Saliva-based rapid detection test paper for periodontal pathogenic bacteria and detection method thereof

Abstract

The application discloses a saliva-based periodontal pathogenic bacteria rapid detection test paper and a detection method thereof, and belongs to the technical field of biomedical detection. The test paper is sequentially provided with a sample pad, a neutralization isolation pad, a combination pad, a mixed transition zone, a slow-flow buffer section, a chromatography carrier and a water absorption pad along a chromatography flow direction. The chromatography carrier is a semi-closed arrow-shaped microfluidic chip, which is internally provided with an NC capillary strip and is provided with a micro-sawtooth flow guide structure, realizes unidirectional flow of liquid and eliminates side wall channeling; independent probe storage areas are arranged in each channel downstream of the combination pad; the test paper is internally provided with blue dextran tracer dye indicating liquid to indicate the progress of a liquid front. Through the combination design of the asymmetric flow resistance of the arrow-shaped channel, the mixed transition zone, the slow-flow buffer section and the balanced micro-column, the guanidine salt removal of the double-layer neutralization isolation pad and the hybrid calibration time sequence coupling, the application realizes high-sensitivity multiplex parallel rapid detection of periodontal pathogenic bacteria without nucleic acid extraction and amplification, and the total time length is not more than 20 minutes, and the application is suitable for oral clinic and home self-detection scenes.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to rapid detection technology for oral pathogenic microorganisms, and particularly to a saliva-based rapid test strip for periodontal pathogens and its detection method. This invention belongs to the cross-application field of microfluidic chips, nucleic acid hybridization, and point-of-care testing, and is suitable for rapid identification and diagnosis of periodontal pathogens in scenarios such as dental clinics, community screening, home self-testing, and epidemiological surveys. Background Technology

[0002] Periodontal disease is one of the most common diseases endangering human oral health, and it is closely related to infection by key periodontal pathogens such as *Porphyromonas gingivalis*, *Tannerella forsythia*, *Treponema denticola*, and *Aggregatibacter actinomycetemcomitans*. Traditional methods for detecting periodontal pathogens mainly include molecular diagnostic techniques such as anaerobic culture identification, polymerase chain reaction (PCR), and real-time quantitative PCR. Although these methods have high sensitivity and specificity, they generally rely on specialized laboratory equipment, trained technicians, and cumbersome pretreatment steps such as nucleic acid extraction and purification, resulting in testing cycles that can take several hours or even days, making it difficult to meet the practical needs of rapid outpatient screening and home self-testing.

[0003] In recent years, rapid test strips based on the lateral chromatography immunoassay principle have been widely used in the field of rapid detection of infectious diseases. However, their application in the field of nucleic acid detection of periodontal pathogens still faces many technical bottlenecks, which are mainly manifested in: The complex matrix effect of saliva samples significantly interferes with detection accuracy. Saliva contains high concentrations of guanidine isothiocyanate (GITC, commonly used as a component of nucleic acid extraction lysis buffer), plasma salts, mucins, extraneous proteins, and abundant non-target microbial nucleic acids. Direct sample loading severely inhibits the efficiency of nucleic acid hybridization reactions. Existing test strip products typically lack effective guanidine salt removal and hybridization environment regulation mechanisms, resulting in low detection sensitivity and high false negative rates.

[0004] Existing lateral chromatography test strips all rely on a simple physical bond between a nitrocellulose (NC) membrane and a backing plate, lacking the ability to actively regulate liquid flow behavior. In traditional test strips, liquid is prone to sidewall channeling and bypass loss at the interface between the NC membrane and the backing plate, resulting in sample waste and signal inhomogeneity. At the same time, the residence time of liquid in the detection line area is too short, leading to low target-probe complex capture efficiency, insufficient signal enrichment, and easy missed detection of weak positive samples.

[0005] Multiplex assays face significant challenges due to uneven fluid distribution and cross-interference between channels. Parallel multi-target assays require equal distribution of a sample across multiple reaction channels. Existing test strips lack effective flow equalization structures, resulting in significant differences in fluid volume between channels. Competitive consumption of target probes distorts the quantitative relationship, leading to poor comparability of test results. Furthermore, sharing the same quality control system across different target channels makes it impossible to independently determine the chromatographic patency and reagent activity of each channel, easily resulting in false negative / positive misjudgments.

[0006] Saliva contains a low load of periodontal pathogens, and the 16S rRNA target requires denaturation to expose the hybridization site. However, existing extraction-free direct amplification systems generally rely on heating equipment or strong chemical denaturants, making them difficult to integrate with test strip chromatography systems.

[0007] Existing test strip products have vague interpretation standards and lack effective chromatographic process indicators and system validity stratification mechanisms. When a single channel malfunctions, the entire strip is often discarded, resulting in resource waste. Furthermore, it is difficult to distinguish between improper sample pretreatment and test strip failure, which seriously affects the reliability of test results and the applicability of the test. Summary of the Invention

[0008] Purpose of the invention The purpose of this invention is to achieve highly sensitive, highly specific, and rapid multiplex parallel detection of 16S rRNA from periodontal pathogens in saliva without the need for nucleic acid extraction and amplification. This is achieved through a layered synergistic design of arrow-shaped microfluidic unidirectional cutoff channels and nitrocellulose capillary strip micro-serrated flow guiding structures, combined with a dead-volume-free sample distribution system consisting of a tapered mixing transition zone, a constant cross-section slow-flow buffer section, and a flow-balancing microcolumn. Furthermore, it utilizes a time-coupling mechanism for guanidine salt removal and hybridization calibration via a double-layered structured neutralizing isolation pad, supplemented by blue dextran tracer dye liquid front indication, multi-channel independent quality control, and layered validity interpretation standards. This allows for complete control over the entire process from sample collection, lysis and denaturation, guanidine salt removal, probe hybridization, homogenization and flow splitting, signal enrichment, to result interpretation. Simultaneously, it solves the technical challenges of uneven liquid distribution between channels, sidewall channel bypass loss, guanidine salt ionization inhibition effect, and difficulty in tracing and interpreting detection failures. This invention is applicable to rapid screening in dental clinics, epidemiological surveys of periodontal disease in communities, home self-examination, and real-time detection of periodontal pathogens in resource-limited scenarios such as polar regions, scientific expeditions, and mobile healthcare. It is especially suitable for applications that require simultaneous differential diagnosis of multiple periodontal pathogens, such as Porphyromonas gingivalis, Forsythia suspensa, Treponema denticulatum, and Actinomyces sympathomimeticum.

[0009] Technical solution To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a rapid test strip for periodontal pathogens based on saliva, wherein the test strip is provided with a sample pad, a neutralizing isolation pad, a conjugate pad, a mixing transition zone, a slow-flow buffer section, a chromatography carrier, and an absorbent pad in sequence along the sample chromatography flow direction; The chromatography carrier is a semi-closed, arrow-shaped microfluidic chip integrally molded from thermoplastic polymers. The material is selected from polymethyl methacrylate, polycarbonate, polydimethylsiloxane, and cyclic olefin copolymers. The complete channels are prepared by hot embossing, ultraviolet nanoimprinting, or injection molding. The channels have an axisymmetric arc-shaped arrow-shaped sidewall structure, which forms an asymmetric flow resistance, allowing the liquid to flow in only one direction. Independent strip-shaped nitrocellulose capillary strips are embedded in the groove at the bottom of the channel. The NC strips are fitted with the groove with a 2%-3% gap and are anchored to the bottom of the groove by interval ultrasonic hot melt spot welding. The spot welding temperature is ≤150℃, and the longitudinal distance between the weld point and the detection line and quality control line is ≥2mm. Micro-serrated flow guiding structures with a pitch of 1-2mm are set on both sides of the NC strips to constrain the liquid flow to adhere to the NC membrane surface and eliminate sidewall channeling and liquid bypass loss. Multiple channels are arranged in parallel, and a balancing micro-column is set at the diversion inlet to distribute the liquid in each channel synchronously and equally. The mixing transition zone is a gradually narrowing flow channel between the conjugate pad and the slow-flow buffer section, which homogenizes the sample and probe mixture through shear flow; a 2-5mm equal cross-section slow-flow buffer section is set between the downstream end of the mixing transition zone and the balance microcolumn; an independent probe reserve area is set for each channel downstream of the conjugate pad to reduce the difference in probe distribution between channels. The detection probe loaded with coupled signal markers on the binding pad specifically recognizes 16S rRNA of periodontal pathogens; a single detection line and a dedicated quality control line are sequentially arranged along the liquid flow direction in each target channel. The detection line fixes the capture probe, and the capture probe specifically binds to the detection probe-16S rRNA complex. The quality control line independently verifies the chromatographic patency and reagent activity of the corresponding channel. The test strip contains a built-in blue dextran tracer dye, which is pre-loaded in lyophilized form at the boundary between the slow-flow buffer section and the chromatography carrier at a concentration ≤0.1%. It enters the chromatography carrier with the liquid front and indicates that the liquid flows through the entire detection line area. The sample pad has a maximum effective capacity of ≤150μL; the neutralization isolation pad is layered, with the upper layer being a guanidine salt removal and adsorption layer, where β-cyclodextrin derivatives are covalently immobilized on the surface of cellulose fibers and capture guanidine ions through molecular inclusion; the lower layer is a hybrid salt calibration layer, loaded with a 10×SSC high-salt buffer system and BSA; the two layers are physically isolated and work together to eliminate the guanidine salt ionization inhibition effect.

[0010] Furthermore, the arrow-shaped microfluidic channel is formed by connecting and splicing several arrow-shaped channel units end to end along the liquid flow direction; a single arrow-shaped channel unit is formed by an axisymmetric arc-shaped wall, with the axis of symmetry of the arc-shaped wall parallel to the liquid flow direction; the arc-shaped wall includes a first sidewall and a second sidewall; along the forward liquid flow direction, the downstream end of the first sidewall and the downstream end of the second sidewall intersect tangentially, forming a guiding angle α towards the downstream of the fluid, which allows the forward fluid to pass with low resistance; the upstream end of the second sidewall intersects tangentially with the upstream end of the first sidewall of the adjacent channel unit, forming a flow-blocking angle φ towards the upstream of the fluid, which causes the reverse-flowing fluid to generate vortex high resistance; the main body of the channel arc-shaped wall structure is complete and independent, and is layered with the NC strip edge micro-serrated guiding structure, with no functional interference between them.

[0011] Furthermore, the guiding angle α is 15°-30°, resulting in low flow resistance when the forward fluid passes through; the obstruction angle φ is 120°-150°, resulting in turbulence and significantly increased flow resistance when the reverse-flowing liquid passes through this angle; the asymmetric flow resistance difference between the two causes the channel to be cut off in one direction, preventing backflow of liquid.

[0012] Furthermore, the inner wall of the microfluidic channel is activated by plasma surface treatment and chemically grafted with carboxyl or amino active modification layers, and the capture probe is covalently and stably fixed. Nitrocellulose capillary strips are independently embedded inside the channel, and these strips are not synchronously imprinted with the polymer substrate. The NC strips are fixed to the bottom of the tank by interval ultrasonic hot melt spot welding, with a spot welding temperature ≤150℃ and a longitudinal distance ≥2mm between the weld points and the detection and quality control spraying areas, ensuring no high-temperature thermal damage to the NC membrane pores. The semi-enclosed arrow-shaped microfluidic channel forming process is selected from at least one of hot imprinting, ultraviolet nanoimprinting, and injection molding. Injection molding is suitable for integrally preparing the channel from a whole substrate, while hot imprinting and ultraviolet nanoimprinting are suitable for secondary processing of pre-made polymer films to form the channel. The micro-serrated structure of the NC strip edge is processed by standard micro-milling and EDM. The mixing transition zone and the 2-5mm slow-flow buffer section are integrally injection molded, eliminating the defects of the split jet.

[0013] Furthermore, the signal marker is selected from at least one of colloidal gold nanoparticles, colored latex microspheres, fluorescent microspheres, and quantum dots; the detection probe is a specific oligonucleotide probe that targets the conserved target sequence of 16S rRNA of periodontal pathogens.

[0014] Furthermore, the periodontal pathogens are selected from at least one of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans.

[0015] Furthermore, when conducting multiplex detection, the chromatography carrier is equipped with multiple parallel, independently arrow-shaped microfluidic target channels with completely symmetrical fluid resistance. A balancing microcolumn is installed at the diversion inlet, and a 2-5 mm equal-section slow-flow buffer section is placed in front to eliminate uneven liquid distribution caused by shear jet. Combined with the downstream matching of each channel, an independent probe reserve area is set up, and the shear flow mixing effect of the gradually narrowing mixing transition zone reduces the competition for probe supply and distribution differences between channels. Only one detection line and one dedicated quality control line are arranged in each independent target channel. Specific capture probes targeting the 16S rRNA of different periodontal pathogens are fixed on the detection lines of different channels, and there is no cross-recognition binding activity between the various capture probes. The same amount of sample is injected into each channel simultaneously, and the detection is differentiated without interference.

[0016] Furthermore, the sample pad is thickened to create a lysis retention zone with a maximum carrying capacity of ≤150μL, and the entire sample is pretreated with lysis denaturation buffer. This lysis denaturation buffer is free of SDS, proteinase K, lysozyme, EDTA, and EGTA, and consists of a ternary chemical lysis system: Triton X-100 at a concentration ≤0.5%, guanidine isothiocyanate, and a nucleic acid denaturant. The surfactant solubilization and guanidine salt dissociation work synergistically to destroy the Gram-negative outer membrane and cell membrane of periodontal pathogens, releasing intracellular 16S rRNA. The nucleic acid denaturant destroys 16S rRNA. The rRNA secondary stem-loop structure exposes the hybridization target sequence and has no component compatibility antagonism. The neutralizing isolation pad has a double-layer physical separation structure: the upper guanidine salt removal adsorption layer has β-cyclodextrin derivatives chemically cross-linked and covalently immobilized on the pad fiber. This β-cyclodextrin does not detach or migrate with liquid rinsing and relies on molecular inclusion to capture residual guanidine ions in the sample, and is not affected by competition from high sodium ions. The lower hybridization salt calibration layer is loaded with a 10×SSC high-salt buffer system and a high concentration of BSA. This layer independently regulates the ionic strength of the system to the optimal range for nucleic acid hybridization, and BSA blocks free impurities. The double-layer structure synergistically eliminates the guanidine salt ionization inhibition effect, and hybridization failure is prevented. The sample pad lysis denaturation buffer also contains a blocking agent, which is selected from at least one of bovine serum albumin, casein, and polyvinylpyrrolidone. This blocking agent inhibits the non-specific adsorption of salivary proteins and impurities.

[0017] Secondly, the present invention also provides a rapid detection method for periodontal pathogens based on saliva, using the above-mentioned test strip, comprising the following steps: S1. Quantitative Sampling, Constrained Predilution, Limited Volume Loading, and Lysis / Denaturation: Use the provided graduated quantitative capillary pipette to collect the subject's original saliva sample, with the target collection volume strictly controlled at 50-75 μL. During collection, visually confirm that the liquid level is consistently within the 50-75 μL range. If the sample volume is less than 50 μL or exceeds 75 μL, discard and resample. Immediately after collection, transfer all saliva in the tube to the provided diluent, repeatedly blowing and aspirating the tube wall at least 3 times to ensure no sample residue remains. Match the dilution factor according to the actual collection volume, strictly controlling the total volume after dilution to ≤150 μL: For 50 μL of saliva, a 3-fold dilution can be used, resulting in a total volume of 150 μL; for 75 μL of saliva, only a 2-fold dilution is allowed, resulting in a total volume of 150 μL. For intermediate sampling volumes of 50-75 μL, match the dilution factor proportionally. A quick reference table of sampling volume and dilution factor is attached to the provided consumables. After dilution and mixing, only 80-120 μL of the sample should be taken. Add 80-120 μL of liquid to the lysis holding area of ​​the sample pad, and discard all remaining diluted sample. This 80-120 μL addition range represents 53%-80% of the 150 μL capacity limit of the sample pad. This range ensures sufficient contact between the lysis buffer and the sample, appropriate reaction system concentration, and efficient bacterial lysis and nucleic acid release. It also reserves volume margin to avoid liquid overflow or flooding caused by operational fluctuations. The sample pad stably retains the sample, simultaneously completing the entire process of bacterial cell disruption, 16S rRNA release, and nucleic acid denaturation. No separate nucleic acid extraction or amplification is required throughout the process. A constant temperature device can be used to maintain 25-37℃ to accelerate the lysis reaction. S2. Layered Guanidine Salt Removal and Hybridization Calibration: The liquid carrying lysed components and denatured rRNA first flows through the upper guanidine salt removal adsorption layer of the neutralization isolation pad. Covalently immobilized β-cyclodextrin encapsulates and captures residual guanidine ions without shedding and contaminating the downstream. It then flows into the lower hybridization salt calibration layer. The 10×SSC buffer system calibrates the hybridization ion strength, and BSA blocks contaminating proteins. Nucleic acid hybridization inhibitors are eliminated layer by layer and stepwise to construct a stable hybridization microenvironment. S3. Dead-volume-free probe hybridization and clear temporal boundaries: After neutralization, the sample liquid permeates into the downstream binding pad via capillary action, thoroughly mixing with the labeled detection probe on the binding pad. The denatured and exposed 16S rRNA of the target sequence undergoes base pairing with the detection probe, generating a detection probe-target nucleic acid complex. This hybridization reaction is completed entirely within the binding pad, taking a maximum of 5-8 minutes. The mixed liquid continuously flows into the gradually narrowing mixing transition zone, achieving homogenization through shear flow. When the liquid reaches the boundary between the slow-flow buffer section and the chromatography carrier, the pre-loaded blue dextran tracer dye dissolves synchronously and migrates with the liquid front. The tracer dye only indicates the liquid flow path and does not represent the complete completion of the probe-target hybridization reaction; there is no synchronous determination equivalence between the two. S4. Homogenization and split chromatography with controllable duration signal enrichment: The homogenized liquid, homogenized by a 2-5mm slow-flow buffer section, is evenly distributed to each symmetrical parallel target channel, eliminating the uneven liquid volume caused by the jet; the NC strip is fixed by low-temperature ultrasonic spot welding without any movement, and the micro-serrations at the edges continuously constrain the liquid to flow completely through the surface of the NC porous medium, avoiding sidewall channel bypass loss, while not damaging the unidirectional flow structure of the main channel; the liquid flows continuously through the detection line area under the action of capillary driving force, and the liquid flow naturally decelerates due to the change in NC membrane pore size and the specific binding and retardation effect of the fixed capture probe on the line. The liquid front flows through the detection line area for no less than 30-60 seconds. The natural fluid retardation effect allows the target-probe complex to be fully intercepted and enriched, forming a stable and reproducible detection signal. The whole process is a spontaneous physicochemical process of chromatography, without the need for manual settling or timing intervention; S5. Quality control binding reaction: Free detection probes that have not bound to the target continue to migrate along a single channel to the downstream dedicated quality control line, specifically bind to the quality control molecule, and generate the corresponding quality control indicator band for the channel. The quality control results of each channel are evaluated independently. S6. Standardized Stratified Result Interpretation and Differentiated Handling of Failure Causes: Simultaneously observe the migration range of the tracer dye, the detection lines of each target channel, and the signals of the dedicated quality control lines, with the aid of a standard quality control colorimetric card for interpretation; Judgment criteria: The tracer dye completely covers the entire detection line area; An effective channel is defined as the dedicated quality control line reaching a preset color development intensity threshold, which is compared with the matching standard colorimetric card, and the color development intensity is not lower than the minimum qualified color development intensity indicated on the colorimetric card; Overall detection effectiveness stratified judgment: For multi-channel chips with a total number of ≥4 channels, the entire detection system is considered effective when the number of effective channels is ≥80% of the total number of channels; for chips with a total number of less than 4 channels, the entire detection system is considered effective when all channels are effective, thus avoiding rounding errors. Ambiguity in the error tolerance rules; Effective channels: Qualitatively determine the corresponding pathogenic bacteria negative / positive based on the presence or absence of bands on the test line, and semi-quantitatively determine the pathogenic bacteria load based on the intensity of band color development, and issue a normal result; Single channel quality control invalid, overall test meets standards: The corresponding target result is uniformly marked as "invalid (local channel abnormality, it is recommended to retest this species)," without discarding the entire card, and the target is retested separately using a single-species test strip with a fixed target capture probe, so as to prevent retest failure caused by mismatch of test strip models; If the same target is invalid for two consecutive retests, it is determined that the test strip batch has defects; The number of effective channels does not reach the corresponding channel number threshold: The overall test is determined to be invalid, which is caused by sample / operation factors, and it is necessary to re-quantitatively sample, match the dilution factor, and retest the entire card.

[0018] Furthermore, the detection is carried out in a constant temperature environment of 25-37℃; the hybridization reaction between the detection probe and denatured 16S rRNA in step S3 takes a maximum of 5-8 minutes; the liquid completes all parallel channel chromatography migration and signal delay enrichment process in step S4, which takes a maximum of 8-12 minutes; the total time of the two steps in sequence does not exceed 20 minutes, with no dead volume delay redundancy; if the ambient temperature is below 25℃, the hybridization and chromatography time can be appropriately extended or a constant temperature auxiliary device can be used to accelerate the reaction; the total time from the completion of saliva sample addition to the completion of valid result interpretation does not exceed 20 minutes.

[0019] Beneficial effects This invention achieves unidirectional zero backflow cutoff of liquid through the asymmetric flow resistance sidewall structure of the arrow-shaped microfluidic channel, eliminating cross-contamination of detection lines and disordered reaction timing caused by reverse backflow, and significantly improving the reliability of multiple detection.

[0020] This invention achieves a firm anchoring of the NC membrane and polymer substrate through a 2%-3% gap fit between the bottom groove of the semi-enclosed microfluidic chip and the NC capillary strip, and interval ultrasonic hot melt spot welding (≤150℃), avoiding thermal damage to the NC membrane pore structure caused by high-temperature spot welding. Combined with the micro-serrated flow guiding structure with a pitch of 1-2mm on both sides of the NC strip, it eliminates the sidewall channeling and liquid bypass loss commonly found in traditional test strips, ensuring that all liquid flows through the detection line area to participate in the reaction, thereby improving detection sensitivity and signal uniformity.

[0021] This invention eliminates uneven liquid distribution between channels caused by jet flow through a combination of a tapered mixing transition zone for shear flow homogenization, a 2-5mm equal-section slow-flow buffer section, and a balanced microcolumn at the diversion inlet. This ensures synchronous and equal-volume injection of samples into each parallel target channel, with the liquid volume difference between channels controlled within 5%. Furthermore, the independent probe reserve area for each channel downstream of the pad avoids competitive consumption when multiple channels share a probe library, improving the accuracy of quantitative relationships in multiplex detection. The built-in blue dextran tracer dye enables visual monitoring of the liquid front propulsion, providing operators with clear indications of when to interpret the flow.

[0022] This invention achieves rapid cell wall disruption and 16S rRNA release and denaturation of periodontal pathogens through a thickened lysis retention zone on the sample pad and a ternary chemical lysis system, eliminating the need for nucleic acid extraction and thermal denaturation equipment throughout the process. Combined with a double-layer physical separation structure of the neutralizing isolation pad, the guanidine salt ionization inhibition effect is eliminated in a time-decoupled manner, creating an optimal ionic microenvironment suitable for nucleic acid hybridization, thus solving the common technical problem of matrix inhibition in direct detection of saliva samples. Simultaneously, the natural retardation effect of the NC membrane ensures that the liquid flow through the detection line area lasts for at least 30-60 seconds, achieving sufficient retention and signal enrichment of the target-probe complex, and improving the detection sensitivity of weakly positive samples.

[0023] This invention solves the problem of difficulty in tracing failures caused by the shared use of quality control lines for each target channel, combined with a stratified validity judgment standard (≥80% of effective channels are valid when there are ≥4 channels in total, and all channels are valid when there are fewer than 4 channels). It achieves stratified handling of target-specific retesting when a single channel is abnormal and whole-card retesting when the whole channel is invalid, forming a closed-loop testing quality assurance system.

[0024] This invention employs a combined quality control strategy of quantitative sampling specifications (50-75 μL), constrained dilution factor matching, and a limited loading volume of 80-120 μL. This strategy standardizes the entire process of sample collection, dilution, and loading, ensuring appropriate concentration of the lysis reaction system and optimal nucleic acid release efficiency. The total detection time does not exceed 20 minutes, achieving true real-time detection and shortening the detection cycle by more than 90% compared to traditional PCR methods. The detection targets cover four core periodontal pathogens: Porphyromonas gingivalis, Forsythia suspensa, Treponema denticulatum, and Actinomyces sympathomimeticum, achieving a one-time comprehensive screening of the main pathogenic bacteria of chronic periodontitis and invasive periodontitis. Attached Figure Description

[0025] Figure 1 This is a schematic cross-sectional view of the overall structure of the test paper of the present invention. Figure 2 This is a schematic diagram illustrating the usage process and layered interpretation logic of the test strip of this invention. Detailed Implementation

[0026] The present invention will now be clearly and completely described in conjunction with the technical solutions in the embodiments thereof. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] In the description of this invention, the terms "upstream," "downstream," "forward," and "reverse" refer to the direction of sample chromatography flow, while "left," "right," "up," and "down" refer to the orientation or positional relationship shown in the accompanying drawings. These terms are used only for the convenience of describing this invention and simplifying the description, and are not intended to 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.

[0028] Example 1 focuses on materials and preparation methods: This embodiment details the material selection, specifications, processing technology, and assembly method of each component of the test strip of the present invention. Using a test strip for the quadruple parallel detection of four periodontal pathogens—Porphyromonas gingivalis, Forsythia suspensa, Treponema denticulatum, and Actinomyces sympathomimeticum—as an example, this embodiment fully illustrates the entire preparation process from raw materials to finished product.

[0029] Materials and proportions: Sample pad: glass cellulose fiber membrane, with a maximum effective liquid carrying capacity of 150 μL; after pretreatment with lysis denaturation buffer, it is dried in a 37°C oven for 12 hours before use.

[0030] Lysis and denaturation buffer formulation: per 100 mL system: Triton X-100 0.5 g; guanidine isothiocyanate 1.0 mol / L; formamide 20% v / v; bovine serum albumin 1.0% w / v; balance 50 mM Tris-HCl buffer. After mixing the above components, filter through a 0.22 μm filter membrane for sterilization and store at 4°C protected from light for later use.

[0031] Neutralizing pad: a double-layer composite structure, with the upper layer being a guanidine salt removal and adsorption layer and the lower layer being a hybrid salt calibration layer. The two layers are physically separated by a hydrophobic porous PTFE separator membrane, and are stacked and hot-pressed together after independent pretreatment.

[0032] Upper guanidine salt removal adsorption layer: cellulose fiber membrane, β-cyclodextrin derivative covalently immobilized on the fiber surface by 1,4-butanediol diglycidyl ether crosslinking agent, with an immobilization amount of about 15 μg / mg fiber; after washing to remove free β-cyclodextrin with pH 7.0 phosphate buffer, it was vacuum dried at 25°C.

[0033] Lower hybrid salt calibration layer: glass cellulose membrane, loaded with 10×SSC buffer system, BSA 2.0% w / v, Tween-20 0.1% v / v; freeze-dried to form a loose and porous dry reserve layer.

[0034] Binding pad: A polyester fiber membrane with a detection probe mixture loaded with coupled colloidal gold nanoparticles.

[0035] Preparation of colloidal gold-probe conjugates: Specific detection probes targeting the 16S rRNA of four periodontal pathogens were mixed with colloidal gold solution at a concentration of 2 μg / mL, incubated with shaking at room temperature for 2 hours, and then BSA was added to a final concentration of 1% w / v for 30 minutes to block. The mixture was then centrifuged at 12000 rpm for 30 minutes, and the precipitate was redissolved in gold labeling preservation solution to OD=10 and stored at 4℃.

[0036] Application amount of pretreatment solution for bonding pad: 100 μL of gold-labeled probe mixture per centimeter width, and dry at 37°C for 2 hours.

[0037] Four independent probe reserve areas are set up with the downstream matching four channels. The corresponding target gold-labeled probes are positioned and sprayed through the slit coating head. The reserve areas are spaced 1.5mm apart.

[0038] Mixing transition zone and slow-flow buffer section: Polydimethylsiloxane prepolymer and curing agent are mixed in a 10:1 ratio and then injection molded to prepare an integrated tapered flow channel and a downstream slow-flow buffer section with a constant cross-section of 2-5mm; the mixing transition zone has an inlet width of 4mm, an outlet width of 2.5mm, a tapering angle of 15°, and a flow channel depth of 0.3mm; the slow-flow buffer section has a length of 3mm and a cross-section of 2.5mm × 0.3mm.

[0039] After being treated with oxygen plasma, the inner walls of the mixing transition zone and the slow-flow buffer section were immersed in a 1% v / v 3-aminopropyltriethoxysilane ethanol solution for 2 hours at room temperature, washed with ethanol, and then cured at 120°C for 30 minutes.

[0040] Chromatography carrier: made of polymethyl methacrylate, four parallel, axisymmetric arc-shaped arrow-shaped microfluidic channels are integrally prepared by injection molding process. The chip has an external size of 20mm×15mm×2mm, a channel width of 0.5mm, and a depth of 0.3mm.

[0041] Injection molding process parameters: barrel temperature 240℃, mold temperature 80℃, injection pressure 120MPa, holding pressure 80MPa, holding time 3s, cooling time 15s.

[0042] Each arrow-shaped channel is composed of 8 arrow-shaped units connected end to end; the guiding angle α in a single unit is 20°, and the obstruction angle φ is 135°.

[0043] The groove at the bottom of the channel is 0.52mm wide and 0.15mm deep.

[0044] After the chip is injection molded, the inner wall of the channel is activated by oxygen plasma and then immersed in a 10% acrylic acid aqueous solution for graft polymerization at 60°C for 2 hours. This introduces a carboxyl active modification layer into the inner wall of the channel for subsequent covalent fixation of the capture probe.

[0045] NC strip embedding and anchoring: A nitrocellulose capillary strip is independently embedded in each groove, with a 2%-3% gap between the strip and the groove. Anchoring is achieved using interval ultrasonic hot-melt spot welding at 120℃, with a weld spacing of 3mm. The longitudinal distance between the weld and the test line / quality control line is ≥2.5mm. After welding, the strip flatness and fit are inspected under a 4x optical microscope.

[0046] Coating of detection and control lines: Using a scribing sprayer, capture probe solution and biotinylated BSA solution are sprayed onto designated areas of the NC strip.

[0047] Capture probe sequence: Pg (Porphyromonas gingivalis): Bio-ACAGTTGGGTCCTATCCG; Tf (Focustanabella): Bio-TGGTAGTCACTTCCGACC; Td (Tetranychus dactylus): Bio-GCGTTGCTGCGAGCTTA; Aa (Accompanied by Actinomycetes): Bio-CCGTAGTTAGCCGTCCC.

[0048] Spray concentration: 1.0 mg / mL of capture probe per detection line, spraying amount 0.5 μL / cm; biotinylated BSA spraying amount for control line 0.5 μL / cm. Dry at 37℃ for 1 hour after spraying.

[0049] NC strip edge micro-serrated structure processing: In the NC strip cutting process, it is formed by one-step punching with a precision die-cutting tool, with a serration depth of 0.05mm.

[0050] Blue dextran tracer dye: Blue dextran 2000 was dissolved in deionized water at a concentration of 0.08% w / v, and 2% sucrose and 0.5% trehalose were added as freeze-drying protectants. A 0.5 μL droplet was precisely spotted at the boundary between the slow-flow buffer section and the chromatography carrier using a microdroplet spotter, and then freeze-dried to form freeze-dried microspheres.

[0051] Absorbent pad: Pure cotton velvet filter paper, no chemical pretreatment.

[0052] Preparation method: S1. Preparation of the sample pad: Cut glass cellulose membranes into 15mm × 4mm pieces. Immerse the cut membrane pieces in the aforementioned lysis and denaturation buffer solution for 10 minutes, ensuring complete saturation. Remove the membrane pieces and place them on a clean stainless steel mesh to drain excess liquid. Then, transfer them to a 37°C forced-air drying oven for 12 hours. After drying, seal the sample pads in an aluminum foil bag and store them at room temperature away from light for later use.

[0053] S2. Preparation of neutralizing isolation pad: S2-1. Preparation of the upper guanidine salt removal adsorption layer: Cut cellulose fiber membranes into 15mm × 4mm sizes. Dissolve hydroxypropyl-β-cyclodextrin in 0.2mol / L NaOH aqueous solution to prepare a 5% w / v solution, and add 1,4-butanediol diglycidyl ether and mix well. Immerse the cellulose membrane in the above crosslinking reaction solution and react in a water bath shaker at 60℃ for 4 hours. After the reaction is complete, remove the membrane and wash it repeatedly with a large amount of deionized water, 0.01mol / L HCl, and deionized water until the pH of the washing solution is neutral. Dry the washed membrane under vacuum at 25℃ for 6 hours and store it in a sealed container.

[0054] S2-2. Preparation of the lower hybrid salt calibration layer: Cut glass cellulose membrane into 15mm × 4mm pieces and immerse them in a mixed solution containing 10×SSC, 2% BSA, and 0.1% Tween-20 for 5 minutes at room temperature. After removing the membrane and draining off excess liquid, freeze-dry it at -40℃ for 12 hours to allow the effective components to be loaded onto the surface of the membrane fibers in a dry, loose form.

[0055] S2-3. Double-layer composite: The upper guanidine salt removal adsorption layer, the hydrophobic porous PTFE isolation membrane, and the lower hybrid salt calibration layer are stacked sequentially from top to bottom, and hot-pressed at 80℃ and 2kg / cm linear pressure for 3 seconds to form an integrated double-layer neutralizing isolation pad.

[0056] S3. Preparation of the conjugate pad: S3-1. Preparation of gold-labeled detection probes: Take 40nm colloidal gold solution and add four detection probes to a final concentration of 2μg / mL, respectively. Incubate at room temperature with shaking for 2 hours. Add BSA to a final concentration of 1% w / v for blocking and continue incubation for 30 minutes. Centrifuge the conjugate solution at 4℃ and 12000rpm for 30 minutes, discard the supernatant, resuspend the precipitate in gold-labeled preservation solution to the original volume, repeat centrifugation and washing once, and finally resuspend to OD=10.

[0057] S3-2. Spraying: The gold-labeled probe mixture was uniformly sprayed onto the polyester fiber membrane using a slit nozzle. The spraying volume was controlled at 100 μL per centimeter of width. The four probes were mixed in equal proportions in the mixture. After drying at 37°C for 2 hours, the concentrated gold-labeled probe solution specific to each target was spot-applied to four independent probe reservoirs on the downstream end of the binding pad using a high-precision positioning spotting device. Each reservoir corresponded to one of the four targets: Pg, Tf, Td, and Aa. After spotting, the membrane was dried again at 37°C for 1 hour and then sealed for storage.

[0058] S4. Fabrication of microfluidic chips: S4-1. Injection Molding: PMMA granules are vacuum dried at 80℃ for 4 hours and then fed into the injection molding machine. The barrel temperature is 240℃, the mold temperature is 80℃, the injection pressure is 120MPa, the holding pressure is 80MPa, the holding time is 3s, and the cooling time is 15s. The resulting chip substrate has a four-channel parallel arrow-shaped microfluidic structure, a bottom groove, a balanced micro-pillar, and a diversion inlet structure.

[0059] S4-2. Carboxylation modification of the channel inner wall: Immerse the injection-molded chip in anhydrous ethanol and ultrasonically clean for 10 minutes, rinse with deionized water and dry with nitrogen. After oxygen plasma treatment, immediately immerse in a 10% acrylic acid aqueous solution and graft polymerize in a 60°C water bath for 2 hours. After removal, wash with plenty of deionized water to remove unreacted monomers and homopolymers, and dry with nitrogen for later use.

[0060] S4-3. NC Strip Cutting and Serration: The NC film is placed on a precision die-cutting machine and cut into 0.5mm × 12mm strips using a custom-made cutter with a 1.5mm pitch micro-serration cutting edge, and a serration depth of 0.05mm. The cut NC strips are stored in a low-humidity environment for later use.

[0061] S4-4. NC Strip Insertion and Spot Welding Anchoring: Insert the cut NC strips one by one into the bottom grooves of each channel. Under an inverted microscope, adjust the strip position so that both ends are flush with the grooves. Use an ultrasonic hot spot welder to weld every 3mm along the strip length, with a total of 3 weld points per channel, ensuring that the longitudinal distance between the weld points and the preset detection line and quality control line area is ≥2.5mm.

[0062] S4-5. Coating of Detection and Control Lines: The chip with the NC stripe fixed by spot welding is mounted on the stage of the scribing and gold spraying instrument. The target capture probe solution is sprayed sequentially at the detection line positions of the corresponding channels at a spraying rate of 0.5 μL / cm. Biotinylated BSA solution is sprayed at the control line positions of each channel. After coating, the chip is dried in an oven at 37℃ for 1 hour.

[0063] S4-6. Tracer dye spotting: Using a microdroplet spotter, the blue dextran lyophilized solution was precisely spotted at the end of the slow-flow buffer section of each channel and the boundary where it overlapped with the chromatography carrier, and then immediately freeze-dried.

[0064] S5. Test strip assembly: In a clean environment, following the flow direction of liquid chromatography, the components prepared in steps S1-S4 are sequentially overlapped onto a PVC backing substrate, with an overlap length of 2 mm. The specific order from top to bottom is: sample pad, neutralization isolation pad, binding pad, mixing transition zone, slow-flow buffer section, chromatography carrier, and absorbent pad. After each layer is stacked, it is repeatedly rolled three times with a 2 kg roller to ensure tight adhesion. The assembled large sheet of test paper is cut into 4 mm wide individual strips, each strip is individually packaged in an aluminum foil bag with desiccant, and sealed for storage.

[0065] The test strip prepared in this embodiment can be used for performance evaluation in subsequent embodiments.

[0066] Example 2 Optimized Formulation I: This embodiment provides an experimental scheme for a rapid saliva-based test strip for periodontal pathogens, focusing on the optimized combination of material ratios and their corresponding detection performance. Based on the preparation process of Example 1, this embodiment optimizes the formulations of the lysis-denaturing buffer and the neutralizing isolation pad to achieve stronger lysis-release efficiency and guanidine salt removal capability. The test strip is prepared using optimized formulation I, and its performance is evaluated.

[0067] Materials and proportions: Sample pad: Same material and specifications as in Example 1, with the lysis denaturation buffer formulation adjusted as follows, per 100 mL system: Triton X-100 0.3% (v / v); Guanidine isothiocyanate (GITC) 1.5 mol / L; Formamide (nucleic acid denaturant) 25% (v / v); Bovine serum albumin (BSA) 1.0% (w / v); Casein 0.2% (w / v); The remaining volume is 50 mM Tris-HCl buffer (pH 7.5).

[0068] Neutralizing isolation pad: Upper guanidine salt removal adsorption layer: cellulose fiber membrane, β-cyclodextrin derivative is sulfobutyl-β-cyclodextrin, loading is 20 μg / mg fiber; crosslinking agent is ethylene glycol diglycidyl ether, reaction conditions are 60℃, 6 hours; after washing with pH 6.5 phosphate buffer, vacuum drying is performed.

[0069] Lower hybrid salt calibration layer: glass cellulose membrane, loaded with 15×SSC buffer system and 0.5% (w / v) polyvinylpyrrolidone, freeze-drying parameters are the same as in Example 1.

[0070] Binding pad: Same as in Example 1, but the probe concentration during gold-labeled probe coupling is increased to 3 μg / mL, and casein is used instead of BSA as the blocking agent to reduce cross-reactivity with salivary mucin.

[0071] Microfluidic chip material: cyclic olefin copolymer replaces PMMA, channels are prepared by hot embossing process, and the inner wall of the channel is treated with ultraviolet / ozone for 15 minutes and then grafted with a hydrophilic coating of polyethylene glycol (PEG) to replace the carboxyl modification.

[0072] NC strip: aperture 6μm, spot welding temperature 130℃, micro-serration pitch 1.0mm.

[0073] Tracer dye: 0.05% (w / v) blue dextran, lyophilization protectant: 3% sucrose + 1% mannitol.

[0074] Other components: Same as in Example 1.

[0075] Preparation method: S1. Preparation of the sample pad: Prepare the solution according to the optimized lysis and denaturation buffer formulation described above, and filter through a 0.22 μm filter membrane. Immerse the glass cellulose membrane for 12 minutes, remove and drain, dry in a forced-air dryer at 37°C for 14 hours, and seal for later use.

[0076] S2. Preparation of neutralizing isolation pad: S2-1. Upper guanidine salt removal of the adsorbed layer: A 6% w / v solution of sulfobutyl-β-cyclodextrin was prepared by dissolving sulfobutyl-β-cyclodextrin in 0.1 mol / L NaOH aqueous solution. Ethylene glycol diglycidyl ether was added, and the mixture was stirred. The cellulose membrane was then immersed in the solution and reacted in a water bath shaker at 60°C for 6 hours. After the reaction was complete, the membrane was washed and dried under vacuum at 25°C for 8 hours.

[0077] S2-2. Lower hybrid salt calibration layer: The glass cellulose membrane was immersed in a mixed solution of 15×SSC, 3% BSA, and 0.5% PVP K30 for 5 minutes, and then freeze-dried at -40℃ for 14 hours.

[0078] S2-3. Double-layer composite: Same as Example 1.

[0079] S3. Preparation of the conjugate pad: S3-1. Preparation of gold-labeled detection probes: Four detection probes were added to a 40nm colloidal gold solution to a final concentration of 3μg / mL, and the solution was shaken at room temperature for 2.5 hours. Casein was added to a final concentration of 1% w / v for blocking for 40 minutes. Centrifugation and washing parameters were the same as in Example 1, and the solution was finally resuspended to OD=10.

[0080] S3-2. Spraying and dotting: The operation is the same as in Example 1. The four probes are mixed in equal proportions and sprayed. Then, the specific gold-labeled probe solutions of each target are dotted in the independent probe reserve area downstream of the pad.

[0081] S4. Fabrication of microfluidic chips: S4-1. Hot stamping: Place the COC sheet in a hot stamping machine, with a mold temperature of 170℃, a pressure of 5MPa, and hold the pressure for 5 minutes. After cooling to 60℃, demold to obtain a chip substrate with a four-channel parallel arrow-shaped structure.

[0082] S4-2. Hydrophilic modification of the inner wall of the channel: The COC chip was treated in a UV / ozone cleaner for 15 minutes, and then immersed in a toluene solution containing 1% PEG-silane coupling agent 2-methoxy polyethylene glycol propyltrimethoxysilane. The reaction was carried out at 60°C for 2 hours. The chip was then washed with toluene, ethanol and deionized water in sequence, and dried with nitrogen.

[0083] S4-3. NC strip cutting and sawing: NC film with 6μm aperture, die-cut sawing pitch of 1.0mm, the rest is the same as in Example 1.

[0084] S4-4. NC strip insertion and spot welding: Same as in Example 1, spot welding temperature 130℃.

[0085] S4-5. Spraying of test lines and quality control lines: The operation is the same as in Example 1.

[0086] S4-6. Tracer dye loading: Blue dextran concentration 0.05%, freeze-drying protectant is 3% sucrose + 1% mannitol, spotting and freeze-drying operations are the same as in Example 1.

[0087] S5. Test strip assembly: Same as in Example 1.

[0088] Example 3 Optimized Formulation II: This embodiment provides an experimental scheme for another saliva-based rapid periodontal pathogen detection test strip, focusing on adjusting the sample pad capacity and signal marker type, and optimizing the geometric parameters of the microfluidic channel to suit fluorescence signal detection mode. In this embodiment, fluorescent microspheres are used as signal markers instead of colloidal gold particles, adapted to a fluorescence reader for quantitative / semi-quantitative detection; simultaneously, the maximum sample pad capacity is adjusted to 120 μL, and the geometric parameters of the microfluidic channel and material ratios are optimized accordingly, with the test strip prepared through optimized formulation II.

[0089] Materials and proportions: Sample pad: Same material and specifications as in Example 1. The lysis and denaturation buffer formulation is as follows, per 100 mL system: Triton X-100 0.5% (v / v); Guanidine isothiocyanate (GITC) 1.2 mol / L; Nucleic acid denaturing aid: 10% ethylene glycol (v / v); Bovine serum albumin 1.5% (w / v); Polyvinylpyrrolidone 0.3% (w / v); The remaining volume is 50 mM Tris-HCl buffer.

[0090] Maximum sample pad capacity: 120 μL.

[0091] Neutralizing and isolating pad: same as in Example 1, but with the upper cellulose fiber membrane thickness 0.5 mm and the lower glass cellulose membrane thickness 0.5 mm.

[0092] Binding pad: Polyester fiber membrane, with detection probes loaded with coupled fluorescent microspheres.

[0093] Preparation of fluorescent microsphere-probe conjugates: Fluorescent microspheres were activated by EDC / NHS, washed by centrifugation, and then the detection probe was added. The reaction was carried out at room temperature for 3 hours. Unreacted carboxyl sites were blocked by ethanolamine, and BSA (1% w / v) was added for 30 minutes. After centrifugation and washing, the microspheres were resuspended in fluorescent microsphere storage solution until OD (350nm) = 5.

[0094] Combined with the amount of pretreatment solution sprayed: 80 μL per centimeter width, and the fluorescent microsphere probe concentrate solution was dotted in the independent probe reserve area.

[0095] The microfluidic chip is made of polycarbonate and is manufactured by injection molding. The flow guiding angle α = 15°, the flow blocking angle φ = 150°, the length of the slow-flow buffer section is 5 mm, and the diameter of the balancing micropillar is 0.3 mm.

[0096] PC injection molding process parameters: barrel temperature 280℃, mold temperature 90℃, injection pressure 140MPa, holding pressure 100MPa, holding time 4s, cooling time 20s.

[0097] The inner wall of the channel is treated with oxygen plasma and then grafted with an amino-active modified layer for subsequent covalent fixation of the capture probe.

[0098] NC strip: aperture 10μm, spot welding temperature 140℃, micro-serration pitch 2.0mm.

[0099] Tracer dye: Blue dextran concentration 0.1%, applied to the leading edge of each channel entrance at the lyophilization site.

[0100] Other components: Same as in Example 1.

[0101] Preparation method: S1. Preparation of the sample pad: Prepare the lysis and denaturation buffer solution according to the formula. Cut the glass cellulose fiber membrane into 12mm × 3.5mm pieces, soak for 10 minutes, remove and drain, and dry at 37℃ for 12 hours for later use.

[0102] S2. Preparation of neutralizing isolation pad: The steps are the same as in Example 1, but the upper layer thickness is 0.5 mm, the lower layer thickness is 0.5 mm, and the composite temperature is adjusted to 75°C.

[0103] S3. Preparation of the conjugate pad: S3-1. Preparation of fluorescent microsphere-probe conjugates: 200 nm time-resolved fluorescent microspheres were placed in a 2 mL centrifuge tube and centrifuged at 10,000 rpm for 15 minutes. The supernatant was discarded, and the microspheres were resuspended in 1 mL of 0.1 M MES buffer. EDC and NHS were added separately, and the reaction was carried out at room temperature for 30 minutes to activate the carboxyl groups. After washing by centrifugation at 10,000 rpm, the microspheres were resuspended in 0.05 M MES buffer. Four detection probes were added to a final concentration of 4 μg / mL, and the reaction was carried out at room temperature for 3 hours. Ethanolamine was added for blocking for 15 minutes, followed by BSA to a concentration of 1% w / v for blocking for 30 minutes. After two centrifugations and washings, the microspheres were resuspended in the fluorescent microsphere storage solution to an OD (350 nm) of 5.

[0104] S3-2. Spraying and Spot Coating: A mixture of four fluorescent microsphere probes in equal proportions was sprayed onto a polyester fiber membrane using a slit coating method at a rate of 80 μL / cm, and dried at 37°C for 2 hours. Downstream independent probe storage areas were spot coated with concentrated solutions of target-specific fluorescent microsphere probes and dried at 37°C for 1 hour.

[0105] S4. Fabrication of microfluidic chips: S4-1. PC Injection Molding: PC granules are vacuum dried at 120℃ for 6 hours before injection molding. The barrel temperature is 280℃, the mold temperature is 90℃, the injection pressure is 140MPa, the holding pressure is 100MPa, the holding time is 4s, and the cooling time is 20s. This produces a chip substrate with channel geometry parameters.

[0106] S4-2. Aminoation modification of the channel inner wall: The PC chip was ultrasonically cleaned in anhydrous ethanol for 10 minutes, rinsed with deionized water, and dried with nitrogen. After oxygen plasma treatment, it was immediately immersed in a 2% 3-aminopropyltriethoxysilane ethanol solution and reacted at room temperature for 4 hours. After removal, it was washed with ethanol and deionized water in sequence, and cured at 120℃ for 30 minutes.

[0107] S4-3. NC strip cutting and sawing: NC film with a pore size of 10μm, die-cut sawing pitch of 2.0mm, the rest is the same as in Example 1.

[0108] S4-4. NC strip insertion and spot welding: Same as in Example 1, spot welding temperature 140℃.

[0109] S4-5. Spraying of test lines and quality control lines: The operation is the same as in Example 1.

[0110] S4-6. Tracer dye loading: Blue dextran concentration 0.1%, spotting and freeze-drying operation as in Example 1.

[0111] S5. Test strip assembly: Same as in Example 1.

[0112] Example 4 Optimized Formulation III: This embodiment provides a third experimental scheme for a rapid saliva-based test strip for periodontal pathogens, targeting dual detection. It simplifies the chip structure, adjusts material ratios, and reduces the cost per test. This embodiment aims for dual parallel detection, simplifying the chip to a dual-channel structure. The formulations of each component are further adjusted based on Embodiment 1, optimizing the detection of two of the most common clinical periodontal pathogens: *Porphyromonas gingivalis* and *Fussetiana*.

[0113] Materials and proportions: Sample pad: Same material as in Example 1, lysis denaturation buffer formulation, per 100 mL system: Triton X-100 0.4% (v / v); Guanidine isothiocyanate (GITC) 0.8 mol / L; Nucleic acid denaturing agent: formamide 15% (v / v); Bovine serum albumin (BSA) 0.8% (w / v); Casein 0.5% (w / v); The remaining volume is 50 mM Tris-HCl buffer.

[0114] Neutralizing isolation pad: Upper guanidine salt removal adsorption layer: cellulose fiber membrane, β-cyclodextrin crosslinked and immobilized with epichlorohydrin, immobilization amount 12 μg / mg fiber; washing solution is pH 7.0 PBS.

[0115] Lower hybrid salt calibration layer: glass cellulose membrane, loaded with 10×SSC buffer system, BSA 1.5% (w / v), PEG20000 0.2% (w / v).

[0116] Binding pad: Polyester fiber membrane loaded with Pg and Tf detection probes coupled with colored latex microspheres.

[0117] Preparation of colored latex-probe conjugates: Red latex microspheres were activated by EDC / NHS and then conjugated with Pg and Tf detection probes, respectively. After being blocked by BSA, they were resuspended in the preservation solution until OD (450nm) = 8.

[0118] Binding pad pretreatment spraying: Pg and Tf colored latex probes are mixed in equal proportions and sprayed onto the binding pad. The downstream independent probe reserve area is dotted with the concentrated solution of each probe.

[0119] Microfluidic chip: made of PMMA, dual-channel parallel structure, flow guiding angle α=25°, flow blocking angle φ=140°, channel width 0.6mm, depth 0.35mm, slow flow buffer section length 2mm, and balancing micropillars are set at the dual-channel diversion inlet.

[0120] Injection molding process: barrel temperature 235℃, mold temperature 75℃, injection pressure 110MPa, holding pressure 70MPa, holding time 3s, cooling time 12s.

[0121] The carboxylation modification of the channel inner wall is the same as in Example 1.

[0122] NC strip: aperture 12μm, spot welding temperature 110℃, micro-serration pitch 1.8mm.

[0123] Absorbent pad: Same as in Example 1.

[0124] Preparation method: S1. Preparation of the sample pad: Prepare the lysis and denaturation buffer solution according to the above formula, and filter through a 0.22 μm filter membrane. Impregnate the glass cellulose membrane for 10 minutes, remove and drain, and dry at 37°C for 12 hours for later use.

[0125] S2. Preparation of neutralizing isolation pad: S2-1. Upper guanidine salt removal of the adsorption layer: Dissolve β-cyclodextrin in 50 mL of 0.5 mol / L NaOH aqueous solution, add 1 mL of epichlorohydrin, and stir to form a homogeneous solution. Immerse the cellulose membrane in the above solution and react at 55 °C for 4 hours. After the reaction is complete, remove the membrane and wash it successively with a large amount of deionized water, 0.01 mol / L HCl, and deionized water until neutral. Dry it under vacuum at 25 °C for 6 hours.

[0126] S2-2. Lower hybrid salt calibration layer: The glass cellulose membrane is immersed in a mixed solution of 10×SSC, 1.5% BSA and 0.2% PEG20000 for 5 minutes, and then freeze-dried at -40℃ for 12 hours.

[0127] S2-3. Double-layer composite: Same as Example 1.

[0128] S3. Preparation of the conjugate pad: S3-1. Preparation of Colored Latex-Probe Conjugates: Red latex microspheres were placed in a centrifuge tube and centrifuged at 12000 rpm for 20 minutes. The supernatant was discarded, and the microspheres were resuspended and washed twice with 0.1 M MES buffer. 3 mg EDC and 5 mg NHS were added, and the mixture was incubated at room temperature for 30 minutes for activation. After centrifugation and washing, the microspheres were resuspended in 0.05 M MES, and Pg and Tf detection probes were added to a concentration of 5 μg / mL, respectively. The mixture was incubated at room temperature for 4 hours. 0.2 M glycine was added for blocking for 30 minutes, followed by BSA blocking for another 30 minutes. After centrifugation and washing, the microspheres were resuspended in storage buffer until OD (450 nm) = 8.

[0129] S3-2. Spraying and Dotting: Equal volumes of Pg and Tf colored latex probes are mixed and sprayed onto the conjugate pad, then dried at 37°C for 2 hours. Pg and Tf probe concentrates are dotted onto the downstream independent probe storage areas respectively.

[0130] S4. Fabrication of microfluidic chips: S4-1. PMMA Injection Molding: With appropriate adjustment of process parameters, a dual-channel parallel arrow-shaped structure chip is formed.

[0131] S4-2-S4-6. Channel modification, NC strip embedding spot welding, detection line spraying, tracer dye spot loading: The operation is the same as in Example 1, wherein the NC strip aperture is 12μm, the spot welding temperature is 110℃, the micro-serration pitch is 1.8mm, and the detection line is sprayed with Pg and Tf capture probes respectively.

[0132] S5. Test strip assembly: Same as in Example 1, but the cutting width is still 4mm.

[0133] Example 5 Control Group: This embodiment provides a control test strip, whose structure differs from the technical solution of this invention in several key aspects, used to compare and demonstrate the performance improvement brought about by the core innovation of this invention. This embodiment is a control example; the test strip adopts a traditional lateral chromatography structure and does not include the arrow-shaped microfluidic unidirectional cutoff channel, NC strip micro-serrated flow guiding structure, mixing transition zone / slow flow buffer section / balanced microcolumn homogenization and diversion system, double-layer neutralizing isolation pad, and blue dextran tracer dye, which are the core features of this invention. It is used for parallel comparison experiments to verify the significant advantages of this invention's technical solution in terms of detection sensitivity, specificity, and ease of operation.

[0134] Materials and proportions: Sample pad: Glass cellulose membrane, same specifications as in Example 1, but only treated with a simple pretreatment solution and does not have simultaneous lysis and denaturation function. The maximum capacity of the sample pad is 200 μL.

[0135] Neutralizing and isolating pad: a single-layer glass cellulose membrane loaded with a 10×SSC buffer system and 1% BSA, without a guanidine salt removal adsorption layer and without a double-layer physical isolation structure.

[0136] Binding pad: The material is the same as in Example 1. The preparation method of the gold-labeled probe conjugate is the same as in Example 1, but the four probes are mixed and sprayed onto the entire binding pad without an independent probe reserve area. The downstream of the binding pad is directly connected to the NC membrane, without a mixing transition zone, a slow-flow buffer section, tracer dye, balance micropillars, or other structures.

[0137] Chromatography carrier: conventional NC membrane, directly adhered to PVC backing plate, without polymer microfluidic channel wrapping, without NC strip edge micro-serrated structure, without ultrasonic spot welding anchoring, without arrow-shaped unidirectional cutoff flow resistance structure, and without balanced microcolumn distribution of liquid flow.

[0138] Absorbent pad: Same as in Example 1.

[0139] Quality control and testing line setup: Four testing lines and one shared quality control line are sequentially sprayed onto the NC membrane along the same liquid flow direction. All testing lines are arranged in series within the same channel and share the same quality control line.

[0140] Preparation method: S1. Preparation of the sample pad: Cut glass cellulose fiber membranes into 15mm×4mm sizes, soak them in a simple pretreatment solution for 10 minutes, remove and drain, and dry at 37℃ for 12 hours for later use.

[0141] S2. Preparation of neutralizing isolation pad: Take a single-layer glass cellulose membrane, immerse it in a mixture of 10×SSC and 1% BSA for 5 minutes, remove and drain, and dry at 37℃ for 6 hours.

[0142] S3. Preparation of the conjugate pad: S3-1. Preparation of gold-labeled probes: Four types of gold-labeled probe conjugates were prepared using the same method as in Example 1.

[0143] S3-2. Spraying: Mix the four gold-labeled probes in equal proportions and spray them evenly onto the polyester fiber membrane at a rate of 100 μL / cm. Dry at 37°C for 2 hours. No separate probe storage area is provided.

[0144] S4. Preparation of NC membrane carriers: S4-1. NC membrane bonding: The conventional NC membrane is bonded to the corresponding position on the PVC backing board using double-sided adhesive, without grooves, spot welding anchoring, or micro-serrated flow guiding structure.

[0145] S4-2. Coating of Detection and Control Lines: Four detection lines and one shared control line are sequentially set on the NC membrane along the liquid flow direction, with a spacing of 2 mm between each line. Dry at 37℃ for 1 hour.

[0146] S5. Test strip assembly: In a clean environment, following standard lateral chromatography test strip assembly methods, the sample pad, neutralization isolation pad, conjugation pad, NC membrane carrier, and absorbent pad are sequentially overlapped onto a PVC backing substrate along the liquid flow direction, with an overlap length of 2 mm. No mixing transition zone, slow-flow buffer section, balancing microcolumn, tracer dye, or microfluidic channel structure is included. After rolling 2 kg strips three times, they are cut into 4 mm wide individual strips and stored in sealed aluminum foil bags.

[0147] Overview of differences between the control group and the experimental group: ; Performance comparison and verification: Take the control test strips from Examples 1-4 and Example 5, and use clinical saliva samples (containing the standard strain of Porphyromonas gingivalis ATCC 33277, at a concentration of 10) that have been determined by qPCR. 5Parallel testing was conducted using CFU / mL, and the evaluation indicators included: positive detection rate (sensitivity); uniformity of test line color development (coefficient of variation CV%); integrity of fluid flow propagation; and concordance rate between multiplex detection results and qPCR. The results showed that the positive detection rate of the test strips in Examples 1-4 was 95%-100%, the CV of the test line color development was <12%, the fluid flow completely covered the test line area, and the concordance rate of multiplex detection was >93%. In contrast, the positive detection rate of the control group test strips was only 60%-70%, the CV of color development was >30%, some test strips showed incomplete fluid flow or channeling, and multiplex detection results showed cross-interference. These comparative results fully validate the synergistic effect of the core technical features of this invention, including the arrow-shaped microfluidic unidirectional cutoff channel, NC strip micro-serrated flow guide, mixing transition zone-slow flow buffer section-balanced microcolumn homogenization and diversion system, double-layer neutralizing isolation pad, and tracer dye indication.

[0148] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A rapid saliva-based test strip for detecting periodontal pathogens, characterized in that, The test strip is sequentially composed of a sample pad, a neutralization isolation pad, a binding pad, a mixing transition zone, a slow-flow buffer section, a chromatography carrier, and an absorbent pad along the sample chromatography flow direction. The chromatography carrier is a semi-closed, arrow-shaped microfluidic chip integrally molded from thermoplastic polymers. The material is selected from polymethyl methacrylate, polycarbonate, polydimethylsiloxane, and cyclic olefin copolymers. The complete channel is prepared by hot embossing, ultraviolet nanoimprinting, or injection molding. The channel has an axisymmetric arc-shaped arrow-shaped sidewall structure, which forms an asymmetric flow resistance, allowing the liquid to flow in only one direction. Independent strip-shaped nitrocellulose capillary strips are embedded in the groove at the bottom of the channel. The NC strips are fitted with the groove with a 2%-3% gap and are anchored to the bottom of the groove by interval ultrasonic hot melt spot welding. The spot welding temperature is ≤150℃, and the longitudinal distance between the weld point and the detection line and quality control line is ≥2mm. Micro-serrated flow guiding structures with a pitch of 1-2mm are set on both sides of the NC strips to constrain the liquid flow to adhere to the NC membrane surface and eliminate sidewall channeling and liquid bypass loss. Multiple channels are arranged in parallel, and a balancing micro-column is set at the diversion inlet to ensure that the liquid in each channel is distributed synchronously and in equal amounts. The mixing transition zone is a gradually narrowing flow channel between the conjugate pad and the slow-flow buffer section, which homogenizes the sample and probe mixture through shear flow; a 2-5mm equal cross-section slow-flow buffer section is set between the downstream end of the mixing transition zone and the balance microcolumn; an independent probe reserve area is set for each channel downstream of the conjugate pad to reduce the difference in probe distribution between channels. The detection probe loaded with coupled signal markers on the binding pad specifically recognizes 16S rRNA of periodontal pathogens; a single detection line and a dedicated quality control line are sequentially arranged along the liquid flow direction in each target channel. The detection line fixes the capture probe, and the capture probe specifically binds to the detection probe-16S rRNA complex. The quality control line independently verifies the chromatographic patency and reagent activity of the corresponding channel. The test strip contains a built-in blue dextran tracer dye, which is pre-loaded in lyophilized form at the boundary between the slow-flow buffer section and the chromatography carrier at a concentration ≤0.1%. It enters the chromatography carrier with the liquid front and indicates that the liquid flows through the entire detection line area. The sample pad has a maximum effective load capacity of ≤150μL; the neutralization isolation pad is layered, with the upper layer being a guanidine salt removal and adsorption layer, and β-cyclodextrin derivatives covalently immobilized on the surface of cellulose fibers, relying on molecular inclusion to capture guanidine ions; The lower layer is a hybrid salt calibration layer, loaded with a 10×SSC high-salt buffer system and BSA; the two layers are physically isolated and work together to eliminate the guanidine salt separation inhibition effect.

2. The rapid saliva-based periodontal pathogen detection test strip according to claim 1, characterized in that, The arrow-shaped microfluidic channel is formed by connecting and splicing several arrow-shaped channel units end to end along the liquid flow direction; a single arrow-shaped channel unit is formed by an axisymmetric arc-shaped wall, the axis of symmetry of the arc-shaped wall is parallel to the liquid flow direction; the arc-shaped wall includes a first sidewall and a second sidewall; Along the forward flow direction of the liquid, the downstream end of the first sidewall and the downstream end of the second sidewall intersect tangentially, forming a guiding angle α towards the downstream of the fluid. This guiding angle allows the forward fluid to pass through with low resistance. The upstream end of the second sidewall intersects the arc of the upstream end of the first sidewall of the adjacent channel unit, forming a flow-blocking angle φ facing upstream of the fluid. This flow-blocking angle causes the reverse-flowing fluid to generate vortex high resistance. The arc-shaped wall structure of the main channel body is complete and independent, and is layered with the micro-serrated flow-guiding structure at the edge of the NC strip, with no functional interference between them.

3. The rapid saliva-based periodontal pathogen detection test strip according to claim 2, characterized in that, The guiding angle α is 15°-30°, resulting in low flow resistance when the fluid passes through in the forward direction. The flow-blocking angle φ is 120°-150°. When the backflowing liquid flows through this angle, turbulence is formed and the flow resistance is greatly increased. The asymmetric flow resistance difference between the two causes the channel to be cut off in one direction, preventing the liquid from flowing back.

4. The rapid saliva-based periodontal pathogen detection test strip according to claim 1, characterized in that, The inner wall of the microfluidic channel is activated by plasma surface treatment and chemically grafted with carboxyl or amino active modification layers to covalently stabilize the capture probe; nitrocellulose capillary strips are independently embedded inside the channel, and these strips are not synchronously imprinted with the polymer substrate; the NC strips are fixed to the bottom of the tank by interval ultrasonic hot melt spot welding, with a spot welding temperature ≤150℃, and the longitudinal distance between the weld point and the detection and quality control spraying area ≥2mm, so that the NC membrane pores are free from high-temperature thermal damage. The semi-enclosed arrow-shaped microfluidic channel forming process is selected from at least one of hot embossing, ultraviolet nanoimprinting, and injection molding; injection molding is suitable for integrally preparing channels from a whole substrate, while hot embossing and ultraviolet nanoimprinting are suitable for secondary processing of pre-made polymer films to form channels; the micro-serrated structure of the NC strip edge is processed by standard micro-milling and EDM; the mixing transition zone and the 2-5mm slow flow buffer section are integrally injection molded, and the flow splitting defect is eliminated.

5. The rapid saliva-based periodontal pathogen detection test strip according to claim 1, characterized in that, The signal marker is selected from at least one of colloidal gold nanoparticles, colored latex microspheres, fluorescent microspheres, and quantum dots; the detection probe is a specific oligonucleotide probe that targets the conserved target sequence of 16S rRNA of periodontal pathogens.

6. The rapid saliva-based periodontal pathogen detection test strip according to claim 1, characterized in that, The periodontal pathogens are selected from at least one of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacteractinomycetemcomitans.

7. The rapid saliva-based periodontal pathogen detection test strip according to claim 1, characterized in that, When conducting multiplex detection, the chromatography carrier is equipped with multiple parallel, independently arrow-shaped microfluidic target channels with completely symmetrical fluid resistance. A balancing microcolumn is installed at the diversion inlet, and a 2-5 mm equal-section slow-flow buffer section is placed in front to eliminate uneven liquid distribution caused by shear jet. An independent probe reserve area is set up in each channel in combination with the downstream pad. The shear flow mixing effect of the gradually narrowing mixing transition zone reduces the competition for probe supply and distribution differences between channels. Only one detection line and one dedicated quality control line are set in each independent target channel. Specific capture probes targeting the 16S rRNA of different periodontal pathogens are fixed on the detection lines of different channels. There is no cross-recognition binding activity between the various capture probes. The same amount of sample is injected into each channel simultaneously, and the detection is carried out without interference.

8. The rapid saliva-based periodontal pathogen detection test strip according to claim 1, characterized in that, The sample pad is thickened to create a lysis retention area, with a maximum liquid capacity of ≤150μL, and the entire sample is pretreated with lysis denaturation buffer. The lysis and denaturation buffer is free of SDS, proteinase K, lysozyme, EDTA, and EGTA, and consists of a ternary chemical lysis system: Triton X-100 at a concentration ≤0.5%, guanidine isothiocyanate, and a nucleic acid denaturant. The surfactant solubilization and guanidine salt separation work synergistically to destroy the Gram-negative outer membrane and cell membrane of periodontal pathogens and release intracellular 16S rRNA. The nucleic acid denaturant destroys the secondary stem-loop structure of 16S rRNA, exposing the hybridization target sequence, and there is no component compatibility antagonism. The neutralizing isolation pad has a double-layer physical separation structure: the β-cyclodextrin derivative in the upper guanidine salt removal adsorption layer is chemically cross-linked and covalently fixed to the pad fiber. The β-cyclodextrin does not fall off or migrate with the liquid, and relies on molecular inclusion to capture residual guanidine ions in the sample, and is not affected by competition from high sodium ions. The lower hybridization salt calibration layer is loaded with a 10×SSC high-salt buffer system and a high concentration of BSA. This layer independently regulates the ionic strength of the system to the optimal range for nucleic acid hybridization, and the BSA blocks free impurities. The bilayer structure synergistically eliminates the guanidine salt ionization inhibition effect, thus preventing hybridization failure. The sample pad lysis and denaturation buffer also contains a blocking agent selected from at least one of bovine serum albumin, casein, and polyvinylpyrrolidone. This blocking agent inhibits the non-specific adsorption of salivary proteins and miscellaneous nucleic acids.

9. A rapid detection method for periodontal pathogens based on saliva, characterized in that, The test strip according to any one of claims 1-8 comprises the following steps: S1. Quantitative sampling, constrained predilution, limited volume loading, and lysis-denaturation: Use the matching graduated capillary pipette to collect the subject's original saliva sample, with the target collection volume strictly controlled at 50-75 μL; during the collection process, visually confirm that the liquid level is stably within the 50-75 μL graduation range. If the sample volume is less than 50 μL or more than 75 μL, discard and resample; immediately after collection, transfer all the saliva in the tube to the matching diluent, and repeatedly blow and aspirate the tube wall more than 3 times to ensure that there is no sample residue on the wall; The dilution factor should be matched according to the actual collection volume, and the total volume after dilution should be strictly controlled to be ≤150μL: 50 μL of saliva can be diluted 3 times, and the total volume after dilution is 150 μL; 75 μL of saliva can only be diluted 2 times, and the total volume after dilution is 150μL; the dilution factor should be matched proportionally for intermediate sampling volumes of 50-75 μL. A quick reference table of sampling volume-dilution factor is attached to the accompanying consumables. After dilution and mixing, only 80-120 μL of the liquid is added dropwise to the lysis retention area of ​​the sample pad, and the remaining diluted sample is discarded. This 80-120 μL dropwise range is 53%-80% of the 150 μL capacity limit of the sample pad. This range ensures sufficient contact between the lysis buffer and the sample, appropriate reaction system concentration, and efficient bacterial lysis and nucleic acid release, while reserving a volume margin to avoid liquid overflow or flooding caused by operational fluctuations. The sample pad stably retains the sample, simultaneously completing the entire process of bacterial cell disruption, 16S rRNA release, and nucleic acid denaturation, without the need for separate nucleic acid extraction and amplification. A constant temperature device can be used to maintain 25-37℃ to accelerate the lysis reaction. S2. Layered Guanidine Salt Removal and Hybridization Calibration: The liquid carrying lysed components and denatured rRNA first flows through the upper guanidine salt removal adsorption layer of the neutralization isolation pad. Covalently immobilized β-cyclodextrin encapsulates and captures residual guanidine ions without shedding and contaminating the downstream. It then flows into the lower hybridization salt calibration layer. The 10×SSC buffer system calibrates the hybridization ion strength, and BSA blocks contaminating proteins. Nucleic acid hybridization inhibitors are eliminated layer by layer and stepwise to construct a stable hybridization microenvironment. S3. No dead volume probe hybridization and well-defined temporal boundaries: The neutralized sample liquid permeates into the downstream conjugate pad through capillary action, and mixes thoroughly with the labeled detection probe on the conjugate pad. The denatured and exposed target sequence of 16S rRNA undergoes base complementary pairing with the detection probe to generate a detection probe-target nucleic acid complex. This hybridization reaction is completed entirely within the conjugate pad and takes a maximum of 5-8 minutes. The mixed liquid continuously flows into the gradually narrowing mixing transition zone, and homogenization is achieved by shear flow. When the liquid reaches the boundary between the slow-flow buffer section and the chromatography support, the pre-loaded blue dextran tracer dye dissolves synchronously and migrates with the liquid front. The tracer dye only shows the liquid flow path and does not represent that the probe and target hybridization reaction is fully completed. There is no synchronous determination equivalence between the two. S4. Homogenization and split chromatography with controllable duration signal enrichment: The homogenized liquid after being homogenized by a 2-5mm slow-flow buffer section is evenly distributed to each symmetrical parallel target channel, eliminating the uneven liquid volume in the channel caused by the jet; the NC strip is fixed by low-temperature ultrasonic spot welding without any movement, and the micro-serrations on the edge continuously constrain the liquid to flow completely through the surface of the NC porous medium, avoiding the loss of sidewall channel bypass, while not damaging the unidirectional flow structure of the main channel; Under the action of capillary driving force, the liquid flows continuously through the detection line area. Due to the change in NC membrane pore size and the specific binding and retardation effect of the fixed capture probe on the line, the liquid flow naturally decelerates. The entire process of the liquid front flowing through the detection line area lasts for no less than 30-60 seconds. The natural fluid retardation effect allows the target-probe complex to be fully trapped and enriched, forming a stable and reproducible detection signal. The entire process is a spontaneous physicochemical process of chromatography, without the need for manual settling or timing intervention. S5. Quality control binding reaction: Free detection probes that have not bound to the target continue to migrate along a single channel to the downstream dedicated quality control line, specifically bind to the quality control molecule, and generate the corresponding quality control indicator band for the channel. The quality control results of each channel are evaluated independently. S6. Standardized stratified result interpretation and differentiated handling of failure causes: Simultaneously observe the migration range of tracer dye, the detection line of each target channel and the dedicated quality control line signal, and use the standard quality control colorimetric card to assist in interpretation; Judgment criteria: The tracer dye completely covers the entire detection line area; the effective channel is defined as the dedicated quality control line reaching the preset color development intensity threshold, which is compared with the matching standard colorimetric card, and the color development intensity is not lower than the minimum qualified color development intensity marked on the colorimetric card; Overall detection validity stratification: For multi-channel chips with a total number of channels ≥ 4, the entire detection system is considered valid when the number of valid channels ≥ 80% of the total number of channels; for chips with a total number of channels less than 4, the entire detection system is considered valid when all channels are valid, thus avoiding ambiguity in the fault tolerance rules caused by decimal rounding. Effective pathway: The presence or absence of bands on the test line is used to qualitatively determine whether the corresponding pathogen is negative or positive; the intensity of the band color development is used to semi-quantitatively determine the pathogen load; and results are issued when they are normal. Single-channel quality control is invalid, but overall testing meets the standards: The corresponding target result is uniformly marked as "invalid (local channel abnormality, it is recommended to retest this bacterial species)", and there is no need to discard the whole card. Instead, a single bacterial species test strip with a fixed target capture probe is used to retest the target separately, thereby preventing retest failure caused by mismatch of test strip models; if the same target is invalid in two consecutive retests, the test strip batch is determined to be defective; If the number of valid channels does not reach the corresponding channel number threshold, the overall test is deemed invalid due to sample / operational factors. It is necessary to resample quantitatively, match the dilution factor, and retest the entire card.

10. The rapid detection method for periodontal pathogens based on saliva according to claim 9, characterized in that, The tests were conducted in a constant temperature environment of 25-37℃; The hybridization reaction between the detection probe and denatured 16S rRNA in step S3 takes a maximum of 5-8 minutes; the liquid-based parallel channel chromatography migration and signal delay enrichment process in step S4 takes a maximum of 8-12 minutes. The total time for the two-step process to be added sequentially does not exceed 20 minutes, with no dead volume delay redundancy. If the ambient temperature is below 25°C, the hybridization and chromatography time can be appropriately extended or a constant temperature auxiliary device can be used to accelerate the reaction. The total time from the completion of saliva sample addition to the completion of valid result interpretation does not exceed 20 minutes.

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