Rapid detection kit and method for streptococcus suis and Graisseria parasuis based on dual LAMP-LFD technology

Abstract

The invention discloses a kit and a method for rapidly detecting streptococcus suis and Graisseria parasuis based on a dual LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) technology, and belongs to the field of molecular biological detection. According to the method, a double LAMP primer combination is used for amplifying a gdh gene of streptococcus suis and an infB gene of Graisseria parasuis, and rapid detection is carried out by combining a transverse flow test strip. The kit comprises a dual LAMP primer combination (SEQ ID No.1-SEQ ID No.8), and an LAMP reaction premix solution, a Bst II DNA polymerase, positive and negative controls, an amplification product diluent (ddH2O) and a transverse flow test strip which can be added according to requirements, and can also comprise internal standard DNA, a genome DNA degrading enzyme and specially designed nanoparticles. By means of the automatic temperature control device, the method can complete the whole process from sample treatment to result judgment within 45 minutes, and is suitable for on-site rapid detection. The invention provides an efficient, simple and convenient detection scheme, rapid diagnosis of the swine streptococcosis and the glaisseria parasuis can be achieved, and effective technical support is provided for prevention and control work of the swine streptococcosis and the glaisseria parasuis.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology detection, and specifically relates to a method for rapidly detecting Streptococcus suis and Glaseria parasuis based on dual LAMP-LFD (loop-mediated isothermal amplification-lateral flow test strip) technology, which is particularly suitable for rapid on-site diagnosis and pig herd health monitoring. Background Art

[0002] Streptococcus suis (SS) and Glaesserella parasuis (GPS) are common and important pathogens in pigs, widely causing clinical symptoms such as sepsis, polyserositis, meningitis, arthritis and pneumonia, and the pathological changes are similar. According to the difference in capsular polysaccharides, Streptococcus suis is divided into 35 serotypes (types 1 to 34 and 1 / 2), of which type 1, type 2, type 1 / 2, type 7, type 9 and type 14 are the main pathogenic serotypes, especially type 2, which is the most prevalent and harmful in pigs. There are as many as 15 serotypes of Glaesserella parasuis, and the serotypes of the prevalent strains in different places are different. The most common serotypes are types 4, 5 and 12, and there is a lack of immune cross-protection between these serotypes. Streptococcus suis and Glassera parasuis are prone to mixed infection. Due to similar symptoms, it is difficult to distinguish them clinically. Therefore, the development of a sensitive, specific and simple detection technology is of great significance for the rapid diagnosis and prevention of Streptococcus suis and Glassera parasuis.

[0003] The current diagnostic methods for Streptococcus suis and Glassera parasuis mainly include epidemiological detection, pathogen isolation and identification, molecular biological detection and serological detection. Among them, the pathogen isolation and identification method is cumbersome and time-consuming, and its clinical application is relatively limited; although conventional PCR technology and fluorescent quantitative PCR technology have high specificity and sensitivity, their equipment costs are high and require professional personnel to operate, which limits their application in clinical sites. Serological detection methods, such as agglutination tests and ELISA, are effective in some cases, but have high requirements for experimental materials and cannot detect Streptococcus suis and Glassera parasuis at the same time.

[0004] As a new type of molecular biological detection method, loop-mediated isothermal amplification (LAMP) has significant advantages such as simple operation, high sensitivity, and no need for complex equipment. Lateral flow test strips (LFD) have an intuitive and convenient way to observe results and are easy to apply on site. The dual LAMP-LFD detection method for Streptococcus suis and Glasseria parasuis has not been reported in the existing literature. Therefore, establishing a dual LAMP-LFD detection method that can simultaneously detect Streptococcus suis and Glasseria parasuis can not only greatly improve the efficiency of rapid pathogen identification, but also provide a more efficient and convenient technical means for the diagnosis, epidemiological investigation and epidemic prevention and control of swine streptococcal disease and Glasser's disease. Summary of the invention

[0005] The technical problem of the present invention is to provide a dual LA MP-LFD kit and a detection method thereof for rapid and accurate detection of Streptococcus suis and Glassera parasuis, so as to achieve rapid diagnosis of Streptococcus suis and Glassera parasuis pathogens, thereby providing technical support for timely prevention and control.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The invention discloses a method for detecting Streptococcus suis and Glassera parasuis based on dual LAMP-LFD, the core of which is to design a LAMP primer combination for Streptococcus suis and Glassera parasuis. The primer combination includes: LAMP primers for amplifying Streptococcus suis: SS-F3 (SEQ ID No. 1), SS-B3 (SEQ ID No. 2), SS-FIP (SEQ ID No. 3) and SS-BIP (SEQ ID No. 4); LAMP primers for amplifying Glassera parasuis: GPS-F3 (SEQ ID No. 5), GPS-B3 (SEQ ID No. 6), GPS-FIP (SEQ ID No. 7) and GPS-BIP (SEQ ID No. 8); wherein the 5' end marker of the primer is one or more of Biotin, 6-FAM, FITC or HRP to improve the sensitivity of detection and the signal visualization effect.

[0008] Furthermore, the primer combination achieves efficient amplification of Streptococcus suis and Glaseria parasuis through LAMP amplification reaction, wherein the 5' ends of SS-FIP and GPS-FIP are respectively labeled with Biotin, and the 5' ends of SS-BIP and GPS-BIP are respectively labeled with 6-FAM and Digoxigenin, further enhancing the specificity and visualization of detection.

[0009] The present invention also provides a detection kit based on dual LAMP-LFD, which contains the above-mentioned dual LAMP primer combination and includes the following components: LAMP reaction premix; Bst II DNA polymerase; amplification product dilution; lateral flow test strips; positive control and negative control; the above components ensure rapid and efficient detection of pathogenic nucleic acids.

[0010] The present invention also provides a method for detecting Streptococcus suis and Glassera parasuis based on dual LAMP-LFD, comprising the following steps: extracting genomic DNA from a sample to be detected; performing a LAMP amplification reaction using the LAMP primer combination described in claim 1; adding the amplification product to a lateral flow test strip, and judging whether the sample contains Streptococcus suis and Glassera parasuis according to the detection result of the test strip.

[0011] Furthermore, the reaction system of the LAMP amplification is: 2 μL of template DNA; 1.6 μL of primer combination; 5 μL of LA MP reaction premix; 2 μL of Bst II DNA polymerase; 14.4 μL of ddH2O; and the reaction procedure of the LAMP amplification is: 62°C, 40 minutes.

[0012] The specific composition of the primer combination is: SS-F3 0.1 μL, SS-B3 0.1 μL, SS-FIP 0.2 μL, SS-BIP 0.2 μL; GPS-F3 0.1 μL, GPS-B3 0.1 μL, GPS-FIP 0.4 μL, GPS-BIP 0.4 μL.

[0013] The present invention provides a detection method based on dual LAMP-LFD and a kit thereof, which has the following technical features:

[0014] (1) Innovative design of dual LAMP-LFD detection primers

[0015] One of the core innovations of the present invention is the design of dual LAMP primers. The primer combination includes specific LAMP primers designed for the gdh gene of Streptococcus suis and the infB gene of Glassera parasuis. The design of the primer combination takes into account the amplification ability of multiple serotypes of pathogens, and can effectively improve the detection accuracy of pathogens of different genotypes.

[0016] The LAMP primers of Streptococcus suis include SS-F3 (SEQ ID No.1), SS-B3 (SEQ ID No.2), SS-FI P (SEQ ID No.3) and SS-BIP (SEQ ID No.4); the LAMP primers of Glassera parasuis include GPS-F3 (SEQ ID No.5), GPS-B3 (SEQ ID No.6), GPS-FIP (SEQ ID No.7) and GPS-BIP (SEQ ID No.8).

[0017] These primers can efficiently and specifically amplify nucleic acids of Streptococcus suis and Glasseria parasuis, and can simultaneously detect two pathogens in the same reaction system. By optimizing the design of primers, the present invention can cover Streptococcus suis and Glasseria parasuis of different serotypes, thereby improving the breadth and accuracy of detection.

[0018] (2) Primer labeling optimization

[0019] Another innovative point is the labeling method of the primers. In order to further improve the sensitivity and visualization effect of the detection, the present invention labels the 5' end of the primer. Specifically, the 5' ends of the primers SS-FIP and GPS-FIP are respectively labeled with Biotin, and the 5' ends of SS-BIP and GPS-BIP are respectively labeled with 6-FAM (fluorescent label) and Digoxigenin (DIG label). These markers play a key role in the identification and detection of LAMP amplification products.

[0020] The combination of fluorescent markers such as Biotin and 6-FAM enables the lateral flow test strip to visually display the amplification results through color changes; DIG labeling further enhances specificity, and through the combination of different markers, two pathogens can be detected in the same reaction. This labeling optimization effectively improves the detection sensitivity, accuracy and convenience of the kit, and is particularly suitable for rapid on-site detection.

[0021] (3) Design of dual LAMP-LFD kit

[0022] The present invention further provides a kit for detecting Streptococcus suis and Glaseria parasuis based on dual LAMP-LFD, the kit comprising the above-mentioned LAMP primer combination and having the following innovative features:

[0023] LAMP reaction premix: The kit includes an optimized LAMP reaction premix, which can work stably under normal field environmental conditions to ensure the high efficiency of the reaction system; Bst II DNA polymerase: Bst II DNA polymerase with high-efficiency amplification performance is used to ensure that the LAMP amplification reaction is completed in a shorter time; Amplification product diluent: used to optimize the detection process of amplification products to avoid interference caused by excessive products; lateral flow test strips: The test strips adopt a surface enhancement design to enhance the signal of the amplification product, making the results more visual and improving the stability of the signal; positive and negative controls: To ensure the reliability of the results, the kit also includes positive and negative controls to ensure the accuracy of the detection process.

[0024] These components included in the kit enable the kit to not only quickly complete the detection of Streptococcus suis and Glasseria parasuis, but also have strong adaptability to field applications, and are particularly suitable for detection needs under different environmental conditions.

[0025] (4) Optimization of dual LAMP-LFD detection method

[0026] In the detection method, the present invention also adds the following innovative optimizations:

[0027] Adaptive temperature control device: The LAMP amplification reaction adopts a new type of high-efficiency heat source temperature control device, which can automatically adjust the reaction temperature to adapt to different samples and environmental conditions, improve the stability of the reaction, and avoid experimental errors caused by environmental temperature fluctuations.

[0028] Rapid detection process: The method of the present invention can complete the entire process from sample processing to result determination within 45 minutes, which is particularly suitable for on-site rapid detection applications. This optimization significantly shortens the detection time of traditional PCR methods, making on-site diagnosis more efficient and convenient.

[0029] (5) Nanoparticle signal enhancement technology

[0030] To further improve the detection sensitivity, the present invention introduces specially designed nanoparticles into the kit to enhance the reaction area signal on the lateral flow test strip. The nanoparticles can significantly improve the sensitivity of low-concentration pathogen detection, ensuring that pathogen nucleic acids can still be accurately detected even when the pathogen concentration is low. This innovation greatly improves the sensitivity and accuracy of the detection, and is particularly suitable for rapid on-site detection and large-scale screening.

[0031] (6) Portable equipment that does not require professional operation

[0032] The kit of the present invention also includes a portable device that can automatically complete DNA extraction, LAMP amplification, lateral flow test strip testing and result reading of the sample, reducing manual operations and improving the efficiency and accuracy of the test. In addition, the device has a wireless data transmission function and can upload the test results to a cloud database in real time, facilitating remote monitoring and data management.

[0033] With this device, users can complete the detection process without professional knowledge. It is suitable for use in pig farms, epidemiological surveys and other scenarios, which improves the universality and practicality of the method.

[0034] (7) Comprehensive application of the method

[0035] The detection method of the present invention can complete the rapid and accurate detection of Streptococcus suis and Glasseria parasuis without the need for professional operators, and has strong adaptability, and is particularly suitable for use in non-laboratory environments such as pig farms and epidemiological survey sites.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] The present invention adopts dual LAMP technology combined with lateral flow test strips, which can simultaneously detect Streptococcus suis and Glasseria parasuis, and the detection results are simple and sensitive; through label optimization, the visualization effect of the signal is improved, so that the detection method can quickly complete the diagnosis on site; the kit is complete in design and easy to operate, suitable for clinical field application, and is particularly suitable for early detection and prevention and control of Streptococcus suis and Glasseria parasuis.

[0038] The present invention realizes the rapid differential diagnosis of Streptococcus suis and Glassera parasuis pathogens by developing a dual LAMP-LFD kit and a detection method thereof, and provides effective technical support for the prevention and control of Streptococcus suis and Glassera parasuis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Example of double LAMP-LFD result judgment

[0040] Figure 1 An example of the result judgment of the dual LAMP-LFD test is shown. A: Positive result, indicating that the sample contains nucleic acids of Streptococcus suis and Glasseria parasuis. B: Negative result, indicating that the sample does not contain nucleic acids of Streptococcus suis and Glasseria parasuis. C: Invalid result, which may be caused by improper operation or reagent problems.

[0041] Figure 2 : Optimization results of SS (A, C, E) and GPS (B, D, F) LAMP reaction conditions

[0042] Figure 2The optimization results of LAMP reaction conditions for Streptococcus suis (SS) and Glassera parasuis (GPS) are shown. M: DL2000 DNA Marker. A and B: Temperature optimization results, 1-8 are temperature settings of 60℃ to 65℃, and 9 is a ddH2O control. C and D: Time optimization results, 1-6 are 20, 25, 30, 35, 40, and 45 minutes of reaction time, and 7 is a ddH2O control. E and F: Primer concentration ratio optimization, 1-6 are concentration ratios of 1:1, 1:2, 1:4, 1:6, 1:8, and 1:10, respectively, and 7 is a ddH2O control.

[0043] Figure 3 :PCR amplification results of recombinant plasmids pMD-SS and pMD-GPS

[0044] Figure 3 The results of PCR amplification using the recombinant plasmids pMD-SS and pMD-GPS as templates are shown. M: DL2000 DNA Marker. 1: PCR amplification results using the pMD-SS recombinant plasmid as template. 2: PCR amplification results using the pMD-GPS recombinant plasmid as template.

[0045] Figure 4 :Dual LAMP-LFD sensitivity detection using recombinant plasmid standards pMD-SS and pMD-GPS as templates

[0046] Figure 4 The sensitivity experiment of dual LAMP-LFD detection using recombinant plasmid standards pMD-SS and pMD-GPS as templates is shown. 1-7: Concentration gradient of pMD-SS and pMD-GPS, from 106 to 100 copies / μL. This figure shows the detection sensitivity of the dual LAMP-LFD kit at different concentrations, proving that it can detect pathogenic nucleic acids as low as 10 copies / μL.

[0047] Figure 5 :Dual LAMP-LFD detection of simulated tissue samples

[0048] Figure 5 The results of the dual LAMP-LFD kit in simulated pig lung tissue samples are shown. 1: Pig lung tissue without Streptococcus suis and Glassera parasuis. 2-6: Streptococcus suis 181.85×10 5 CFU and Glassera parasuis 222.2×10 5 CFU. This figure verifies the detection ability of the dual LAMP-LFD kit in simulated tissue samples with different concentrations.

[0049] Figure 6 :Dual LAMP-LFD kit specific detection

[0050] Figure 6 The results of the dual LAMP-LFD kit for specific detection of different pathogens are shown. 1-6: Streptococcus suis types 1, 2, 7, 9, 14, 16. 7-8: Haemophilus parasuis types 5, 4. 9: Enterococcus faecalis. 10: Streptococcus agalactiae. 11: Pasteurella multocida. 12: Actinobacillus pleuropneumoniae. 13: Mycoplasma hyorhinis. 14: Streptococcus pyogenes. 15: Streptococcus pneumoniae. 16: Haemophilus parasuis type 12. 17: Positive control. 18: Negative control.

[0051] Figure 6 The test kit was demonstrated to have high specificity for Streptococcus suis and Glasserella parasuis, and had no cross-reaction with other common pathogens.

[0052] Figure 7 :Detection of dual LAMP-LFD kit in different instruments

[0053] Figure 7 The performance of the dual LAMP-LFD kit in different detection instruments is shown. The figure illustrates the consistency of the kit's detection results in common experimental equipment (such as PCR instruments, metal baths, water baths, etc.), demonstrating the multi-device adaptability and stability of the kit. DETAILED DESCRIPTION

[0054] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0055] The present invention provides a rapid detection kit and method for Streptococcus suis and Glassera parasuis based on dual LAMP-LFD technology. In the embodiments, various serotypes of Streptococcus suis and Glassera parasuis and other common pig pathogens are used as controls for experimental verification to ensure the sensitivity, specificity and stability of the dual LAMP-LFD kit.

[0056] The bacterial strains used in this example are all stored and supplied by the China Veterinary Drug Administration, specifically including: Streptococcus suis: type 1 CVCC2937 strain, type 2 CVCC9740 strain, type 7 CVCC563 strain, type 9 CVCC989 strain, type 14 CVCC 212 strain, and type 16 CVCC223 strain.

[0057] Glassella parasuis: type 4 strain CVCC156, type 5 strain CVCC167, type 12 strain CVCC134.

[0058] Other common pig pathogens: Mycoplasma hyorhinis strain CVCC679, Enterococcus faecalis strain CVCC1927, Streptococcus agalactiae strain CVCC586, Pasteurella multocida strain CVCC390, Actinobacillus pleuropneumoniae strain CVCC259, Streptococcus pyogenes strain CVCC593, and Streptococcus pneumoniae strain CVCC1929.

[0059] The above bacterial strains were used to verify the specificity, sensitivity and wide applicability of the kit of the present invention.

[0060] Example 1 Design of LAMP primer set

[0061] The selection of target genes is one of the important factors that affect the success of LAMP detection. The present invention selects the gdh gene of Streptococcus suis and the infB gene of Glassera parasuis as target genes. The selection basis of these two genes and the corresponding LAMP primer design are described in detail below:

[0062] Selection of gdh gene of Streptococcus suis: The gdh gene of Streptococcus suis encodes the key virulence factor of the pathogen, glutamate dehydrogenase (GDH). This gene shows extremely high conservation in different serotypes of Streptococcus suis, with sequence similarity as high as 96% to 100%. Therefore, the gdh gene is an ideal target gene for rapid diagnosis of Streptococcus suis.

[0063] Selection of infB gene of G. parasuis: The infB gene of G. parasuis is its specific marker gene and has been widely used in species phylogeny studies. In molecular identification, the infB gene can effectively distinguish G. parasuis from other related species. Existing studies have shown that the infB gene is highly conserved in G. parasuis and is an ideal diagnostic target.

[0064] Based on the high conservatism and specificity of these two genes, the present invention designs two pairs of LAMP primers for gdh and infB genes. Each pair of primers includes an outer primer and an inner primer, and the inner primer is labeled to enhance the sensitivity and visualization of the detection. The specifically designed LAMP primer set is shown in the following table:

[0065] Table 1 Two pairs of LAMP primer sets

[0066]

[0067] Streptococcus suis primer set: SS-F3 and SS-B3 are outer primers used to initiate the amplification reaction of the gdh gene.

[0068] SS-FIP and SS-BIP are inner primers, labeled with Biotin and 6-FAM, respectively. These markers help to improve the sensitivity and visualization of the detection and ensure the accurate detection of LAMP reaction products.

[0069] Primer set for G. parasuis: GPS-F3 and GPS-B3 are outer primers used to initiate the amplification reaction of the infB gene.

[0070] GPS-FIP and GPS-BIP are internal primers, labeled with Biotin and Digoxigenin, respectively, which further improve the specificity and sensitivity of the detection.

[0071] Through the above design, the LAMP primer combination of the present invention can efficiently and specifically amplify the nucleic acid sequences of Streptococcus suis and Glaseria parasuis. When performing LAMP amplification, it can achieve simultaneous detection of the two pathogens, and intuitively determine whether the two pathogens are present through the test results of the lateral flow test strip.

[0072] Primer labeling: Different labels (Biotin, 6-FAM, and Digoxigenin) were introduced on the inner primers (SS-FIP and GPS-FIP) and inner primers (SS-BIP and GPS-BIP) to enhance the visualization of the amplification products and make the detection results more intuitive and accurate.

[0073] Dual detection capability: By performing dual LAMP amplification on two pathogens (Streptococcus suis and Glassera parasuis), the present invention can simultaneously detect two pathogens in the same reaction system, greatly improving the detection efficiency and reducing the detection time and cost.

[0074] The above design ensures the application of the present invention in clinical sites, and can effectively and quickly detect Streptococcus suis and Glaseria parasuis, thereby improving the detection sensitivity, specificity and ease of operation.

[0075] Example 2 Optimization of LAMP reaction temperature, reaction time and primer concentration ratio

[0076] This example aims to improve the sensitivity and accuracy of the dual LAMP-LFD detection method by optimizing the temperature, reaction time and primer concentration ratio of the LAMP reaction. These parameters are optimized using the single control variable method to ensure that the reaction conditions are suitable for the efficient amplification of Streptococcus suis and Glasera parasuis.

[0077] LAMP reaction temperature optimization: In the LAMP amplification process, the reaction temperature is one of the key factors affecting the amplification efficiency and specificity. In order to optimize the reaction temperature, the temperature range of 60°C to 65°C was set in this embodiment. By optimizing the temperature conditions, the ladder bands of the LAMP amplification products were observed and the amplification effect was evaluated. The experimental results showed that when the reaction temperature was 62°C, the LAMP amplification products showed clear and typical ladder bands, and the amplification effect was the best (see Figure 2 (A, B).

[0078] LAMP reaction time optimization: Reaction time is an important parameter that determines the stability of the amplified product and the reaction efficiency. In order to optimize the reaction time, this example set a reaction time of 20 minutes to 45 minutes, and evaluated the quality and stability of the amplified product. The experimental results showed that when the reaction time was 40 minutes or more, the LAMP amplified product tended to be stable, and the bands were clearly visible (see Figure 2 (C, D).

[0079] Optimization of primer concentration ratio: The primer concentration ratio in the LAMP reaction has an important influence on the sensitivity and specificity of the amplification. By optimizing the concentration ratio between the outer primers (F3, B3) and the inner primers (FIP, BIP), the efficiency and specificity of the amplification reaction can be improved. This embodiment sets different primer concentration ratios (F3:FIP and B3:BIP), which are 1:1, 1:2, 1:4, 1:6, 1:8, and 1:10, respectively, and compares them. The experimental results show that when the primer concentration ratio is 1:4, the LAMP amplification band effect is the best, the band is clearer, and the amplification effect is more ideal than other concentration ratios (see Figure 2 (E, F).

[0080] Final optimization of the dual LAMP reaction system: Based on the above optimization experimental results, the optimal LAMP reaction system and reaction procedure for dual LAMP-LFD detection of Streptococcus suis and Glassera parasuis were finally determined. The final reaction system configuration is: template DNA: 2μL (sample DNA / positive control / negative control); primer combination: 1.6μL (SS-F3 0.1μL, SS-B3 0.1μL, SS-FIP 0.2μL, SS-BIP 0.2μL, GPS-F3 0.1μL, GPS-B3 0.1μL, GPS-FIP 0.4μL, GPS-BIP 0.4μL); LAMP reaction premix: 5μL, Bst II DNA polymerase: 2μL, ddH2O: 14.4μL; reaction procedure: 62℃, 40 minutes.

[0081] Lateral flow test strip detection: After the reaction is completed, take 5-10 μL of LAMP amplification product, dilute it 20 times with diluent, mix it well, take 80 μL of the diluted reaction product and drop it into the sample well of the lateral flow test strip, and record the test results in the reading area on the test strip within 15 minutes.

[0082] The results of the test strips are interpreted according to the following standards: If positive bands appear in the quality control area (C), **test area 1 (T1) and test area 2 (T2), the sample to be tested contains nucleic acids of Streptococcus suis and Glaseria parasuis; If positive bands appear in the quality control area (C) and **test area 1 (T1), and no bands appear in test area 2 (T2), the sample to be tested contains only nucleic acids of Streptococcus suis; If positive bands appear in the quality control area (C) and **test area 2 (T2), , and no band appears in detection zone 1 (T1), the sample to be tested contains only the nucleic acid of Glassera parasuis; if a positive band appears in the quality control zone (C), and no band appears in detection zone 1 (T1) and detection zone 2 (T2), the sample to be tested does not contain nucleic acid of Streptococcus suis and Glassera parasuis; if no positive band appears in the quality control zone (C), the test is invalid, and whether positive bands appear in detection zone 1 (T1) and detection zone 2 (T2) is considered invalid.

[0083] The structure and working principle of the lateral flow test strip: Sample pad: used to absorb and transfer sample liquid; Conjugate pad: contains antibodies or markers that bind to the amplification product; Nitrocellulose (NC membrane): serves as a carrier for reaction and signal formation; Absorption pad: used to absorb excess liquid, ensure liquid flow and facilitate the display of results.

[0084] On the test strip, the NC membrane's **Detection Zone 1 (T1) and Detection Zone 2 (T2)** are immobilized with anti-nucleic acid marker antibodies 1 and 2 that specifically bind to the amplified products of Streptococcus suis and Glassera parasuis, respectively, and are labeled with 6-FAM and Biotin and Digoxigenin and Biotin, respectively. **The quality control zone (C)** is used to determine whether the sample is sufficient and confirm whether the reaction is normal. If no band is displayed in the quality control zone, the test is considered invalid.

[0085] Through the optimization experiment of this embodiment, the dual LAMP reaction system and temperature control program were finally determined, and clear detection results can be obtained at different pathogen concentrations (including pathogen nucleic acids as low as 10 copies / μL), and accurate differential diagnosis of Streptococcus suis and Glassera parasuis is ensured. The dual LAMP-LFD detection method and kit of the present invention can quickly and easily complete the detection on site, and has extremely high application value.

[0086] Example 3 Construction of recombinant plasmid standards

[0087] This example aims to construct recombinant plasmid standards of Streptococcus suis and Glassera parasuis for sensitivity testing and standardization of dual LAMP-LFD detection. The target gene was amplified by PCR, and the amplified product was cloned into the pMD18-T vector to finally construct recombinant plasmids pMD-SS and pMD-GPS.

[0088] PCR amplification and recombinant plasmid construction: In order to construct the recombinant plasmid standard, we selected the gdh gene of Streptococcus suis and the infB gene of Glassera parasuis as target genes. These genes are highly conserved in the two pathogens and are ideal targets for LAMP detection.

[0089] PCR amplification was performed using the primers shown in Table 2. The specific primer sequences and amplified fragments are as follows:

[0090] Table 2 Primers for constructing recombinant plasmid standards

[0091]

[0092] Through PCR amplification, we successfully obtained the amplified products for the gdh gene and the infB gene. The amplified products were detected by 1% agarose gel electrophoresis, and the results showed that the sizes of the amplified fragments were consistent with the expectations, which were 1347 bp (gdh) and 1360 bp (infB) respectively (see Figure 3 ).

[0093] Recombinant plasmid construction: The PCR amplification product was recovered by a DNA gel recovery kit and connected to the pMD18-T vector. Then, it was transformed into competent E. coli cells and preliminarily screened by bacterial liquid PCR identification method. The correctness of the constructed recombinant plasmids pMD-SS and pMD-GPS was verified by sequencing.

[0094] Determination of recombinant plasmid concentration and calculation of copy number: In order to standardize the detection and perform sensitivity test, this example uses an ultraviolet spectrophotometer (NanoDrop) to determine the concentration of the two recombinant plasmids and converts them into copy numbers. The results show that the concentrations of the two recombinant plasmids pMD-SS and pMD-GPS are 1×1010 copies / μL, respectively, which provides a standardized template for subsequent sensitivity experiments.

[0095] Figure 3 :PCR amplification results of recombinant plasmids pMD-SS and pMD-GPS

[0096] Figure 3 The PCR amplification results of the recombinant plasmids pMD-SS and pMD-GPS are shown. M: DL 2000 DNA Marker, 1: pMD-SS recombinant plasmid amplification product, 2: pMD-GPS recombinant plasmid amplification product, the amplified target fragments are consistent with the expectations, 1347 bp (gdh) and 1360 bp (infB), respectively.

[0097] Through this example, we successfully constructed recombinant plasmid standards (pMD-SS and pMD-GPS) of Streptococcus suis and Glassera parasuis, and measured their concentrations and copy numbers. This standard provides a key reference for the sensitivity verification of the subsequent dual LAMP-LFD detection method and ensures the standardization and accuracy of the detection method.

[0098] Example 4 Construction of dual LAMP-LFD kit

[0099] This embodiment aims to construct a rapid detection kit and method for Streptococcus suis and Glaseria parasuis based on dual LAMP-LFD technology. The kit contains a series of optimized components, which can achieve rapid, simple and efficient pathogen detection, and is particularly suitable for rapid on-site detection.

[0100] The kit comprises at least the following main components: a dual LAMP primer combination (SEQ ID No. 1 to SEQ ID No. 8): the combination comprises specific primers for the gdh gene of Streptococcus suis and the infB gene of Glassera parasuis, and can simultaneously amplify the nucleic acids of the two pathogens in the same reaction system.

[0101] LAMP reaction premix: contains the buffer, Mg 2+ Ions, dNTPs and other components provide the necessary chemical environment for LA MP amplification reaction.

[0102] Bst II DNA polymerase: used for DNA synthesis in amplification reactions. Bst II polymerase has good isothermal amplification properties and is suitable for LAMP reactions.

[0103] Positive control (Streptococcus suis and Glassera parasuis DNA): used to ensure the accuracy and reliability of the test. The positive control DNA is extracted from Streptococcus suis and Glassera parasuis, respectively, and can be used to confirm the effectiveness of the detection method.

[0104] Negative control (ddH2O): used to verify the background situation that the reaction system does not contain the target nucleic acid to ensure the accuracy and specificity of the kit.

[0105] Amplification product diluent (ddH2O): used to dilute the amplification product for subsequent lateral flow test strip detection.

[0106] Lateral flow test strips: used for visualization and interpretation of results. The test strips contain antibodies suitable for pathogen nucleic acid markers, which can visually display whether the sample contains nucleic acids of Streptococcus suis and Glaseria parasuis through color changes.

[0107] This kit is designed to be simple and can complete the entire process from sample processing to result reading on site. The specific steps include:

[0108] Sample preparation: Extract genomic DNA from the sample to be tested from the pig. The sample may include lung, meninges, joints or other tissue samples.

[0109] LAMP amplification reaction: The extracted DNA was added to the LAMP reaction system together with the dual LAMP primer combination, and an isothermal amplification reaction was performed at 62°C using Bst II DNA polymerase to amplify specific gene regions of Streptococcus suis and Glaseria parasuis.

[0110] Result detection: After the reaction is completed, add the amplified product to the sample well of the lateral flow test strip, and determine whether the target pathogen is contained by the reaction result of the detection area on the test strip. The test strip will display the result based on whether there is a corresponding positive band.

[0111] The dual LAMP-LFD kit of the present invention has the following innovations:

[0112] Dual LAMP primer combination: capable of simultaneously detecting Streptococcus suis and Glaseria parasuis. The dual LAMP amplification system saves time and cost and improves the detection throughput.

[0113] Optimized lateral flow test strip design: The test strip uses surface enhancement technology to achieve higher signal strength and better stability, thus ensuring accurate detection even at low pathogen concentrations.

[0114] No equipment required: The test kit is easy to operate and does not require complex equipment for on-site testing. It is suitable for rapid on-site testing, such as in pig farms and clinical diagnosis.

[0115] High sensitivity and high specificity: Through optimization of primer labeling, design of reaction system and enhancement of lateral flow test strips, the kit can obtain clear detection results even at pathogen nucleic acid concentrations as low as 10 copies / μL.

[0116] The following is an example of the components of the kit: dual LAMP primer combination (SS-F3, SS-B3, SS-FIP, SS-BIP, GPS-F3, GPS-B3, GPS-FIP, GPS-BIP): 1.6 μL in total; LAMP reaction premix: 5 μL; Bst II DNA polymerase: 2 μL; positive control (Streptococcus suis and Glaseria parasuis DNA): 1 μL; negative control (ddH2O): 1 μL; amplification product dilution (ddH2O): appropriate amount; lateral flow test strip: according to the configuration of the kit.

[0117] The kit is easy to use, can significantly shorten the detection time and improve the accuracy of on-site detection, and has broad application prospects.

[0118] Example 5 Determination of sensitivity of dual LAMP-LFD kit

[0119] This example aims to verify the sensitivity of the dual LAMP-LFD kit by performing sensitivity tests on positive plasmids with different concentration gradients to determine the lowest pathogen nucleic acid concentration that the kit can accurately detect.

[0120] In this experiment, the previously constructed recombinant plasmid standards pMD-SS (gdh gene of Streptococcus suis) and pMD-GPS (infB gene of Glassera parasuis) were used. These two positive plasmids were mixed in equal volumes and diluted 10-fold to obtain plasmid solutions of different concentrations. The plasmid concentration ranged from 1×10 7 copies / μL to 1×100 copies / μL.

[0121] Experimental steps:

[0122] Plasmid mixing and dilution: Take equal volumes of pMD-SS and pMD-GPS positive plasmids, mix them and dilute them 10-fold to generate plasmid solutions of different concentrations, ranging from 1×10 7 copies / μL to 1×100 copies / μL.

[0123] LAMP reaction: The mixed plasmid solutions of different concentrations were used as templates and added to the dual LAMP reaction system. Each reaction system was configured according to the previously determined optimization scheme, including: 2 μL template DNA, 1.6 μL primer combination, 5 μL LAMP reaction premix, 2 μL Bst II DNA polymerase, 14.4 μL ddH2O, the reaction program was set to 62°C, and the reaction time was 40 minutes.

[0124] Lateral flow test strip detection: After the reaction is completed, add the LAMP amplification product to the sample well of the lateral flow test strip and record the test results after 15 minutes. The intensity of the bands in the reaction area (T1 and T2) of the test strip is used to determine whether the sample contains the target pathogen nucleic acid.

[0125] The experimental results show that the dual LAMP-LFD kit can accurately detect pathogenic nucleic acids with a minimum concentration of 10 copies / μL, regardless of whether the mixed plasmids of Streptococcus suis or Glaseria parasuis are used. This indicates that the dual LAMP-LFD kit of the present invention has high sensitivity and can effectively detect pathogenic nucleic acids as low as 10 copies / μL.

[0126] Figure 4: Sensitivity results of dual LAMP-LFD detection

[0127] Figure 4 The sensitivity test results of the dual LAMP-LFD kit are shown. 1-7: The concentration gradient of pMD-SS and pMD-GPS ranges from 1×107 to 1×100 copies / μL.

[0128] The results showed that the kit could still accurately detect pathogenic nucleic acids at a concentration of 10 copies / μL. The test results verified the high sensitivity of the dual LAMP-LFD kit in the detection of low-concentration pathogenic nucleic acids, which is suitable for various field applications, especially in the rapid diagnosis of swine streptococcal disease and parasuis Glaser's disease.

[0129] Through the sensitivity test of this experiment, the dual LAMP-LFD kit of the present invention can reliably detect pathogenic nucleic acids of Streptococcus suis and Glasera parasuis as low as 10 copies / μL, demonstrating the high sensitivity and effectiveness of the kit in rapid on-site diagnosis.

[0130] Example 6 Dual LAMP-LFD kit sensitivity determination in simulated tissue samples

[0131] This example aims to verify the detection sensitivity of the dual LAMP-LF D kit in actual samples by using simulated tissue samples of Streptococcus suis and Glassera parasuis. The sensitivity and effectiveness of the detection kit for pathogens were tested by adding different concentrations of Streptococcus suis and Glassera parasuis bacterial liquid to the simulated samples.

[0132] First, Streptococcus suis and Glaseria parasuis were inoculated into different culture media: Streptococcus suis: inoculated into 10 mL of tryptic soy broth (TSB) containing 5% newborn calf serum. Glaseria parasuis: inoculated into 10 mL of TSB medium containing 5% newborn calf serum and 0.1% NAD. The two bacterial cultures were cultured at 37°C and 180 r / min for 12 hours. After culture, the bacterial cultures were diluted to 10 using a continuous 10-fold dilution method. -7 According to the experimental requirements, different concentrations of bacterial solution (10 -5 , 10 -6 , 10 -7 ) and dripped onto TSA medium (TSA) containing 5% newborn calf serum and TSA medium containing 5% newborn calf serum and 0.1% NAD. Use a bacterial stick to evenly spread the bacterial solution on the medium, continue to culture at 37°C for 12 hours, and then count the colonies. The experiment was repeated three times to ensure the reliability of the results.

[0133] Through culture and colony counting, we obtained the bacterial count results of Streptococcus suis and Glaseria parasuis at different dilutions, as shown in the following table:

[0134] Table 3 Count results of Streptococcus suis and Parasuis glauca

[0135]

[0136] From Table 3, we can see that in 10 -7 At the dilution concentration of 1.3, the average colony count of Streptococcus suis was 2 CFU / mL, and the average colony count of Glassera parasuis was 5 CFU / mL. These results show that the dual LAMP-LFD kit can detect extremely low concentrations of pathogens.

[0137] 0.25 g of pig lung tissue sample was taken, ground and then added with 100 μL of diluted mixed bacterial solution of Streptococcus suis and Glaseria parasuis. Subsequently, genomic DNA was extracted using a commercial kit and then detected using the dual LAMP-LFD kit of the present invention.

[0138] The experimental results showed that when the dual LAMP-LFD kit was used to detect diluted samples, the minimum detected bacterial counts were 18 CFU (Streptococcus suis) and 22 CFU (Glaserella parasuis). This indicates that the dual LAMP-LFD kit has a high sensitivity to pathogens in simulated tissue samples and can effectively detect low concentrations of pathogens.

[0139] Figure 5 :Dual LAMP-LFD detection results of simulated tissue samples

[0140] Figure 5 The results of the dual LAMP-LFD kit on simulated tissue samples with different concentrations of Streptococcus suis and Glaseria parasuis were shown. The results showed that the kit was able to detect Streptococcus suis and Glaseria parasuis at 10 -7 Accurate detection results were still obtained at low concentrations, further verifying its high sensitivity.

[0141] Through sensitivity testing of simulated tissue samples, the dual LAMP-LFD kit of the present invention showed excellent sensitivity, and was able to accurately detect pathogenic nucleic acids as low as 18 CFU / mL (Streptococcus suis) and 22 CFU / mL (Glaserella parasuis), without interference from the complex background of tissue samples. The performance of the kit in actual samples further demonstrated its potential for rapid on-site diagnosis of Streptococcus suis and Glasserella parasuis.

[0142] Example 7 Dual LAMP-LFD Kit Specificity Detection

[0143] This example aims to verify the specificity of the dual LAMP-LFD kit, test whether it can accurately distinguish S. suis from G. parasuis, and exclude cross-reactions with other porcine pathogens. To this end, we selected multiple S. suis serotypes, common porcine pathogens, and recombinant plasmids as templates for dual LAMP-LFD detection.

[0144] In this experiment, nucleic acids of the following pathogens were used as templates: Streptococcus suis: type 1, type 2, type 7, type 9, type 14, type 16; Parasuis Glassera: type 4, type 5, type 12; other common swine pathogens: Mycoplasma hyorhinis, Enterococcus faecalis, Streptococcus agalactiae, Pasteurella multocida, Actinobacillus pleuropneumoniae, Streptococcus pyogenes, Streptococcus pneumoniae. The nucleic acids of these pathogens were used to verify whether the dual LAMP-LFD kit can specifically detect Streptococcus suis and Parasuis Glassera, and does not cross-react with other pathogens. Positive control: A mixture of recombinant plasmids pMD-SS (S. suis gdh gene) and pMD-GPS (Parsuis Glassera infB gene) was used. Negative control: ddH2O was used as a negative control to ensure the accuracy of the test results.

[0145] The required template DNA (nucleic acids of Streptococcus suis, Glasseria parasuis, and other pathogens) was added to each reaction system, and the LAMP reaction was performed using an optimized dual LAMP primer combination. The reaction temperature was 62°C, the reaction time was 40 minutes, and a lateral flow test strip was used for detection to determine whether the sample contained nucleic acids of Streptococcus suis or Glasseria parasuis.

[0146] The experimental results showed that the dual LAMP-LFD kit could accurately detect nucleic acids of Streptococcus suis and Glasserella parasuis, and other porcine pathogens such as Mycoplasma hyorhinis, Enterococcus faecalis, Streptococcus agalactiae, Pasteurella multocida, Actinobacillus pleuropneumoniae, Streptococcus pyogenes and Streptococcus pneumoniae showed no cross-reaction (see Figure 5 ).

[0147] These results indicate that the dual LAMP-LFD kit of the present invention has excellent specificity and can effectively distinguish Streptococcus suis, different serotypes of Streptococcus suis, and Glaseria parasuis from other common pig pathogens, thus ensuring the accuracy and reliability of the detection results.

[0148] Figure 5 :Specificity detection results of dual LAMP-LFD kit

[0149] Figure 5The specific detection results of the dual LAMP-LFD kit are shown, using nucleic acids of different S. suis serotypes and other common swine pathogens as templates, and the detection results are as follows: 1-6: S. suis types 1, 2, 7, 9, 14, and 16; 7-8: Glaseria parasuis types 4, 5, and 12; 9: Mycoplasma hyorhinis; 10: Enterococcus faecalis; 11: Streptococcus agalactiae; 12: Pasteurella multocida; 13: Actinobacillus pleuropneumoniae; 14: Streptococcus pyogenes; 15: Streptococcus pneumoniae; 16: positive control (pMD-SS and pMD-GPS mixture); 17: negative control (ddH2O).

[0150] As can be seen from the figure, the kit only shows positive bands in the detection area of ​​Streptococcus suis and Glaseria parasuis, and no signals are generated for other pathogens. This proves the specificity of the kit and its ability to accurately distinguish the target pathogens.

[0151] This experiment verified the high specificity of the dual LAMP-LFD kit, which can accurately detect Streptococcus suis and Glasserella parasuis without cross-reaction with other common swine pathogens. This kit provides a reliable and efficient tool for rapid on-site diagnosis of Streptococcus suis and Glasserella parasuis, and has broad application prospects.

[0152] Example 8 Repeatability and applicability test

[0153] This example aims to verify the repeatability and applicability of the dual LAMP-LFD kit. Through inter-batch and intra-batch tests, as well as comparison of reaction effects on different instruments, the stability and reliability of the kit in practical applications are further confirmed.

[0154] In order to verify the repeatability of the test kit, three different batches of diagnostic reagents were prepared in this embodiment. Each batch of reagents was prepared according to the standard operating procedure to ensure the consistency of the quality and concentration of the reagents. By performing intra-batch and inter-batch testing on these three batches of reagents, we can verify the consistency and reliability between different batches.

[0155] During the test, we used each batch of reagents to detect Streptococcus suis and Glaseria parasuis, and used positive and negative controls to ensure the accuracy of the experimental results. The experimental results showed that the test results of inter-batch tests and intra-batch tests were consistent, further verifying the good repeatability of the kit. Whether it was repeated tests in the same batch of reagents or comparisons between different batches of reagents, the results showed high consistency, indicating that the detection performance of the kit is stable and suitable for large-scale applications.

[0156] In order to verify the applicability of the dual LAMP-LFD kit in different devices, the same batch of reagents was tested on three common heating devices in this example: PCR instrument: a standard device commonly used in molecular biology experiments; metal bath: a common laboratory constant temperature device; water bath: a convenient device for isothermal amplification reactions.

[0157] The reaction temperature of each device was set to 62°C and the reaction time was 40 minutes, which is consistent with standard operation. The experimental results showed that the dual LAMP-LFD kit was able to successfully perform LAMP amplification on these three different devices, and the test results were consistent in the determination of the results of the lateral flow test strips. Whether in a PCR instrument, a metal bath or a water bath, the reaction effect of the kit was good, and the results were consistent and there was no difference.

[0158] Figure 7 :Detection results of dual LAMP-LFD kit in different instruments

[0159] Figure 7 The detection effect of the dual LAMP-LFD kit in different devices is shown. A, B, C: The results of the dual LAMP reaction in a PCR instrument, a metal bath, and a water bath, respectively. On these three devices, the LAMP amplification products can produce clear and obvious bands, and the results on the lateral flow test strips are consistent.

[0160] Through the verification of inter-batch and intra-batch tests and the applicability tests on different devices, this example shows that the dual LAMP-LFD kit has good repeatability and high applicability. The kit can work stably on different batches and different devices, providing consistent test results, ensuring the reliability and flexibility of the kit in practical applications. The stability and applicability of the kit make it have broad application prospects in field diagnosis and large-scale epidemiological surveys.

[0161] Example 9 Clinical Application

[0162] This example aims to verify the feasibility and accuracy of the dual LAMP-LFD kit in the clinical diagnosis of swine streptococcal disease and parasuis Glaserasis. By testing 22 clinical samples, the dual LAMP-LFD method of the present invention was compared with the test results of the reference national standard method (GB / T 19915.5-2005 "Multiple PCR Detection Method for Streptococcus suis Type 2" and GB / T 34750-2017 "Detection Method for Haemophilus parasuis") to evaluate its accuracy and reliability.

[0163] 22 different types of samples from pigs were collected from the clinic, including: pleural effusion, lung tissue, nasal swabs. For each sample, the dual LAMP-LFD kit and the reference national standard method (PCR) were used to detect Streptococcus suis (SS) and Glasseria parasuis (GPS), respectively. Dual LAMP-LFD detection method: The dual LAMP-LFD kit according to the present invention was used for detection, and the results were determined by LAMP amplification and lateral flow test strips. Reference national standard method: GB / T 19915.5-2005 "Multiple PCR Detection Method for Streptococcus suis Type 2" and GB / T 34750-2017 "Detection Method for Haemophilus parasuis" were used for comparison.

[0164] The experimental results showed that the results of the dual LAMP-LFD method and the national standard method were as follows: SS positive rate: 40.90% (9 / 22), consistent with the national standard method (40.90%). GPS positive rate: 50.00% (11 / 22), consistent with the national standard method (50.00%). Mixed infection rate (simultaneous infection of Streptococcus suis and Glaseria parasuis): 18.18% (3 / 22), consistent with the national standard method.

[0165] Table 4 Clinical sample test results

[0166]

[0167] *SS: GB / T 19915.5-2005 “Multiplex PCR Detection Method for Streptococcus suis Type 2”, *GPS: GB / T 34750-2017 “Detection Method for Haemophilus parasuis”.

[0168] By comparing the test results of 22 clinical samples, the following conclusions can be drawn: The dual LAMP-LFD method has completely consistent results with the national standard method, indicating that this method has the same accuracy as the traditional PCR method. The dual LAMP-LFD kit of the present invention can effectively and quickly detect Streptococcus suis and Glasseria parasuis, and is suitable for different types of clinical samples. The dual LAMP-LFD method provides a sensitive, specific and simple detection scheme, which can provide strong support for the early diagnosis and prevention of streptococcal disease and Glasseria parasuis, and is particularly suitable for rapid on-site detection.

[0169] The results showed that the dual LAMP-LFD detection method not only has high sensitivity and accuracy, but also is consistent with the existing standard method and has broad clinical application prospects.

Claims

1. A rapid detection kit and method for Streptococcus suis and Glaseria parasuis based on dual LAMP-LFD technology, characterized in that: The method comprises the following steps: (1) extracting genomic DNA of a sample to be detected; (2) using the DNA extracted in step (1) as a template, and using two sets of LAMP primers to perform a LAMP amplification reaction, wherein: the LAMP primers used to amplify Streptococcus suis are: SS-F3 (SEQ ID No. 1), SS-B3 (SEQ ID No. 2), SS-FIP (SEQ ID No. 3) and SS-BIP (SEQ ID No. 4); the LAMP primers used to amplify Glaseria parasuis are: GPS-F3 (SEQ ID No. 5), GPS-B3 (SEQ ID No. 6), GPS-FIP (SEQ ID No. 7) and GPS-BIP (SEQ ID No. 8); and the 5' end marker of the primer combination is one or more of Biotin, 6-FAM, FITC or HRP to enhance the sensitivity and visualization effect of detection; (3) combining the amplification product with a lateral flow test strip, and judging whether the sample to be detected contains Streptococcus suis or Glaseria parasuis according to the detection result of the test strip.

2. The method according to claim 1, characterized in that The primers in the LAMP amplification reaction system are designed to complement multiple sequence regions, providing amplification capabilities for multiple serotypes of Streptococcus suis and Glaseria parasuis, and the primer combination is optimized to improve the detection accuracy of pathogens of different genotypes.

3. A rapid detection kit and method for Streptococcus suis and Glaseria parasuis based on dual LAMP-LFD technology, characterized in that: The kit comprises: the LAMP primer combination as claimed in claim 1; a stable LAMP reaction premix solution capable of stably operating in a field environment with temperature fluctuations; and a surface-enhanced lateral flow test strip to enhance signal visualization and improve detection sensitivity.

4. The kit according to claim 3, characterized in that The kit also includes a genomic DNA degrading enzyme to ensure that the sample does not contain any non-specific DNA that may interfere with the detection, thereby improving the specificity of the kit.

5. The kit according to claim 3 or 4, characterized in that The kit further includes specially designed nanoparticles for enhancing the reaction area signal on the lateral flow test strip, and the nanoparticles can significantly improve the sensitivity of detection, especially in the detection of low-concentration pathogens.

6. A rapid detection kit and method for Streptococcus suis and Glaseria parasuis based on dual LAMP-LFD technology, characterized in that: The LAMP amplification reaction procedure includes: (1) using a new type of high-efficiency heat source temperature control device to automatically adjust the reaction temperature to adapt to different samples and environmental conditions; (2) after the amplification reaction is completed, directly combining the amplification product with an improved lateral flow test strip to analyze the presence of pathogenic nucleic acid through visual band detection; wherein the method can complete the entire process from sample processing to result determination within 45 minutes, and is particularly suitable for on-site rapid detection.

7. The method according to claim 6, characterized in that The reaction temperature range of the LAMP amplification is 60° C. to 65° C., and the specificity and sensitivity of the amplification product are optimized by adjusting the Mg2+ ion concentration in the amplification reaction.

8. The kit or method according to any one of claims 1 to 7, characterized in that The kit and method can complete rapid and accurate dual pathogen detection without the need for professional operation, and have strong adaptability for field application, especially when used in pig farms and epidemiological survey sites.

9. A rapid detection kit and method for Streptococcus suis and Glaseria parasuis based on dual LAMP-LFD technology, characterized in that: The kit further includes a portable device that can automatically complete DNA extraction, LA MP amplification, lateral flow test strip testing and result reading of the sample. The device can ensure the consistency and stability of the kit under various operating environments through an intelligent control system.

10. The kit according to claim 9, characterized in that The portable device has a wireless data transmission function and can upload the test results to a cloud database in real time, thereby realizing remote monitoring and data management of the test results of Streptococcus suis and Glasseria parasuis, and improving the efficiency of pig health management.

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