Rapid detection card for oral health states of dogs and cats as well as preparation process and application of rapid detection card

Abstract

The invention discloses a dog and cat oral health state rapid detection card and a preparation technology and application thereof, the dog and cat oral health state rapid detection card comprises a proper handle and a detection area, the detection area is fixed at one end of the handle, and the detection area is loaded with a first detection reagent and a second detection reagent; the first detection reagent and the second detection reagent respectively comprise DTNB serving as a color developing agent and TNBS serving as a color developing agent, the first detection reagent and the second detection reagent are arranged on the detection area in a partitioned mode of a first area and a second area, and the first area and the second area are arranged at intervals in a parallel strip-shaped mode. The method has the beneficial effects that markers in two anaerobic bacteria metabolites of mercaptan and amino acid are simultaneously detected through DTNB and TNBS, the limitation of single index detection is overcome, the sensitivity is high, more accurate and earlier anaerobic bacteria concentration judgment is realized, signals can be captured at the initial stage of anaerobic bacteria metabolic activity, the early warning effect is achieved, and the method is suitable for popularization and application. The detection operation is simple, convenient and rapid, and professional equipment or a complex sampling process is not needed.

Description

Technical Field

[0001] This invention relates to the field of pet health testing technology, and more specifically, to a rapid oral health status testing card for dogs and cats, its preparation process, and its application. Background Technology

[0002] Currently, the main methods for assessing the oral health of dogs and cats include:

[0003] Visual examination: It is simple but highly subjective and can easily miss early lesions.

[0004] Traditional microbial culture methods require 3-7 days of strict anaerobic culture, are cumbersome to operate, time-consuming, and have limited sensitivity.

[0005] PCR / gene chip technology: high detection accuracy, but requires expensive instruments and professional operators, and is not suitable for home or on-site rapid screening.

[0006] Post-anesthesia instrument examination: This requires anesthetizing the animal, which carries certain risks, and the process is complex and costly.

[0007] Some existing patents, such as Chinese invention patent application number 201110008841.3, mainly determine the concentration of anaerobic bacteria by detecting the concentration of a single thiol. This method has the following limitations: the detection results are not precise enough, it cannot detect other combinations of markers besides specific thiols; and it requires waiting for the anaerobic bacteria to multiply to a certain scale and for metabolites to accumulate to a high concentration before effective detection is possible, resulting in a relatively delayed detection window. Furthermore, the reaction is a basic liquid-phase reaction, requiring no special equipment for convenient use.

[0008] Furthermore, according to the AAHA Dental Care Guidelines for Dogs and Cats (2019), oral diseases are among the most common health problems in dogs and cats, significantly impacting their quality of life and overall health. The guidelines emphasize the link between oral health and systemic diseases, such as the close relationship between periodontal disease and heart disease, kidney disease, and diabetes. However, a tool that can quickly, non-invasively, and accurately assess oral health in a clinical or home setting is currently lacking.

[0009] Other studies (Penlington et al., Folia Veterinaria, 2019) have shown a significant correlation between canine periodontal disease and various systemic diseases (such as cardiovascular disease and liver disease), further highlighting the importance of early oral health screening. However, current detection methods struggle to achieve rapid, painless, and non-invasive screening.

[0010] Another Chinese patent (CN217212566U) describes a device for detecting bad breath in pet dogs and cats, which uses an extraction mechanism (syringe, trachea, and suction bulb) to collect and analyze oral gases. However, this requires a complex sampling mechanism and is inconvenient to operate.

[0011] Furthermore, US Patent 8,815,152 B2 discloses a test strip for detecting thiol compounds, which assesses oral health by detecting the concentration of thiols in oral fluid. While this technology offers the possibility of rapid detection, it still has the following shortcomings:

[0012] Relying solely on a single thiol indicator is not sensitive enough; it requires waiting for anaerobic bacteria to multiply to a certain scale and for metabolites to accumulate to a high concentration before it can be effectively detected, resulting in a relatively delayed detection window.

[0013] It has limited sensitivity and specificity for early lesions;

[0014] It has not been optimized for the oral physiology of dogs and cats, thus its applicability is limited.

[0015] Therefore, there is a need to develop a detection tool that can quickly and sensitively detect anaerobic bacteria without waiting for them to multiply to a certain scale or for metabolites to accumulate to a high concentration, thus enabling early detection within the window period. At the same time, it should be easy to operate, require no special equipment, and allow for early diagnosis of oral health in dogs and cats without anesthesia. Summary of the Invention

[0016] To address the shortcomings of existing technologies, the present invention aims to provide a rapid detection card for canine and feline oral health status, its preparation process, and its application.

[0017] To achieve the above objectives, the present invention provides the following technical solution:

[0018] A rapid oral health status test card for dogs and cats includes a handle and a test area. The handle is a rectangular sheet structure that is easy to insert into the mouth of dogs and cats. The test area is fixed to one end of the handle and is loaded with a first test reagent and a second test reagent.

[0019] The first detection reagent contains the colorimetric reagent DTNB (5,5′-dithiobis(2-nitrobenzoic acid)), which reacts with thiol compounds produced by the metabolism of anaerobic bacteria in the oral cavity of dogs and cats to produce a color change;

[0020] The second detection reagent contains the colorimetric reagent TNBS (2,4,6-trinitrobenzenesulfonic acid), which reacts with primary amine compounds produced by the metabolism of anaerobic bacteria in the oral cavity of dogs and cats to produce a color change;

[0021] The first detection reagent and the second detection reagent are arranged in a partitioned manner on the detection area as a first region and a second region, and the first region and the second region are arranged alternately in a parallel strip shape;

[0022] The concentration of DTNB in ​​the first test reagent is 2.52–5.05 mmol / L, and the concentration of TNBS in the second test reagent is 0.171–0.513 mmol / L.

[0023] The present invention is further configured such that the preferred concentration of DTNB in ​​the first detection reagent is 3.03 mmol / L, and the preferred concentration in the second detection reagent is 0.342 mmol / L.

[0024] The present invention is further configured such that the first detection reagent further includes a base liquid one for maintaining the stability, solubility and reactivity of DTNB, and the second detection reagent further includes a base liquid two for maintaining the stability, solubility and reactivity of TNBS; the pH value of both the first detection reagent and the second detection reagent is 7.5–8.5.

[0025] The present invention is further configured such that the base solution is based on a phosphate buffer solution with a pH of 8.0 and is compounded with ≤5% ethanol and 5% glycerol;

[0026] The second base solution is preferably a phosphate buffer solution with a pH of 8.0, and the second base solution contains no ethanol or has an ethanol content of ≤5%.

[0027] The invention is further configured such that the detection area is a rectangular pad, the four corners of the pad and the four corners of the handle are rounded, the pad is made of a composite porous membrane of polyethylene and polypropylene, and the handle is made of polyvinyl chloride so that the handle has sufficient rigidity to be inserted into the mouth of dogs and cats.

[0028] The present invention is further configured such that the length of the detection area is 7.1±0.05mm, the width is 6.4±0.1mm, and the thickness is 0.25±0.05mm; the length of the handle is 70±0.1mm, the width is 6.4±0.1mm, and the thickness is 1.0±0.01mm.

[0029] A manufacturing process for a rapid oral health status detection card specifically for dogs and cats includes the following steps:

[0030] S1, Loading of the first detection reagent (DTNB):

[0031] S11. Cover the detection area pad with a perforated mask that matches the shape of the first area, so that the perforated window of the mask is precisely aligned with the first area to be sprayed.

[0032] S12. Load the first test reagent into the atomizing precision spray gun, and spray it evenly on the cutout window of the mask under the conditions of air pressure 0.2-0.4MPa, spraying distance 5-10cm, and moving speed 10-20mm / s, so that the first test reagent is deposited only on the first area exposed by the cutout window on the mask.

[0033] S13. After completing the spraying of all the first areas on the inspection area, carefully remove the mask and transfer the pad used as the inspection area to the drying oven. Dry it in the dark at 25-30℃ for 10-15 minutes, and then cool it after drying.

[0034] S2, Second Detection Reagent (TNBS) Loading:

[0035] S21 uses another mask to cover the pad after it has been processed and cooled in step S13. The mask includes a cutout window that is consistent with the shape of the second region, so that this window of the mask corresponds to the second region of the detection area.

[0036] S22. After replacing the precision spray gun or thoroughly cleaning the original precision spray gun and its internal tubing, load the second test reagent and spray the second area on the test area under the conditions of air pressure 0.2-0.4MPa, spraying distance 5-10cm, and moving speed 10-20mm / s.

[0037] S23. After spraying all the second areas on the detection area in sequence, remove the mask. Transfer the pad loaded with the first and second test reagents to a drying oven and dry it at 25°C in the dark for 10-15 minutes to thoroughly dry the detection area, thus completing the preparation of the detection area.

[0038] S3. Attach the detection area to one end of the handle to complete the preparation of the detection card.

[0039] The use of a rapid oral health status test card for dogs and cats in the preparation of a home self-testing tool for non-diagnostic purposes.

[0040] The use of a rapid oral health status test card for dogs and cats in the preparation of a test tool to assist veterinarians in assessing oral health risks in dogs and cats.

[0041] By adopting the above technical solution, the beneficial effects of the present invention are as follows:

[0042] 1. By simultaneously detecting biomarkers in anaerobic metabolites of thiols and ammonia using DTNB and TNBS, the limitations of single-index detection are overcome. This method is highly sensitive and can produce obvious color changes in the early stages of anaerobic metabolic activity, thus advancing the detection window and enabling more accurate and earlier determination of anaerobic bacterial concentration. It can capture signals in the early stages of anaerobic metabolic activity, achieving an early warning effect.

[0043] 2. The test is simple and quick, requiring no professional equipment or complicated sampling procedures. It can be easily completed by pet owners at home. From the moment the test area touches the oral cavity to the completion of colorimetric interpretation, the total time is approximately 40-60 seconds.

[0044] 3. The test card has a reasonable structural design. The handle length and test area size are optimized for the oral anatomy of dogs and cats. The corners are rounded to ensure safe and comfortable use. No anesthesia is required, making it safe and non-invasive.

[0045] 4. By comparing with the standard colorimetric card, a semi-quantitative assessment of the concentration of oral anaerobic bacteria can be achieved. The results are clear and intuitive, easy for non-professionals to interpret, and the results are intuitive and reliable.

[0046] 5. Clinically validated, the test results of this test card are highly correlated with the actual periodontal health status of dogs and cats. Its semi-quantitative grading results can effectively assist veterinarians in conducting rapid risk assessment and graded management, providing an intuitive and reliable basis for early intervention, and are highly practical. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the structure of an embodiment of the present invention.

[0048] Figure 2 This is a top view of the test card.

[0049] Figure 3 This is a schematic diagram of the structure of the first and second regions in the detection area.

[0050] Figure 4 This is a schematic diagram of the structure of a standard colorimetric card.

[0051] In the diagram: handle 1, detection area 2, first area 21, second area 22. Detailed Implementation

[0052] The following describes specific embodiments and references. Figures 1 to 4 The present invention will be described in further detail below, but the embodiments of the invention are not limited thereto. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0053] Example 1: A rapid oral health status detection card for dogs and cats, comprising a handle 1 and a detection area 2, the structure of which is as follows: Figure 1 and Figure 2As shown, handle 1 is a rectangular sheet structure that is easy to insert into the mouth of dogs and cats. Detection area 2 is a rectangular pad that is fixed to one end of handle 1. The four corners of the pad and the four corners of handle 1 are rounded to ensure safety when inserted into the mouth of dogs and cats, avoid scratching the inner wall of the mouth, and increase safety during use. The length of detection area 2 is 7.1±0.05mm, the width is 6.4±0.1mm, and the thickness is 0.25±0.05mm. This thickness range ensures sufficient saliva absorption while ensuring rapid and clear color development. The length of handle 1 is 70±0.1mm, the width is 6.4±0.1mm, and the thickness is 1.0±0.01mm. The pad is made of a composite porous membrane of polyethylene and polypropylene, and handle 1 is made of polyvinyl chloride, giving handle 1 sufficient rigidity to facilitate insertion into the mouth of dogs and cats, and ensuring that dogs and cats accept it calmly and use it conveniently.

[0054] The detection area 2 is loaded with a first detection reagent and a second detection reagent. The first and second detection reagents are arranged in a partitioned manner in the detection area 2, namely a first region 21 and a second region 22, and the first region 21 and the second region 22 are arranged alternately in a parallel strip pattern (e.g., Figure 3 As shown in the figure, the color development results of the first region 21 and the second region 22 are displayed independently and used together to interpret the total concentration of anaerobic bacterial metabolites.

[0055] The first test reagent contains the colorimetric reagent DTNB (5,5′-dithiobis(2-nitrobenzoic acid)), which reacts with thiol compounds in the metabolites of anaerobic bacteria in the oral cavity of dogs and cats to produce a color change, generating yellow DTNB. - Anions, with varying color intensities, reflect thiol concentration levels, helping to determine the concentration of anaerobic bacteria in the oral environment of dogs and cats.

[0056] The second detection reagent contains the chromogenic agent TNBS (2,4,6-trinitrobenzenesulfonic acid), which reacts specifically with primary amine compounds produced by the metabolism of anaerobic bacteria in the oral cavity of dogs and cats to produce a color change, forming a stable yellow trinitrophenylamine derivative. TNBS has better solubility in a weakly alkaline aqueous phase, avoiding or significantly reducing the use of organic solvents. The resulting chromogenic product is stable and develops rapidly. Both thiols and amino acid metabolites are important chemical markers of anaerobic bacterial activity in the oral cavity of dogs and cats; the combined detection of both can improve the sensitivity and specificity of identifying oral abnormalities.

[0057] In this invention, the color displayed in detection area 2 is consistent with that of a standard colorimetric card (e.g., Figure 4The standard colorimetric chart (as shown) is used for comparison. It includes multiple color gradient levels (such as light yellow, medium yellow, dark yellow, and yellowish-brown), each corresponding to different oral anaerobic bacteria concentration risks. For example, 'negative', 'positive+', 'positive++', and 'positive+++' represent low to high concentrations of anaerobic bacteria, respectively. The concentration and health status of oral anaerobic bacteria are determined based on the color intensity.

[0058] Standard colorimetric card grading table:

[0059]

[0060] The concentration of DTNB in ​​the first test reagent is 2.52–5.05 mmol / L, and the concentration of TNBS in the second test reagent is 0.171–0.513 mmol / L. In this embodiment, preferably, the concentration of DTNB in ​​the first test reagent is 3.03 mmol / L, and the concentration of TNBS in the second test reagent is 0.342 mmol / L.

[0061] The first detection reagent also includes a base solution one for maintaining the stability of DTNB; the second detection reagent also includes a base solution two for maintaining the solubility and reactivity of TNBS. To ensure safety for animal use and stability of the colorimetric system, this invention uses a weakly alkaline aqueous phase system as the base buffer system for both types of colorimetric reagents. The pH values ​​of both the first and second detection reagents are controlled between 7.5 and 8.5, preferably pH=8.0.

[0062] Base solution one is a composite buffer stabilization system. Based on phosphate-buffered saline (PBS) at pH 8.0, it is a weakly alkaline system formed by mixing 0.05M Na₂HPO₄ and 0.05M KH₂PO₄ in a 3:2 volume ratio. Base solution one may further contain ≤5% (v / v) ethanol to improve spraying and film formation uniformity, and approximately 5% (v / v) glycerol to enhance film flexibility and maintain humidity, preventing DTNB from drying and deactivating in the cured film. The combination of PBS and glycerol in base solution one can inhibit the hydrolysis of disulfide bonds in DTNB under weakly alkaline conditions, thereby improving the long-term stability of DTNB under solid-phase loading.

[0063] Base solution two is used to dissolve TNBS and maintain its colorimetric reactivity. It is preferably prepared using phosphate-buffered saline (PBS) at pH 8.0 or other equivalent weakly alkaline aqueous buffer systems. To improve animal safety and maintain TNBS solubility, base solution two contains little to no ethanol (≤5%, e.g., 3%). When base solution two contains ethanol, this very small amount is used to improve TNBS dispersion and film formation, and is not used as the primary solvent. Within the pH range of 7.5–8.5, especially close to pH 8.0, TNBS can rapidly undergo nucleophilic substitution reactions with primary amine compounds in oral samples, resulting in stable and interpretable yellow derivatives. This weakly alkaline system balances TNBS solubility, reactivity, and long-term stability, and significantly reduces the irritation risk that high concentrations of organic solvents may pose.

[0064] In this embodiment, the pH value of the first detection reagent is 7.5, and the pH value of the second detection reagent is 8.0. Base solution one is based on a phosphate buffer solution of 0.05M Na₂HPO₄:0.05M KH₂PO₄ = 3:2 (v / v), and contains ≤5% ethanol and 5% glycerol; base solution two is based on a phosphate buffer solution with pH 8.0, and ≤5% ethanol may be added as needed for film formation, but high-concentration ethanol or other highly volatile organic solvents are not used.

[0065] Preparation of the first test reagent:

[0066] Preparation of phosphate buffer (PBS, pH 8.0 standard system): Prepare 100 mL of 0.05 M Na₂HPO₄ solution and 100 mL of 0.05 M KH₂PO₄ solution. Take 60 mL of the Na₂HPO₄ solution and mix it with 40 mL of KH₂PO₄ solution to obtain 100 mL of phosphate buffer with a pH of approximately 8.0, which will be used as the dissolution medium for DTNB.

[0067] Dissolve DTNB (preparation of solution A): Weigh 0.4g of DTNB and add it to 100mL of PBS. Gently mix at room temperature until completely dissolved (do not heat). The solution is pale yellow; this is called solution A. Solution A should be stored away from light at 4°C.

[0068] Prepare base solution one (weakly alkaline PBS stabilized system): Take PBS (pH 8.0, 0.05M Na2HPO4: 0.05M KH2PO4 = 3:2) as the basic buffer system, add: ≤5% (v / v) ethanol (to improve film uniformity) and 5% (v / v) glycerol (to maintain humidity and enhance film flexibility), mix well and you will get base solution one.

[0069] Preparation of the first detection reagent (DTNB working solution): Take 15 mL of solution A, add the base solution and dilute to 50 mL, mix gently to obtain the first detection reagent with a final concentration of 3.03 mmol / L. The solution is colorless and transparent. The DTNB working solution should be stored away from light at 4°C.

[0070] Preparation of the second test reagent:

[0071] To prepare a weakly alkaline aqueous solution of TNBS directly: Weigh 0.1g of TNBS and add it to an appropriate amount of 0.05M PBS with pH=8.0. Stir gently in the dark until completely dissolved.

[0072] Dilute and prepare the second detection reagent: Adjust the volume of the above TNBS solution to 50 mL, with a final concentration of 0.342 mmol / L. If improved film formation is required, ≤5% (v / v) ethanol can be added, but ethanol should not be used as the main solvent. After preparation, mix thoroughly and store at 4°C protected from light.

[0073] The colorimetric reaction mechanism of the first and second detection reagents:

[0074] DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) is a colorimetric reagent with high specificity for thiol groups. Under weakly alkaline conditions (pH 7.5–8.5), DTNB reacts with thiol compounds (–SH, such as methanethiol, hydrogen sulfide-derived thiol compounds, etc.) produced by anaerobic bacteria in the oral environment of dogs and cats through a thiol-disulfide bond exchange reaction, producing yellow DTNB. - The anion-based product exhibits a maximum absorption peak at 412 nm, and its color intensity is positively correlated with the concentration of thiol metabolites. Therefore, DTNB can be used for qualitative / semi-quantitative indication of the levels of thiol anaerobic bacterial metabolites in the oral cavity.

[0075] TNBS (2,4,6-trinitrobenzenesulfonic acid) primarily reacts with primary amine groups (-NH2) in the sample, including the terminal α-amino groups of primary amine compounds or peptides released during anaerobic bacterial metabolism, as well as the ε-amino groups of lysine residues. TNBS forms stable dinitrophenyl (DNP) derivatives via nucleophilic substitution, a reaction that also produces a distinct yellow color, with the degree of color change correlated with the concentration of the amino marker. Slightly alkaline conditions (pH 7.5–8.5) promote the reaction kinetics of TNBS and enhance the colorimetric efficiency.

[0076] The reason for combining DTNB and TNBS on the same detection card is that they recognize different substrate types: DTNB is specific to thiol groups, while TNBS is specific to primary amine compounds. They correspond to different key chemical categories in anaerobic bacterial metabolites, exhibiting independent colorimetric mechanisms and complementary reaction targets. Since there is no chemical reaction competition or conflicting colorimetric principles within the colorimetric system, they can be used together in a physically spaced partitioned manner within the same detection zone 2, thereby achieving multi-dimensional indication of the anaerobic bacterial metabolic environment.

[0077] Although the substrate specificity of the two chromogenic reagents does not interfere with each other, mixing the first and second detection reagents in the same reaction region may still have adverse effects, including:

[0078] Potential cross-reaction: Some metabolites or peptides in the oral cavity may contain both thiol and amino groups. If the two reagents coexist in the same reaction liquid phase, double derivatization or color superposition may occur, reducing the specificity of interpretation.

[0079] The trade-off caused by the difference in optimal pH: Although the optimal reaction conditions for DTNB and TNBS are both weakly alkaline, their specific optimal pH values ​​differ slightly; it is difficult for a mixed system to simultaneously meet the optimal reaction environment for both, which may affect the colorimetric reaction rate and endpoint stability.

[0080] Difficulty in identifying colorimetric products: Both types of colorimetric reactions produce yellow products. If there is no spatial partitioning, it is difficult to distinguish the source of color development, which is not conducive to determining whether the increase is due to thiol markers or amino markers, thus reducing the specificity and interpretability of the detection.

[0081] For the reasons mentioned above, the present invention loads DTNB and TNBS onto discrete first regions 21 and second regions 22 within the detection area 2, respectively, in a spatial partitioning manner so that the two colorimetric reactions can proceed independently in a separated environment, thereby ensuring the accuracy, specificity and identifiability of the colorimetric reaction.

[0082] A manufacturing process for a rapid oral health status detection card specifically for dogs and cats includes the following steps:

[0083] S1, Loading of the first detection reagent (DTNB):

[0084] S11. Cover the detection area 2 pad with a perforated mask that matches the shape of the first area 21, so that the perforated window of the mask is precisely aligned with the first area 21 to be sprayed.

[0085] S12. The first test reagent is loaded into the atomizing precision spray gun and uniformly sprayed onto the hollow window of the mask under the conditions of air pressure 0.3 MPa, spraying distance 8 cm and spray gun moving speed 15 mm / s, so that the first test reagent is deposited only on the first area 21 exposed by the hollow window on the mask.

[0086] S13. After completing the spraying of all the first areas 21 on the test area 2 in sequence, carefully remove the mask and transfer the pad used as the test area 2 to the drying oven. Dry it in the dark at 25-30℃ for 10-15 minutes, and then cool it after drying.

[0087] S2, Second Detection Reagent (TNBS) Loading:

[0088] S21 uses another mask to cover the pad after it has been processed and cooled in step S13. The mask includes a cutout window that is consistent with the shape of the second region 22, so that this window of the mask corresponds to the second region 22 of the detection area 2.

[0089] S22. After replacing the precision spray gun or thoroughly cleaning the original precision spray gun and its internal pipeline, load the second test reagent and spray the second area 22 on the test area 2 under the conditions of air pressure 0.3 MPa, spraying distance 8 cm and spray gun moving speed 15 mm / s.

[0090] S23. After spraying all the second regions 22 on the detection area 2 in sequence, remove the mask. Transfer the pad loaded with the first and second test reagents to the drying oven and dry it at 25°C in the dark for 10-15 minutes to thoroughly dry the detection area 2, thereby completing the preparation of the detection area 2.

[0091] S3. Attach the medical-grade adhesive from the detection area 2 to one end of the handle 1 to complete the preparation of the test card.

[0092] In this invention, before preparing the test card, the air conditioner is turned on to ensure that the preparation environment is below 25°C and kept dry and cool. Two layers of thick paper towels are laid on the clean preparation table to absorb any reagents that may splash out, keeping the table clean and dry.

[0093] Performance verification showed that the detection card prepared in this embodiment has a detection sensitivity of 0.1 mmol / L for thiols. At this concentration, a distinct light yellow color development (corresponding to the '+' level on the colorimetric card) can be observed, indicating that it can provide early warning in the early stage of anaerobic bacterial metabolic activity.

[0094] The method of using the rapid detection card of this invention includes the following steps:

[0095] S1 Insert the test area 2 of the test card into the dog's or cat's mouth, so that it comes into contact with the gum margin or oral mucosa to collect saliva and gingival crevicular fluid. Before inserting the test area 2 into the dog's or cat's mouth, ensure that the dog or cat has not eaten within 0.5-2 hours before the test.

[0096] S2 Take out the test card and wait 5-30 seconds for the test area 2 to fully develop color. The color of the test area 2 of the test card of this invention is stable within 5 minutes after development, and the color gradation does not fade or spread significantly. This provides the operator with sufficient time for colorimetric interpretation and ensures the accuracy and repeatability of the test results.

[0097] S3 compares the color displayed in detection area 2 with the standard colorimetric card (e.g., ...). Figure 4 By comparing the colors shown, the concentration of anaerobic bacteria in the oral cavity can be determined based on the color intensity, thereby assessing the oral health status of dogs and cats.

[0098] When using the rapid test card of the present invention, it can be directly inserted into the pet's mouth for testing. The test operation is simple and fast, requiring no professional equipment or complicated sampling procedures. It can be easily completed by pet owners at home. From the moment the test area 2 contacts the mouth to the completion of colorimetric interpretation, the total time is approximately 40-60 seconds. It provides a test card that does not require sampling, can be used directly in the pet's mouth, and displays test results quickly.

[0099] When using the detection card of this invention, hold handle 1 firmly to prevent pets from biting it. If the pet strongly resists, stop using the card. The detection card of this invention is a disposable item and should be properly disposed of after use, keeping it away from pets or children.

[0100] Comparative Example 1: The difference from Example 1 is that the detection area 2 of this test card is loaded only with DTNB chromogenic agent from the first test reagent, with a concentration of 3.03 mmol / L. Detection area 2 does not contain TNBS chromogenic agent. This comparative example is used to verify the indicative ability of the single thiol chromogenic system for oral anaerobic bacterial metabolites and its performance difference with the dual chromogenic system.

[0101] Comparative Example 2: The difference from Example 1 is that the detection area 2 of this test card is loaded only with the TNBS chromogenic agent from the second detection reagent, with a concentration of 0.342 mmol / L. Detection area 2 does not contain the DTNB chromogenic agent. This comparative example is used to verify the reaction effect of the single primary amine compound chromogenic system and its difference in sensitivity and risk indication capability compared with the DTNB / TNBS combined system.

[0102] The test cards prepared in Example 1, Comparative Example 1, and Comparative Example 2 were used to test a 3-year-old cat whose oral health was initially diagnosed by a veterinarian. The colorimetric results were compared with the standard colorimetric card, and the results are shown in Table 1:

[0103]

[0104] The results of the experiments on dogs were the same as those on cats.

[0105] The test cards prepared in Example 1, Comparative Example 1, and Comparative Example 2 were used to test a 3-year-old cat that had been preliminarily diagnosed by a veterinarian as having a mild risk of periodontitis. The colorimetric results were compared with the standard colorimetric card, and the results are shown in Table 2.

[0106]

[0107] The results of the experiments on dogs were the same as those on cats.

[0108] The test cards prepared in Example 1, Comparative Example 1, and Comparative Example 2 were used to test a 3-year-old cat that had been preliminarily diagnosed by a veterinarian as having a moderate risk of periodontitis. The colorimetric results were compared with the standard colorimetric card, and the results are shown in Table 3.

[0109]

[0110] The results of the experiments on dogs were the same as those on cats.

[0111] Based on Tables 1, 2, and 3, this result indicates that DTNB reagent alone has a weak colorimetric reaction intensity, and the interpretation result is a low concentration (based on the "+" on the standard colorimetric card). This underestimates the actual metabolic activity level of anaerobic bacteria in the cat's oral cavity, and the detection window is delayed, which may lead to misjudgment of periodontitis risk or insufficient diagnostic sensitivity.

[0112] Experimental results showed that when detecting cats at mild periodontitis risk, the test cards containing only DTNB and TNBS showed a "low concentration" level, significantly lower than the "medium concentration" level shown by the test card using both DTNB and TNBS. Similarly, when detecting cats at severe periodontitis risk, the test cards containing only DTNB and TNBS showed a "medium concentration" level, significantly lower than the "high concentration" level shown by the test card using both DTNB and TNBS, indicating a lag in the detection window when using a single chromogenic agent. Therefore, the test card using both DTNB and TNBS demonstrates a higher overall sensitivity to the metabolites of anaerobic bacteria in the feline oral cavity compared to DTNB alone, resulting in a more significant colorimetric reaction and facilitating earlier identification of oral health risks. This invention enables the simultaneous detection of biomarkers in two types of anaerobic bacterial metabolites, thiols and primary amines, using DTNB and TNBS. This overcomes the limitations of single-index detection and achieves high sensitivity. It can produce obvious color changes in the early stages of anaerobic bacterial metabolic activity, advance the detection window, and enable more accurate and earlier determination of anaerobic bacterial concentration. It can capture signals in the early stages of anaerobic bacterial metabolic activity, achieving an early warning effect.

[0113] Example 2: The difference between this example and Example 1 is that the concentration of DTNB in ​​the first test reagent and the concentration of TNBS in the second test reagent are different. In this example, the concentration of DTNB in ​​the first test reagent is 2.52 mmol / L and the concentration of TNBS in the second test reagent is 0.171 mmol / L.

[0114] Example 3: The difference between this example and Example 1 is that the concentration of DTNB in ​​the first test reagent and the concentration of TNBS in the second test reagent are different. In this example, the concentration of DTNB in ​​the first test reagent is 5.05 mmol / L and the concentration of TNBS in the second test reagent is 0.513 mmol / L.

[0115] Using the rapid test cards prepared in Examples 1, 2, and 3, a 3-year-old cat with a preliminary veterinary diagnosis of oral health was tested. The colorimetric results were compared with the standard colorimetric card, and the results are shown in Table 4.

[0116]

[0117] Using the rapid test cards prepared in Examples 1, 2, and 3, a 3-year-old cat initially diagnosed by a veterinarian as having a mild risk of periodontitis was tested. The colorimetric results were compared with the standard colorimetric card, and the results are shown in Table 5.

[0118]

[0119] Using the rapid test cards prepared in Examples 1, 2, and 3, a 3-year-old cat initially diagnosed by a veterinarian as having a moderate risk of periodontitis was tested. The colorimetric results were compared with the standard colorimetric card, and the results are shown in Table 6.

[0120]

[0121] Combining Tables 4, 5, and 6, the results indicate that Example 1 (DTNB 3.03 mmol / L + TNBS 0.342 mmol / L) performed best in terms of specificity, sensitivity, and discrimination: it showed high specificity on healthy samples with no false positives, produced clear and distinguishable color changes on samples with mild lesions, clearly identified them, achieved early warning, had good sensitivity, and its color gradient could accurately reflect changes in oral health status from healthy to mild to moderate, with obvious color gradation and good discrimination.

[0122] Example 2 (DTNB 2.52mmol / L + TNBS 0.171mmol / L) lacks sensitivity and cannot accurately identify mild and moderate lesions, thus failing to achieve early warning and accurate differentiation of the severity of oral inflammation.

[0123] Example 3 (DTNB 5.05mmol / L + TNBS 0.513mmol / L) has poor specificity, produces false positives on healthy samples, and cannot accurately distinguish between healthy and mild disease states.

[0124] Example 4

[0125] This embodiment provides a rapid oral health status test card specifically for dogs and cats. This test card can be used as a home self-testing tool for non-diagnostic purposes, or as a testing tool to assist veterinarians in assessing oral health risks in dogs and cats.

[0126] This embodiment provides the use of a rapid oral health status test card for dogs and cats in the preparation of a home self-testing tool for non-diagnostic purposes, or in the preparation of a test tool to assist veterinarians in assessing oral health risks in dogs and cats.

[0127] Example 5: Verification of Detection Sensitivity of Detection Card

[0128] The test card prepared in Example 1 (batch number: 2025082001) was used to test the thiol standard solutions of gradient concentrations.

[0129]

[0130] The results showed that the detection limit for thiols was 0.1 mmol / L, and a distinct pale yellow color (corresponding to the '+' level on the standard colorimetric card) was observed in detection zone 2 at this concentration. This demonstrates that the present invention can effectively detect anaerobic bacterial metabolites at extremely low concentrations, exhibiting high sensitivity and an early detection window.

[0131] Example 6: Verification of Repeatability and Reproducibility of Test Card Detection

[0132] To verify the reliability of the test card, 0.2 mmol / L, 1.0 mmol / L, and 10 mmol / L thiol standards were tested using the same batch (batch number: 2025082001). The test results are as follows:

[0133]

[0134] Using test cards from different production batches (batch numbers: 2025031001, 2025073001, 2025082001), thiol standards at concentrations of 0.2 mmol / L, 1.0 mmol / L, and 10 mmol / L were tested. The results are as follows:

[0135]

[0136] Statistical results show that the colorimetric consistency of intra-batch repeatability and inter-batch reproducibility is both higher than 95%. This proves that the test card described in this invention not only has good repeatability, but also exhibits stable and consistent quality across different batches, demonstrating its reliability for industrial production.

[0137] Example 7: Stability Verification of the Test Card

[0138] The test card prepared in Example 1 (batch number: 2025082001) was placed at 45°C for 30 days, and the 0.2 mmol / L thiol standard was tested. The test results are as follows:

[0139]

[0140] After 30 days, the test results ('+' level) for 0.2 mmol / L thiol standard were completely consistent with those at day 0, showing no significant performance degradation. Based on the accelerated stability test results, the shelf life of this test card under normal storage conditions is estimated to be up to 24 months. This indicates that the product of this invention has excellent storage stability and can meet the needs of commercial distribution and daily storage.

[0141] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0142] In this invention, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," "link," and "fix" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection, a direct connection, or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any ordinary changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid oral health status detection card specifically for dogs and cats, characterized in that, It includes a handle (1) and a detection area (2). The handle (1) is a rectangular sheet structure that is easy to insert into the mouth of a dog or cat. The detection area (2) is fixed to one end of the handle (1). The detection area (2) is loaded with a first detection reagent and a second detection reagent. The first detection reagent contains the colorimetric reagent DTNB (5,5′-dithiobis(2-nitrobenzoic acid)), which reacts with thiol compounds produced by the metabolism of anaerobic bacteria in the oral cavity of dogs and cats to produce a color change; The second detection reagent contains the colorimetric reagent TNBS (2,4,6-trinitrobenzenesulfonic acid), which reacts with primary amine compounds produced by the metabolism of anaerobic bacteria in the oral cavity of dogs and cats to produce a color change; The first detection reagent and the second detection reagent are arranged in the detection area (2) in the form of a first region (21) and a second region (22), and the first region (21) and the second region (22) are arranged in parallel strips. The concentration of DTNB in ​​the first test reagent is 2.52–5.05 mmol / L, and the concentration of TNBS in the second test reagent is 0.171–0.513 mmol / L.

2. The rapid detection card according to claim 1, characterized in that, The preferred concentration of DTNB in ​​the first detection reagent is 3.03 mmol / L, and the preferred concentration in the second detection reagent is 0.342 mmol / L.

3. The rapid detection card according to claim 1, characterized in that, The first test reagent further includes a base solution one for maintaining the stability, solubility and reactivity of DTNB, and the second test reagent further includes a base solution two for maintaining the stability, solubility and reactivity of TNBS; the pH values ​​of the first test reagent and the second test reagent are both 7.5–8.

5.

4. The rapid detection card according to claim 3, characterized in that, The base solution is based on a phosphate buffer solution with a pH of 8.0 and is compounded with ≤5% ethanol and 5% glycerol. The second base solution is preferably a phosphate buffer solution with a pH of 8.0, and the second base solution contains no ethanol or has an ethanol content of ≤5%.

5. The rapid detection card according to claim 1, characterized in that, The detection area (2) is a rectangular pad. The four corners of the pad and the four corners of the handle (1) are rounded. The pad is made of a composite porous membrane of polyethylene and polypropylene. The handle (1) is made of polyvinyl chloride, so that the handle (1) has sufficient hardness to be inserted into the mouth of dogs and cats.

6. The rapid detection card according to claim 5, characterized in that, The length of the detection area (2) is 7.1±0.05mm, the width is 6.4±0.1mm, and the thickness is 0.25±0.05mm; the length of the handle (1) is 70±0.1mm, the width is 6.4±0.1mm, and the thickness is 1.0±0.01mm.

7. A manufacturing process for a rapid detection card according to any one of claims 1-6, characterized in that, Includes the following steps: S1, Loading of the first detection reagent (DTNB): S11. Cover the detection area (2) pad with a cutout mask that matches the shape of the first area (21) so that the cutout window of the mask is precisely aligned with the first area (21) to be sprayed. S12. Load the first test reagent into the atomizing precision spray gun, and spray it evenly on the hollow window of the mask under the conditions of air pressure 0.2-0.4MPa, spraying distance 5-10cm, and moving speed 10-20mm / s, so that the first test reagent is deposited only on the first area (21) exposed by the hollow window on the mask. S13. After completing the spraying of all the first areas (21) on the test area (2) in sequence, carefully remove the mask and transfer the pad that serves as the test area (2) to the drying oven. Dry it in the dark at 25-30℃ for 10-15 minutes, and then cool it after drying. S2, Second Detection Reagent (TNBS) Loading: S21 uses another mask to cover the pad after it has been processed and cooled in step S13. The mask includes a cutout window that is consistent with the shape of the second region (22), so that this window of the mask corresponds to the second region (22) of the detection area (2). S22. After replacing the precision spray gun or thoroughly cleaning the original precision spray gun and its internal pipeline, load the second test reagent and spray the second area (22) on the test area (2) under the conditions of air pressure 0.2-0.4MPa, spraying distance 5-10cm, and moving speed 10-20mm / s. S23. After spraying all the second areas (22) on the detection area (2) in sequence, remove the mask. Transfer the pad loaded with the first and second test reagents to the drying oven and dry it at 25°C in the dark for 10-15 minutes to thoroughly dry the detection area (2), thereby completing the preparation of the detection area (2). S3. Glue the detection area (2) to one end of the handle (1) to complete the preparation of the detection card.

8. Use of the rapid test card according to any one of claims 1-6 in the preparation of a home self-testing tool for non-diagnostic purposes.

9. Use of the rapid test card according to any one of claims 1-6 in the preparation of a test tool for assisting veterinarians in assessing oral health risks in dogs and cats.

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