A Novel Method to Map Small RNAs with High Resolution.

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers. Graphic abstract: Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.

Impact of Divalent Metal Ions on Regulation of Trans-Cleavage Activity of CRISPR-Cas13a: A Combined Experimental and Computational Study.

CRISPR-LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg2+ to maintain its nuclease activity. However, the effect of other divalent metal ions on its trans-cleavage activity remains less explored. Herein, we addressed this issue by combining experimental and molecular dynamics simulation analysis. In vitro studies showed that both Mn2+ and Ca2+ could replace Mg2+ as cofactors of LbuCas13a. In contrast, Ni2+ , Zn2+ , Cu2+ , or Fe2+ inhibits the cis- and trans-cleavage activity, while Pb2+ does not affect it. Importantly, molecular dynamics simulations confirmed that calcium, magnesium, and manganese hydrated ions have a strong affinity to nucleotide bases, thus stabilizing the conformation of crRNA repeat region and enhancing the trans-cleavage activity. Finally, we showed that combination of Mg2+ and Mn2+ can further enhance the trans-cleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.

Harnessing the LdCsm RNA Detection Platform for Efficient microRNA Detection.

In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, which is very complex for miRNAs. Recently, the development of CRISPR-based nucleic acid detection tools has brought new promises to efficient miRNA detection. Three CRISPR systems can be explored for miRNA detection, including type III, V, and VI, among which type III (CRISPR-Cas10) systems have a unique property as they recognize RNA directly and cleave DNA collaterally. In particular, a unique type III-A Csm system encoded by Lactobacillus delbrueckii subsp. bulgaricus (LdCsm) exhibits robust target RNA-activated DNase activity, which makes it a promising candidate for developing efficient miRNA diagnostic tools. Herein, LdCsm was tested for RNA detection using fluorescence-quenched DNA reporters. We found that the system is capable of specific detection of miR-155, a microRNA implicated in the carcinogenesis of human breast cancer. The RNA detection system was then improved by various approaches including assay conditions and modification of the 5'-repeat tag of LdCsm crRNAs. Due to its robustness, the resulting LdCsm detection platform has the potential to be further developed as a better point-of-care miRNA diagnostics relative to other CRISPR-based RNA detection tools.

Rapid point-of-care detection of SARS-CoV-2 RNA with smartphone-based upconversion luminescence diagnostics.

Accurate COVID-19 screening via molecular technologies is still hampered by bulky instrumentation, complicated procedure, high cost, lengthy testing time, and the need for specialized personnel. Herein, we develop point-of-care upconversion luminescence diagnostics (PULD), and a streamlined smartphone-based portable platform facilitated by a ready-to-use assay for rapid SARS-CoV-2 nucleocapsid (N) gene testing. With the complementary oligo-modified upconversion nanoprobes and gold nanoprobes specifically hybridized with the target N gene, the luminescence resonance energy transfer effect leads to a quenching of fluorescence intensity that can be detected by the easy-to-use diagnostic system. A remarkable detection limit of 11.46 fM is achieved in this diagnostic platform without the need of target amplification, demonstrating high sensitivity and signal-to-noise ratio of the assay. The capability of the developed PULD is further assessed by probing 9 RT-qPCR-validated SARS-CoV-2 variant clinical samples (B.1.1.529/Omicron) within 20 min, producing reliable diagnostic results consistent with those obtained from a standard fluorescence spectrometer. Importantly, PULD is capable of identifying the positive COVID-19 samples with superior sensitivity and specificity, making it a promising front-line tool for rapid, high-throughput screening and infection control of COVID-19 or other infectious diseases.

Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA).

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence.

Synthesis and Characterization of Silica Coated Magnetite Nanoparticle Clusters for Viral RNA Extraction

Magnetite nanoparticles or iron oxide nanoparticles are the most explored magnetic nanoparticles till recent times, particularly due to their attractive properties for biomedical applications such as viral RNA extraction. The physical and chemical properties of magnetite nanoparticles and their clusters largely depend on the synthesis method and chemical structure. Co-precipitation method was used to synthesize magnetite nanoparticles at varying process parameters. The nanoparticles were coated with silicon-oxide using Stober method at different deposition durations. These particles were characterized by X-Ray Diffraction, Scanning Electron Microscopy, vibrating sample Magnetometer and PCR testing to study the phases formed, morphology, size, magnetic properties and RNA extraction efficiency. The synthesized magnetite nanoparticles were in the range of 10 to 100 nm;suitable for super-para-magnetic behavior. The maximum saturation magnetization achieved for synthesized paramagnetic beads was 56 emu and RNA extraction efficiency was more than 80% as compared to commercial beads. © 2022 Trans Tech Publications Ltd, Switzerland.

Complementing RNA Detection with Pharmaceutical Monitoring for Early Warning of Viral Outbreaks through Wastewater-Based Epidemiology

Wastewater-based epidemiology using viral nucleic acids to predict community viral outbreaks has many challenges, including differences in viral shedding of infected individuals and interference from the wastewater matrix. In this study, we demonstrate that monitoring pharmaceutical residues in untreated sewage provides complementary information that correlates with future occurrences of viral outbreaks. We monitored 63 pharmaceutically active compounds, including antivirals used to treat COVID-19 and influenza and over-the-counter drugs commonly used to relieve the symptoms of infection. Weekly sampling was conducted at four municipal sewage treatment plants in Western New York. Residues of drugs associated with managing COVID-19 symptoms were detected, including azithromycin (1.99-5.00 μg/L), chloroquine (0.01-33.00 μg/L), hydroxychloroquine (0.05-30.54 μg/L), and lopinavir (13.75-181.20 μg/L). A significant correlation (p < 0.001) was observed between the total COVID-19-related drugs detected and the 5-day rolling averages of reported cases. Acetaminophen concentrations spiked approximately 2.5 weeks before a spike in SARS-CoV-2 RNA copies in all wastewater treatment plants sampled. The results suggest over-the-counter analgesic concentrations, in particular, acetaminophen in raw sewage to be used to complement viral RNA data as an early warning system for effective management of viral outbreaks at the community level. © 2022 American Chemical Society.

Telomere G-triplex lights up Thioflavin T for RNA detection: new wine in an old bottle.

Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT-based method. We believe this ht-G3/ThT-based label-free strategy could be widely applied for RNA detection.

RT-LAMP assay combining multi-fluorescent probes for SARS-CoV-2 RNA detection and variant differentiation.

Simple and accurate testing tools for SARS-CoV-2 viral RNA detection are essential for the prevention of the spread of the virus and timely governmental actions. Herein, we present a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of ORF1ab and N gene fragments of SARS-CoV-2 in one pot. Using two primer sets and two molecular beacon (MB) probes respectively labelled with different fluorophore, positive results were obtained with a limit of detection of 20 and 2 copies/µL for ORF1ab and N gene fragments, respectively. Moreover, the RT-LAMP based assay was applied to detect single-site differences in S genes using two one-step displacement (OSD) probes targeting wild-type and mutant (P681R mutation was chosen as model) genes. Through that, the wild type strain and P681R mutant variant were well distinguished from each other, and a preliminary observation was also made on other mutations at this site such as P681H. The proposed method has high sensitivity for quantification and high specificity for mutation differentiation. In addition, it does not require accurate sophisticated thermal cycler instrumentation and can be used in clinical settings in resource-limited regions.

A comparison of precipitation and filtration-based SARS-CoV-2 recovery methods and the influence of temperature, turbidity, and surfactant load in urban wastewater.

Wastewater-based epidemiology (WBE) has become a complimentary surveillance tool during the SARS-CoV-2 pandemic. Viral concentration methods from wastewater are still being optimised and compared, whilst viral recovery under different wastewater characteristics and storage temperatures remains poorly understood. Using urban wastewater samples, we tested three viral concentration methods; polyethylene glycol precipitation (PEG), ammonium sulphate precipitation (AS), and CP select™ InnovaPrep® (IP) ultrafiltration. We found no major difference in SARS-CoV-2 and faecal indicator virus (crAssphage) recovery from wastewater samples (n = 46) using these methods, PEG slightly (albeit non-significantly), outperformed AS and IP for SARS-CoV-2 detection, as a higher genome copies per litre (gc/l) was recorded for a larger proportion of samples. Next generation sequencing of 8 paired samples revealed non-significant differences in the quality of data between AS and IP, though IP data quality was slightly better and less variable. A controlled experiment assessed the impact of wastewater suspended solids (turbidity; 0-400 NTU), surfactant load (0-200 mg/l), and storage temperature (5-20 °C) on viral recovery using the AS and IP methods. SARS-CoV-2 recoveries were >20% with AS and <10% with IP in turbid samples, whilst viral recoveries for samples with additional surfactant were between 0-18% for AS and 0-5% for IP. Turbidity and sample storage temperature combined had no significant effect on SARS-CoV-2 recovery (p > 0.05), whilst surfactant and storage temperature combined were significant negative correlates (p < 0.001 and p < 0.05, respectively). In conclusion, our results show that choice of methodology had small effect on viral recovery of SARS-CoV-2 and crAssphage in wastewater samples within this study. In contrast, sample turbidity, storage temperature, and surfactant load did affect viral recovery, highlighting the need for careful consideration of the viral concentration methodology used when working with wastewater samples.

A novel microfluidic RNA chip for direct, single-nucleotide specific, rapid and partially-degraded RNA detection.

Direct RNA detection is critical for providing the RNA insights into gene expression profiling, noncoding RNAs, RNA-associated diseases and pathogens, without reverse transcription. However, classical RNA analysis usually requires RT-PCR, which can cause bias amplification and quantitation errors. To address this challenge, herein we report a microfluidic RNA chip (the microchip prototype) for direct RNA detection, which is primarily based on RNA extension and labeling with DNA polymerase. This detection strategy is of high specificity (discriminating against single-nucleotide differences), rapidity, accuracy, nuclease resistance, and reusability. Further, we have successfully detected disease-associated RNAs in clinical samples, demonstrating its great potentials in biomedical research and clinical diagnosis.

Reliable Diagnostics of SARS-CoV-2 Infections Using One- and Two-Gene Molecular Tests for a Viral RNA Detection-Results Questioning Previous Observations.

SARS-CoV-2 is a new virus from the Coronaviridae family and its rapid spread is now the most important medical problem worldwide. Currently used tests vary in the number and selection of SARS-CoV-2 target genes. Meanwhile, the choice of the appropriate target gene may be important in terms of a reliable detection of a viral RNA. As some researchers questioned the sensitivity of the monogenic VIASURE SARS-CoV-2 S gene Real Time PCR Detection Kit (CerTest Biotec, Zaragoza, Spain) in mid-2020, the aim of the study was to evaluate the usefulness of this kit, used along with the BD MAX™ System (Becton Dickinson, East Rutherford, NJ, USA), and compare the results with two-gene Bosphore Novel Coronavirus (2019-nCoV) Detection Kit v1 (Anatolia Diagnostics and Biotechnology Products Inc., Istanbul, Turkey). Both tests were carried out on 306 nasopharyngeal/oropharyngeal swabs. The consistent results (72 positive and 225 negative results found simultaneously in both kits) were obtained for 297 (97.1%) samples altogether, while discrepancies between the results of the evaluated tests were observed for nine (2.9%) specimens. There were no statistically significant differences between the method used and the frequency of positive results. Both tests, targeted at detecting one and two genes, are effective in SARS-CoV-2 RNA detection.

High-sensitivity and versatile plasmonic biosensor based on grain boundaries in polycrystalline 1L WS2 films.

Structural defects play an important role in exploitation of two-dimensional layered materials (2DLMs) for advanced biosensors with the increasingly high sensitivity and low detection limit. Grain boundaries (GBs), as an important type of structural defect in polycrystalline 2DLM films, potentially provide sufficient active defect sites for the immobilization of bioreceptor units via chemical functionalization. In this work, we report the selective functionalization of high-density GBs with complementary DNA receptors, via gold nanoparticle (AuNP) linkers, in wafer-scale polycrystalline monolayer (1L) W(Mo)S2 films as versatile plasmonic biosensing platforms. The large surface area and GB-rich nature of the polycrystalline 1L WS2 film enabled the immobilization of bioreceptors in high surface density with spatial uniformity, while the AuNPs perform not only as bioreceptor linkers, but also promote detection sensitivity through surface plasmon resonance enhancement effect. Therefore, the presented biosensor demonstrated highly sensitive and selective sub-femto-molar detection of representative RNA sequences from the novel coronavirus (RdRp, ORF1ad and E). This work demonstrates the immense potential of AuNP-decorated GB-rich 2DLMs in the design of ultra-sensitive biosensing platforms for the detection of biological targets beyond RNA, bringing new opportunities for novel healthcare technologies.

An innovative approach for the non-invasive surveillance of communities and early detection of SARS-CoV-2 via solid waste analysis.

The diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection requires the detection of viral RNA by reverse transcription-polymerase chain reaction (RT-qPCR) performed mainly using nasopharyngeal swabs. However, this procedure requires separate analysis per each individual, performed in advanced centralized laboratory facilities with specialized medical personnel. In this study, an alternative approach termed "solid waste-based surveillance (SWBS)" was explored, in order to investigate SARS-CoV-2 infection in small communities through the indirect sampling of saliva left on waste. Sampling was performed at 20 different sites in Italy during the second peak of COVID-19. Three swabs were positive for SARS-CoV-2 using a published RT-qPCR protocol targeting the non-structural protein 14 region, and the viral load ranged 4.8 × 103-4.0 × 106 genome copies/swab. Amino acid substitutions already reported in SARS-CoV-2 sequences circulating in Italy (A222V and P521S) were detected in two positive samples. These findings confirmed the effectiveness of SWBS for non-invasive and dynamic SARS-CoV-2 surveillance.

Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription-free exponential amplification reaction, RTF-EXPAR.

A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription-free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.

Investigating the Presence of SARS CoV-2 in Free-Living and Captive Animals.

Due to SARS CoV-2 recombination rates, number of infected people and recent reports of environmental contamination, the possibility of SARS CoV-2 transmission to animals can be expected. We tested samples of dominant free-living and captive wildlife species in Croatia for the presence of anti-SARS CoV-2 antibodies and viral RNA. In total, from June 2020 until February 2021, we tested blood, muscle extract and fecal samples of 422 free-living wild boars (Sus scrofa), red foxes (Vulpes vulpes) and jackals (Canis aureus); blood and cloacal swabs of 111 yellow-legged gulls (Larus michahellis) and fecal samples of 32 zoo animals. A commercially available ELISA (ID.Vet, France) and as a confirmatory test, a surrogate virus neutralization test (sVNT; GenScript, Netherlands) were used. Fecal samples were tested for the presence of viral RNA by a real-time RT-PCR protocol. Fifteen out of 533 (2.8%) positive ELISA results were detected; in wild boars (3.9%), red foxes (2.9%) and jackals (4.6%). However, the positive findings were not confirmed by sVNT. No viral RNA was found. In conclusion, no spillover occurred within the investigated period (second COVID-19 wave). However, further investigation is needed, especially regarding wildlife sample features for serological tests.

Evaluation of the persistence of SARS-CoV-2 (ATCC® VR-1986HK™) on two different food contact materials: flow pack polyethylene and polystyrene food trays.

Even though SARS-CoV-2's primary transmission pathway is person-to-person, the role played by surfaces and food contact materials in carrying viral RNA should be further explored. For this purpose, the study aimed to investigate the persistence of SARS-CoV-2 using the strain ATCC® VR-1986HK™ on flow pack polyethylene (FPP) and polystyrene food trays (PFT). Samples of FPP and PFT were contaminated with heat-inactivated SARS-CoV-2 and were incubated at a temperature of 24 ± 1 °C and at controlled relative humidity (RH 65%). The experimental design included analyses at the time 0, 3, 6, 12, 24, 36, 48 and after every 24 h until the viral RNA was no longer detectable. The results showed a significant decrease (P < 0.001) in viral copy numbers on PFT within 3 h (24% reduction) and, at 72 h, the viral RNA had fallen below the limit of detection. Regarding the FPP, it was necessary to wait 24 h for a significant decrease (P = 0.015) in the viral load (14% reduction), while the detection threshold was reached at 96 h. These findings showed that the viral RNA persists longer on flow pack polyethylene samples than on polystyrene food trays, thus highlighting the importance of material characteristics in the persistence of SARS-CoV-2.

Verification and Validation of SARS-CoV-2 Assay Performance on the Abbott m2000 and Alinity m Systems.

We verified the analytical performance of the Abbott RealTime SARS-CoV-2 assay on the m2000 system and compared its clinical performance to the CDC 2019-nCoV real-time PCR diagnostic panel and the Thermo Fisher TaqPath RT-PCR COVID-19 kit. We also performed a bridging study comparing the RealTime SARS-CoV-2 assay with the new Abbott Alinity m SARS-CoV-2 assay. A number of standards, reference materials, and commercially available controls were used for the analytical verification to confirm the limit of detection, linearity, and reproducibility. We used nasopharyngeal (NP) swab specimens collected in saline for the clinical verification and bridging studies. Overall, we found 91.2% positive percent agreement (PPA; 95% confidence interval [CI] = 76.2 to 98.14%) and a 100% negative percent agreement (NPA; 95% CI = 97.97 to 100%) between the results of the RealTime SARS-CoV-2 and CDC tests with 217 NP specimens (P = 0.13). We found a PPA of 100% (95% CI = 90.26 to 100%) and an NPA of 95.15% (95% CI = 83.47 to 99.4%) between the results of the RealTime and TaqPath tests with 77 NP specimens (P = 0.24). Finally, we tested 203 NP swab specimens for SARS-CoV-2 on the m2000 on the Alinity m systems. The PPA and NPA were 92.2% (95% CI = 85.3 to 96.59%) and 92% (95% CI = 84.8 to 96.5%), respectively (P = 0.4). Although cycle number (Cn) values obtained for the concordant positive samples were highly correlated (R2 = 0.95), the Cn values were on average 14.14 higher on the Alinity m system due to the unread cycles with the RealTime SARS-CoV-2 assay.

Reverse transcription lesion-induced DNA amplification: An instrument-free isothermal method to detect RNA.

One challenge in point-of-care (POC) diagnostics is the lack of room-temperature methods for RNA detection based on enzymatic amplification and visualization steps. Here we perform reverse transcription lesion-induced DNA amplification (RT-LIDA), an isothermal amplification method that only requires T4 DNA ligase. RT-LIDA involves the RNA-templated ligation of DNA primers to form complementary DNA (cDNA) followed by toehold-mediated strand displacement of the cDNA and its exponential amplification via our isothermal ligase chain reaction LIDA. Each step is tuned to proceed at 28 °C, which falls within the range of global room temperatures. Using RT-LIDA, we can detect as little as ∼100 amol target RNA and can distinguish RNA target from total cellular RNA. Finally, we demonstrate that the resulting DNA amplicons can be detected colorimetrically, also at room temperature, by rapid, target-triggered disassembly of DNA-modified gold nanoparticles. This integrated amplification/detection platform requires no heating or visualization instrumentation, which is an important step towards realizing instrument-free POC testing.