Stem-loop Research Articles

NMR 1H,19F-based screening of the four stem-looped structure 5_SL1-SL4 located in the 5'-untranslated region of SARS-CoV 2 RNA.

Development of new antiviral medication against the beta-coronavirus SARS-CoV-2 (SCoV2) is actively being pursued. Both NMR spectroscopy and crystallography as structural screening technologies have been utilised to screen the viral proteome for binding to fragment libraries. Here, we report on NMR screening of elements of the viral RNA genome with two different ligand libraries using 1H-NMR-screening experiments and 1H and 19F NMR-screening experiments for fluorinated compounds. We screened against the 5'-terminal 119 nucleotides located in the 5'-untranslated region of the RNA genome of SCoV2 and further dissected the four stem-loops into its constituent RNA elements to test specificity of binding of ligands to shorter and longer viral RNA stretches. The first library (DRTL-F library) is enriched in ligands binding to RNA motifs, while the second library (DSI-poised library) represents a fragment library originally designed for protein screening. Conducting screens with two different libraries allows us to compare different NMR screening methodologies, describe NMR screening workflows, validate the two different fragment libraries, and derive initial leads for further downstream medicinal chemistry optimisation.

Intramolecular aptamer switches.

Aptamer switches as effective biosensing tools have become a focal point of research in engineered aptasensors. Intramolecular aptamer switches are more versatile, affordable, and simpler than classical "open-close" and strand displacement-based aptamer switches. Recently, many new aptamers with an overall hairpin structure have been reported. In this study, intramolecular aptamer switches were developed by adding new base pairs to the end of aptamers. The additional nucleotides can pair with the internal domains of the aptamer, causing a change in its conformation from the original secondary structure without a target. When a target binds to an aptamer, a marked change in the structure of the aptamer is expected. As models for testing this intramolecular aptamer switch idea, aptamers of oxytetracycline (OTC), 17β-estradiol (E2), and adenosine were employed. When the additional base pairs are too long, binding the target to the aptamer becomes more challenging. This research offers valuable insights into the development of intramolecular aptamer switches and their potential applications in biosensor design.

Degradation and detection of organophosphorus pesticides based on peptides and MXene-peptide composite materials.

Safety problems caused by organophosphorus pesticide (OP) residues are constantly occurring, so the development of new methods for the degradation and detection of OPs is of great scientific significance. In the present study, β-sheet peptides and β-hairpin peptides for catalyzing the hydrolysis of OPs were designed and synthesized. The peptide sequences with the highest hydrolytic activity (EHSGGVTVDPPLTVEHSAG) were screened by investigating the effect of the location of the active sites of the peptide and the peptide's structure on the degradation of OPs. In addition, the relationship between the peptides' conformation and hydrolytic activity was further analyzed based on density functional theory calculations. The noncovalent interactions of the peptides with the OPs and the electrostatic potential on the molecular surface and molecular docking properties were also investigated. It was found that peptides with approximate active amino acids consisting of the catalytic triad and with the hairpin structure had enhanced hydrolytic activity toward the hydrolysis of OPs. To develop an electrochemical sensor technique to detect OPs, the conductive MXene (Ti3C2) material was first immobilized with a caffeic acid monolayer via enediol-metal complex chemistry and then bound with the β-hairpin peptide (EHSGGVTVDPPLTVEHSAG) via carboxy-amine condensation chemistry between the -COOH of caffeic acid and the -NH2 of the peptide to prepare a MXene-peptide composite. Then, the prepared composite was modified on the surface of a glassy carbon electrode to construct an electrochemical sensor for the detection of OPs. The developed technique could be used to monitor OPs within 15 min with a two orders of linear working range and with a detection limit of 0.15 μM. Meanwhile, the sensor showed good reliability for the detection of OPs in real vegetables.

Peptide nucleic acids can form hairpins and bind RNA-binding proteins.

RNA-binding proteins (RBPs) are a major class of proteins that interact with RNAs to change their fate or function. RBPs and the ribonucleoprotein complexes they constitute are involved in many essential cellular processes. In many cases, the molecular details of RBP:RNA interactions differ between viruses, prokaryotes and eukaryotes, making prokaryotic and viral RBPs good potential drug targets. However, targeting RBPs with small molecules has so far been met with limited success as RNA-binding sites tend to be extended, shallow and dynamic with a mixture of charged, polar and hydrophobic interactions. Here, we show that peptide nucleic acids (PNAs) with nucleic acid-like binding properties and a highly stable peptide-like backbone can be used to target some RBPs. We have designed PNAs to mimic the short RNA stem-loop sequence required for the initiation of prokaryotic signal recognition particle (SRP) assembly, a target for antibiotics development. Using a range of biophysical and biochemical assays, the designed PNAs were demonstrated to fold into a hairpin structure, bind the targeted protein and compete with the native RNA hairpin to inhibit SRP formation. To show the applicability of PNAs against other RBPs, a PNA was also shown to bind Nsp9 from SARS-CoV-2, a protein that exhibits non-sequence-specific RNA binding but preferentially binds hairpin structures. Taken together, our results support that PNAs can be a promising class of compounds for targeting RNA-binding activities in RBPs.

GPU-accelerated non-dominated sorting genetic algorithm III for maximizing protein production

<abstract><p>Maximizing protein expression levels poses a major challenge in bioengineering. To increase protein expression levels, numerous factors, including codon bias, codon context bias, hidden stop codons, homologous recombination, suitable guanine-cytosine ratio, and hairpin loop structure, are crucial and quantified by six objective functions: CAI, CPB, HSC, HD, GC3, and SL. Optimizing these six objectives simultaneously constitutes a multi-objective optimization problem, aiming to identify the favorable Pareto solutions rather than a singular optimal solution. However, achieving satisfactory solutions requires numerous cycles and solutions, thus leading to a large number of functional evaluations. While there are frameworks for multi-objective optimization problems, they often lack efficient support for objective function computation in protein encoding. In this paper, we proposed a method to design a set of coding sequences (CDSs) based on non-dominated sorting genetic algorithm III (NSGA-III), accelerated using NVIDIA graphical processing units (GPUs). Experimental results indicated that our method is 15,454 times faster than the Pymoo framework and is evaluated using 100 solutions and 100 cycles. Since our GPU implementation facilitated the use of larger solutions and more cycles, we were able to design a superior set of CDSs by increasing solutions to 400 and cycles to 12,800. In addition, our NSGA-III-based method consistently surpassed the NSGA-II approach when the number of cycles exceeded 3200 by utilizing 100 solutions. Finally, we observed that a gradual reduction of the mutation probability as the number of cycles increased yielded better quality results than maintaining a fixed mutation probability.</p></abstract>

Lung Epithelial Regnase-1 Dampens Local Immune Response but Does Not Worsen Susceptibility to Klebsiella pneumoniae.

Klebsiella pneumoniae (KP) presents a global health threat, leading to significant morbidity and mortality due to its multidrug-resistant profile and the limited availability of therapeutic options. To eliminate KP lung infection, the host initiates a robust inflammatory response. One of the host's mechanisms for mitigating excessive inflammation involves the RNA-binding protein regnase-1 (Reg1, MCPIP1, or ZC3H12A). Reg1 has an RNA binding domain that recognizes stem-loop structures in the 3' untranslated region of various proinflammatory transcripts, leading to mRNA decay. However, excessive suppression of inflammation by Reg1 results in suboptimal KP control. Reg1 deficiency within the nonhematopoietic compartment confers resistance to KP in the lung. Given that lung epithelium is crucial for KP resistance, we hypothesized that selective deletion of Reg1 in lung epithelial cells might enhance proinflammatory signals, leading to a better control of KP. Our transcriptomic analysis of epithelial cells in KP-infected wild-type mice revealed the presence of three distinct alveolar type 2 cell (AT2) subpopulations (conventional, inflammatory, and cycling) and enrichment of Reg1 in inflammatory AT2 cells. We conditionally deleted Reg1 in lung AT2 cells (ΔReg1), which amplified the local inflammatory response in the lung and increased macrophage cell numbers compared with controls. However, when ΔReg1 mice were subjected to KP infection, there were no significant differences in bacterial burden or survival compared with controls. These findings suggest that the local inflammatory response enhanced by Reg1 deletion in AT2 cells is insufficient to control KP infection.

Identification of the Csr global regulatory system mediated by small RNA decay in Aeromonas salmonicida.

We investigated the presence and functionality of the carbon storage regulator (Csr) system in Aeromonas salmonicida SWSY-1.411. CsrA, an RNA-binding protein, shared 89% amino acid sequence identity with Escherichia coli CsrA. CsrB/C sRNAs exhibited a typical stem-loop structure, with more GGA motifs, which bind CsrA, than E. coli. CsrD had limited sequence identity with E. coli CsrD; however, it contained the conserved GGDEF and EAL domains. Functional analysis in E. coli demonstrated that the Csr system of A. salmonicida influences glycogen biosynthesis, biofilm formation, motility, and stability of both CsrB and CsrC sRNAs. These findings suggest that in A. salmonicida, the Csr system affects phenotypes like its E. coli counterpart. In A. salmonicida, defects in csr homologs affected biofilm formation, motility, and chitinase production. However, glycogen accumulation and protease production were unaffected. The expression of flagellar-related genes and chitinase genes was suppressed in the csrA-deficient A. salmonicida. Northern blot analysis indicated the stabilization of CsrB and CsrC in the csrD-deficient A. salmonicida. Similar to that in E. coli, the Csr system in A. salmonicida comprises the RNA-binding protein CsrA, the sRNAs CsrB and CsrC, and the sRNA decay factor CsrD. This study underscores the conservation and functionality of the Csr system and raises questions about its regulatory targets and mechanisms in A. salmonicida.

Structural Analysis of the Hepatitis B Virus RNA Encapsidation Signal ε by Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE).

RNA structure is crucial for RNA function, including in viral cis-elements such as the hepatitis B virus (HBV) RNA encapsidation signal ε. Interacting with the viral polymerase ε mediates packaging of the pregenomic (pg) RNA into capsids, initiation of reverse transcription, and it affects the mRNA functions of pgRNA. As free RNA, the 61-nucleotide (nt) ε sequence adopts a bipartite stem-loop structure with a central bulge and an apical loop. Due to stable Watson-Crick base pairing, this was already predicted by early RNA folding programs and confirmed by classical enzymatic and chemical structure probing. A newer, high-resolution probing technique exploits the selective acylation of solvent-accessible 2'-hydroxyls in the RNA backbone by electrophilic compounds such as 2-methylnicotinic acid imidazolide (NAI), followed by mapping of the modified sites by primer extension. This SHAPE principle has meanwhile been extended to numerous applications. Here we provide a basic protocol for NAI-based SHAPE of isolated HBV ε RNA which already provided insights into the impact of mutations, and preliminarily, of polymerase binding on the RNA structural dynamics. While the focus is on NAI modification, we also briefly cover target RNA preparation by in vitro transcription, primer extension using a radiolabeled primer, and analysis of the resulting cDNAs by denaturing polyacrylamide gelelectrophoresis (PAGE). Given the high tolerance of SHAPE chemistry to different conditions, including applicability in live cells, we expect this technique to greatly facilitate deciphering the conformational dynamics underlying the various functions of the ε element, especially in concert with the recently solved three-dimensional structure of the free RNA.

Hairpin self-assembly powered by exonuclease III for highly sensitive and cross-validated miRNA-155 detection.

Cancer is one of the most important causes of human death and poses a serious threat to human health. As a cancer biomarker, microRNA-155 (miRNA-155) is highly expressed in various types of cancer tissues and is involved in the proliferation of tumor cells. Therefore, developing a miRNA-155 detection technology with high specificity and sensitivity is of great significance for the early detection, accurate treatment and prognostic evaluation of tumors. Here, we developed a fluorescence detection method using exonuclease III-assisted target cycling and catalytic hairpin assembly (CHA) as a signal amplification technique. This study developed a biosensor for the detection of miRNA-155, utilizing a DNA hairpin (Hp) for target recognition and generating double-stranded DNA (dual-Hp-T). The 3' flat end of the double-stranded DNA can be cleaved by exonuclease III to achieve the target cycle, and a large amount of single-stranded DNA (fuel) can trigger CHA to achieve signal amplification. Simultaneously, the fluorescence resonance energy transfer (FRET) of signal probes with different fluorescence labels on H1 and H2 ends occurs with the CHA reaction. The two fluorescence signals obtained can be used to cross-validate the experimental results. The biosensor exhibits excellent performance of high recovery, high sensitivity and high operability, which can achieve the specific detection of miRNA-155 with a detection limit as low as 8.3 pM. Additionally, the detection efficacy in a human serum environment is also highly satisfactory. This technology provides strong technical support for the development of nucleic acid probes and the diagnosis and treatment of cancer, demonstrating significant practical application value.

SABER-FISH in Hydractinia.

In situ hybridization allows the detection of nucleic acid sequences in fixed cells and tissues. The gelatinous nature of cnidarians and Hydractinia demands extensive and exhausting protocols to detect RNA transcripts with traditional methods (e.g., colorimetric in situ hybridization). Signal amplification by exchange reaction (SABER) fluorescence in situ hybridization (FISH) enables simplifying and multiplex imaging of RNA targets in a rapid and cost-effective manner. In one enzymatic reaction, SABER-FISH uses a strand-displacing polymerase and catalytic DNA hairpin to generate FISH probes with adjustable signal amplification, allowing highly sensitive detection of nucleic acids and reducing the number of required probes. Here I describe the methodology to detect transcripts within the cells of Hydractinia by SABER-FISH in whole-mount samples.

DNA-directed formation of plasmonic core-satellite nanostructures for quantification of hepatitis C viral RNA.

Hepatitis C virus (HCV) continues to be a significant public health challenge, affecting an estimated 71 million people globally and posing risks of severe liver diseases. Despite advancements in treatments, diagnostic limitations hinder the global elimination efforts targeted by 2030. This study introduces an innovative diagnostic approach, integrating catalytic hairpin assembly (CHA) with plasmonic core-satellite gold nanoparticle (AuNP) assemblies, to enable sensitive and specific detection of HCV RNA. We optimized the stoichiometry of DNA hairpins to form highly stable three-way junctions (3WJs), minimizing non-specific reactions in an enzyme-free, isothermal amplification process. The resulting dual-transduction biosensor combines colorimetric and surface-enhanced Raman spectroscopy (SERS) techniques, utilizing the Raman reporter malachite green isothiocyanate (MGITC) for signal generation. Our system targets a conserved 23-nucleotide sequence within the HCV 5'-UTR, essential for RNA replication, facilitating pan-genotypic HCV detection that complements direct-acting antiviral strategies. We evaluated the biosensor's efficacy using fluorescence spectroscopy, native PAGE, AFM, and TEM. Findings indicate that the 60 nm core AuNPs surrounded by 20 nm satellite AuNPs achieved a ten-fold increase in sensitivity over the 10 nm satellites, detecting HCV RNA concentrations as low as 1.706 fM. This sensitivity is crucial, given the extremely low viral loads present during early infection stages. Our research demonstrates the promise of enzyme-free molecular biosensors for HCV, with the potential to provide cost-efficient, rapid, point-of-care testing, although further sensitivity enhancements are needed to address the challenges of early-stage detection.

Combined detection of hepatitis B virus surface antigen and hepatitis B virus DNA using a DNA sensor.

Hepatitis B virus (HBV) infection is associated with the progression of liver disease. Occult HBV infection (OBI) is defined as the existence of detectable HBV DNA in HBV surface antigen negative individuals. However, HBV DNA is negative in serum while HBV surface antigen remains positive in incompletely cured chronic HBV infection. Hence, combined detection of HBV surface antigen and HBV DNA is essential for the accurate detection and rehabilitation of HBV infection. Therefore, a multiplex detection strategy based on branched DNA nanostructures was developed. The single-stranded segment of a branched DNA nanostructure (segments of S4 and S2) assembled by four single-stranded DNA was hybridized with the aptamer of HBV surface antigen and DNA hairpin to construct a DNA nanosensor, which can achieve high specificity identification and highly sensitive fluorescence responses to the targets. The detection limits of the developed nanosensor for HBV and HBV DNA are 50 pM and 5 nM, respectively. The combined detection of HBV surface antigen and HBV DNA provides a new insight for more thorough diagnostic evaluation of HBV infection and rehabilitation.

A chimeric hairpin DNA aptamer-based biosensor for monitoring the therapeutic drug bevacizumab.

The accurate and rapid detection of specific antibodies in blood is very important for efficient diagnosis and precise treatment. Conventional methods often suffer from time-consuming operations and/or a narrow detection range. In this work, for the rapid determination of bevacizumab in plasma, a series of chimeric hairpin DNA aptamer-based probes were designed by the modification, labeling and theoretical computation of an original aptamer. Then, the dissociation constant of the modified hairpin DNA to bevacizumab was measured and screened using microscale thermophoresis. The best chimeric hairpin DNA aptamer-based probe was then selected, and a one-step platform for the rapid determination of bevacizumab was constructed. This strategy has the advantages of being simple, fast and label-free. Because of the design and screening of the hairpin DNA, as well as the optimization of the concentration and electrochemical parameters, a low detection limit of 0.37 pM (0.054 ng mL-1) with a wide linear range (1 pM-1 μM) was obtained. Finally, the rationally constructed biosensor was successfully applied to the determination of bevacizumab in spiked samples, and it showed good accuracy and precision. This method is expected to truly realize accurate and rapid detection of bevacizumab and provides a new idea for the precise treatment of diseases.

A two-layer circuit cascade-based DNA machine for highly sensitive miRNA imaging in living cells.

Sensitive detection of microRNA (miRNA), one of the most promising biomarkers, plays crucial roles in cancer diagnosis. However, the low expression level of miRNA makes it extremely urgent to develop ultrasensitive and highly selective strategies for quantification of miRNA. Herein, a DNA machine is rationally constructed for amplified detection and imaging of low-abundance miRNA in living cells based on the toehold-mediated strand displacement reaction (TMSDR). The isothermal and enzyme-free DNA machine with low background leakage is fabricated by integrating two DNA circuits into a cascade system, in which the output of one circuit serves as the input of the other one. Once the DNA machine is transfected into breast cancer cells, the overexpressed miRNA-203 initiates the first-layer circuit through TMSDR, leading to the concentration variation of fuel strands, which further influences the assembly of hairpin DNA in the second-layer circuit and the occurrence of fluorescence resonance energy transfer (FRET) for fluorescence imaging. Benefiting from the cascade of the two-layer amplification reaction, the proposed DNA machine acquires a detection limit down to 4 fM for quantification of miR-203 and a 10 000-fold improvement in amplification efficiency over the single circuit. Therefore, the two-layer circuit cascade-based DNA machine provides an effective platform for amplified analysis of low-abundance miRNA with high sensitivity, which holds great promise in biomedical and clinical research.

Efficient and accurate detection of GC-associated miR-96-5p using a competitive lateral flow method based on SERS.

To facilitate rapid, efficient, and accurate detection of miR-96-5p associated with gastric cancer (GC), we developed a bioanalytical platform by integrating surface-enhanced Raman spectroscopy with lateral flow assay (SERS-LFA). With these SERS-LFA strips, miR-96-5p within the specimen competed with Au rhombic dodecahedron (AuRD) conjugated single-stranded DNA (ssDNA) to bond to the immobilized hairpin DNA (hpDNA) probe on the T line. Consequently, higher abundance of miR-96-5p led to reduced conjugation of AuRD on the T line, thereby resulting in diminished SERS intensity. The biosensor exhibited a detection time of approximately 30 min and demonstrated a low limit of detection (LOD) for miR-96-5p in PBS buffer solution, down to 3.7 fM. To validate its clinical utility for the early diagnosis of patients with different degrees of gastric lesions, we performed quantitative evaluations in cohorts that included healthy individuals, patients with mild intraepithelial neoplasia, patients with severe intraepithelial neoplasia, as well as patients diagnosed with GC. The results obtained from the SERS-LFA strips were in agreement with those obtained from the quantitative real-time polymerase chain reaction (qRT-PCR). Given the accomplishments, this biosensor has significant potential for the clinical diagnosis of GC, offering a promising avenue for timely detection and improved patient prognoses.

Monitoring Real-Time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism.

RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dynamic and subject to rapid changes in response to environmental cues, temperature in particular. Thermosensitive RNA secondary structures have been harnessed by multiple organisms to survey their temperature environment and to adjust gene expression accordingly. It is thus highly desirable to observe RNA structural changes in real time over a range of temperatures. Multiple approaches have been developed to study structural dynamics, but many of these require extensive processing of the RNA, large amounts of RNA input, and/or cannot be applied under physiological conditions. Here, we describe the use of a dually fluorescently labeled RNA oligonucleotide (containing a predicted hairpin structure) to monitor subtle RNA structural dynamics in vitro by Förster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy. These approaches can be employed under physiologically relevant conditions over a range of temperatures and with RNA concentrations as low as 200nM; they enable us to observe RNA structural dynamics in real time and to correlate these dynamics with changes in biological processes such as translation.

Handgrip-Ring Structure Sensing Probe Assisted Multiple Signal Amplification Strategy for Sensitive and Label-Free Single-Stranded Nucleic Acid Analysis.

Precise and efficient identification of single-stranded nucleic acids is crucial for both pathological research and early diagnosis of diseases, such as cancers. Therefore, we have devised a novel biosensor that utilizes an elegantly designed handgrip-ring structure sensing probe to enhance the detection sensitivity and reduce background signals. The handgrip-ring structure sensing probe combines ring padlock-based target recognition and hairpin structure probe-based signal amplification. The target sequences form a binding interaction with the ring padlock in the sensing probe, leading to the elongation of the single-stranded chain with the assistance of polymerase. This elongation step results in the release of the hairpin probe, triggering a signal amplification process. This design significantly minimized the potential discrepancies that may occur during the signal amplification process, hence bestowing the approach with a low level of background signals. By utilizing this innovative design, the current biosensor demonstrates a remarkable ability to detect miRNA with a limit as low as 376 aM and single-stranded DNA sequences with a limit as low as 45.3 aM. In addition, it possesses exceptional discrimination capabilities. The efficacy of this approach in diagnosing targets was also effectively proved by the rational redesign of the ring padlock.

Signal amplified colorimetric nucleic acid detection based on autocatalytic hairpin assembly.

Herein, a nucleic acid assay based on autocatalytic hairpin assembly (ACHA) was proposed. In this system, two split G-quadruplex sequences were integrated into H1 and H2, respectively. And a DNA strand with the same sequence to target DNA was integrated into the assistant hairpin H3. In the presence of target DNA, the hairpin structure of H1 was opened and catalytic hairpin assembly (CHA) was activated, and then a series of DNA assembly steps based on the toehold-mediated DNA strand displacement were triggered and the product H1-H2 with sticky ends on both sides was formed. On the one side of H1-H2, the split two G-quadruplex sequences were close enough to form the intact G-quadruplex for the signal readout. At the same time, two sticky ends on the other side of H1-H2 hybridized with H3 and a new sticky end with the sequence same to the target DNA was exposed, which can immediately trigger the autocatalytic hairpin assembly reaction, and then the reaction rate of CHA was effectively accelerated and the colorimetric signal was significantly amplified. This ACHA signal amplified strategy has been successfully applied for the rapid and colorimetric nucleic acid detection.

PH-dependent complex formation with TAR RNA and DNA: application towards logic gates.

Nucleic acid-based logic gates have shown great potential in biotechnology, medicine as well as diagnostics. Herein, we have constructed pH-responsive logic devices by utilizing HIV-1 TAR hairpins in combination with a thiazole peptide that exhibits turn-on fluorescence upon interacting with TAR RNA or DNA. Based on this, INHIBIT-AND and YES-INHIBIT-AND logic gates were constructed in parallel. The pH alteration leads to conformational changes of the hairpin structure, enabling the construction of a multi-reset reusable logic system which could be developed for in vitro sensing of the HIV-1 viral RNA.

Naked-eye detection of plant viral disease using polymerase chain reaction amplification and DNAzyme.

Plant viral diseases can seriously affect the yield and quality of crops. In this work, a convenient and highly sensitive biosensor for the visual detection of plant viral disease is proposed by the PCR-induced generation of DNAzyme. In the absence of nucleic acid for a target plant virus, the primers prohibited the production of G-quadruplex by forming a hairpin structure. However, PCR amplification occurred and generated a number of specific PCR products with free G-quadruplex sequences at both ends in the presence of the target cDNA. A catalytically active G-quadruplex DNAzyme was formed with the help of K+ and hemin, resulting in the formation of colored products visible to the naked eye and a strong absorbance by the addition of ABTS2- and H2O2. The absorbance and the logarithm of target cDNA concentrations showed a good linear relationship in the range of 10 fM-1.0 nM, with a linear regression equation of A = 0.1402 lg c + 0.3761 (c: fM) and a detection limit of 0.19 fM. This method was successfully applied to the analysis of emerging tobacco mosaic virus (TMV) infections in tobacco leaf samples collected in the field due to its flexibility and convenience, indicating a potential application for the early detection of plant viral disease.