Sensitive RNA Detection Research Articles

One-tube target amplification-free CRISPR-Cas13 autocatalytic system

Clustered regularly interspaced short palindromic repeats (CRISPR) diagnostics have shown great promise for nucleic acid testing. However, CRISPR/Cas13a-based RNA sensing is hindered by the absence of accurate, fast and sensitive assays. Current Cas-based methods generally involve two separate steps: target-amplification and Cas-detection. This two-step method complicates detection, causes cross-contamination, and is difficult to deploy. Therefore, it is necessary to develop a facile one-step Cas13a-based RNA detection assay. We present a one-tube target amplification-free CRISPR-Cas13a autocatalytic circuit system that combines the Cas13a nuclease, T4 polynucleotide kinase, Bsu DNA polymerase and T7 RNA polymerase with a universally designed combinatorial DNA probe to create a one-pot assay for isothermal RNA detection. In this assay, after target RNA recognition, the cis-cleavage and trans-cleavage activities of Cas13 are activated, which leads to the initiation of a Cas13a autocatalytic reaction, resulting in an exponentially amplifying detection signal, thus enabling the detection of approximately 23.01 (95 % CI:16.95-41.23) copies/µL RNA in 45 min. A facile, easy-to-deploy, and one-tube Cas13a-based autocatalytic reaction system was constructed for rapid and highly sensitive RNA detection for the first time. This Cas13a-based autocatalytic system offers not only a versatile platform for directly detecting RNA targets but also a route toward point-of-care nucleic acid testing.

CRISPR/Cas12a-Powered Amplification-Free RNA Diagnostics by Integrating T7 Exonuclease-Assisted Target Recycling and Split G-Quadruplex Catalytic Signal Output.

Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for RNA detection often rely on reverse transcription (RT) and DNA amplification or involve a time-consuming procedure and poor sensitivity. Herein, we proposed a CRISPR/Cas12a-enabled amplification-free assay for rapid, specific, and sensitive RNA diagnostics. This assay, which we termed T7/G4-CRISPR, involved the use of a T7-powered nucleic acid circuit to convert a single RNA target into numerous DNA activators via toehold-mediated strand displacement reaction and T7 exonuclease-mediated target recycling amplification, followed by activating Cas12a trans-cleavage of the linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion to the input RNA target. We first performed step-by-step validation of the entire assay process and optimized the reaction parameters. Using the optimal conditions, T7/G4-CRISPR was capable of detecting as low as 3.6 pM target RNA, obtaining ∼100-fold improvement in sensitivity compared with the most direct Cas12a assays. Meanwhile, its excellent specificity could discriminate single nucleotide variants adjacent to the toehold region and allow species-specific pathogen identification. Furthermore, we applied it for analyzing bacterial 16S rRNA in 40 clinical urine samples, exhibiting a sensitivity of 90% and a specificity of 100% when validated by RT-quantitative PCR. Therefore, we envision that T7/G4-CRISPR will serve as a promising RNA sensing approach to expand the toolbox of CRISPR-based diagnostics.

Quantitative analysis of SARS-CoV-2 RNA in wastewater and evaluation of sampling frequency during the downward period of a COVID-19 wave in Japan

Wastewater-based epidemiology (WBE) is a practical approach for detecting the presence of SARS-CoV-2 infections and assessing the epidemic trend of the coronavirus disease 2019 (COVID-19). The purpose of this study was to evaluate the minimum sampling frequency required to properly identify the COVID-19 trend during the downward epidemic period when using a highly sensitive RNA detection method. WBE was conducted using the Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids (EPISENS-S), a highly sensitive SARS-CoV-2 RNA detection method, at nine neighboring wastewater treatment plants (WWTPs). These WWTPs were in the same prefecture in Japan, and they had different sewer types, sampling methods, and sampling frequencies. The overall detection rate of SARS-CoV-2 RNA was 97.8 % during the entire study period when the geometric means of new COVID-19 cases per 100,000 inhabitants were between 3.3 and 7.7 in each WWTP. The maximum SARS-CoV-2 RNA concentration in wastewater was 2.14 × 104 copies/L, which corresponded to pepper mild mottle virus (PMMoV)–normalized concentrations of 6.54 × 10−3. We evaluated the effect of sampling frequencies on the probability of a significant correlation with the number of newly reported COVID-19 cases by hypothetically reducing the sampling frequency in the same dataset. When the wastewater sampling frequency occurred 5, 3, 2, and 1 times per week, these results exhibited significant correlations of 100 % (5/5), 89 % (8/9), 85 % (23/27), and 48 % (13/27), respectively. To achieve significant correlation with a high probability of over 85 %, a minimum sampling frequency of twice per week is required, even if sampling methods and sewer types are different. WBE using the EPISENS-S method and a sampling frequency of more than twice a week can be used to properly monitor COVID-19 wave epidemic trends, even during downward periods.

Chromogenic Visualization of Single RNA Molecules In Situ with Duplex Capability by Rolling Circle Amplification with Alkaline Phosphatase.

Visualizing spatial patterns of gene expression by optical microscopy at single-molecule resolution represents a long-standing challenge for imaging and molecular engineering technologies. In this study, we developed a method for visualizing mRNA with duplex capability by optical microscopy using rolling circle amplification with streptavidin-modified alkaline phosphatase (SA-ALP) to provide highly selective and sensitive RNA detection. ALP-based RNA detection provides comparable sensitivity and specificity to fluorescence-based in situ assays and similar performance to the current RNAscope technique for single-molecule RNA detection, but with improved ease of operation. This versatile and relatively user-friendly method of single-molecule RNA visualization can also overcome common problems of background interference. Our findings show that the red spots generated by the Fast Red staining in situ are readily quantified by image analysis, even in samples with heavy melanin deposition, supporting the clinical translation of this assay to improve diagnostic assays for recalcitrant tissues. This system was adaptable for duplex assays with multiple probes and multiple stains, which is ALP with horseradish peroxidase to produce red and brown signals to simultaneously visualize two different RNA targets. The duplex assay can be successfully applied to quantify mRNA expression from two genes in situ within single cells and multiple cell types. With the advantages of high sensitivity and low hardware requirements, this versatile and user-friendly method of RNA visualization may enable low-resource institutions to conduct previously inaccessible diagnostic or research questions about the in situ expression and distribution of RNAs at single-molecule resolution.

A universal and specific RNA biosensor via DNA circuit-mediated PAM-independent CRISPR/Cas12a and PolyA-rolling circle amplification

Point of care testing (POCT) has important clinical significance for the diagnosis and prognosis evaluation of diseases. At present, the biosensor based on CRISPR/Cas12a has become a powerful diagnostic tool due to its high sensitivity. However, CRISPR/Cas12a requires PAM sequence to recognize target double strand and only can recognize specific sequence, so it is not universal. The current RNA detection techniques either lack consideration for specificity and universality, are expensive and difficult, or both. Therefore, it is crucial to create a CRISPR/Cas12a-based RNA detection system that is easy to use, cheap, specific, and universal in order to further its use in molecular diagnostics. Here, we established a DNA circuit-mediated PAM-independent CRISPR/Cas12a coupled PolyA-rolling circle amplification for RNA detection biosensor, namely DCPRBiosensor. The DCPRBiosensor not only functions as a simple, inexpensive, and highly sensitive RNA detection sensor, but it also boasts innovative specificity and universality features. More importantly, DCPRBiosensor removes the PAM restriction of CRISPR/Cas12a. The DCPRBiosensor's detection limit reached 100 aM and it had a linear relationship between 100 aM and 10 pM. We detected four piRNAs to verify the universality and stability of DCPRBiosensor. Then, we verified that DCPRBiosensor has good discrimination ability for single-base mismatch. Finally, we successfully detected piRNA in DLD-1 and HCT-116 cells and urine mixed samples within 4.5 h. In conclusion, we believe that DCPRBiosensor will have a substantial impact on both the development of CRISPR/as12a′s applications and the investigation of the clinical value of piRNA.

Harnessing the CRISPR-Cas13d System for Protein Detection by Dual-Aptamer-Based Transcription Amplification.

CRISPR-based biosensing technology has been emerging as a revolutionary diagnostic tool for many disease-related biomarkers. In particular, RspCas13d, a newly identified RNA-guided Cas13d ribonuclease derived from Ruminococcus sp., has shown great promise for accurate and sensitive detection of RNA due to its RNA sequence-specific recognition and robust collateral trans-cleavage activity. However, its diagnostic utility is limited to detecting nucleic-acid-related biomarkers. To address this limitation, herein we present a proof-of-concept demonstration of a target-responsive CRISPR-Cas13d sensing system for protein biomarkers. This system was rationally designed by integrating a dual-aptamer-based transcription amplification strategy with CRISPR-Cas13d (DATAS-Cas13d), in which the protein binding initiates in-vitro RNA transcription followed by the activation of RspCas13d. Using a short fluorescent ssRNA as the signal reporter and cardiac troponin I (cTnI) as the model analyte, the DATAS-Cas13d system showed a wide linear range, low detection limit, and high specificity for the detection of cTnI in buffer and human serum. Thanks to the facile integration of various bioreceptors into the DATAS-Cas13d system, the method could be adapted to detecting a broad range of clinically relevant protein biomarkers, and thus broaden the medical applications of Cas13d-based diagnostics.

RNA-based isothermal amplification technology and its clinical application in pathogen infection

Abstract It is very important to detect pathogenic bacteria, viruses, or fungi in a patient’s secretion or body fluid samples as soon as possible to determine the patient’s recovery. For certain pathogens, the amount of ribosomal RNA copies contained is often tens of thousands of times higher than the amount of DNA copies, so the detection of RNA has higher sensitivity. In addition, whether for DNA pathogens or RNA pathogens, the direct detection of ribonucleic acid transcribed by pathogens in vivo can distinguish active infection or past infection, can eliminate the influence of residual DNA of pathogens that have died in the lesions, and can also avoid excessive medical interventions for transient infections, which is of great significance in the field of infectious pathogen detection. Isothermal amplification technology played important roles in molecular diagnosis because of its significant advantages. Highly sensitive RNA detection can be achieved by both direct transcription amplification and indirect amplification based on reverse transcription. Direct transcription amplification technologies relies on reverse transcriptase and T7 RNA polymerase to achieve linear transcription amplification of RNA on one-step; while the indirect amplification technology depends on a reverse transcriptional process at the beginning of the reaction. Both methods have outstanding advantages in clinical application, and commercial kits and commercial all-in-one machines based on these principles have been put into clinical use. This review mainly introduces the clinical application of isothermal amplification technologies in the detection of RNA pathogens and the main difficulties faced at this stage. It is hoped to provide insightful ideas for the construction of pathogen RNA detection technology to meet the needs of point-of-care testing in the future.

Light Scattering Technology-Combined Ligation-Dependent Loop-Mediated Isothermal Amplification (LL-LAMP) for Sensitive Detection of RNA.

Loop-mediated isothermal amplification (LAMP) has been widely used in nucleic acid assay because of its high specificity, sensitivity, and isothermal property. However, the complexity of amplification product detection is still a major challenge for its wide applications. Herein, we developed a light scattering technology-assisted, low-cost, and simple detection manner of LAMP products without expensive reagents and complicated instruments. Only needing to add a kind of strong acid to the amplification products, the amplification products can aggregate into large particles in a strongly acidic medium, and large particles can produce strong light scattering, which shows a good proportional relationship with the number of amplification products in a wide range. The proposed method shows excellent sensitivity and high specificity that can quantify RNA as low as 100 aM with a single-base resolution.

Robust Recombinant Expression of Human Placental Ribonuclease Inhibitor in Insect Cells.

Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in E. coli, the robust expression is complicated by maintaining the redox potential and solubility by various expression tags. In the present paper we describe the expression of RI in baculovirus-infected High Five cells in large scale utilizing a modified transfer vector combining the beneficial properties of Profinity Exact Tag and pONE system. The recombinant RI is expressed at a high level in a fusion form, which is readily cleaved during on-column chromatography. A subsequent anion exchange chromatography was used as a polishing step to yield 12 mg native RI per liter of culture. RI expressed in insect cells shows higher thermal stability than the commercially available RI products (mainly produced in E. coli) based on temperature-dependent RNase inhibition studies. The endotoxin-free RI variant may also be applied in future therapeutics as a safe additive to increase mRNA stability in mRNA-based vaccines.

Fluorophore–PNA–Quencher/Quencher–DNA probe for miRNA detection

Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly sensitive RNA detection methods are required to determine miRNA expression levels and intracellular localization. In this study, we designed new double-stranded peptide nucleic acid (PNA)/DNA probes consisting of a fluorophore–PNA–quencher (fPq) and a quencher–DNA (qD) for miR-221 detection. We optimized the fPq structure, PNA–DNA hybrid length, and hybrid position. The resultant fPq-2/qD-6b probe was a 6-bp hybrid probe with a 10-base fPq and a 6-base qD. The signal-to-background ratios of the probes showed that fPq-2/qD-6b had a higher target sensitivity than fPq (PNA beacon)-type and fP/qD-type probes. The results of the detection limit and target specificity indicate that the fPq/qD probe is promising for RNA detection in both cells and cell extracts as well as for miRNA diagnosis.

Collaborative Equilibrium Coupling of Catalytic DNA Nanostructures Enables Programmable Detection of SARS-CoV-2.

Accessible and adaptable nucleic acid diagnostics remains a critical challenge in managing the evolving COVID‐19 pandemic. Here, an integrated molecular nanotechnology that enables direct and programmable detection of SARS‐CoV‐2 RNA targets in native patient specimens is reported. Termed synergistic coupling of responsive equilibrium in enzymatic network (SCREEN), the technology leverages tunable, catalytic molecular nanostructures to establish an interconnected, collaborative architecture. SCREEN mimics the extraordinary organization and functionality of cellular signaling cascades. Through programmable enzyme–DNA nanostructures, SCREEN activates upon interaction with different RNA targets to initiate multi‐enzyme catalysis; through system‐wide favorable equilibrium shifting, SCREEN directly transduces a single target binding into an amplified electrical signal. To establish collaborative equilibrium coupling in the architecture, a computational model that simulates all reactions to predict overall performance and optimize assay configuration is developed. The developed platform achieves direct and sensitive RNA detection (approaching single‐copy detection), fast response (assay reaction is completed within 30 min at room temperature), and robust programmability (across different genetic loci of SARS‐CoV‐2). When clinically evaluated, the technology demonstrates robust and direct detection in clinical swab lysates to accurately diagnose COVID‐19 patients.

Sensitive detection of RNA based on concatenated self-fuelled strand displacement amplification and hairpin-AgNCs.

Herein, a self-fuelled amplification strategy (SFAS) is proposed, in which two strand displacement amplification (SDA) processes were concatenated for the proliferation of ssDNA. The ssDNA then initiated a polymerase action and caused the destruction of hairpin-templated silver nanoclusters (AgNCs), resulting in decreased fluorescence for sensing miRNA-21. This SFAS-based sensor is less complicated in design and facile in operation, because of the easy concatenation of SDA and mutual enzymes used in the signal output process. The sensitivity of this SFAS-based miRNA sensor was 1.78 × 10-11 M with a linear relationship in the range 0.02-1.0 × 10-9 M, and the recoveries of this method ranged from 82.07% to 106.58% with an average RSD of 10.96%.

Clinical characteristics of recovered COVID-19 patients with re-detectable positive RNA test.

BackgroundThe characteristics, significance and potential cause of positive SARS-CoV-2 diagnoses in recovered coronavirus disease 2019 (COVID-19) patients post discharge (re-detectable positive, RP) remained elusive.MethodsA total of 262 COVID-19 patients discharged from January 23 to February 25, 2020 were enrolled into this study. RP and non-RP (NRP) patients were grouped according to disease severity, and the characterization at re-admission was analyzed. SARS-CoV-2 RNA and plasma antibody levels were measured, and all patients were followed up for at least 14 days, with a cutoff date of March 10, 2020.ResultsA total of 14.5% of RP patients were detected. These patients were characterized as young and displayed mild and moderate conditions compared to NRP patients while no severe patients were RP. RP patients displayed fewer symptoms but similar plasma antibody levels during their hospitalization compared to NRP patients. Upon hospital readmission, these patients showed no obvious symptoms or disease progression. All 21 close contacts of RP patients were tested negative for viral RNA and showed no suspicious symptoms. Eighteen out of 24 of RNA-negative samples detected by the commercial kit were tested positive for viral RNA using a hyper-sensitive method, suggesting that these patients were potential carriers of the virus after recovery from COVID-19.ConclusionsOur results indicated that young patients, with a mild diagnosis of COVID-19 are more likely to display RP status after discharge. These patients show no obvious symptoms or disease progression upon re-admission. More sensitive RNA detection methods are required to monitor these patients. Our findings provide information and evidence for the management of convalescent COVID-19 patients.

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells.

Reliable extraction and sensitive detection of RNA from human peripheral blood mononuclear cells (PBMCs) is critical for a broad spectrum of immunology research and clinical diagnostics. RNA analysis platforms are dependent upon high-quality and high-quantity RNA; however, sensitive detection of specific responses associated with high-quality RNA extractions from human samples with limited PBMCs can be challenging. Furthermore, the comparative sensitivity between RNA quantification and best-practice protein quantification is poorly defined. Therefore, we provide herein a critical evaluation of the wide variety of current generation of RNA-based kits for PBMCs, representative of several strategies designed to maximize sensitivity. We assess these kits with a reverse transcription quantitative PCR (RT-qPCR) assay optimized for both analytically and diagnostically sensitive cell-based RNA-based applications. Specifically, three RNA extraction kits, one post-extraction RNA purification/concentration kit, four SYBR master-mix kits, and four reverse transcription kits were tested. RNA extraction and RT-qPCR reaction efficiency were evaluated with commonly used reference and cytokine genes. Significant variation in RNA expression of reference genes was apparent, and absolute quantification based on cell number was established as an effective RT-qPCR normalization strategy. We defined an optimized RNA extraction and RT-qPCR protocol with an analytical sensitivity capable of single cell RNA detection. The diagnostic sensitivity of this assay was sufficient to show a CD8+ T cell peptide epitope hierarchy with as few as 1 × 104 cells. Finally, we compared our optimized RNA extraction and RT-qPCR protocol with current best-practice immune assays and demonstrated that our assay is a sensitive alternative to protein-based assays for peptide-specific responses, especially with limited PBMCs number. This protocol with high analytical and diagnostic sensitivity has broad applicability for both primary research and clinical practice.

L-DNA-tagged fluorescence in situ hybridization for highly sensitive imaging of RNAs in single cells.

We report an effective fluorescence in situ hybridization strategy, named l-DNA tagged FISH (LT-FISH), for highly sensitive RNA detection in fixed cultured cells. LT-FISH includes two-step hybridization processes with a l-d chimera oligonucleotide probe and a fluorescence-labeled PCR product tethering a l-DNA tag. The degree of fluorescence enhancement, depending on the length of PCR products, was up to 14-fold when the 606 bp product was used. Endogenous mRNA and miRNA in cancer cells were visualized by utilizing this l-DNA-mediated signal amplification technique.

A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells.

Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose a concept of cell-permeable fluorogenic dimer of sulforhodamine B dyes (Gemini-561) and corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The unprecedented brightness and photostability together with high affinity of this complex allowed, for the first time, direct fluorescence imaging in live mammalian cells of RNA polymerase-III transcription products as well as messenger RNAs labelled with a single copy of the aptamer, i.e. without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.

Axonal mRNA transport and translation at a glance.

Localization and translation of mRNAs within different subcellular domains provides an important mechanism to spatially and temporally introduce new proteins in polarized cells. Neurons make use of this localized protein synthesis during initial growth, regeneration and functional maintenance of their axons. Although the first evidence for protein synthesis in axons dates back to 1960s, improved methodologies, including the ability to isolate axons to purity, highly sensitive RNA detection methods and imaging approaches, have shed new light on the complexity of the transcriptome of the axon and how it is regulated. Moreover, these efforts are now uncovering new roles for locally synthesized proteins in neurological diseases and injury responses. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of how axonal mRNA transport and translation are regulated, and discuss their emerging links to neurological disorders and neural repair.

High sensitive RNA detection by one-step RT-PCR using the genetically engineered variant of DNA polymerase with reverse transcriptase activity from hyperthermophilies

One-step RT-PCR has not been widely used even though some thermostable DNA polymerases with reverse transcriptase (RT) activity were developed from bacterial and archaeal polymerases, which is owing to low cDNA synthesis activity from RNA. In the present study, we developed highly-sensitive one-step RT-PCR using the single variant of family A DNA polymerase with RT activity, K4polL329A (L329A), from the hyperthermophilic bacterium Thermotoga petrophila K4 or the 16-tuple variant of family B DNA polymerase with RT activity, RTX, from the hyperthermophilic archaeon Thermococcus kodakarensis. Optimization of reaction condition revealed that the activities for cDNA synthesis and PCR of K4polL329A and RTX were highly affected by the concentrations of MgCl2 and Mn(OCOCH3)2 as well as those of K4polL329A or RTX. Under the optimized condition, 300 copies/μl of target RNA in 10 μl reaction volumes were successfully detected by the one-step RT-PCR with K4polL329A or RTX, which was almost equally sensitive enough compared with the current RT-PCR condition using retroviral RT and thermostable DNA polymerase. Considering that K4polL329A and RTX are stable even at 90-100°C, our results suggest that the one-step RT-PCR with K4polL329A or RTX is more advantageous than the current one.

RNA detection with C2c2

The discovery of two distinct endonuclease activities in the C2c2 protein explains how template RNAs are processed in type VI CRISPR systems and enables the development of a sensitive RNA detection system.

Electrochemical RNA genosensors for toxic algal species: enhancing selectivity and sensitivity

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system.