, DNA-binding dyes like Ethidium Bromide(EtBr), and SYBR have been employed to detect amplified products. However, these dyes present several drawbacks, limiting the widespread adoption of LAMP in diagnostic applications. To address this limitation, a novel dye called tinto rangTM has been introduced with promising applications in nucleic acid analysis. In this study, we present an evaluation of tinto rangTM's performance in comparison to commercially available dyes commonly used in LAMP assays. One of the unique advantages of tinto rangTM is its capability to facilitate the analysis of amplified nucleic acid products through three distinct methods: visual color change, changes in relative fluorescence, and turbidity. In addition to assessing tinto rangTM's performance, we also investigated various approaches to tackle the issue of non-specific amplification, which has been a common challenge in LAMP reactions. The experimental results unequivocally demonstrate that tinto rangTM outperforms pH-indicating and other DNA-binding dyes used in LAMP assays, making it a superior alternative. This study paves the way for the broader adoption of tinto rangTM in various applications, including point-of-care diagnostics, disease monitoring, and molecular biology research.

FBPP: software to design PCR primers and probes for nucleic acid base detection of foodborne pathogens

Foodborne pathogens can be found in various foods, and it is important to detect foodborne pathogens to provide a safe food supply and to prevent foodborne diseases. The nucleic acid base detection method is one of the most rapid and widely used methods in the detection of foodborne pathogens; it depends on hybridizing the target nucleic acid sequence to a synthetic oligonucleotide (probes or primers) that is complementary to the target sequence. Designing primers and probes for this method is a preliminary and critical step. However, new bioinformatics tools are needed to automate, specific and improve the design sets to be used in the nucleic acid‒base method. Thus, we developed foodborne pathogen primer probe design (FBPP), an open-source, user-friendly graphical interface Python-based application supported by the SQL database for foodborne pathogen virulence factors, for (i) designing primers/probes for detection purposes, (ii) PCR and gel electrophoresis photo simulation, and (iii) checking the specificity of primers/probes.

Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays.

The use of lateral flow assays to detect nucleic acid targets has many applications including point-of-care diagnostics, environmental monitoring, and food safety. A sandwich format, similar to that in protein immunoassays, is often used to capture the target nucleic acid sequence with an immobilized complementary strand anchored to a substrate, and then to visualize this event using a complementary label nucleic acid bound to a nanoparticle label. A critical component of high-sensitivity nucleic acid detection is to utilize high-density capture surfaces for the effective capture of target nucleic acid. Multiple methods have been reported, including the use of streptavidin-based protein anchors that can be adsorbed to the lateral flow substrate and that can utilize the high-affinity streptavidin-biotin linkage to bind biotinylated nucleic acid capture sequences for subsequent target nucleic acid binding. However, these protein anchors have not been systematically characterized for use in the context of nucleic acid detection. In this work, we characterize several protein-based anchors on nitrocellulose for (i) capturing the robustness of the attachment of the protein anchor, (ii) capturing nucleic acid density, and (iii) targeting nucleic acid capture. Further, we demonstrate the signal gains in target nucleic acid hybridization made by increasing the density of capture nucleic acid on a nitrocellulose substrate using multiple applications of protein loading onto nitrocellulose. Finally, we use our high-density capture surfaces to demonstrate high-sensitivity nucleic acid detection in a lateral flow assay (in the context of a SARS-CoV-2 sequence), achieving a LOD of approximately 0.2 nM.

PNA-Pdx: Versatile Peptide Nucleic Acid-Based Detection of Nucleic Acids and SNPs.

Monitoring diseases caused by pathogens or by mutations in DNA sequences requires accurate, rapid, and sensitive tools to detect specific nucleic acid sequences. Here, we describe a new peptide nucleic acid (PNA)-based nucleic acid detection toolkit, termed PNA-powered diagnostics (PNA-Pdx). PNA-Pdx employs PNA probes that bind specifically to a target and are then detected in lateral flow assays. This can precisely detect a specific pathogen or genotype genomic sequence. PNA probes can also be designed to invade double-stranded DNAs (dsDNAs) to produce single-stranded DNAs for precise CRISPR-Cas12b-based detection of genomic SNPs without requiring the protospacer-adjacent motif (PAM), as Cas12b requires PAM sequences only for dsDNA targets. PNA-Pdx identified target nucleic acid sequences at concentrations as low as 2 copies/μL and precisely detected the SARS-CoV-2 genome in clinical samples in 40 min. Furthermore, the specific dsDNA invasion by the PNA coupled with CRISPR-Cas12b precisely detected genomic SNPs without PAM restriction. Overall, PNA-Pdx provides a novel toolkit for nucleic acid and SNP detection as well as highlights the benefits of engineering PNA probes for detecting nucleic acids.

A dual fragment triggered DNA ladder nanostructure based on logic gate and dispersion-to-localization catalytic hairpin assembly for efficient fluorescence assay of SARS-CoV-2 and H1N1

DNA nanotechnology has been widely utilized in the construction of various functional nanostructures. However, most DNA nanostructures have the shortcomings of low response rate and serious background leakage. Herein, we proposed the conception of AND logic gate cascaded dispersion-to-localization catalytic hairpin assembly (AND gate-DLCHA) for the fabrication of novel DNA ladder nanostructures. In our design, the entropy-driven AND logic gate can precisely recognize two fragments of the target nucleic acid sequences. After AND logic gate activation by target nucleic acids, dispersion-to-localization catalytic hairpin assembly was initiated. Consequently, tremendous DNA ladder nanostructures were generated and the response signal was rapidly enhanced, which can be used for rapid and amplied detection of nucleic acids. Taking advantage of the sensitivity and specificity of AND gate-DLCHA strategy, the fluorescence sensors were established and successfully applied in ultrasensitive assay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (H1N1) within 45 min with the limit of detection (LOD) as low as 66 copies mL−1 (SARS-CoV-2) and 33 copies mL−1 (H1N1), which showed perspectives in pathogen identification and biomedical application. The high selectivity and reliability of established sensors was attributed to the dual-fragment analysis. Meanwhile, the sensors possessed minimal leakage and greatly enhanced signal to background (S/B) ratio owing to substrate transduction from dispersion into colocalization. This rationally developed logic gate cascaded dispersion-to-localization catalytic hairpin assembly strategy presented a new approach for the development of DNA nanostructures.

CRISPR/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform

Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, H1N1, H3N2, IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.

Ultrasensitive and Specific Clustered Regularly Interspaced Short Palindromic Repeats Empowered a Plasmonic Fiber Tip System for Amplification-Free Monkeypox Virus Detection and Genotyping.

The urgent necessity for highly sensitive diagnostic tools has been accentuated by the ongoing mpox (monkeypox) virus pandemic due to the complexity in identifying asymptomatic and presymptomatic carriers. Traditional polymerase chain reaction-based tests, despite their effectiveness, are hampered by limited specificity, expensive and bulky equipment, labor-intensive operations, and time-consuming procedures. In this study, we present a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based diagnostic platform with a surface plasmon resonance-based fiber tip (CRISPR-SPR-FT) biosensor. The compact CRISPR-SPR-FT biosensor, with a 125 μm diameter, offers high stability and portability, enabling exceptional specificity for mpox diagnosis and precise identification of samples with a fatal mutation site (L108F) in the F8L gene. The CRISPR-SPR-FT system can analyze viral double-stranded DNA from mpox virus without amplification in under 1.5 h with a limit of detection below 5 aM in plasmids and about 59.5 copies/μL when in pseudovirus-spiked blood samples. Our CRISPR-SPR-FT biosensor thus offers fast, sensitive, portable, and accurate target nucleic acid sequence detection.

Fanzor is a eukaryotic programmable RNA-guided endonuclease

RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR–Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage1. Although a few eukaryotic RNA-guided systems have been studied, including RNA interference2 and ribosomal RNA modification3, it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported4,5. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity4,6. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins4,7, raising the possibility that eukaryotes are also equipped with CRISPR–Cas or OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolve the structure of Spizellomyces punctatus Fz at 2.7 Å using cryogenic electron microscopy, showing the conservation of core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life.

Real-Time Quantitative PCR: Primer Design, Reference Gene Selection, Calculations and Statistics.

Real-time quantitative PCR is a technique that can measure the content of the target nucleic acid sequence of interest in a given sample. It is mainly divided into absolute and relative quantitative methods. The relative quantification is mainly used in gene expressions for functional genomic and transcriptome studies. However, to use this technology accurately, there are some key points to master. First, specific primers need to be designed to ensure amplification of the gene of interest (GOI). Second, the appropriate reference gene or reference gene combination has to be selected. Finally, scientific gene expression level calculations and statistics are required to obtain accurate results. Therefore, this work proposes a workflow for relative quantitative PCR and introduces the relevant points so that beginners can better understand and use this technology.

Advances in Electrochemical Biosensor Technologies for the Detection of Nucleic Acid Breast Cancer Biomarkers.

Breast cancer is the second leading cause of cancer deaths in women worldwide; therefore, there is an increased need for the discovery, development, optimization, and quantification of diagnostic biomarkers that can improve the disease diagnosis, prognosis, and therapeutic outcome. Circulating cell-free nucleic acids biomarkers such as microRNAs (miRNAs) and breast cancer susceptibility gene 1 (BRCA1) allow the characterization of the genetic features and screening breast cancer patients. Electrochemical biosensors offer excellent platforms for the detection of breast cancer biomarkers due to their high sensitivity and selectivity, low cost, use of small analyte volumes, and easy miniaturization. In this context, this article provides an exhaustive review concerning the electrochemical methods of characterization and quantification of different miRNAs and BRCA1 breast cancer biomarkers using electrochemical DNA biosensors based on the detection of hybridization events between a DNA or peptide nucleic acid probe and the target nucleic acid sequence. The fabrication approaches, the biosensors architectures, the signal amplification strategies, the detection techniques, and the key performance parameters, such as the linearity range and the limit of detection, were discussed.

PH-Responsive and Reversible A-Motif-Based DNA Hydrogel: Synthesis and Biosensing Application.

Functional DNA hydrogels with various motifs and functional groups require perfect sequence design to avoid cross-bonding interference with themselves or other structural sequences. This work reports an A-motif functional DNA hydrogel that does not require any sequence design. A-motif DNA is a noncanonical parallel DNA duplex structure containing homopolymeric deoxyadenosines (poly-dA) strands that undergo conformation changes from single strands at neutral pH to a parallel duplex DNA helix at acidic pH. Despite this and other advantages over other DNA motifs like no cross-bonding interference with other structural sequences, the A-motif has not been explored much. We successfully synthesized a DNA hydrogel by using an A-motif as a reversible handle to polymerize a DNA three-way junction. The A-motif hydrogel was initially characterized by electrophoretic mobility shift assay, and dynamic light scattering, which showed the formation of higher-order structures. Further, we used imaging techniques like atomic force microscopy and scanning electron microscope to validating its hydrogel like highly branched morphology. pH-induced conformation transformation from monomers to gel is quick and reversible, and was analysed for multiple acid-base cycles. The sol-to-gel transitions and gelation properties were further examined in rheological studies. The use of the A-motif hydrogel in the visual detection of pathogenic target nucleic acid sequence was demonstrated for the first time in a capillary assay. Moreover, pH-induced hydrogel formation was observed in situ as a layer over the mammalian cells. The proposed A-motif DNA scaffold has enormous potential in designing stimuli-responsive nanostructures that can be used for many biological applications.

Cas12a/blocker DNA-based multiplex nucleic acid detection system for diagnosis of high-risk human papillomavirus infection

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins are an innovative tool in molecular diagnostics owing to their high specificity and modularity for target nucleic acid sequences. However, the sequence-indiscriminate trans-cleavage activity of the Cas protein renders multiplex detection challenging. In this study, we developed a Cas12a-based multiplex detection system by designing blocker DNA complementary to reporter DNA, which enables the simultaneous detection of two genes with a single Cas protein in a single reaction. As a proof of concept, we chose high-risk human papillomavirus (HPV) 16 and 18 as the model targets and incorporated recombinase polymerase amplification (RPA) and transcription reactions to achieve high accuracy and sensitivity. Using the proposed system, we detected the genes of both HPV 16 and 18 down to 1 aM within 80 min under isothermal conditions. We validated the performance of the system in detecting genomic DNA from various cell lines and clinical samples from cervical cancer patients with high specificity. The proposed system facilitated rapid multiplex detection of high-risk HPVs in a single reaction tube with only Cas12a, thus representing a more user-friendly and economical alternative to previous Cas protein-based multiplex detection assays. The proposed system has considerable potential for point-of-care testing and could be expanded to detect various nucleic acid biomarkers.

European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development.

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development.

Molecular Mechanisms Underlying CRISPR/Cas-Based Assays for Nucleic Acid Detection

Applied to investigate specific sequences, nucleic acid detection assays can help identify novel bacterial and viral infections. Most up-to-date systems combine isothermal amplification with Cas-mediated detection. They surpass standard PCR methods in detection time and sensitivity, which is crucial for rapid diagnostics. The first part of this review covers the variety of isothermal amplification methods and describes their reaction mechanisms. Isothermal amplification enables fast multiplication of a target nucleic acid sequence without expensive laboratory equipment. However, researchers aim for more reliable results, which cannot be achieved solely by amplification because it is also a source of non-specific products. This motivated the development of Cas-based assays that use Cas9, Cas12, or Cas13 proteins to detect nucleic acids and their fragments in biological specimens with high specificity. Isothermal amplification yields a high enough concentration of target nucleic acids for the specific signal to be detected via Cas protein activity. The second part of the review discusses combinations of different Cas-mediated reactions and isothermal amplification methods and presents signal detection techniques adopted in each assay. Understanding the features of Cas-based assays could inform the choice of an optimal protocol to detect different nucleic acids.

Utilization of the Tapestri Platform for Quantification of Single Cell Vector Copy Number in CAR T Cell Products

Chimeric antigen receptor (CAR) T cell immunotherapies have been transformative solutions to treat both adult and pediatric patients with a variety of cancers. Given that CAR T cell therapies involve genetic alterations of a patient's T cells by introduction of the CAR into host cells using lentiviral vectors, the quality of CAR T cell therapies are extensively regulated to warrant safety. Key safety and efficacy attributes need to be accurately measured prior to reintroducing modified T cells back into patients, including CAR transgene copy number or viral vector copy number (VCN). We have previously compared digital PCR (dPCR) strategies to the historical standard release assay of quantitative PCR (qPCR) for VCN quantification given that dPCR is known to provide absolute quantification of target nucleic acid sequences with many advantages over qPCR in regards to simplicity, reproducibility, lower limit of detection, and definitive quantification. We were able to demonstrate that dPCR produces accurate, precise, and reproducible CAR T cell VCN measurements when compared to qPCR. However, these conventional PCR methods measuring vector copy number are either population averaged or involve labor-intensive and time-consuming single cell colony picking and outgrowth, with respective drawbacks in resolution and sample representation. Here, we introduce the Tapestri platform, to measure thousands of single cell level VCN with accuracy and precision. The Tapestri platform workflow is enabled by its two droplet steps. First, thousands of cells are each microfluidically encapsulated in a first droplet where lysis of the cells occur, releasing DNA from its heterochromatin state for uniform DNA interrogation. Then, the individual lysates are uniquely barcoded and target sequences amplified via multiplexed PCR inside each droplet. The final products are sequenced on an Illumina sequencing instrument. Single cell VCN Tapestri data was generated and orthogonally compared with dPCR and matches with the bulk measurement on clonal Jurkat cell lines (VCN=2) mixed with control cell lines (VCN=0) at a range of different ratios with accuracy and precision. We demonstrate that the Tapestri platform provides absolute quantification of CAR transgenes at the single cell level and has the potential to provide more precise measurements of CAR T cell therapeutic product attributes prior to release to patients. Future studies will evaluate the utility of this Tapestri platform in measuring both CAR transgene copies and surface expression/density of the CAR at the single cell level, further improving upon current CAR T cell product release assays.

Selective and Reversible Ligand Assembly on the DNA and RNA Repeat Sequences in Myotonic Dystrophy.

Small molecule targeting of DNA and RNA sequences has come into focus as a therapeutic strategy for diseases such as myotonic dystrophy type 1 (DM1), a trinucleotide repeat disease characterized by RNA gain-of-function. Herein, we report a novel template-selected, reversible assembly of therapeutic agents in situ via aldehyde-amine condensation. Rationally designed small molecule targeting agents functionalized with either an aldehyde or an amine were synthesized and screened against the target nucleic acid sequence. The assembly of fragments was confirmed by MALDI-MS in the presence of DM1-relevant nucleic acid sequences. The resulting hit combinations of aldehyde and amine inhibited the formation of r(CUG)exp in vitro in a cooperative manner at low micromolar levels and rescued mis-splicing defects in DM1 model cells. This reversible template-selected assembly is a promising approach to achieve cell permeable and multivalent targeting via in situ synthesis and could be applied to other nucleic acid targets.

A CRISPR/Cas12a-empowered surface plasmon resonance platform for rapid and specific diagnosis of the Omicron variant of SARS-CoV-2.

The outbreak of the COVID-19 pandemic was partially due to the challenge of identifying asymptomatic and presymptomatic carriers of the virus, and thus highlights a strong motivation for diagnostics with high sensitivity that can be rapidly deployed. On the other hand, several concerning SARS-CoV-2 variants, including Omicron, are required to be identified as soon as the samples are identified as ‘positive’. Unfortunately, a traditional PCR test does not allow their specific identification. Herein, for the first time, we have developed MOPCS (Methodologies of Photonic CRISPR Sensing), which combines an optical sensing technology-surface plasmon resonance (SPR) with the ‘gene scissors’ clustered regularly interspaced short palindromic repeat (CRISPR) technique to achieve both high sensitivity and specificity when it comes to measurement of viral variants. MOPCS is a low-cost, CRISPR/Cas12a-system-empowered SPR gene-detecting platform that can analyze viral RNA, without the need for amplification, within 38 min from sample input to results output, and achieve a limit of detection of 15 fM. MOPCS achieves a highly sensitive analysis of SARS-CoV-2, and mutations appear in variants B.1.617.2 (Delta), B.1.1.529 (Omicron) and BA.1 (a subtype of Omicron). This platform was also used to analyze some recently collected patient samples from a local outbreak in China, identified by the Centers for Disease Control and Prevention. This innovative CRISPR-empowered SPR platform will further contribute to the fast, sensitive and accurate detection of target nucleic acid sequences with single-base mutations.

JMM profile: Loop-mediated isothermal amplification (LAMP): for the rapid detection of nucleic acid targets in resource-limited settings.

Loop-mediated isothermal amplification (LAMP) is a rapid alternative to PCR, in which the reaction occurs at one temperature and uses a polymerase with high displacement activity, e.g. Bacillus stearothermophilus DNA polymerase I (Bst) or homologues. Since the discovery of LAMP in 2000, several applications have been developed to employ this technique in the rapid detection of nucleic acid targets and enhance its performance. Improvements to the LAMP technique and a variety of innovative detection methods have led to its application for a wide range of targets in medical and veterinary microbiology, particularly in resource-poor settings. The key advantages of LAMP-based diagnostics include the ability to rapidly detect target nucleic acid sequences within 30 min and its ease of use, facilitating its application in field, bedside, pen-side, point-of-care and point-of-need diagnostic settings. LAMP can be a valuable tool to aid in the detection and management of disease outbreaks.

Upconversion-mediated CRISPR-Cas12a biosensing for sensitive detection of ochratoxin A.

The CRISPR-Cas system has achieved breakthrough applications in the field of molecular diagnostics. CRISPR/Cas12a can accurately identify subtle changes in a target nucleic acid sequence and has a wide range of applications such as in highly sensitive detection methods. In this study, an upconversion-magnetic probe-DNA-Fe3O4 probe was designed to replace traditional fluorescent probes using nucleic acid aptamers to design a biosensing method powered by CRISPR/Cas12a. The CRISPR/Cas12a technology can be widely used for sensitive, rapid, and stable detection of ochratoxin A. In our sensing strategy, the ochratoxin A capture probe was used to capture ochratoxin A and release the Cas protein activation chain, further triggering the single-stranded DNA degradation activity of CRISPR/Cas12a and removal of the fluorescent probe to generate a fluorescent signal. Compared with traditional commercial kits, our method was more rapid and exhibited comparable detection capability.