A Simplified Amplification-Free Strategy with Lyophilized CRISPR-CcrRNA System for Drug-Resistant Salmonella Detection.

Drug resistance in pathogenic bacteria has become a major threat to global health. The misuse of antibiotics has increased the number of resistant bacteria in the absence of rapid, accurate, and cost-effective diagnostic tools. Here, an amplification-free CRISPR-Cas12a time-resolved fluorescence immunochromatographic assay (AFC-TRFIA) is used to detect drug-resistant Salmonella. Multi-locus targeting in combination crRNA (CcrRNA) is 27-fold more sensitive than a standalone crRNA system. The lyophilized CRISPR system further simplifies the operation and enables one-pot detection. Induction of nucleic acid fixation via differentially charged interactions reduced the time and cost required for flowmetric chromatography with enhanced stability. The induction of nucleic acid fixation via differentially charged interactions reduces the time and cost required for flowmetric chromatography with enhanced stability. The platform developed for the detection of drug-resistant Salmonella has an ultra-sensitive detection limit of 84 CFU mL-1 within 30 min, with good linearity in the range of 102 -106 CFU mL-1 . In real-world applications, spiked recoveries range from 76.22% to 145.91%, with a coefficient of variation less than 10.59%. AFC-TRFIA offers a cost-effective, sensitive, and virtually equipment-independent platform for preventing foodborne illnesses, screening for drug-resistant Salmonella, and guiding clinical use.

An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA.

Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a "sample-in-answer-out" single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.

CRISPR‐Cas Biochemistry and CRISPR‐Based Molecular Diagnostics

Polymerase chain reaction (PCR)‐based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR‐mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point‐of‐care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR‐based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics‐target preamplification‐free detection, microRNA (miRNA) testing, non‐nucleic‐acid detection, and SARS‐CoV‐2 detection‐are also covered.

An aM-level sensitive cascade CRISPR-Dx system (ASCas) for rapid detection of RNA without pre-amplification.

The CRISPR/Cas system is known as one of the directions of the next generation of mainstream molecular diagnostic technology. However, most current CRISPR/Cas molecular diagnostics still rely on the pre-amplification of nucleic acid due to the limited sensitivity of CRISPR/Cas alone, which has no significant advantage over commercial Taqman-PCR and TwistAmp® Exo kits. Herein, we report an aM-level sensitive cascade CRISPR-Dx system (ASCas) that eliminates nucleic acid pre-amplification, thus avoiding aerosol contamination and greatly reducing the testing environment and personnel skill requirements for molecular diagnostics. Most importantly, the Cas13a nucleases with high sensitivity and trans-cleavage efficiency can rapidly cleaved RNA bubbles on the hybridized cascade probe at low concentration target RNA detection, which results in the destruction of the cascade probe and releases a large amount of trigger DNA for further signal amplification of secondary Cas12a reactions. Therefore, the ASCas system achieves amplification-free, ultra-sensitivity (1 aM), and ultra-fast (20 min) RNA detection. In addition, the ASCas system replaces the complicated screening process of primers and probes with the programmed Cas13a-crRNA design so that a suitable detection system can be constructed more quickly and straightforwardly for the mutation-prone SARS-CoV-2 virus.

CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics.

Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) testing, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.

A SERS-signalled, CRISPR/Cas-powered bioassay for amplification-free and anti-interference detection of SARS-CoV-2 in foods and environmental samples using a single tube-in-tube vessel.

The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans-cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans-cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.

Nanomechanical assay for ultrasensitive and rapid detection of SARS-CoV-2 based on peptide nucleic acid.

The massive global spread of the COVID-19 pandemic makes the development of more effective and easily popularized assays critical. Here, we developed an ultrasensitive nanomechanical method based on microcantilever array and peptide nucleic acid (PNA) for the detection of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) RNA. The method has an extremely low detection limit of 0.1 fM (105 copies/mL) for N-gene specific sequence (20 bp). Interestingly, it was further found that the detection limit of N gene (pharyngeal swab sample) was even lower, reaching 50 copies/mL. The large size of the N gene dramatically enhances the sensitivity of the nanomechanical sensor by up to three orders of magnitude. The detection limit of this amplification-free assay method is an order of magnitude lower than RT-PCR (500 copies/mL) that requires amplification. The non-specific signal in the assay is eliminated by the in-situ comparison of the array, reducing the false-positive misdiagnosis rate. The method is amplification-free and label-free, allowing for accurate diagnosis within 1 h. The strong specificity and ultra-sensitivity allow single base mutations in viruses to be distinguished even at very low concentrations. Also, the method remains sensitive to fM magnitude lung cancer marker (miRNA-155). Therefore, this ultrasensitive, amplification-free and inexpensive assay is expected to be used for the early diagnosis of COVID-19 patients and to be extended as a broad detection tool. Electronic Supplementary Material: Supplementary material (experimental section, N gene sequences and all nucleic acid sequences used in the study, Figs. S1-S6, and Tables S1-S3) is available in the online version of this article at 10.1007/s12274-022-4333-3.

High-intensity vector signals for detecting SARS-CoV-2 RNA using CRISPR/Cas13a couple with stabilized graphene field-effect transistor.

False detection of SARS-CoV-2 is detrimental to epidemic prevention and control. The scalar nature of the detected signal and the imperfect target recognition property of developed methods are the root causes of generating false signals. Here, we reported a collaborative system of CRISPR-Cas13a coupling with the stabilized graphene field-effect transistor, providing high-intensity vector signals for detecting SARS-CoV-2. In this collaborative system, SARS-CoV-2 RNA generates a "big subtraction" signal with a right-shifted feature, whereas any untargets cause the left-shifted characteristic signal. Thus, the false detection of SARS-CoV-2 is eliminated. High sensitivity with 0.15 copies/µL was obtained. In addition, the wide concerned instability of the graphene field-effect transistor for biosensing in solution environment was solved by the hydrophobic treatment to its substrate, which should be a milestone in advancing it's engineering application. This collaborative system characterized by the high-intensity vector signal and amazing stability significantly advances the accurate SARS-CoV-2 detection from the aspect of signal nature.

Amplification‐Free Detection of SARS‐CoV‐2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field‐Effect Transistors

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) systems have recently received notable attention for their applications in nucleic acid detection. Despite many attempts, the majority of current CRISPR‐based biosensors in infectious respiratory disease diagnostic applications still require target preamplifications. This study reports a new biosensor for amplification‐free nucleic acid detection via harnessing the trans‐cleavage mechanism of Cas13a and ultrasensitive graphene field‐effect transistors (gFETs). CRISPR Cas13a‐gFET achieves the detection of SARS‐CoV‐2 and respiratory syncytial virus (RSV) genome down to 1 attomolar without target preamplifications. Additionally, we validate the detection performance using clinical SARS‐CoV‐2 samples, including those with low viral loads (Ct value >30). Overall, these findings establish our CRISPR Cas13a‐gFET among the most sensitive amplification‐free nucleic acid diagnostic platforms to date.

Rücktitelbild: Amplification‐Free Detection of SARS‐CoV‐2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field‐Effect Transistors

Der Nachweis von Nukleinsäuren spielt eine wichtige Rolle in der medizinischen Diagnostik, der Umweltüberwachung und der Lebensmittelsicherheit. In ihrem Forschungsartikel (e202203826) entwickelten Xue Gao, Yi Zhang und Mitarbeiter einen neuen Biosensor für den amplifikationsfreien Nukleinsäurenachweis, indem sie den trans‐Spaltungsmechanismus von Cas13a und ultrasensitive Graphen‐Feldeffekttransistoren (gFETs) nutzten. Die Abbildung zeigt die Cas13a‐vermittelte RNA‐trans‐Spaltung auf der gFET‐Oberfläche für die Sensorsignalübertragung.

Recent advances in nucleic acid analysis and detection with microfluidic and nanofluidics

Microfluidic- and nanofluidics-based nucleic acid sensing and analysis have become of interest to the public, especially during the current COVID pandemic. In this chapter, we provide a comprehensive review of recent research dedicated to the advances of nucleic acid analysis and detection including various polymerase chain reaction platforms, isothermal target amplification methods, and emerging amplification-free methods, such as optofluidics sensing, electrochemical sensing, thermal sensing, and advanced microscopy for label-free DNA/RNA analysis. The future advancement and prospects of nucleic acid analysis are also discussed. © 2022 Elsevier B.V. All rights reserved.

Amplification-free detection of SARS-CoV-2 using gold nanotriangles functionalized with oligonucleotides.

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.

Rapid and High-Throughput SARS-CoV-2 RNA Detection without RNA Extraction and Amplification by Using a Microfluidic Biochip.

The ongoing outbreak of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has spread globally and poses a threat to public health and National economic development. Rapid and high-throughput SARS-CoV-2 RNA detection without the need of RNA extraction and amplification remain a key challenge. In this study, a new SARS-CoV-2 RNA detection strategy using a microfluidic biochip for the rapid and ultrasensitive detection of SARS-CoV-2 without RNA extraction and amplification was developed. This new strategy takes advantage of the specific SARS-CoV-2 RNA and probe DNA reaction in the microfluidic channel, fluorescence signal regulation by nanomaterials, and accurate sample control by the microfluidic chip. It presents an ultralow limit of detection of 600 copies mL-1 in a large linear detection regime from 1 aM to 100 fM. Fifteen samples were simultaneously detected in 40 min without the need for RNA purification and amplification. The detection accuracy of the strategy was validated through quantitative reverse transcription polymerase chain reaction (qRT-PCR), with a recovery of 99-113 %. Therefore, the SARS-CoV-2 RNA detection strategy proposed in this study can potentially be used for the quantitative diagnosis of viral infectious diseases.

Clustered Regularly Interspaced Short Palindromic Repeats-Mediated Amplification-Free Detection of Viral DNAs Using Surface-Enhanced Raman Spectroscopy-Active Nanoarray.

Nucleic acid biomarkers have been widely used to detect various viral-associated diseases, including the recent pandemic COVID-19. The CRISPR-Cas-based trans-activating phenomenon has shown excellent potential for developing sensitive and selective detection of nucleic acids. However, the nucleic acid amplification steps are typically required when sensitive and selective monitoring of the target nucleic acid is needed. To overcome the aforementioned challenges, we developed a CRISPR-Cas12a-based nucleic acid amplification-free biosensor by a surface-enhanced Raman spectroscopy (SERS)-assisted ultrasensitive detection system. We integrated the activated CRISPR-Cas12a by viral DNA with a Raman-sensitive system composed of ssDNA-immobilized Raman probe-functionalized Au nanoparticles (RAuNPs) on the graphene oxide (GO)/triangle Au nanoflower array. Using this CRISPR-based Raman-sensitive system improved the detection sensitivity of the multiviral DNAs such as hepatitis B virus (HBV), human papillomavirus 16 (HPV-16), and HPV-18 with an extremely low detection limit and vast detection range from 1 aM to 100 pM without the amplification steps. We suggest that this ultrasensitive amplification-free detection system for nucleic acids can be widely applied to the precise and early diagnosis of viral infections, cancers, and several genetic diseases.

Rapid and unamplified identification of COVID-19 with morpholino-modified graphene field-effect transistor nanosensor.

SARS-CoV-2 RNA is identified as a pivotal player to bolster energizing zones of COVID-19 detection. Herein, we develop a rapid and unamplified nanosensing platform for detection of SARS-CoV-2 RNA in human throat swab specimens. A gold nanoparticle (AuNP)-decorated graphene field-effect transistor (G-FET) sensor was fabricated, after which complementary phosphorodiamidate morpholino oligos (PMO) probe was immobilized on the AuNP surface. This sensor allowed for highly sensitive testing of SARS-CoV-2 RdRp as PMO does not have charges, leading to low background signal. Not only did the method present a low limit of detection in PBS (0.37 fM), throat swab (2.29 fM), and serum (3.99 fM), but also it achieved a rapid response to COVID-19 patients' samples within 2 min. The developed nanosensor was capable of analyzing RNA extracts from 30 real clinical samples. The results show that the sensor could differentiate the healthy people from infected people, which are in high agreement with RT-PCR results (Kappa index = 0.92). Furthermore, a well-defined distinction between SARS-CoV-2 RdRp and SARS-CoV RdRp was also made. Therefore, we believe that this work provides a satisfactory, attractive option for COVID-19 diagnosis.