Molecular Beacon Probes Research Articles

Paired dCas9 design as a nucleic acid detection platform for pathogenic strains.

The wide application of molecular beacon probes in specific DNA detection, especially in the fast prototyping of pathogen DNA detection kits in point-of-care diagnostics, has been hindered by the nonflexible choice of target sequences and the unstable fluorophore output. We developed an in vitro DNA detection system consisting of a pair of dCas9 proteins linked to split halves of luciferase, named the Paired dCas9 (PC) reporter. Co-localization of the reporter pair to a ~46bp target sequence defined by two single guide RNAs (sgRNAs) activated luciferase which subsequently generated highly intensified luminescent signals. Combined with an array design and statistical analyses, the PC reporter system could be programmed to access sequence information across the entire genome of the pathogenic Mycobacterium tuberculosis H37Rv strain. These findings suggest great potential for the PC reporter in effective and affordable in vitro nucleic acid detection technologies. In this article we highlighted the systems design from our previous researchworkon the PC reporter (Zhang et al, 2015)with a focuson methodology.

Graphene-oxide-based bioassay for the fluorometric determination of agrC gene transcription in methicillin-resistant Staphylococcus aureus that uses nicking-enzyme-assisted target recycling and a hybridization chain reaction

Herein, we report the development of a graphene-oxide-based (GO-based) fluorescent bioassay for determining agrC gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). The design is based on nicking-enzyme-assisted (Nb.BbvcI-assisted) target recycling amplification (NATR) and a hybridization chain reaction (HCR). The system consists of a helper probe (HP), a molecular beacon (MB) probe, four hairpins, and endonuclease Nb.BbvcI, which plays a role in target recycling and signal amplification. In the absence of the target, all of the carboxyfluorescein-labeled (FAM-labeled) hairpins are adsorbed through π-stacking interactions onto the surface of GO, resulting in FAM signal quenching. When the target is added, three nucleic acid chains hybridize together to form a triple complex that is recognized by Nb.BbvCI. The MB probe is then cleaved by Nb.BbvCI to generate an HP/target complex and two new DNA fragments; the former is hybridized to another MB probe and enters the next round of reaction. The two newly reproduced DNA fragments induce a HCR with the assistance of hairpins 1–4 to create double-stranded DNA (dsDNA) products. These dsDNA products are repelled by GO and generate strong fluorescence at excitation/emission wavelengths of 480/514 nm. Importantly, synergy between FAM and the dsDNA-SYBR Green I duplex structure led to significantly amplified fluorescence and enhanced sensitivity. The bioassay showed a detection limit of 7.5 fM toward the target and a good linearity in the 10 fM to 100 pM range. The developed method was applied to monitor biofilm formation and study the mechanism of drug action, with satisfactory results obtained.

Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes

BackgroundRNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on a double-stranded molecular beacon method. This RNA virus probe contains two base sequences—a recognition strand that binds to the specific domain of CSFV N2 or SARS-CoV-2 N, with a fluorophore (FAM) labeled at the 5′ end, and a complementary strand (CSFV-Probe B or SARS-CoV-2-Probe B), combined with a quencher (BHQ2) labeled at the 3′ end.ResultsUsing linear molecular beacon probe technology, the detection limit of the RNA virus probe corresponding to CSFV and SARS-CoV-2 were as low as 0.28 nM and 0.24 nM, respectively. After CSFV E2 and SARS-CoV-2 N genes were transfected into corresponding host cells, the monitoring of RNA virus probes showed that fluorescence signals were dramatically enhanced in a concentration- and time-dependent manner. These results were supported by those of quantitative (qRT-PCR) and visualization (confocal microscopy) analyses. Furthermore, CSF-positive swine samples and simulated SARS-CoV-2 infected mouse samples were used to demonstrate their applicability for different distributions of viral nucleic acids in series tissues.ConclusionsThe proposed RNA virus probe could be used as a PCR-free, cost-effective, and rapid point-of-care (POC) diagnostic platform for target RNA virus detection, holding great potential for the convenient monitoring of different RNA viruses for early mass virus screening.Graphical

Evaluation of real-time NASBA assay for the detection of SARS-CoV-2 compared with real-time PCR.

Purpose In January 2020, the COVID-19 pandemic started and has severely affected all countries around the world. The clinical symptoms alone are not sufficient for a proper diagnosis. Thus, molecular tests are required. Various institutes and researchers developed real-time PCR-based methods for the detection of the virus. However, the method needs expensive equipment. In the present study, we developed a real-time NASBA assay for the detection of SARS-CoV-2.MethodsPrimers and molecular beacon probes for RdRp and N genes were designed. In silico analysis showed that primers and the probes were specific for SARS-CoV-2. The standard samples with known copy numbers of the virus were tested using the NASBA assay and an FDA-approved real-time PCR kit. A series of standard samples were prepared and tested. Clinical sensitivity, precision analysis, and clinical assessment of the assay were performed.ResultsThe limit of detection of the assay was 200 copies/mL. The clinical sensitivity of the assay was 97.64%. The intra-assay and inter-assay for both N and RdRp genes were less than 5% and 10%, respectively. Clinical assessment of the assay showed that the positive agreement rate and negative agreement rate of the assays were determined to be 97.64% and 100%, respectively.ConclusionsThe results of the present study show that the developed real-time NASBA is a sensitive and specific method for the detection of SARS-CoV-2 and is comparable with real-time PCR. NASBA is an isothermal signal amplification method, and if stand-alone fluorescent readers are available, the real-time NASBA can be used without the need for expensive thermocyclers. In addition compared to other isothermal methods like LAMP, the primer design is straightforward. Thus, real-time NASBA could be a suitable method for inexpensive SARS-CoV-2 detection.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11845-022-03046-2.

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.

Novel probe-based melting curve assays for the characterization of fluoroquinolone resistance in Mycoplasma genitalium.

Background Mycoplasma genitalium infection is a sexually transmitted infection that has rapidly become resistant to mainstay treatments. While individualized treatment approaches have been recommended and adopted for macrolides, individualized therapy for fluoroquinolones has not yet been explored, due to a lack of commercial molecular assays and a lack of confidence in specific mutations associated with resistance. In another recent study, we defined a clear role and diagnostic utility in focusing on the absence of resistance mutations to inform microbial cure with fluoroquinolone antimicrobials.MethodsWe developed two proof-of-concept molecular tests that focus on detection of M. genitalium and characterization of WT parC sequences that are strongly linked to fluoroquinolone susceptibility.ResultsWe screened a total of 227 M. genitalium-positive samples using novel molecular beacon and dual hybridization probe assays. These assays were able to detect M. genitalium and characterize fluoroquinolone susceptibility in 143/227 (63%) samples, based on clear differences in melting peak temperatures. The results of these molecular assays were in 100% agreement with ‘gold standard’ Sanger sequencing. Additionally, WT parC sequences were readily distinguished from M. genitalium samples harbouring parC mutations of known or suspected clinical significance. The ability of the assays to successfully characterize fluoroquinolone susceptibility and resistance was reduced in low M. genitalium load samples.ConclusionsThese proof-of-concept assays have considerable potential to improve individualized treatment approaches and rationalize tests of cure for M. genitalium infection. The ability to initiate individualized treatment in up to two-thirds of cases will enhance antimicrobial stewardship for this challenging pathogen.

Construction of Dual-Target Recognition-Based Specific MicroRNA Detection Method for Acute Pancreatitis Analysis.

MicroRNAs (miRNAs) play crucial roles in regulating various biological processes and are considered promising biomarkers for clinical diagnosis and therapy of acute pancreatitis. Herein, we present a duplex-specific nuclease (DSN enzyme) and DNAzyme-assisted fluorescent miRNA detections assay that can provide improved detection specificity due to a design of dual-target recognition and a comparable sensitivity. The dual-target recognitions are composed of (i) miRNA unfold hairpin structure toehold to form DNA-RNA duplex, among which the DNA section will be digested by DSN enzyme, releasing miRNA to participant in a next recycle. (ii) After DNAzyme-based nicking site formation in loop section of molecular beacon (MB), miRNA can bind with the loop section of MB and gradually unfold MB probe, generating fluorescence signals. With this general principle, distinct discrimination capability towards even one base pair mismatch of homogenous miRNA is obtained, showing a promising prospect in clinical diagnosis and therapy of acute pancreatitis.

Establishment of a rapid method for skipjack tuna (Katsuwonus pelamis) authentication using molecular beacons in loop-mediated isothermal amplification

One major drawback to the traditional loop-mediated isothermal amplification (LAMP) detection methods is the increased likelihood of detecting false-positive signals derived from non-specific amplification. Molecular beacon (MB) is increasingly being used in many applications and the MB-LAMP assay has proved itself as a target-specific method. The present work selected skipjack tuna as a case study, and developed a novel MB-LAMP assay for rapid species authentication. Specifically, the optimal MB structure includes 13 nucleobases in the loop region (binding specifically to loop primer LF) and 5 nucleobases in the stem region. For the established MB-LAMP assay, in the presence of the amplicons, the MB probe LFP-1 hybridizes to its target and forms a double helix. The change in conformation separates the quencher from the fluorophore, thereby resulting in the fluorescence release. The novel MB-LAMP assay has proved its specificity and can detect as little as 0.5 pg of skipjack tuna DNA.

Enzyme-embedded ZIF-8/DNA nanocomposite probe for live cells imaging and sensitive detection of ATP

Adenosine triphosphate (ATP) is the direct supplier of energy in almost all biological processes. The intracellular imaging and sensitive detection of ATP will conduce to elucidate its underlying molecular mechanisms of physiological and pathological functions. Herein, a nanocomposite probe was constructed by adsorbing functionalized DNA onto the surface of ZIF-8 pre-embedded with DNA polymerases and nicking endonuclease for live cells imaging and sensitive detection of ATP. The functionalized DNA contained the molecular beacon and recognition probe which with an aptamer sequence, two cleavage sites of nicking enzyme and two template sequences. After uptake into cells, the nanocomposite probe was biodegraded in acid lysosome, releasing the embedded enzymes and surface-adsorbed functionalized DNA. The specific binding of recognition probe to ATP triggered the double-cycle cascade polymerization nicking reaction with the assistance of embedded enzymes, bringing a significant enhancement of fluorescence signal. Based on this, the imaging of ATP in human cancer cells (HeLa, A549 and MCF-7 cells) was successfully implemented. The expression level changes of ATP induced by oligomycin or Ca2+ in HeLa cells could also be efficiently monitored. Furthermore, the sensitive quantification detection of ATP in vitro was achieved with a limit of detection of 7.7 nM. These results showed this enzyme-embedded ZIF-8/DNA nanocomposite probe will be a promising tool for ATP-associated biomedical research.

Molecular beacon-based DNA tetrahedrons for APE 1 activity detection in living cells

Human apurine/apyrimidine endonuclease (APE) is a multifunctional enzyme that affects the occurrence and development of cancer through DNA repair and redox regulation. It has been identified as a tumor marker for detection and prognosis and drug target due to its overexpression in a variety of cancers. However, the accurate detection of APE 1 activity is still a huge challenge. Traditional approaches exist some disadvantages, including low sensitivity, complex operation and professional equipment requirements. Therefore, it is necessary to develop a fast, sensitive and simple biosensor to detect APE 1 activity in vivo and in vitro. In this report, a new tetrahedral molecular beacon (TMB) probe is designed for the detection of APE 1 activity. This probe not only has the advantage that the tetrahedron can enter the cells without a transfection agent, but also has the advantages of simple operation and low cost. The extracellular detection limit of TMB is as low as 0.0026 U/mL, and this program has successfully achieved APE 1 intracellular imaging to distinguish cancer cells overexpressing APE 1 from normal cells. In addition, the probe which is an effective mean for screening APE 1 inhibitor can also monitor the response of APE 1 to different drugs. All in all, our probe can be successfully applied to detect APE 1 activity in vivo and vitro, and can achieve living cells drug screening. And this probe also provides a general method for the detection of other endogenous biological targets in living cells.

A persistent luminescence resonance energy transfer-based molecular beacon probe for the highly sensitive detection of microRNA in biological samples

Herein, a time-resolved luminescence resonance energy transfer (TR-LRET) molecular beacon (MB) probe employing persistent luminescence nanoparticles (PLNPs) as the energy donors was first constructed, and further designed for microRNA21 (miR21) sensing. This probe (named as PLNPs-MB) was facilely fabricated by covalent bioconjugation between poly-(acrylic acid) (PAA) modified near-infrared (NIR) emissive PLNPs i.e. ZnGa2O4:Cr3+ and functionalized MB oligonucleotide (5′-NH2 and 3′-BHQ3). Accordingly, PLNPs and BHQ3 were in close proximity to each other, leading to the occurrence of LRET and obvious persistent luminescence (PL) quenching. In the presence of miR21, loop of the PLNP-MB was hybridized, accompanying BHQ3 away from PLNPs and the restraint of LRET process. As a result, PL of the PLNPs was recovered, which built the foundation of miR21 quantification. The probe provided a linear response range from 0.1 to 10 nM for miR21 detection. Quantification limit of this probe was competitive and about 1–2 orders of magnitude lower than that of other reported MB probes for nucleic acid. Moreover, the proposed probe was successfully adopted for miR21 detection in biological fluids (human serum, cell extraction). This work also provided a sensitive detection nanoplatform for other targets through modifying diverse MBs onto the surface of PLNPs.

Abstract 579: Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis

Abstract Background Digital PCR (dPCR) is a promising method for liquid biopsies and is able to quantify nucleic acids more sensitively than real-time PCR. However, dPCR has a large fluctuation in the fluorescence intensity of the droplets or wells due to insufficient PCR in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping. Methods A sample is digitized into a silicon chip with up to ~104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that are complimentary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, dye color of the probe, and melting temperature (Tm). Because the Tm of the PCR products does not considerably depend on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping. Results The concept was confirmed by simultaneously identifying wild-type KRAS, BRAF and, eight mutants of these genes (G12D, G12R, G12V, G13D, G12A, G12C, G12S, and V600E) through four-color melting curve analysis. G12D, G12V and G12R-mutant KRAS alleles were detected with FAM-labeled probes, G13D-mutant KRAS allele and wildtype KRAS allele were detected with HEX-labeled probes, G12S and G12C-mutant KRAS alleles were detected with Cy5-labeled probes, and V600E-mutant BRAF allele and wildtype BRAF allele were detected with CAL Fluor Red 610-labeled probes. The results showed that the peaks of the Tm distributions of the DNA groups were separated by 3.5 °C on average, while the standard deviation of the Tm distributions was 0.23 °C on average. The difference in Tm was 15 times larger than the standard deviation, enabling more accurate genotyping than when using the fluorescence intensity. Conclusion We have proposed a measurement method that combines dPCR with melting curve analysis, and successfully performed the multiplexed genotyping assay. We plan to evaluate the effect of this method on measurement using clinical specimens of pancreatic cancer patients in the future. Citation Format: Junko Tanaka, Tatsuo Nakagawa, Takeshi Ishida, Yoshio Kamura, Akiko Shiratori, Kunio Harada, Chihiro Uematsu. Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 579.

Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection.

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.

Effect of TRPV4 Polymorphisms on the Development of Airway Response to Increased Inspiratory Load

Alongside with inflammation, increased airway resistance is a key feature of obstructive lung diseases. The data accumulated up to date suggest that bronchoconstriction commonly observed in asthma or COPD is not only a symptom but serves as an important factor of the diseases’ pathogenesis. Mechanical deformation of the airways occurring during the episodes of bronchoconstriction or accompanying chronic airflow limitation may per se promote respiratory inflammation and remodeling, thus, forming a vicious cycle. The precise molecular mechanisms triggering the response to mechanical stress in the airways have not been established so far. In this regard, a particular attention is drawn to TRP channels – polymodal receptors sensitive to many physical and chemical stimuli including stretching and compression at cellular level. TRPV4 is a mechanosensitive channel abundantly expressed in the airway epithelium that was previously proved to participate in stretch-induced respiratory responses during mechanical ventilation. The aim of this study was to establish the effects of SNPs located in TRPV4 region on the airway response induced by inspiratory resistive breathing. The study enrolled 122 patients with asthma, 82 patients with COPD and 54 healthy subjects. All the participants underwent 5-min isocapnic (5% CO2) inspiratory resistive breathing with load at 45% of maximum inspiratory pressure. Specific airway conductance (sGaw) was measured by body plethysmography before and after the inspiratory load. Relative change (Δ) in sGaw by ≥20% was considered as a significant response. Six SNPs (rs7139451, rs11068434, rs3825394, rs3825396, rs6606743, rs3742034) were genotyped by LATE-PCR with melting analysis of molecular beacon probes. All the studied SNPs were in Hardy-Weinberg equilibrium. SNP rs3825396 located in intronic region of TRPV4 exerted significant effect on the airway response to mechanical stress. In general sample, carriers of the AG genotype had higher ΔsGaw (9.0 (-8.5-30.6)%) in comparison with the GG homozygotes (-6.4 (-20.0-11.9)%, p=0.01). This trend was evident in the subgroups of healthy subjects, asthma and COPD patients, although the difference was not significant due to reduced number of observations. Carriers of the AA genotype were absent both in our sample and in reference HapMap population. The AG genotype was found in 5.4%, 13.3% and 25.0% of subjects with ΔsGaw ≤-20%, -20%<ΔsGaw<20% and ΔsGaw ≥20%, respectively (p=0.01). The difference was even more significant when comparing only between cases with ΔsGaw ≥20% and ΔsGaw ≤-20% (p=0.007). This association retained after adjustment for sex, age and diagnosis in logistic regression model (adjusted OR 5.2 95%CI (1.3-20.6), p=0.02). We did not found significant associations with the airway response to mechanical stress for the other studied SNPs. In conclusion, we established that TRPV4 rs3825396 SNP may influence reaction of the respiratory tract to increased inspiratory load. This association seems to be independent of the presence of the obstructive airway pathology, although further verification on larger sample is required.

A universal fluorescent sensing system for pathogen determination based on loop-mediated isothermal amplification triggering dual-primer rolling circle extension

The great challenge for loop-mediated isothermal amplification (LAMP) is target-specific detection. However, the most popular fluorogenic probe method needs complicated sequence design and large reagent consumption. Rolling circle amplification (RCA), known for its simplicity, is presumably compatible with LAMP in single-tube reaction detection based on Bst DNA polymerase to realize universality. Therefore, this work combined LAMP with RCA through strand displacement strategy to determine pathogens. Specifically, pathogen gene initiated LAMP process, displacing two single strands (S1 and S2) hybridized with LAMP loop primers for dual-primer rolling circle extension. Then, Molecular beacon (MB) probe paired to RCA amplicons emitting fluorescence signal for real-time detection. For detecting different pathogens, LAMP primers were specifically designed without changing the detection strategies through same RCA set. The established LAMP-RCA technique quantitatively detected invA and malB ranging from 102 to 106 copies μL−1, also demonstrating the universality. Meanwhile, S. typhimurium without genomic DNA extraction was directly quantified ranging from 102 to 3.16 × 104 CFU μL−1, with a detection limit of 32 CFU μL−1. With good selectivity, this work was successfully applied for S. typhimurium determination in 10 % milk, indicating that the established method had considerable potential in complex samples.

Sequence-Specific Detection of ORF1a, BRAF, and ompW DNA Sequences with Loop Mediated Isothermal Amplification on Lateral Flow Immunoassay Strips Enabled by Molecular Beacons.

Loop-mediated isothermal amplification (LAMP) holds great potential for point-of-care (POC) diagnostics due to its speed and sensitivity. However, differentiation between spurious amplification and amplification of the target sequence is a challenge. Herein, we develop the use of molecular beacon (MB) probes for the sequence-specific detection of LAMP on commercially available lateral flow immunoassay (LFIA) strips. The detection of three unique DNA sequences, including ORF1a from SARS-CoV-2, is demonstrated. In addition, the method is capable of detecting clinically relevant single-nucleotide polymorphisms (BRAF V600E). For all sequences tested, the LFIA method offers similar sensitivity to fluorescence detection using a qPCR instrument. We also demonstrate the coupling of the method with solid-phase microextraction to enable isolation and detection of the target sequences from human plasma, pond water, and artificial saliva. Lastly, a 3D printed device is designed and implemented to prevent contamination caused by opening the reaction containers after LAMP.

Xpert MTB/XDR: a 10-Color Reflex Assay Suitable for Point-of-Care Settings To Detect Isoniazid, Fluoroquinolone, and Second-Line-Injectable-Drug Resistance Directly from Mycobacterium tuberculosis-Positive Sputum.

We describe the design, development, analytical performance, and a limited clinical evaluation of the 10-color Xpert MTB/XDR assay (CE-IVD only, not for sale in the United States). This assay is intended as a reflex test to detect resistance to isoniazid (INH), fluoroquinolones (FLQ), ethionamide (ETH), and second-line injectable drugs (SLIDs) in unprocessed sputum samples and concentrated sputum sediments which are positive for Mycobacterium tuberculosis The Xpert MTB/XDR assay simultaneously amplifies eight genes and promoter regions in M. tuberculosis and analyzes melting temperatures (Tm s) using sloppy molecular beacon (SMB) probes to identify mutations associated with INH, FLQ, ETH, and SLID resistance. Results can be obtained in under 90 min using 10-color GeneXpert modules. The assay can differentiate low- versus high-level resistance to INH and FLQ as well as cross-resistance versus individual resistance to SLIDs by identifying mutation-specific Tm s or Tm patterns generated by the SMB probes. The assay has a limit of detection comparable to that of the Xpert MTB/RIF assay and successfully detected 16 clinically significant mutations in a challenge set of clinical isolate DNA. In a clinical study performed at two sites with 100 sputum and 214 clinical isolates, the assay showed a sensitivity of 94% to 100% and a specificity of 100% for all drugs except for ETH compared to that of sequencing. The sensitivity and specificity were in the same ranges as those of phenotypic drug-susceptibility testing. Used in combination with a primary tuberculosis diagnostic test, this assay should expand the capacity for detection of drug-resistant tuberculosis near the point of care.

High-Coverage Nucleic Acid Probes for Distinguishing SARS-COV-2 from Influenza

There is an urgent need for rapid, accurate, and ultrasensitive diagnostics to identify respiratory viruses. Amplification-free detection of nucleic acids provides a promising technique for rapid diagnosis of viral infections. The objective of this study was to design, characterize, and optimize mutation-resistant molecular beacon probes for the detection of influenza versus SARS-CoV-2 viruses. High-coverage probe cocktails were computationally designed using our “fast evaluation of viral emerging risks” (FEVER) pipeline.

Post-COVID mucormycosis: Ascertainment of the pathological diagnostic approach.

Post-COVID mucormycosis: Ascertainment of the pathological diagnostic approach.

Manufacturing Reusable NAND Logic Gates and Their Initial Circuits for DNA Nanoprocessors.

DNA-based computers can potentially analyze complex sets of biological markers, thereby advancing diagnostics and the treatment of diseases. Despite extensive efforts, DNA processors have not yet been developed due, in part, to limitations in the ability to integrate available logic gates into circuits. We have designed a NAND gate, which is one of the functionally complete set of logic connectives. The gate's design avoids stem-loop-folded DNA fragments, and is capable of reusable operations in RNase H-containing buffer. The output of the gate can be translated into RNA-cleaving activity or a fluorescent signal produced either by a deoxyribozyme or a molecular beacon probe. Furthermore, three NAND-gate-forming DNA strands were crosslinked by click chemistry and purified in a simple procedure that allowed ≈1013 gates to be manufactured in 16 h, with a hands-on time of about 30 min. Two NAND gates can be joined into one association that performs a new logic function simply by adding a DNA linker strand. Approaches developed in this work could contribute to the development of biocompatible DNA logic circuits for biotechnological and medical applications.