Amplification primers, kits, and applications for rapid detection of Salmonella.

Abstract

This invention discloses a primer, kit, and application for rapid detection of Salmonella. The detection kit consists of a rapid nucleic acid amplification reagent, a fluorescent immunochromatographic test strip, and a running buffer. The rapid nucleic acid amplification reagent includes a graphene-containing amplification buffer, Salmonella-specific amplification primers, and quality control materials. The fluorescent immunochromatographic test strip consists of a sample pad, a binding pad coated with a fluorescent microsphere antibody (a marker at one end of the DNA amplification product) conjugate, an NC membrane containing a detection line (a marker at the other end of the DNA amplification product) and a control line, absorbent paper, and a PVC adhesive plate. After the rapid nucleic acid amplification reaction is completed, the running buffer is added, mixed, and then dropped onto the sample application window of the test strip. After 2 minutes, the fluorescence signal is read using a fluorescence analyzer. The entire process can be completed within 30 minutes. This Salmonella rapid detection kit is simple, rapid, sensitive, and provides reliable results, making it beneficial for practical applications.

Description

Technical Field

[0001] This invention relates to an amplification primer, a kit, and a method for rapid detection of Salmonella, belonging to the technical fields of molecular biology detection technology. Background Technology

[0002] Salmonella is one of the most common Gram-negative bacteria causing food poisoning and gastrointestinal infections, containing over 2,600 serotypes capable of causing infection in a variety of hosts. Sources of Salmonella infection primarily include milk, eggs, meat (poultry, beef), vegetables, and fresh fruits, causing tens of millions of infections globally each year, resulting in significant economic and social losses. Furthermore, Salmonella is also a major pathogenic microorganism in animal feed and pet food. The safety of these feeds is not only crucial for animal health but also for the health of those handling animal-derived foods or pet food.

[0003] To reduce Salmonella outbreaks and diseases associated with food and feed products, multi-stage detection and control are necessary from farm to table. Currently, standard methods for Salmonella detection and identification mainly include bacterial isolation and biochemical identification. While culture-based bacterial isolation yields highly reliable results, the procedures are complex, time-consuming (typically 5-7 days), and cross-biochemical reactions can occur between different species within the Enterobacteriaceae family. Immunological methods, such as serological testing, are simple and rapid, but their sensitivity and specificity still need improvement. Highly sensitive and specific nucleic acid amplification methods, such as polymerase chain reaction (PCR), are widely used for detecting pathogens like Salmonella. However, the interpretation of test results mainly relies on traditional agarose gel electrophoresis, which requires the use of toxic nucleic acid dyes and other reagents, raising concerns about experimental safety. Real-time quantitative PCR and other technologies can quantitatively detect targets without the use of toxic reagents, but their high instrument and reagent costs have limited their widespread use in grassroots and practical testing work. Therefore, establishing a molecular analytical method that does not require expensive equipment and reagents and is suitable for point-of-care testing (POCT) of Salmonella is of great significance for effectively controlling the spread of Salmonella.

[0004] According to currently available literature and patents, nucleic acid-based Salmonella detection technologies include conventional PCR, quantitative real-time PCR, digital PCR, immunoPCR, isothermal amplification (such as loop-mediated isothermal amplification, recombinase polymerase amplification, etc.), and their derivative technologies. These methods all have their own limitations, such as reliance on agarose gel electrophoresis (which poses safety risks), strict operational requirements, high cost of detection reagents and instruments, and high requirements for primer design or the need for multiple primer screenings.

[0005] In recent years, colloidal gold test strip detection technology based on immune reactions has been gradually applied to nucleic acid detection, such as the colloidal gold method. This method is convenient and rapid, allowing for colorimetric interpretation; however, the complex structures of primer dimers and polymers in the amplification products can easily lead to false positive results. Therefore, effectively addressing the interference of complex structural products in the amplification products on the interpretation of the amplification products is one of the key aspects of test strip detection technology. Furthermore, to better facilitate the early detection of pathogens, the sensitivity of nucleic acid detection technology based on colloidal gold test strips urgently needs to be improved.

[0006] Therefore, there is an urgent need in this field for a rapid Salmonella test kit that is simple to operate, sensitive, fast, user-friendly, and reliable, as well as a non-diagnostic test method. Summary of the Invention

[0007] Purpose of the Invention: To address the problems of complex procedures, long time, high cost, limited application conditions, and failure to meet POCT requirements in the application of existing detection technologies for Salmonella detection, the first objective of this invention is to provide primers for rapid amplification of the Salmonella genome. The second objective of this invention is to provide a kit containing amplification primers for rapid detection of Salmonella. The third objective of this invention is to provide a method for rapid detection of Salmonella using the amplification primers or kit.

[0008] Technical solution: The amplification primers for rapid detection of Salmonella described in this invention are designed based on the specific conserved region of the Salmonella bcfC gene, including a forward primer bcf-F and a reverse primer bcf-R. The sequence of the forward primer bcf-F is shown in SEQ ID NO: 1, and the sequence of the reverse primer bcf-R is shown in SEQ ID NO: 2.

[0009] Furthermore, the 5' or 3' ends of both the forward and reverse primers can be labeled with markers, including phosphorylation, biotin, digoxigenin, amino, carboxyl, thiol groups, or primer-modified fluorescent groups, so that the amplified DNA double-stranded product contains different markers at both ends.

[0010] Furthermore, the primers can be modified with fluorescent groups including FITC, FAM, Cy3, Cy5, HEX, JOE, ROX, TAMRA, Fluorescein, TET, R110, or Texas Red.

[0011] Furthermore, the marker is preferably placed on the 5' end of the forward primer bcf-F or the reverse primer bcf-R.

[0012] The present invention provides a kit comprising the amplification primers for rapid detection of Salmonella as described in the present invention.

[0013] Furthermore, the kit also includes graphene, amplification buffer, Bst DNA polymerase, dNTPs, polyethylene glycol, betaine, enzyme-free water, positive control, and negative control.

[0014] Furthermore, the graphene includes graphene oxide, reduced graphene, graphene quantum dots, and functionalized modified graphene.

[0015] Furthermore, functionalized graphene includes single-layer or multi-layer graphene, porous graphene, graphene nanotubes, graphene nanosheets, nanographene powder, nitrogen-doped graphene, carboxylated graphene, aminographene, mercaptographene, or graphene film.

[0016] Furthermore, the molar ratio of the forward primer bcf-F to the reverse primer bcf-R in the kit is 1:1 to 1:2.

[0017] Furthermore, the concentrations of both the forward primer bcf-F and the reverse primer bcf-R are 0.1–30 μM.

[0018] Furthermore, the kit also includes fluorescent immunochromatographic test strips and running buffer.

[0019] Furthermore, the running buffer comprises 0.001–1 M of tris(hydroxymethyl)aminomethane (Tris), 0.001–0.5 M of ethylenediaminetetraacetic acid (EDTA), and 0.001–0.8 M of sodium chloride.

[0020] The present invention also includes a method for rapid detection of Salmonella, wherein the detection method utilizes the amplification primers for rapid detection of Salmonella described in the present invention or the kit described therein, and employs a fluorescence detection method to detect Salmonella.

[0021] Further, the method includes the following steps: adding the amplification primers for rapid detection of Salmonella described in this invention or the kit described in this invention to the test solution for amplification reaction; after the amplification reaction is completed, adding running buffer to the reaction tube, mixing by pipetting, allowing it to stand, and reading the values ​​using a fluorescence analyzer. If the fluorescence signal value is greater than 122, it indicates that the sample contains Salmonella; if the fluorescence signal value is less than or equal to 122, it indicates that the sample does not contain Salmonella.

[0022] Furthermore, during the amplification analysis reaction, the amplification primers are subjected to a two-step rapid temperature cycle. The first step is the auxiliary denaturation temperature, ranging from 70 to 78°C, and the second step is the amplification reaction temperature, ranging from 55 to 70°C. The reaction time for each step is 1 to 10 seconds. The number of cycles is 35 to 45.

[0023] Furthermore, the temperature of the first step is 74-76°C, the temperature of the second step is 58-62°C, and the reaction time of each step is 1-3 seconds.

[0024] Further, add 80–90 μL of running buffer to the reaction tube and let it stand for at least 2 minutes.

[0025] The primers and amplification method of this invention have significant advantages over existing technologies, including simplicity, speed, sensitivity, accuracy, user-friendliness, and suitability for point-of-care testing (POCT). Furthermore, this method can amplify target sequences of 20 or more nucleotides and exhibits no cross-reactivity with other enteropathogenic microorganism genomes. Primer design can be flexibly adjusted based on Salmonella serotype changes in the future, offering convenience, speed, and strong clinical applicability, possessing technical advantages not found in bacterial culture, immunological methods, or PCR.

[0026] The fluorescent detection kit and method for rapid detection of Salmonella described in this invention can complete the rapid detection of Salmonella nucleic acid within 30 minutes. It has advantages such as convenient operation, simplicity and speed, high sensitivity, strong specificity, digital interpretation, and suitability for point-of-care testing (POCT), which are beneficial for practical applications.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0028] (1) Short detection time: The amplification reaction only takes about 27 minutes, and the process from nucleic acid amplification to result determination can be completed within 30 minutes.

[0029] (2) Simple and safe operation: The amplification reaction can be completed by a conventional PCR instrument, and there is no need to observe the results through the complex and potentially dangerous agarose electrophoresis experiment.

[0030] (3) Effectively avoid potential false positive problems: The graphene-based reaction system can effectively avoid potential primer dimer interference with fluorescence signal.

[0031] (4) Supports visual judgment: The test strip test results can be directly observed using fluorescence imaging equipment for qualitative analysis.

[0032] (5) High sensitivity: Micro- and nano-sized fluorescent microspheres have significant advantages such as large specific surface area, good fluorescence stability and anti-photobleaching ability, which can effectively improve detection sensitivity compared with colloidal gold.

[0033] (6) High specificity: The primers of this invention amplify highly efficient and specific amplification, specifically amplifying only the Salmonella genome, without cross-reactivity with other intestinal microbial genomes.

[0034] (7) Wide applicability: The rapid nucleic acid amplification method and its detection kit have wide applicability. There are no special requirements for the target sequence. Conventional primers can be used for amplification, and the application prospects are broad.

[0035] (8) Suitable for point-of-care testing (POCT): This invention uses fluorescent immunochromatographic test strips for point-of-care quantitative testing, which is simple, fast and highly sensitive, and suitable for third-party medical testing institutions and grassroots point-of-care testing.

[0036] In summary, the Salmonella detection kit and detection method of the present invention have the advantages of short processing time, simple operation, and ability to amplify short sequences. They provide new technical support for the screening and detection of Salmonella by units such as food supervision, medical and health care, animal husbandry and veterinary medicine, disease prevention and control, and entry-exit inspection and quarantine. They have broad market prospects and significant social and economic benefits. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the structure of the fluorescent immunochromatographic test strip in this invention;

[0038] Figure 2 This is a diagram showing the experimental results of the candidate amplification primers for Salmonella in this invention;

[0039] Figure 3 This is a diagram showing the experimental results of primers designed based on the Salmonella bcfC and invA genes in this invention;

[0040] Figure 4 This is a diagram showing the verification results of the role of graphene material in the amplification reaction in this invention;

[0041] Figure 5 This is a schematic diagram of the fluorescence detection results of the positive control (Salmonella reference strain) in this invention;

[0042] Figure 6 This is a schematic diagram of the fluorescence detection results of the negative control (sterile double-distilled water) in this invention;

[0043] Figure 7 This is a comparison image of the dark-field imaging results of the Salmonella positive and negative control test strips in this invention;

[0044] Figure 8 This is a graph showing the repeatability results of the method for rapid detection of positive and negative controls in this invention.

[0045] Figure 9 This is a graph showing the sensitivity results of the Salmonella fluorescent quantitative test strip detection method of the present invention;

[0046] Figure 10 This is a diagram showing the specificity results of the Salmonella fluorescent quantitative test strip detection method of the present invention;

[0047] Figure 11This is a graph showing the anti-interference test results of the Salmonella fluorescent quantitative test strip detection method of the present invention. Detailed Implementation

[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0049] Example 1: Design of primers for rapid amplification of Salmonella

[0050] Based on the publicly available whole genome sequences of Salmonella (bcfC) from the GenBank database of the Center for Biotechnology Information (Salmonella Typhimurium_CP007581, Salmonella Enteritidis_CP099973, Salmonella Worthington_NZ_CP039503, Salmonella Agona_NZ_CP100736, Salmonella Pullorum_CP075028, Salmonella California_NZ_CP028900, Salmonella Infantis_NZ_CP019202), whole genome multiple sequence alignment analysis was performed using molecular biology tools such as BLASTn, BRIG, and DNASTAR to screen for Salmonella genome-specific conserved genes and sequences. Based on the screened conserved genes bcfC and invA, rapid amplification primers were designed using Primer Premier 5.0, Oligo 6.0, and the online tool NUPACK (http: / / www.nupack.org / ). The designed primers were further validated using BLASTn and Primer-BLAST searches. Primer information is as follows:

[0051] Table 1. Alternative rapid amplification primers designed based on specific conserved regions of Salmonella.

[0052]

[0053] Salmonella reference strain ATCC14028 (concentration approximately 5 × 10⁻⁶) 6Using the CFU / mL genome as a template, the 20 μL reaction system included: 2 μL of 10× amplification buffer (components included 200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.6–8.8), 2 μL of dNTPs (containing dGTP, dCTP, dATP, and dTTP, all at a concentration of 10 mM), 1 μL of liquid polyethylene glycol, 1 μL of betaine (10 mM), 2 μL each of forward primer bcf-F (10 μM) and reverse primer bcf-R (10 μM), 0.8 μL of Bst DNA polymerase (8000 U / mL), 1 μL of graphene dispersion (20 ng / μL), 1 μL of EvaGreen (20×), 3 μL of template, and 4.2 μL of enzyme-free water. Prepare the reaction mixture in miniature reaction tubes, and include a negative control (sterile double-distilled water). Use the primers listed in the table above for simultaneous experiments. After mixing the reaction mixture in each tube, briefly centrifuge and place the tubes in a standard PCR instrument. The reaction program is: 74℃ for 1 second, 59℃ for 1 second, for a total of 40 cycles. After the reaction, add 80 μL of running buffer to each tube, mix thoroughly by pipetting, and then add a drop of fluorescent immunochromatographic strip (e.g., [insert sample here]). Figure 1 (As shown in the image) Sample loading window. Let stand for 2 minutes, then read the fluorescence signal using a portable fluorescence analyzer. Results are as follows... Figure 2 and Figure 3 As shown.

[0054] Figure 2 This is a diagram showing the experimental results of the candidate amplification primers for Salmonella in this invention; Figure 3 This is a diagram showing the experimental results of primers designed based on the Salmonella bcfC and invA genes in this invention; (The diagram is from...) Figure 2 and Figure 3 It was observed that, except for the weak signal of the positive control amplification product from the second primer pair based on bcfC (bcfC-F2 / R2), both the first primer pair based on the bcfC gene (bcfC-F / R) and the primers for the invA gene effectively amplified the positive control, producing amplification products with high fluorescence signal values. Furthermore, the negative control product from the invA gene also exhibited a high fluorescence signal value, while the negative control product from the first primer pair based on the bcfC gene (bcfC-F / R) showed a lower fluorescence signal value (the fluorescence signal curve peak was close to the x-axis). Therefore, the first primer pair based on the bcfC gene (bcfC-F / R) is preferred as the amplification primer for subsequent experiments.

[0055] Example 2: Establishment of a rapid fluorescent detection method for Salmonella

[0056] (1) Verification of the role of graphene in the amplification reaction

[0057] To verify the role of graphene, this experiment used a graphene dispersion (20 ng / μL) for verification. The reaction system was divided into two groups:

[0058] Group without graphene: Salmonella reference strain ATCC14028 (concentration approximately 5 × 10⁻⁶) 6 Using a CFU / mL genome as a template, a 20 μL reaction system consisted of: 2 μL of 10× amplification buffer (components included 200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.6–8.8), 2 μL of dNTPs (containing dGTP, dCTP, dATP, and dTTP, all at 10 mM), 1 μL of liquid polyethylene glycol, 1 μL of betaine (10 mM), 2 μL each of primers F and R (10 μM), 0.8 μL of Bst DNA polymerase (8000 U / mL), 1 μL of Eva Green (20×), 3 μL of template, and 5.2 μL of enzyme-free water. The reaction system was prepared in microtubes, with a negative control (sterile double-distilled water) included. After mixing the reaction tube containing the reaction mixture, centrifuge briefly and place the tube in a standard PCR instrument. The reaction program is: 74℃ for 1 second, 59℃ for 1 second, for a total of 40 cycles. After the reaction, add 80 μL of running buffer to the reaction tube, mix well by pipetting, and then add a drop into the sample window of the fluorescent immunochromatographic test strip. Let stand for 2 minutes, and then read the fluorescence signal using a portable fluorescence analyzer.

[0059] The graphene-added group: Salmonella reference strain ATCC14028 (concentration approximately 5 × 10⁻⁶) 6Using a CFU / mL genome as a template, a 20 μL reaction system consisted of: 2 μL of 10× amplification buffer (components included 200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.6–8.8), 2 μL of dNTPs (containing dGTP, dCTP, dATP, and dTTP, all at 10 mM), 1 μL of liquid polyethylene glycol, 1 μL of betaine (10 mM), 2 μL each of primers F and R (10 μM), 0.8 μL of Bst DNA polymerase (8000 U / mL), 1 μL of graphene dispersion (20 ng / μL), 1 μL of Eva Green (20×), 3 μL of template, and 4.2 μL of enzyme-free water. The reaction system was prepared in microtubes, with a negative control (sterile double-distilled water) included. After mixing the reaction tube containing the reaction mixture, centrifuge briefly and place the tube in a standard PCR instrument. The reaction program is: 74℃ for 1 second, 59℃ for 1 second, for a total of 40 cycles. After the reaction, add 80 μL of running buffer to the reaction tube, mix well by pipetting, and then add a drop into the sample window of the fluorescent immunochromatographic test strip. Let stand for 2 minutes, and then read the fluorescence signal using a portable fluorescence analyzer.

[0060] The results are as follows Figure 4 As shown, Figure 4 This is a diagram showing the verification results of the role of graphene materials in the amplification reaction in this invention; (The diagram is from...) Figure 4 It was found that when the reaction system did not contain graphene, the amplification products of both the positive and negative controls exhibited high fluorescence signal values. When graphene was added to the reaction system, the fluorescence signals of the amplification products of both the Salmonella reference strain genome and the negative control decreased, and the negative control no longer showed a visible fluorescence peak (near the x-axis). These results indicate that the graphene-based reaction system can effectively enhance the contrast between positive and negative results in the method of this invention, which is beneficial for practical applications.

[0061] (2) Establishment of a rapid fluorescent detection method for Salmonella

[0062] Salmonella reference strain ATCC14028 (concentration approximately 5 × 10⁻⁶) 6Using a CFU / mL genome as a template, a 20 μL reaction system consisted of: 2 μL of 10× amplification buffer (components included 200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.6–8.8), 2 μL of dNTPs (containing dGTP, dCTP, dATP, and dTTP, all at 10 mM), 1 μL of liquid polyethylene glycol, 1 μL of betaine (10 mM), 2 μL each of primers F and R (10 μM), 0.8 μL of Bst DNA polymerase (8000 U / mL), 1 μL of graphene dispersion (20 ng / μL), 1 μL of Eva Green (20×), 3 μL of template, and 4.2 μL of enzyme-free water. The reaction system was prepared in microtubes, with a negative control (sterile double-distilled water) included. After mixing the reaction tube containing the reaction mixture, centrifuge briefly and place the tube in a standard PCR instrument. The reaction program is: 74℃ for 1 second, 59℃ for 1 second, for a total of 40 cycles. After the reaction, add 80 μL of running buffer to the reaction tube, mix well by pipetting, and then add a drop into the sample window of the fluorescent immunochromatographic test strip. Let stand for 2 minutes, and then read the fluorescence signal using a portable fluorescence analyzer.

[0063] like Figure 5 and Figure 6 As shown, Figure 5 This is a schematic diagram of the fluorescence detection results of the positive control (Salmonella reference strain) in this invention; Figure 6 This is a schematic diagram of the fluorescence detection results of the negative control (sterile double-distilled water) in this invention; Figure 5 and Figure 6 It can be seen that, using the optimized amplification primers based on the bcfC gene, the fluorescence signal value of the positive control can reach over 20,000, while the fluorescence signal value of the negative control is lower (close to the x-axis).

[0064] Figure 7 Dark-field imaging results after detecting the amplification products of the positive and negative controls using fluorescent immunochromatographic strips. For example... Figure 7 As shown, the test strip for the positive control shows bright bands on both the detection line and the control line, while the test strip for the negative control only shows a bright band on the control line. Experimental results indicate that the result interpretation method of this invention is beneficial for practical applications.

[0065] Example 3: Repeatability test of rapid fluorescent detection method for Salmonella

[0066] Salmonella reference strain ATCC14028 (concentration approximately 5 × 10⁻⁶) 6Using a CFU / mL genome as a template, a 20 μL reaction system consisted of: 2 μL of 10× amplification buffer (components included 200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.6–8.8), 2 μL of dNTPs (containing dGTP, dCTP, dATP, and dTTP, all at 10 mM), 1 μL of liquid polyethylene glycol, 1 μL of betaine (10 mM), 2 μL each of primers F and R (10 μM), 0.8 μL of Bst DNA polymerase (8000 U / mL), 1 μL of graphene dispersion (20 ng / μL), 1 μL of Eva Green (20×), 3 μL of template, and 4.2 μL of enzyme-free water. The reaction system was prepared in microtubes, with a negative control (sterile double-distilled water) included. After mixing the reaction tube containing the reaction mixture, centrifuge briefly and place the tube in a standard PCR instrument. The reaction program is: 74℃ for 1 second, 59℃ for 1 second, for a total of 40 cycles. After the reaction, add 80 μL of running buffer to the reaction tube, mix well by pipetting, and then add a drop into the sample window of the fluorescent immunochromatographic test strip. Let stand for 2 minutes, and then read the fluorescence signal using a portable fluorescence analyzer.

[0067] The results are as follows Figure 8 As shown in Table 2, Figure 8 This is a graph showing the repeatability results of the rapid detection of positive and negative controls using the method of the present invention. Figure 8 As shown in Table 2, after 10 amplification tests on the Salmonella reference strain ATCC14028 genome (positive control) and the negative control (sterile double-distilled water), the fluorescence signals of the amplification products of both samples were relatively stable (coefficients of variation were approximately 0.06 and 0.22, respectively), which is beneficial for practical applications. Meanwhile, the cutoff value (mean fluorescence signal of the negative control amplification product plus 3 times the standard deviation) was calculated to be 122 based on the fluorescence signal value of the negative control amplification product.

[0068] Table 2 Comparison of positive and negative control results of the rapid Salmonella detection method of this invention

[0069]

[0070] Note: CV represents the coefficient of variation.

[0071] Example 4: Sensitivity test of the Salmonella rapid fluorescent detection kit

[0072] This embodiment tests the sensitivity of the rapid fluorescent detection kit of the present invention to detect Salmonella, using Salmonella reference strain ATCC14028 from approximately 5 × 10⁻⁶. 6 CFU / mL was serially diluted 10-fold to 5 × 10⁻⁶.-1 CFU / mL. The reaction system and procedure are shown below:

[0073] The 20 μL amplification reaction system included: 2 μL of 10× amplification buffer (components included 200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.6–8.8), 2 μL of dNTPs (containing dGTP, dCTP, dATP, and dTTP, all at a concentration of 10 mM), 1 μL of liquid polyethylene glycol, 1 μL of betaine (10 mM), 2 μL each of primers F and R (10 μM), 0.8 μL of Bst DNA polymerase (8000 U / mL), 1 μL of graphene dispersion (20 ng / μL), 1 μL of EvaGreen (20×), 3 μL of template, and 4.2 μL of enzyme-free water. The reaction system was prepared in microtubes, with a negative control (sterile double-distilled water) included. After mixing the reaction tube containing the reaction mixture, centrifuge briefly and place the tube in a standard PCR instrument. The reaction program is: 74℃ for 1 second, 59℃ for 1 second, for a total of 40 cycles. After the reaction, add 80 μL of running buffer to the reaction tube, mix well by pipetting, and then add a drop into the sample window of the fluorescent immunochromatographic test strip. Let stand for 2 minutes, and then read the fluorescence signal using a portable fluorescence analyzer.

[0074] Test results as follows Figure 9 As shown, Figure 9 This is a graph showing the sensitivity results of the Salmonella fluorescent quantitative test strip detection method of the present invention. Figure 9 In the middle: the concentrations of nucleic acid standard templates corresponding to the fluorescence amplification curves are approximately 5 × 10⁻⁶. 6 CFU / mL, 5×10 5 CFU / mL, 5×10 4 CFU / mL, 5×10 3 CFU / mL, 5×10 2 CFU / mL, 5×10 1 CFU / mL and 5×100 CFU / mL, with fluorescence signal values ​​all greater than 122, while 5×10 -1 No obvious curve was observed (near the x-axis) for CFU / mL and the negative control, and the fluorescence signal value was less than 122. Therefore, the detection sensitivity of this kit is determined to be 5 × 100 CFU / mL, indicating high sensitivity and application value.

[0075] Example 5: Specificity test of the Salmonella rapid fluorescent detection kit

[0076] The following strains of Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, Salmonella typhimurium, Salmonella choleraesuis, Salmonella Indiana, Salmonella paratyphi B, Salmonella turkey, Salmonella Sanftenburg, Salmonella duck, Escherichia coli, Klebsiella pneumoniae, Shigella flexneri, Shigella sonnei, Proteus vulgaris, Proteus mirabilis, Staphylococcus aureus, Bacillus subtilis, and Pasteurella multocida reference (isolated) strains were cultured, genomic DNA was extracted, and rapid amplification was performed according to the optimized reaction system and reaction conditions determined in Example 2.

[0077] The 20 μL amplification reaction system included: 2 μL of 10× amplification buffer (components included 200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.6–8.8), 2 μL of dNTPs (containing dGTP, dCTP, dATP, and dTTP, all at a concentration of 10 mM), 1 μL of liquid polyethylene glycol, 1 μL of betaine (10 mM), 2 μL each of primers F and R (10 μM), 0.8 μL of Bst DNA polymerase (8000 U / mL), 1 μL of graphene dispersion (20 ng / μL), 1 μL of EvaGreen (20×), 3 μL of template, and 4.2 μL of enzyme-free water. The reaction system was prepared in microtubes, with a negative control (sterile double-distilled water) included. After mixing the reaction tube containing the reaction mixture, centrifuge briefly and place the tube in a standard PCR instrument. The reaction program was: 74℃ for 1 second, 59℃ for 1 second, for a total of 40 cycles. After the reaction, add 80 μL of running buffer to the reaction tube, mix well by pipetting, and then add a drop into the sample window of the fluorescent immunochromatographic test strip. Let stand for 2 minutes, and then read the fluorescence signal using a portable fluorescence analyzer. The results are as follows. Figure 10 As shown in Table 3.

[0078] Figure 10 This is a specificity result diagram of the Salmonella fluorescent quantitative test strip detection method of the present invention. Figure 10 As shown in Table 3, only 10 Salmonella reference strains showed specific fluorescence curves (fluorescence signal values ​​greater than 122), while the 9 non-Salmonella reference / isolated strains and the negative control showed no visible fluorescence curve peaks (fluorescence signal values ​​less than 122) (Table 3).

[0079] The results show that the rapid fluorescence detection method of the present invention has good specificity and high detection application value.

[0080] Table 3 Results of the test for the specificity evaluation of this invention

[0081]

[0082] Note: In the results column of the table, "+" indicates positive and "-" indicates negative.

[0083] Example 6: Interference resistance test of the Salmonella rapid fluorescent detection kit

[0084] Salmonella enteritidis ATCC13076, Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, and Shigella sonnei CMCC51592 were inoculated into brain and heart broth medium and cultured. The culture medium was then diluted 1000-fold with sterile double-distilled water, and bacterial genomic DNA was extracted using a commercially available bacterial genomic DNA extraction kit. The extracted genomic DNA was divided into two samples: Sample A was a 1:1:1:1 mixture of Salmonella enteritidis, Escherichia coli, Klebsiella pneumoniae, and Shigella sonnei reference strains; Sample B was a 1:1:1:1 mixture of Escherichia coli, Klebsiella pneumoniae, and Shigella sonnei reference strains. A negative control (sterile double-distilled water) was also included. Rapid amplification was performed according to the optimized reaction system and conditions determined in Example 2.

[0085] The 20 μL amplification reaction system consisted of: 2 μL of 10× amplification buffer (components included 200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.6–8.8), 2 μL of dNTPs (containing dGTP, dCTP, dATP, and dTTP, all at 10 mM), 1 μL of liquid polyethylene glycol, 1 μL of betaine (10 mM), 2 μL each of primers F and R (10 μM), 0.8 μL of Bst DNA polymerase (8000 U / mL), 1 μL of graphene dispersion (20 ng / μL), 1 μL of Eva Green (20×), 3 μL of template, and 4.2 μL of enzyme-free water. The reaction system was prepared in microtubes, with a negative control (sterile double-distilled water) included. After mixing the reaction tube containing the reaction mixture, centrifuge briefly and place the tube in a standard PCR instrument. The reaction program is: 74℃ for 1 second, 59℃ for 1 second, for a total of 40 cycles. After the reaction, add 80 μL of running buffer to the reaction tube, mix well by pipetting, and then add a drop into the sample window of the fluorescent immunochromatographic test strip. Let stand for 2 minutes, and then read the fluorescence signal using a portable fluorescence analyzer.

[0086] The results are as follows Figure 11 As shown, Figure 11 This is a graph showing the anti-interference test results of the Salmonella fluorescent quantitative test strip detection method of the present invention. Figure 11 As can be seen, the detection line of the amplified product of sample A shows a clear fluorescence signal, while the fluorescence signals of the detection lines of sample B and the negative control are weak (below 122). The experimental results show that the method of the present invention has good anti-interference ability.

[0087] Example 7: Assembly of the detection kit

[0088] The Salmonella-specific amplification primers designed based on the bcfC gene (the forward primer with biotin modified at the 5' end and the reverse primer with digoxigenin modified at the 5' end) as shown in Example 1 were synthesized by a biotechnology company. Both the forward and reverse primers were diluted to 10 μM with sterile double-distilled water and mixed in equal volumes to obtain the detection primers. Salmonella ATCC14028 (concentration not less than 5 × 10⁻⁶) was extracted using a commercially available bacterial genomic DNA extraction kit. 5 Genomic DNA (CFU / mL) was used as a positive control; nucleic acid amplification reagents included: 10× amplification buffer (components of 200mM Tris-HCl, 100mM (NH4)2SO4, 500mM KCl, 20mM MgSO4, 1% Triton X-100, pH 8.6–8.8), dNTPs (containing dGTP, dCTP, dATP, and dTTP, each at 10mM), Bst DNA polymerase (8U / μL), betaine (10mM), liquid polyethylene glycol, graphene dispersion (20ng / μL), amplification primers for rapid detection of Salmonella, enzyme-free water, positive and negative controls (sterile double-distilled water), and running buffer. The running buffer consisted of 0.01M tris(hydroxymethyl)aminomethane (Tris), 0.01M ethylenediaminetetraacetic acid (EDTA), and 0.05M sodium chloride (NaCl). The fluorescent immunochromatographic test strip consists of a base plate, absorbent paper, a nitrocellulose membrane, a conjugate pad, and a sample pad. The nitrocellulose membrane is adhered to the base plate, with one end connected to the absorbent paper and the other end attached to the conjugate pad. The sample pad overlaps the conjugate pad. The nitrocellulose membrane has a biotin-coated control line and a detection line parallel to the control line, coated with anti-digoxigenin antibody. A streptavidin-labeled fluorescent microsphere probe (5 mg / mL) is sprayed onto the conjugate pad, resulting in a probe-immobilized conjugate pad and a treated sample pad. The test strip is housed in a cartridge with a sample application window and a signal reading window on the top cover. The sample application window corresponds to the sample pad, and the signal reading window corresponds to the control line and the detection line.

[0089] The reagents and products mentioned above are packaged together and accompanied by the product instruction manual (including product storage conditions, reaction procedures and result determination methods, etc.) to assemble the Salmonella nucleic acid rapid detection kit described in this invention.

[0090] Example 8: Validation of Clinical Bacterial Isolate Samples

[0091] The reagent kit in Example 7 was used to test and validate 181 clinical bacterial isolates preserved in the laboratory using the method in Example 1. Of these samples, 45 were identified as positive for Salmonella according to the method in the Animal Product Testing Technology Standard for Salmonella Detection in Food (GB4789.4-2016). The test results according to the method of this invention are shown in Table 4. The detection method provided by this invention detected 45 bacterial isolates as positive for Salmonella, and the results are consistent with the national standard detection method. The validation results indicate that the detection method established by the reagent kit of this invention has good clinical application value.

[0092] Table 4. Validation results of collected samples

[0093]

Claims

1. An amplification primer for rapid detection of Salmonella, characterized by, The amplification primer is designed based on the specific conserved region of the Salmonella bcfC gene, and includes a forward primer bcf-F and a reverse primer bcf-R, wherein the sequence of the forward primer bcf-F is shown as SEQ ID NO: 1, the sequence of the reverse primer bcf-R is shown as SEQ ID NO: 2, the 5' end of the forward primer bcf-F and / or the 5' end of the reverse primer bcf-R is labeled with a label, and the label includes phosphorylation, biotin, digoxin, amino, carboxyl, sulfydryl or a primer-modifiable fluorescent group, so that the two ends of the amplified DNA double-stranded product contain different labels.

2. A kit characterized in that, The kit includes the amplification primer for rapidly detecting Salmonella, graphene, a fluorescent immunochromatography test paper and a running buffer as claimed in claim 1, wherein the graphene includes graphene oxide, reduced graphene or graphene quantum dots, and the running buffer includes 0.001-1 M tris-hydroxymethyl aminomethane, 0.001-0.5 M ethylenediaminetetraacetic acid and 0.001-0.8 M sodium chloride.

3. The kit of claim 2, wherein The kit further includes an amplification buffer, a Bst DNA polymerase, dNTPs, polyethylene glycol, betaine, enzyme-free water, a positive control and a negative control.

4. The kit of claim 2, wherein The kit has a molar ratio of the forward primer bcf-F to the reverse primer bcf-R of 1:1-1:2, and the concentration of the forward primer bcf-F and the reverse primer bcf-R is 0.1-30 μM.

5. A method for rapid detection of Salmonella for non-diagnostic purposes, characterized in that, The detection method is to detect Salmonella by using the amplification primer designed based on the specific conserved region of the Salmonella bcfC gene and a fluorescent detection method, wherein the amplification primer includes a forward primer bcf-F and a reverse primer bcf-R, the sequence of the forward primer bcf-F is shown as SEQ ID NO: 1, the sequence of the reverse primer bcf-R is shown as SEQ ID NO: 2, the 5' end of the forward primer bcf-F and / or the 5' end of the reverse primer bcf-R is labeled with a label, and the label is a primer-modifiable fluorescent group, so that the two ends of the amplified DNA double-stranded product contain different labels.

6. The method for rapid detection of Salmonella according to claim 5, characterized in that, The method includes the following steps: adding the amplification primer for rapidly detecting Salmonella into a to-be-tested liquid to perform an amplification reaction, adding a running buffer into a reaction tube after the amplification reaction is completed, blowing and mixing uniformly, standing, reading a value by a fluorescent analyzer, and determining that the to-be-tested sample contains Salmonella if the fluorescent signal value is greater than 122, or determining that the to-be-tested sample does not contain Salmonella if the fluorescent signal value is less than or equal to 122.

7. The method for rapid detection of Salmonella according to claim 5, characterized in that, When performing the amplification analysis reaction, the reaction temperature of the amplification primer is two-step temperature rapid circulation, the first-step temperature is an auxiliary denaturation temperature ranging from 70-78℃, the second-step temperature is an amplification reaction temperature ranging from 55-70℃, the reaction time of each step is 1-10 seconds, and the cycle number is 35-45.

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