Detection of pathogenic biomarker from plant tissues using tearing and steeping methods (TSM).

Abstract

The present disclosure is directed to methods of releasing and detecting biomarkers, and more particularly methods of releasing biomarkers using a tearing and steeping methodology, followed by detection of the released biomarkers.

Description

DETECTION OF PATHOGENIC BIOMARKER FROM PLANT TISSUES USING TEARING AND STEEPING METHODS (TSM).CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims priority to U.S Provisional Application Serial No. 63 / 673,884 filed July 22, 2024, which is incorporated by reference in its entirety.BACKGROUND OF THE INVENTION1 . FIELD OF THE INVENTION

[0002] The present disclosure is directed to methods of releasing and detecting biomarkers, and more particularly methods of releasing biomarkers using a tearing and steeping methodology, followed by detection of the released biomarkers.2. BRIEF DESCRIPTION OF THE RELATED ART

[0003] Infection in plants is caused by multiple pathogens, like bacteria, viruses, fungi and nematodes. During the progression of infections these plants in response to the infection / diseases release some genetic materials (DNA / RNA), specific hormone molecules, proteins (enzymes, structural proteins and signal molecules) or metabolites (sugar, alkaloids, organic acids). These released particles from the pathogenic plants are considered as molecular biomarkers. The early detection and analysis of molecular biomarkers can help in early diagnosis of plant diseases, monitoring the disease progressions, understanding the host-pathogen interactions and developing multiple strategies for disease management and protection of crops. The detection of pathogenic biomarkers from the infected plants in a very early stage can also play a pivotal role in growth of plant and increase the crop-productions annually subsequently helping in preserving the resources and maintaining the economic stability of the country.

[0004] The molecular biomarkers can be released by multiple techniques depending on the type of samples and type of biomarkers. The common techniques used to date include mechanical shearing using homogenizer, chemical extractions using detergents or organic solvents, by the process of enzymatic degradation, applying high frequencysound, heat treatment and other pressure-based techniques to disrupt the cells from the infected tissues / parts. Although above mentioned state of art might have shown some results in detection of plant pathogens but are time consuming, expensive, depend on multiple variables and not sensitive. Accordingly, there is a need in the art for additional methods to efficiently and easily release detectable biomarkers.SUMMARY OF THE INVENTION

[0005] In one aspect, the present invention is directed to a method for preparing a sample, comprising providing one or more plant tissues; tearing the plant tissue to form torn plant tissue; and immersing the torn plant tissue in an aqueous solvent to extract compounds from said plant tissues to form said sample.

[0006] In another aspect, the present invention is directed to a method for analyzing a sample, comprising providing one or more plant tissues for analysis; tearing the plant tissue to form torn plant tissue; immersing the torn plant tissue in an aqueous solvent to extract compounds from said plant tissues and form said sample; and analyzing the sample.

[0007] These and other aspects will become evident upon reading the following description alon with the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 shows a sample processed using TSM technique from leaves with different PVY genomic concentration.

[0009] Figure 2 shows negative tuber spiked with Purified PVY RNA, and sample processed using TSM methods.

[0010] Figure 3 shows samples prepared by using current available protocol and by using TSM and running through market available lateral flow device and device with a Bionano sensor device (BNS).

[0011] Figure 4 shows a comparison between sample preparation using Mechanical- Shearing and TSM methodology.DETAILED DESCRIPTION OF THE INVENTION

[0012] To overcome the limitations of traditional methodologies, the present inventors have invented a tearing and steeping method (TSM). This invented technique simplified the process of releasing biomarkers in a solution with minimal disruption of plant tissues through semi-permeable membranes. This methodology does not depend on the multiple variables like different concentration of chemicals, reagents, solutions, extraction equipment, temperature / pressure gradients for releasing the molecular biomarkers from the given plan tissues or samples, thus increases the reliability of results obtained. The method emphasizes specific time interval for steeping the plant tissue in each solution as it has been optimized for releasing target molecules / bio markers within a precise timeframe, for the accuracy and sensitivity of the detection. The meticulous approach designed in the protocol increases the yield and quality of an extracted sample for experimental as well as downstream analysis. This novel method not only releases molecular biomarkers in a very efficient way but also detects the presence of genomic concentration in the given sample and opens the avenues for monitoring the plant pathogens. The advantages of this invented methodology are listed below: o Minimal and Rapid sample preparation process o No ambiguous protocol o No trained personnel required o Cost-effective o User-friendly o Clear sample extracts o Can be used in different temperature settings o Convenient for different settings from Laboratory to Field.

[0013] The success of any field-deployable diagnostic method depends not just on sensitivity and specificity, but also on its consistency across users and settings. In agricultural environments, where diagnostic tasks may be carried out by individuals with varying levels of technical expertise and under widely differing environmental conditions — such consistency becomes even more critical. The Tearing and Steeping Method (TSM) of the present invention offers an operator-independent, highly reproducible approach to sample preparation that can be universally applied to detect abroad range of plant pathogens. This feature not only enhances its diagnostic value but also supports its potential as a globally scalable platform.

[0014] At its core, the TSM involves two simple steps: (1 ) gently tearing plant tissue such as leaves, tubers, stems, or roots to slightly disrupt the cellular surface, and (2) immersing the sample in ultrapure water aqueous solvent for a specified time interval (typically 3-15 minutes). This process enables the release of soluble molecular biomarkers — including DNA, RNA, proteins, peptides, and metabolites — into solution, without the need for grinding, mechanical shearing, enzymatic digestion, or buffer-based chemical lysis. As used herein, the term “tearing” means the act of breaking apart a material, such as plant matter, by force but without the aid of a machine ora cutting tool. In one embodiment, tearing may be done by hand. In another embodiment, tearing may include crushing, for example with a blunt instrument.

[0015] Unlike conventional methods that rely heavily on user proficiency, the TSM protocol is straightforward, with almost no learning curve. There are no precise mechanical forces to apply, no risk of over- or under-homogenization, and no requirement for trained personnel. The tearing action does not require specialized tools or consistency in pressure, making it highly replicable across different users. Once the plant tissue is steeped, the resulting extract is automatically suitable for downstream applications. This simplicity and standardization are essential for large-scale deployment across farms, greenhouses, seed certification stations, and remote agricultural research facilities.

[0016] Experimental comparisons between users of different skill levels have shown that TSM performs uniformly across operators. In validation studies using Potato virus Y (PVY) as a test case, multiple team members with little or no lab training — were able to extract samples and achieve nearly identical diagnostic outputs when analyzed via RT-qPCR and a biosensor platform. The consistency in Cq values and biosensor signal intensities between users confirmed the protocol’s robustness. More importantly, similar consistency was observed across various plant matrices including leaf, tuber, and stem tissues.

[0017] This reproducibility is not limited to PVY or virus detection. TSM has been demonstrated to work generically across different pathogen classes. Whether the target is fungal chitinases, bacterial DNA, nematode proteins, or viral RNA, the passivediffusion-based release mechanism works the same. By merely steeping the torn plant tissue in ultrapure water, soluble biomarkers from diverse pathogens can be liberated into solution for subsequent detection. As such, TSM does not require customization for different disease agents, making it a truly universal method for front-end sample preparation.

[0018] Another important aspect of TSM is its environmental resilience. Unlike methods that rely on temperature-controlled reagents, enzymes, or vacuum filtration systems, TSM functions effectively over a wide temperature range — from 0°C to 45°C. This flexibility is vital for real-world agricultural scenarios, where laboratory-grade infrastructure is not always accessible. Whether implemented on a tropical plantation, in a desert greenhouse, or during cold-season testing in temperate climates, TSM has proven reliable.

[0019] In terms of workflow, the method integrates seamlessly with multiple downstream detection platforms. Because the extracts contain minimal debris and few inhibitors, they are immediately compatible with technologies such as RT-qPCR, for quantitative detection of nucleic acids, electrochemical and impedance biosensors, for direct real-time molecular detection; lateral flow assays, for simple field-use diagnostics, next-generation multiplexed platforms, for high-throughput pathogen screening, and the like.

[0020] This compatibility arises from TSM’s ability to avoid introducingvariability through chemical lysis buffers or aggressive physical processing. Since the only variable is the duration of steeping — and that is already standardized — samples from different users, locations, or times can be reliably compared and interpreted.

[0021] Furthermore, TSM supports multiplex pathogen detection. Given its ability to simultaneously release a spectrum of biomolecules (RNA, DNA, proteins, small metabolites), a single extract can be analyzed against multiple diagnostic targets. This is especially useful in complex infections or co-infections — such as viral-fungal complexes or viral-vector-pathogen triads — where multiple organisms must be detected to understand disease etiology.

[0022] From a regulatory and logistics standpoint, TSM also reduces the risk of contamination and sample loss. The method does not rely on complex consumables or lab tools, making it easier to train personnel and replicate across large-scale agriculturalprograms. Because the sample prep process is so low-tech, adoption is not limited to advanced diagnostic labs; rather, it can be rapidly deployed in developing regions, low- resource environments, and by extension workers or farmers directly in the field.

[0023] Ultimately, TSM’s operator independence is not just a convenience — it is a foundational feature that makes it viable for real-world use. Diagnostic technologies that require high skill or consistent lab environments are difficult to scale. TSM, by contrast, aligns with the principles of accessibility, scalability, and robustness, allowing any user — anywhere — to contribute to real-time plant disease surveillance.

[0024] In plant pathogen diagnostics, sensitivity is paramount — especially for early-stage infections, asymptomatic carriers, and low-titer pathogen loads. However, achieving such sensitivity is only possible when the upstream sample preparation delivers clean, inhibitor-free extracts. Traditional approaches such as mechanical grinding, enzymatic digestion, or buffer-based extraction often compromise this requirement by releasing large quantities of cellular debris, polysaccharides, phenolics, and other secondary metabolites that hinder analytical performance. The Tearing and Steeping Method (TSM) of the present invention, in contrast, offers a minimal-disruption alternative that significantly reduces debris, preserves biomarker integrity, and enhances sensitivity across a wide range of detection platforms and pathogen types.

[0025] The core principle of TSM is simple: plant tissues — be they leaves, tubers, stems, seeds, roots, or fruits — are gently torn or sliced and immersed in ultrapure water for a defined steeping period, typically ranging from 3 to 15 minutes. This allows for passive diffusion of soluble molecular biomarkers such as nucleic acids (RNA, DNA), proteins, peptides, and even metabolic byproducts. Importantly, this process avoids forceful mechanical disruption, meaning that tough structural components like lignin, cellulose, and large fibrous debris remain contained within the tissue matrix rather than being released into solution. This alone represents a major advantage in plant molecular diagnostics, where clarity of extract is often the bottleneck to reliable detection.

[0026] This low-debris nature of TSM was empirically validated using Potato virus Y (PVY) as a model pathogen. Samples prepared via TSM, even from high-starch-content tubers or phenolic-rich leaves, resulted in clear, particulate-free extracts. When tested using both RT-qPCR and a proprietary bionanosensor platform, these extracts yielded significantly higher signal strength compared to those prepared with conventionalmethods like mechanical shearing or buffer-based maceration. Notably, PVY detection was achieved at very low concentrations — equivalent to Cq values of 38 or higher — well beyond the limit at which lateral flow assays or even standard PCR methods typically fail.

[0027] What makes this finding particularly important is that PVY is merely a demonstration case. The advantage of TSM is generic and not pathogen-specific. Whether the target biomarker is viral RNA, bacterial DNA, fungal chitinase proteins, or nematode-secreted peptides, the method remains the same: tear, steep, extract. This agnostic applicability allows TSM to serve as a universal front-end to multiple downstream diagnostic platforms. For example, biosensors using electrochemical impedance spectroscopy (EIS), ELISA kits, lateral flow devices, or even CRISPR-based systems all benefit from reduced background interference and clearer analyte signals when starting from a TSM-prepared extract.

[0028] Another critical advantage is the elimination of complex filtration or centrifugation steps. These post-processing steps are often required in traditional sample prep workflows to remove unwanted solids or inhibitors. Not only do they add time, cost, and potential sample loss, but they also introduce another point of variability — especially in field environments where power sources, cold storage, and lab equipment may be unavailable. TSM sidesteps this entirely. Extracts are immediately usable in portable formats and require no further treatment. This simplicity is a major enabler for in-field diagnostics, especially when real-time disease management decisions are necessary.

[0029] Moreover, the gentle nature of the TSM approach preserves the structural and functional integrity of sensitive biomolecules. RNA, for instance, is notoriously prone to degradation under mechanical stress or in the presence of RNases released during homogenization. By reducing tissue trauma and eliminating complex chemical buffers, TSM minimizes the risk of enzymatic degradation. Additionally, the steeping medium — ultrapure water — acts as a natural stabilizer, and short steeping durations further limit hydrolysis or oxidative breakdown. This is especially beneficial for pathogens that produce low-copy-number RNA or where early-stage detection is mission-critical.

[0030] TSM’s clean extracts also demonstrate improved stability during storage and transportation. Because the sample contains fewer contaminants and does not rely on volatile solvents or labile reagents, it can be more easily freeze-dried, filtered through point-of-care cartridges, or directly loaded into portable diagnostic readers. Fordecentralized labs or mobile units conducting wide-area pathogen surveillance, this dramatically increases operational efficiency and reliability.

[0031] In comparative trials, false-negative rates for lateral flow assays and even RT- qPCR were significantly reduced when using TSM-prepared samples. This was particularly evident in tuber samples, where conventional extraction methods often fail due to matrix complexity. In some cases, lateral flow assays failed entirely to detect PVY in samples where Cq values were still within the detectable range — simply because the sample matrix inhibited antibody binding or blocked colorimetric flow. TSM eliminated these artifacts, providing consistently reliable results across multiple platforms.

[0032] Lastly, TSM enhances biosensor performance in measurable ways. Biosensors, especially those based on carbon nanotubes or graphene, require minimal surface contamination for accurate signal transduction. Debris or residual plant compounds can physically block probe sites or introduce electrochemical noise. In contrast, TSM samples exhibit minimal fouling and allow for robust probe-target interaction. The result is a sharper signal response, lower background, and greater confidence in both qualitative and quantitative assays.

[0033] In summary, the Tearing and Steeping Method of the invention represents a foundational improvement in plant pathogen diagnostics. By producing clean, debris- free extracts, it unlocks the full potential of modern molecular detection technologies and biosensors. Its ability to enhance sensitivity, reduce false negatives, and ensure compatibility with a wide array of pathogens makes it a universal, future-ready solution for agricultural diagnostics. Whether deployed in a high-throughput lab or a remote crop field, TSM delivers the kind of performance that enables early detection, targeted intervention, and ultimately, better food security outcomes worldwide. The Tearing and Steeping Method of the present invention is a advantageously applicable and useragnostic approach to sample preparation for plant pathogen diagnostics. Its reproducibility, simplicity, and compatibility across detection platforms and pathogen types make it a critical innovation for improving global plant disease monitoring, early intervention, and food security.EXAMPLES

[0034] 1 . Sample Preparation:

[0035] A) Tearing and Steeping Method (TSM) a). Leaf sample preparation• Tear leaf in few folds then insert into a tube provided• Add 10-40ml of an aqueous solvent into the tube. The aqueous solvent can be plain water, or a solution of water with buffers, salts, or other ingredients if desired. In one preferred embodiment, ultra-pure water is used.• Steep the leaf in tube with Ultrapure water for given time interval. Any interval may be used, for example 1-60 minutes. In one embodiment, a 3-15 minutes interval is used.• Extract 2-5ml of sample from the tube.• Use the sample for down stream analysis. b). Tuber sample preparationFortuber sample, the flesh as well as peel of the tuber (e.g., potato) can be utilized.• Sample preparation from peel of tuber• Peel the tuber sample.• Insert the tuber peel in a tube• Add 10-40 ml of ultra-pure water provided in tube.• Steep a peel in tube with ultra-pure water for given time interval (3-15 minutes)• Extract 2-5 ml of sample from the tube.• Use samples for further analysis.• Sample preparation from slice of tuber• Cut the tuber thin slice• Insert tuber slice in a tube• Add 10-40 ml of ultra-pure water provided in tube.• Steep a peel in tube with ultra-pure water for given time interval (3-15 minutes)• Extract 2-5 ml of sample from the tube• Use the sample for further analysis. c) Other Plant tissue sample preparation:• Cut a thin slice of the plant tissue from the plant (stem, root, fruit etc.)• Insert the tissue slice in a tube• Add 10-40 ml of ultra-pure water provided in tube.• Steep a peel in tube with ultra-pure water for given time interval (3-15 minutes)• Extract 2-5 ml of sample from the tube• Use the sample for further analysis.

[0036] B) Sample preparation by Crushing methodsLeaf Sample• Weigh whole leaf (symptomatic / asymptomatic)• Take extraction bag with mesh lining (Agdia company)• Use blunt edge object to crush the sample• Extract the sample without debris using a dropper provided• Use the sample for further analysis.Tuber Sample• Take a tuber / seeds (symptomatic / asymptomatic)• Weigh the thin slice of tuber around 1 gm• Take extraction bag with mesh lining (Agdia company)• Use blunt edge object to crush the sample• Extract the sample without debris using a dropper provided• Use the sample for further analysis.

[0037] C). Sample preparation using Crushing and filtration.Leaf Sample• Weigh whole leaf (symptomatic / asymptomatic)• Take extraction bag with mesh lining (Agdia company)• Use blunt edge object to crush the sample• Use the 0.45um filter syringe and extract the sample• Use the sample for further analysis.Tuber Sample• Take a tuber / seeds (symptomatic / asymptomatic)• Weigh the thin slice of tuber around 1 gm• Take extraction bag with mesh lining (Agdia company)• Use blunt edge object to crush the sample• Use the 0.45um filter syringe and extract the sample• Use the sample for further analysis.

[0038] 2. Result and Data:

[0039] As an example of the TSM technique, samples were prepared using the TSM technique to detect Potato Virus Y (PVY virus) from infected plant parts. The plant parts used for the sample preparation were leaves and tubers. Similarly, samples were prepared using currently available protocols to detect PVY from infected tubers and leaves. The prepared samples using current protocols and TSM technique were run through Real time qPCR (RT-qPCR), lateral flow devices and in a patented Bio-Nano Sensor (BNS) device described in U.S. Patent No. 9,919,922. The data obtained were compared and analyzed further to verify the sensitivity and specificity of the TSM technique. TSM is the better solution for detecting the lowest limit of detection in both BNS and RT-qPCR, than other mechanical shearing methods currently available.

[0040] As shown in Figures 1 and 2, the samples prepared using TSM method were run through the RT-qPCR and in a patented Bio-nano sensor (BNS) device described in U.S. Patent No. 9,919,922. The data obtained shows that with lower viral genomic concentration, lower the signal strength obtained from BNS. This signifies the sample prepared using TSM technique is highly significant in detecting the lower genomic concentration. This data also signifies the sample prepared by TSM is sensitive to both RT-qPCR and BNS technologies.

[0041] As shown in Figure 3, samples prepared using current available protocol and using TSM method were run through a lateral flow device to detect molecular biomarkers present in positive PVY samples. Similarly, the prepared samples were also run through a device containing bio-nano-sensor (BNS). Sample preparation usingTSM method gives results similar to data from current available protocols. The experiment shown above in figure 3 explains that the later flow devices are not sensitive enough to detect infectionfrom plants with low genomic concentration. Even in presence of higher genomic concentration in infected plants, the lateral flow device couldn’t detect the biomarkers from samples prepared by TSM and simultaneously the signal strength from the sample prepared by current available protocols was weak compared to obtained Cq values.

[0042] To verify the experiment, a comparison study was conducted between samples preparation using mechanical shearing and TSM methodology. In figure 4 shows the comparison between the sample preparation using mechanical shearing methods and TSM methodology. From the data obtained, it is concluded that sample preparation using TSM methodology is highly efficient for downstream analysis as well as for other molecular technologies. The sample prepared by using mechanical shearing shows lower amplification and lower signal strengths. This low amplification may be because of the buffers / chemicals used in the extraction process, thus with clear ultra-pure water and no other variables, TSM possess detection of biomarkers more efficiently than other sample preparation techniques. This indicates TSM methodology is highly sensitive and specific for further process.References:• SINHA, SAION KUMAR. Bionanosensor detection device. US-9919922-B2, filed October 01 ,2009 and issued March 20,2018.• da Silva, W., Kutnjak, D., Xu, Y., Xu, Y., Giovannoni, J., Elena, S. F., & Gray, S. (2020). Transmission modes affect the population structure of potato virus Y in potato. PLoS pathogens, 76(6), e1008608.• Agdia Inc. is the leading provider of test kits for plant pathogens and transgenic (GMO) traits, (n.d.). https: / / www.agdia.com /

Claims

CLAIMSWHAT IS CLAIMED IS:1 . A method for preparing a sample, comprising: providing one or more plant tissues; tearing the plant tissue to form torn plant tissue; immersing the torn plant tissue in an aqueous solvent to extract compounds from said plant tissues to form said sample.

2. The method of claim 1 , wherein said one or more plant tissues comprise plant leaves, tubers, stems, roots, seeds, flowers, fruit, or combinations thereof.

3. The method of claim 1 , wherein said tearing step further comprises crushing.

4. The method of claim 1 , wherein said aqueous solvent is ultrapure water.

5. The method of claim 1 , wherein the immersing step takes place from 1 to 60 minutes.

6. The method of claim 5, wherein the immersing step takes place from 3 to 15 minutes.

7. A method for analyzing a sample, comprising: providing one or more plant tissues for analysis;tearing the plant tissue to form torn plant tissue; immersing the torn plant tissue in an aqueous solvent to extract compounds from said plant tissues and form said sample; and analyzing the sample.

8. The method of claim 7, wherein said one or more plant tissues comprise plant leaves, tubers, stems, roots, seeds, fruit, or combinations thereof.

9. The method of claim 7, wherein said tearing step comprises crushing.

10. The method of claim 7, wherein said aqueous solvent is ultrapure water.

11. The method of claim 7, wherein the immersing step takes place from 1 to 60 minutes.

12. The method of claim 11 , wherein the immersing step takes place from 3 to 15 minutes.

13. The method of claim 7, wherein said analyzing step analyzes the sample for biomarkers.

14. The method of claim 13, wherein said biomarkers comprise DNA, RNA, proteins, peptides, enzymes, metabolites, and combinations thereof.

15. The method of claim 7, wherein said analyzing step analyzes the sample for pathogens.

16. The method of claim 15, wherein said pathogens comprise bacteria, viruses, fungi, nematodes, and combinations thereof.

17. The method of claim 7, wherein said analyzing step is selected from RT-qPCR, electrochemical and impedance biosensors, lateral flow assays, high-throughput pathogen screening, and combinations thereof.

Images