Multiplexing Imaging of Closely Located Single-Nucleotide Mutations in Single Cells via Encoded in situ PCR.

Mutation accumulation in RNAs results in closely located single-nucleotide mutations (SNMs), which is highly associated with the drug resistance of pathogens. Imaging of SNMs in single cells has significance for understanding the heterogeneity of RNAs that are related to drug resistance, but the direct "see" closely located SNMs remains challenging. Herein, we designed an encoded ligation-mediated in situ polymerase chain reaction method (termed enPCR), which enabled the visualization of multiple closely located SNMs in bacterial RNAs. Unlike conventional ligation-based probes that can only discriminate a single SNM, this method can simultaneously image different SNMs at closely located sites with single-cell resolution using modular anchoring probes and encoded PCR primers. We tested the capacity of the method to detect closely located SNMs related to quinolone resistance in the gyrA gene of Salmonella enterica (S. enterica), and found that the simultaneous detection of the closely located SNMs can more precisely indicate the resistance of the S. enterica to quinolone compared to the detection of one SNM. The multiplexing imaging assay for SNMs can serve to reveal the relationship between complex cellular genotypes and phenotypes.

Multiple analyses of main flavor components in reconstituted tobacco and transfer behavior of their key substances during heating.

Reconstituted tobacco (RT) is a product made by reprocessing tobacco waste, experiencing a growing demand for heat-not-burn products. The purpose of this study is to analyze the main flavor ingredients in RT aerosol, as well as the transfer behavior of key flavor substances from substrates to aerosol and the concentrations of these compounds in the substrate after heating. First, we demonstrated that the odor of four RT aerosol samples could be distinguished using an electronic nose. Through non-targeted analysis, 93 volatile compounds were detected by gas chromatography-mass spectrometry, and 286 non/semi-volatile compounds were identified by ultra-high-performance liquid electrophoresis chromatography-mass spectrometry in aerosol. Furthermore, we found that the formation of RT aerosol involves primarily evaporation and distillation, however, the total content delivered from unheated RT samples to aerosol remains relatively low due to compound volatility and cigarette filtration. Thermal reactions during heating indicated the pyrolysis of chlorogenic acid to generate catechol and resorcinol, while Maillard reactions involving glucose and proline produced 2,3-dihydro-3,5-dihydroxy-6-methyl-4h-pyran-4-one. The study highlighted that heating RT at approximately 300°C could mitigate the production of harmful substances while still providing a familiar sensory experience with combusted tobacco.

Precise, Sensitive Detection of Viable Foodborne Pathogenic Bacteria with a 6-Order Dynamic Range via Digital Rolling Circle Amplification.

The presence of viable pathogenic bacteria in food can lead to serious foodborne diseases, thus posing a risk to human health. Here, we develop a digital rolling circle amplification (dRCA) assay that enables the precise and sensitive quantification of viable foodborne pathogenic bacteria. Directly targeting pathogenic RNAs via a ligation-based padlock probe allows for precisely discriminating viable bacteria from dead one. The one-target-one-amplicon characteristic of dRCA enables high sensitivity and a broad quantitative detection range, conferring a detection limit of 10 CFU/mL and a dynamic range of 6 orders. dRCA can detect rare viable bacteria, even at a proportion as low as 0.1%, which is 50 times more sensitive than the live/dead staining method. The high sensitivity for detecting viable bacteria accommodates dRCA for assessing sterilization efficiency. Based on the assay, we found that, for pasteurization, slightly elevating the temperature to 68 °C can reduce the heating time to 10 min, which may minimize nutrient degradation caused by high-temperature exposure. The assay can serve as a precise tool for estimating the contamination by viable pathogenic bacteria and assessing sterilization, which facilitates food safety control.

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.

Single-Cell Imaging of mRNA by Target RNA-Initiated RCA.

We present a powerful method for direct mRNA detection based on ligation-based recognition and in situ amplification, capable of single-cell imaging mRNA at single-nucleotide and single-molecule resolution. Attributed to the use of Splint R ligase that can ligate padlock probe with RNA as target template, this method can efficiently detect mRNA in the absence of reverse transcription. This method enables spatial localization and correlation analysis of gene expression in single cells, which helps us to elucidate gene function and regulatory mechanisms.

Recent advances in enzyme-free and enzyme-mediated single-nucleotide variation assay in vitro.

Single-nucleotide variants (SNVs) are the most common type variation of sequence alterations at a specific location in the genome, thus involving significant clinical and biological information. The assay of SNVs has engaged great awareness, because many genome-wide association studies demonstrated that SNVs are highly associated with serious human diseases. Moreover, the investigation of SNV expression levels in single cells are capable of visualizing genetic information and revealing the complexity and heterogeneity of single-nucleotide mutation-related diseases. Thus, developing SNV assay approaches in vitro, particularly in single cells, is becoming increasingly in demand. In this review, we summarized recent progress in the enzyme-free and enzyme-mediated strategies enabling SNV assay transition from sensing interface to the test tube and single cells, which will potentially delve deeper into the knowledge of SNV functions and disease associations, as well as discovering new pathways to diagnose and treat diseases based on individual genetic profiles. The leap of SNV assay achievements will motivate observation and measurement genetic variations in single cells, even within living organisms, delve into the knowledge of SNV functions and disease associations, as well as open up entirely new avenues in the diagnosis and treatment of diseases based on individual genetic profiles.

DNAzyme-activated CRISPR/Cas assay for sensitive and one-pot detection of lead contamination.

Hazardous lead ions (Pb2+) even at a minute level can pose side effects on human health, highlighting the need for tools for trace Pb2+ detection. Herein, we present a DNAzyme-activated CRISPR assay (termed DzCas12T) for sensitive and one-pot detection of lead contamination. Using an extension-bridged strategy eliminates the need for separation to couple the DNAzyme recognition and CRISPR reporting processes. The tandem design endowed the DzCas12T assay with high specificity and sensitivity down to the pM-level. This assay has been used to detect lead contamination in food and water samples, indicating the potential for monitoring lead-associated environmental and food safety.

In Situ Cas12a-Based Allele-Specific PCR for Imaging Single-Nucleotide Variations in Foodborne Pathogenic Bacteria.

In situ profiling of single-nucleotide variations (SNVs) can elucidate drug-resistant genotypes with single-cell resolution. The capacity to directly "see" genetic information is crucial for investigating the relationship between mutated genes and phenotypes. Fluorescence in situ hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR (ARMS-PCR) method that allows the visualization of SNVs related to quinolone resistance inside cells. The capacity of discriminating SNVs is enhanced by incorporating optimized mismatched bases in the allele-specific primers, thus allowing to specifically amplify quinolone-resistant related genes. After in situ ARMS-PCR, we employed a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling specific binding of fluorophore-labeled DNA probes. The method allows to precisely quantify quinolone-resistant Salmonella enterica in the bacterial mixture. Utilizing this method, we investigated the survival competition capacity of quinolone-resistant and quinolone-sensitive bacteria toward antimicrobial peptides and indicated the enrichment of quinolone-resistant bacteria under colistin sulfate stress. The in situ Cas12a-based ARMS-PCR method holds the potential for profiling cellular phenotypes and gene regulation with single-nucleotide resolution at the single-cell level.

Visual genetic typing of glioma using proximity-anchored in situ spectral coding amplification.

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels. The ligation-based padlock probe can discriminate one-nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module-based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one-time labelling, with low cost and simple operation. One-target-one-amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single-molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.

In vivo imaging of mitochondrial DNA mutations using an integrated nano Cas12a sensor.

Mutations in mitochondrial DNA (mtDNA) play critical roles in many human diseases. In vivo visualization of cells bearing mtDNA mutations is important for resolving the complexity of these diseases, which remains challenging. Here we develop an integrated nano Cas12a sensor (InCasor) and show its utility for efficient imaging of mtDNA mutations in live cells and tumor-bearing mouse models. We co-deliver Cas12a/crRNA, fluorophore-quencher reporters and Mg2+ into mitochondria. This process enables the activation of Cas12a's trans-cleavage by targeting mtDNA, which efficiently cleave reporters to generate fluorescent signals for robustly sensing and reporting single-nucleotide variations (SNVs) in cells. Since engineered crRNA significantly increase Cas12a's sensitivity to mismatches in mtDNA, we can identify tumor tissue and metastases by visualizing cells with mutant mtDNAs in vivo using InCasor. This CRISPR imaging nanoprobe holds potential for applications in mtDNA mutation-related basic research, diagnostics and gene therapies.

Single-Cell Genotyping of Single-Nucleotide Mutations Using In Situ Allele-Specific Loop-Mediated Isothermal Amplification.

Single-nucleotide mutations (SNMs) in the bacterial genome may cause antibiotic resistance. The visualization of SNMs can indicate antibiotic resistance phenotypes at the single-cell level but remains challenging. Herein, we proposed an in situ allele-specific isothermal amplification proceeded inside cells, allowing us to image bacterial genes with single-nucleotide resolution. The primer for loop-mediated isothermal amplification (LAMP) was designed with artificial mismatch bases to serve as an allele-specific probe, endowing LAMP to specifically amplify genes with SNMs. Due to the high amplification efficiency of LAMP, the method termed AlleLAMP can generate high gain for imaging SNMs and precisely quantify mutated quinolone-resistant Salmonella in bacterial mixture. We utilized AlleLAMP to survey the selection of antibiotic resistance under the preservative stress and found that the mutant quinolone-resistant strain owned a survival advantage over the wild-type quinolone-sensitive strain under the stress of preservatives. AlleLAMP can serve as a single-cell tool for analyzing the relationship between bacterial genotype and phenotype.

Species-Level Monitoring of Key Bacteria in Fermentation Processes Using Single-Nucleotide Resolved Nucleic Acid Assays Based on CRISPR/Cas12.

Microorganisms can determine the flavor and quality of fermented food, such as Baijiu, which is produced via Daqu fermentation. Therefore, monitoring key microorganisms during fermentation is important for ensuring high-quality fermented food. Here, we report a single-nucleotide resolved nucleic acid assay based on the CRISPR/Cas12 system, enabling the quantification of Bacillus amyloliquefaciens, a key microorganism in Daqu fermentation at the species level. The assay employs an amplification-refractory mutation system derived from PCR to analyze minor genetic differences between different Bacillus species. The utilization of CRISPR/Cas12 further guaranties the specificity of identifying the PCR amplicon and enables the quantification of Bacillus amyloliquefaciens via end-measurement fluorescence. Compared to conventional qPCR, the assay allows for species-level detection of bacteria, thus enabling the precise detection of the Bacillus strain that yields high-level 2,3,5,6-tetramethylpyrazine. The assay promises the precise monitoring of bacterial growth and contribution to flavor during Daqu fermentation, thus facilitating fermented food quality control.

Precise in-field molecular diagnostics of crop diseases by smartphone-based mutation-resolved pathogenic RNA analysis.

Molecular diagnostics for crop diseases can guide the precise application of pesticides, thereby reducing pesticide usage while improving crop yield, but tools are lacking. Here, we report an in-field molecular diagnostic tool that uses a cheap colorimetric paper and a smartphone, allowing multiplexed, low-cost, rapid detection of crop pathogens. Rapid nucleic acid amplification-free detection of pathogenic RNA is achieved by combining toehold-mediated strand displacement with a metal ion-mediated urease catalysis reaction. We demonstrate multiplexed detection of six wheat pathogenic fungi and an early detection of wheat stripe rust. When coupled with a microneedle for rapid nucleic acid extraction and a smartphone app for results analysis, the sample-to-result test can be completed in ~10 min in the field. Importantly, by detecting fungal RNA and mutations, the approach allows to distinguish viable and dead pathogens and to sensitively identify mutation-carrying fungicide-resistant isolates, providing fundamental information for precision crop disease management.

Wash-Free and High-Contrast Imaging of Single-Nucleotide Variation in Single Cells Using Transcription-Amplified Light-Up RNA Aptamer.

Single-nucleotide variation (SNV) imaging can indicate cellular heterogeneity and spatial pattern, but it remains challenging to produce high-gain signal while also yielding single-nucleotide resolution. Herein, we developed a light-up strategy for visualizing SNVs based on transcription amplification, enabling wash-free and high-contrast imaging of SNVs inside cells. The discrimination of SNVs is achieved by ligase-assisted transcription reaction. Employing a light-up RNA aptamer as a reporter eliminates nonspecific probe binding and the washing process and contributes to a 2-fold improvement of signal gain compared to that using the fluorescence in situ hybridization (FISH) method. The method allowed us to precisely quantify drug-resistant strains in the bacteria mixture and identify drug-resistant Salmonella enterica (S. enterica) isolated from poultry farm. Using this approach, we explored the colonization features of drug-resistant and drug-sensitive S. enterica in the mice intestinal tract and screened the prebiotics for Salmonella colonization inhibition. The SNV imaging method promises for the interrogation of genotypes in physiological and pathological states at the single-cell level.

Antibiotic resistance mechanism and diagnosis of common foodborne pathogens based on genotypic and phenotypic biomarkers.

The emergence of antibiotic-resistant bacteria due to the overuse or inappropriate use of antibiotics has become a significant public health concern. The agri-food chain, which serves as a vital link between the environment, food, and human, contributes to the large-scale dissemination of antibiotic resistance, posing a concern to both food safety and human health. Identification and evaluation of antibiotic resistance of foodborne bacteria is a crucial priority to avoid antibiotic abuse and ensure food safety. However, the conventional approach for detecting antibiotic resistance heavily relies on culture-based methods, which are laborious and time-consuming. Therefore, there is an urgent need to develop accurate and rapid tools for diagnosing antibiotic resistance in foodborne pathogens. This review aims to provide an overview of the mechanisms of antibiotic resistance at both phenotypic and genetic levels, with a focus on identifying potential biomarkers for diagnosing antibiotic resistance in foodborne pathogens. Furthermore, an overview of advances in the strategies based on the potential biomarkers (antibiotic resistance genes, antibiotic resistance-associated mutations, antibiotic resistance phenotypes) for antibiotic resistance analysis of foodborne pathogens is systematically exhibited. This work aims to provide guidance for the advancement of efficient and accurate diagnostic techniques for antibiotic resistance analysis in the food industry.

Rapid and Sensitive Detection of Fungicide-Resistant Crop Fungal Pathogens Using an Isothermal Amplification Refractory Mutation System.

Fungicide abuse leads to the emergence of fungicide-resistant fungal pathogens, thus posing a threat to agriculture and food safety. Here, we developed an isothermal amplification refractory mutation system (termed iARMS) allowing us to resolve genetic mutations, enabling rapid, sensitive, and potentially field-applicable detection of fungicide-resistant crop fungal pathogens. iARMS yielded a limit of detection of 25 aM via a cascade signal amplification strategy of recombinase polymerase amplification (RPA) and Cas12a-mediated collateral cleavage at 37 °C within 40 min. Specificity for fungicide-resistant Puccinia striiformis (P. striiformis) detection was guaranteed by RPA primers and the flexible sequence of gRNA. The iARMS assay allowed us to detect as low as 0.1% cyp51-mutated P. striiformis that showed resistance to the demethylase inhibitor (DMI), which was 50 times more sensitive than the sequencing techniques. Thus, it is promising for the discovery of rare fungicide-resistant isolates. We applied iARMS to investigate the emergence of fungicide-resistant P. striiformis in western China and found that its proportion was over 50% in Qinghai, Sichuan, and Xinjiang Province. iARMS can serve as a molecular diagnostic tool for crop diseases and facilitate precision plant disease management.

CRISPR-based biosensors for pathogenic biosafety.

Pathogenic biosafety is a worldwide concern. Tools for analyzing pathogenic biosafety, that are precise, rapid and field-deployable, are highly demanded. Recently developed biotechnological tools, especially those utilizing CRISPR/Cas systems which can couple with nanotechnologies, have enormous potential to achieve point-of-care (POC) testing for pathogen infection. In this review, we first introduce the working principle of class II CRISPR/Cas system for detecting nucleic acid and non-nucleic acid biomarkers, and highlight the molecular assays that leverage CRISPR technologies for POC detection. We summarize the application of CRISPR tools in detecting pathogens, including pathogenic bacteria, viruses, fungi and parasites and their variants, and highlight the profiling of pathogens' genotypes or phenotypes, such as the viability, and drug-resistance. In addition, we discuss the challenges and opportunities of CRISPR-based biosensors in pathogenic biosafety analysis.

Recent Advances in Cardiovascular Disease Biosensors and Monitoring Technologies.

Cardiovascular disease (CVD) causes significant mortality and remains the leading cause of death globally. Thus, to reduce mortality, early diagnosis by measurement of cardiac biomarkers and heartbeat signals presents fundamental importance. Traditional CVD examination requires bulky hospital instruments to conduct electrocardiography recording and immunoassay analysis, which are both time-consuming and inconvenient. Recently, development of biosensing technologies for rapid CVD marker screening attracted great attention. Thanks to the advancement in nanotechnology and bioelectronics, novel biosensor platforms are developed to achieve rapid detection, accurate quantification, and continuous monitoring throughout disease progression. A variety of sensing methodologies using chemical, electrochemical, optical, and electromechanical means are explored. This review first discusses the prevalence and common categories of CVD. Then, heartbeat signals and cardiac blood-based biomarkers that are widely employed in clinic, as well as their utilizations for disease prognosis, are summarized. Emerging CVD wearable and implantable biosensors and monitoring bioelectronics, allowing these cardiac markers to be continuously measured are introduced. Finally, comparisons of the pros and cons of these biosensing devices along with perspectives on future CVD biosensor research are presented.

Precise Authenticity of Quinoa, Coix Seed, Wild Rice and Chickpea Components Using Optimized TaqMan Real-Time PCR.

Functional food such as, quinoa, coix seed, wild rice and chickpea have experienced rapidly increasing demand globally and exhibit high economic values. Nevertheless, a method for rapid yet accurate detection of these source components is absent, making it difficult to identify commercially available food with labels indicating the presence of relevant components. In this study, we constructed a real-time quantitative polymerase chain reaction (qPCR) method for rapid detection of quinoa, coix seed, wild rice and chickpea in food to identify the authenticity of such food. Specific primers and probes were designed with 2S albumin genes of quinoa, SAD genes of coix seed, ITS genes of wild rice and CIA-2 genes of chickpea as the target genes. The qPCR method could specifically identify the four wild rice strains, yielding, LODs of 0.96, 1.14, 1.04 and 0.97 pg/µL quinoa, coix seed, wild rice and chickpea source components, respectively. Particularly, the method allowed the identification of the target component with content below 0.01%. A total of 24 commercially available food samples of different types were detected by using the method and the results indicate that the developed method is applicable to the detection of different food matrices, as well as authenticity verification in deeply processed food.

CRISPR-based nucleic acid diagnostics for pathogens.

Pathogenic infection remains the primary threat to human health, such as the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutated variants, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we firstly describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges involved in on-site diagnostic assays and present emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics.