A kit for identifying brucella gene deletion vaccine and naturally infected strain and application

Abstract

The application discloses a kit for identifying Brucella gene deletion vaccine and natural infection strain and application and belongs to the field of pathogenic bacteria detection. Bp26 The kit comprises polyethylene glycol monomethyl ether thiol-closed black gold glass fiber photo-thermal material, a biotin and 6-FAM double-labeled specific primer pair for VirB12 genes and / or Bp26 lateral flow test strips of the genes. VirB 12 The application combines black gold glass fiber-mediated photo-thermal PCR technology and immunochromatography test strips, and successfully establishes a Brucellosis differential diagnosis method integrating rapid nucleic acid amplification and visual result interpretation, wherein the method is short in time, simple in operation, and can interpret the result by naked eye, has good specificity, sensitivity and repeatability, and provides a brand-new technical scheme for the differential diagnosis of Brucellosis field strain infection and deletion strain vaccine immunization at the grassroots level and on site.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection, and in particular to a kit for differentiating Brucella gene-deleted vaccines and naturally occurring strains, and its application. Background Technology

[0002] Brucellosis ( Brucellosis ) is caused by Brucella ( Brucella Brucellosis is an important zoonotic infectious disease caused by Brucella bacteria, which is widespread globally. Due to its intracellular parasitic nature, Brucella often presents as a chronic or latent infection, making it easily overlooked in its early stages. When detected in the middle or later stages, prevention and treatment face significant challenges. Based on these characteristics and the harm it causes, brucellosis has been listed by the World Health Organization as one of the seven "neglected zoonotic diseases" that threaten human health and contribute to poverty.

[0003] Currently, the prevention and control of brucellosis mainly relies on vaccination, culling of infected animals, and routine monitoring. However, grassroots veterinary laboratories and remote pastoral areas lack rapid, simple, and low-cost on-site testing methods, leading to significant difficulties in early detection and timely response to outbreaks. Existing laboratory testing methods include bacterial isolation and culture, routine serological tests (such as RBPT and SAT), and molecular biological methods (such as conventional PCR and qPCR). Among these, bacterial isolation and culture is the "gold standard," but it is cumbersome, has high biosafety requirements, a long culture cycle of 3-7 days, and cannot distinguish between wild-type virus infection and vaccine immunization. Routine serological methods are prone to cross-reactivity, and their DIVA (Differentiating Infected from Vaccinated Animals) capability is severely insufficient in the context of large-scale vaccination. With the development of diagnostic technologies in the field of molecular biology, nucleic acid amplification technology has been widely used in animal disease detection. Design using deleted genes as targets can effectively solve the DIVA problem, but conventional PCR has low sensitivity (approximately 10). 5 The detection rate of Brucella nucleic acid is 1000 copies / μL, and requires equipment such as gel electrophoresis. Real-time quantitative PCR (qPCR), while highly sensitive, relies on sophisticated instruments and professional personnel, making it difficult to promote in resource-limited areas. Therefore, developing a rapid, sensitive, portable, and visualized method for Brucella nucleic acid identification and detection is of great significance for improving grassroots and field detection capabilities and achieving the identification and control goals of "early diagnosis, early isolation, and early treatment".

[0004] Molecular detection technology is rapidly developing, and its application value in fields such as disease diagnosis, environmental monitoring, and food safety is becoming increasingly prominent. As a cutting-edge direction in molecular detection, point-of-care testing (POCT) has advantages such as ease of operation, rapid detection, and high equipment portability. Traditional PCR uses a heating block to cycle the temperature of the reaction solution in a plastic tube, resulting in low heat transfer efficiency and a single reaction typically exceeding 1 hour. Furthermore, its large instrument size makes it difficult to meet the portability and rapid response requirements of POCT. In recent years, the application of plasmon photothermal effects has provided a new approach to solving these problems. This effect refers to the generation of localized surface plasmon resonance (LSPR) in plasmon nanomaterials under photoexcitation, thereby efficiently converting light energy into heat energy and exhibiting rapid photothermal conversion characteristics. Introducing this characteristic into conventional PCR, replacing traditional heating methods with photothermal heating, can effectively improve the temperature change rate of the reaction system. Based on this principle, photothermal polymerase chain reaction (Photothermal PCR) utilizes the photothermal effect of plasmonic nanomaterials to achieve rapid temperature cycling of the PCR reaction solution, significantly shortening amplification time and improving equipment portability. It is a novel rapid nucleic acid amplification technology. Combining photothermal heating with other detection methods shows promising application prospects; however, there are currently no reports of using photothermal PCR-LFD to differentiate between Brucella gene-deleted vaccines and naturally occurring strains. Summary of the Invention

[0005] The purpose of this invention is to provide a kit and its application for identifying Brucella gene-deleted vaccines and naturally occurring strains, thereby addressing the problems existing in the prior art. This invention combines black-gold glass fiber-mediated photothermal PCR technology with immunochromatographic test strips to identify Brucella gene-deleted vaccines. Bp26 and VirB 12 Using this method as a target, a novel differential diagnostic method for brucellosis, integrating rapid nucleic acid amplification and visual result interpretation, has been successfully established. This method is quick, simple to operate, and the results can be interpreted with the naked eye. It has good specificity, sensitivity, and repeatability, providing a new technical solution for the differential diagnosis of wild-type brucellosis infection and deletion strain vaccine immunization at the grassroots level and on-site.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a kit for differentiating Brucella gene-deleted vaccines and naturally occurring strains, the kit comprising polyethylene glycol monomethyl ether thiol-blocked black gold glass fiber photothermal material, and a kit targeting... Bp26 Genes and / or VirB12 Biotin- and 6-FAM double-labeled specific primer pairs for genes and lateral flow detection test strips; The Bp26 The nucleotide sequences of the gene-specific primer pairs are shown in SEQ ID NO. 5-6. VirB12 The nucleotide sequences of the gene-specific primer pairs are shown in SEQ ID NO.11-12.

[0007] Preferably, the preparation method of the polyethylene glycol monomethyl ether thiol-blocked black gold glass fiber photothermal material includes the following steps: Glass fibers were immersed in a mixed solution of chloroauric acid and trisodium citrate and subjected to an in-situ reduction reaction at room temperature for 80-120 minutes to load gold nanoparticles onto the surface of the glass fibers. After cleaning, the fibers were sealed with a polyethylene glycol monomethyl ether mercapto solution for more than 12 hours, and then cleaned, dried and cut to obtain the final product.

[0008] Preferably, the kit further includes a PCR reaction premix, a positive control, and a negative control; The PCR reaction premix includes Takara Z-Taq, 10×Z-Taq buffer, dNTPS, and enzyme-free water.

[0009] This invention also provides the use of the kit in any of the following: (1) Application in the preparation of products for differentiating Brucella gene-deleted vaccines and naturally occurring strains; (2) Application in the preparation of diagnostic products for brucellosis; (3) In the preparation and detection Bp26 Genes and VirB 12 Applications of genes in products.

[0010] Preferably, the kit uses photothermal PCR technology and lateral flow test strips to identify Brucella gene-deleted vaccines and naturally occurring strains, and the identification method includes the following steps: Using the DNA of the sample to be tested as a template, the method described above will be employed. Bp26 Gene-specific primer pairs or the aforementioned VirB12 Gene-specific primer pairs were used to construct a PCR reaction solution, which was then dropped onto the surface of a black gold glass fiber photothermal material and encapsulated to form a reaction microcavity. The top part of the lateral flow test strip and the K-type thermocouple are fixed above the black gold glass fiber optical material that has been pre-filled with PCR reaction solution, while white light LED is provided from below the black gold glass fiber optical material to perform photothermal PCR amplification. The amplified product is released onto a lateral flow test strip for chromatographic reaction. The results are interpreted by visually observing the color development of the test line and control line. Among them, when the sample to be tested is simultaneously amplified Bp26 Genes and VirB12When the gene is amplified, it is determined to be Brucella wild-type virus infection; when only the gene is amplified... VirB12 Genetic Bp26 When no amplification band is observed in the gene, it is determined to be... Bp26 Gene deletion vaccine immunization; when only the gene is amplified Bp26 Genetic VirB12 When no amplification band is observed in the gene, it is determined to be... VirB12 Immunization with gene-deleted vaccines.

[0011] Preferably, the PCR reaction solution comprises the following components: 0.1-0.3 μL of 0.5 U Z-Taq DNA polymerase, 0.2 μL each of upstream and downstream primers with a final concentration of 0.1-0.3 μM, 1 μL of 10×Z-Taq buffer, 0.8 μL of dNTP mixture, 0.3 μL of template DNA, and enzyme-free water to a final volume of 10 μL.

[0012] Preferably, the photothermal PCR amplification program is as follows: denaturation at 98℃ for 1 second, annealing and extension combined at 61-63℃, for 40-45 cycles; wherein Bp26 The gene annealing extension conditions were 61℃ for 2 seconds, with a cycle count of 40. VirB12 The gene annealing extension conditions were 63℃ for 1s, with a cycle number of 45.

[0013] Preferably, the method for preparing the lateral flow test strip includes the following steps: Anti-6-FAM polyclonal antibody and biotinylated bovine serum albumin were immobilized on nitrocellulose membranes to form detection lines and control lines, respectively, and dried at 37°C for 1 h. Streptavidin-conjugated gold nanoparticles were sprayed onto the conjugate pad. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad were then sequentially attached to the backing plate, and the test strips were cut to obtain the test strips.

[0014] Preferably, the interpretation criteria for the chromatographic reaction results are as follows: when both the test line and the control line show color, it is determined that the corresponding target gene is positive, that is, the natural infection is positive; when only the control line shows color and the test line does not show color, it is determined that the corresponding target gene is negative, that is, the corresponding gene-deleted vaccine strain provides immunity; when the control line does not show color, it is determined that the test is invalid.

[0015] The present invention also provides a portable detection device for identifying Brucella gene-deleted vaccines and naturally occurring strains, comprising: A white LED light source module is disposed below the black gold glass fiber optical material to provide photothermal excitation; Temperature sensing and feedback module, which includes a K-type thermocouple and a signal converter that are closely attached to the central region of the black gold glass fiber optical material; The microcontroller unit, which uses a chip, controls the LED irradiance intensity and fan switching through pulse width modulation, thereby achieving closed-loop temperature cycle control. And lateral chromatography test strips, which are used for the visual interpretation of amplification products.

[0016] The present invention discloses the following technical effects: The BGGF prepared in this invention can achieve saturated loading of gold nanostructures after a 100-minute reaction, and can generate a highly efficient plasmon photothermal effect under low-cost white LED irradiation. After surface blocking treatment with m-PEG-SH (MW: 5000), its inhibitory effect on PCR reaction can be eliminated, and the amplification efficiency is comparable to that of conventional PCR instruments. Based on BGGF, this invention uses a vaccine strain with a deleted gene ( ΔBp26、ΔVirB 12 Using lateral flow detection strips as the detection target, a photothermal PCR-LFD differential diagnostic method was constructed, successfully realizing the differential diagnosis of Brucella wild-type virus infection. ΔBp26、ΔVirB 12 The precise differentiation of immunization with gene-deleted vaccine strains, showing positive signals in wild-type strains while all vaccine strain samples are negative, effectively solves the industry problem of insufficient DIVA capability of conventional serological methods in the context of large-scale vaccine immunization. This characteristic can avoid unnecessary culling of infected animals due to false positive results, significantly reduce prevention and control costs, and provide key technical support for the accurate monitoring and eradication of brucellosis.

[0017] The photothermal PCR-LFD diagnostic method established in this invention compresses the total time for nucleic acid amplification and detection to approximately 17 minutes. It eliminates the need for complex procedures such as gel electrophoresis, allowing results to be interpreted visually. This method combines speed, sensitivity, portability, visualization, and DIVA identification capabilities, providing a powerful technical means for rapid detection of brucellosis at the grassroots level and in the field. With continuous improvement and optimization, this method is expected to be widely applied in the prevention and control of brucellosis, helping to achieve the prevention and control goals of "early diagnosis, early isolation, and early treatment." Simultaneously, this technology platform can also be extended to the rapid detection of other animal diseases, possessing broad prospects for industrialization and application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram illustrating the detection principle of photothermal PCR-LFD; Figure 2Optimization and characterization of BGGF; (a) Optical images of BGGF prepared at different reaction times; (b) SEM images; (c) SEM images of BGGF after 80 min of reaction (100µm, 2µm, 1µm); (d) Inhibitory effects of different fibers on PCR reaction ( Bp26 (Left) and VirB 12 (Right), 1: Marker; 2: Negative control; 3: Positive control; 4: Glass fiber; 5: BGGF; 6: m-PEG-SH (MW: 5000) blocked BGGF); (e) Infrared thermal images of GF and BGGF under white LED illumination; Figure 3 for Bp26 (a) and VirB 12 (b) Primer screening for gene photothermal PCR; In Figure a, M is the marker, and 1-8 are respectively... Bp26 Positive and negative test results for primers 1-4 of the gene; in Figure b, M is the marker, and 1-8 are... VirB 12 Positive and negative test results for primers 1-4 of the gene; Figure 4 for Bp26 (a) and VirB 12 (b) Optimized reaction temperature for gene annealing and extension merging steps. Agarose gel electrophoresis results; M: Marker; 1-18 are negative and positive detection results for annealing temperatures of 55, 56, 57, 58, 59, 60, 61, 62, and 63 °C, respectively. Figure 5 for Bp2 6(a) and VirB 12 (b) Agarose gel electrophoresis results of primer concentration optimization for gene photothermal PCR; M: Marker; 1-5: final primer concentrations of 0.1, 0.15, 0.2, 0.25, and 0.3 µM, respectively; Figure 6 for Bp26 (a) and VirB 12 (b) Optimization of gene photothermal PCR enzyme dosage and agarose gel electrophoresis results; M: Marker; 1-3: 0.1, 0.2, and 0.3 µL of polymerase per reaction; Figure 7 for Bp26 (a) and VirB 12 (b) Optimization of gene photothermal PCR cycle number and agarose gel electrophoresis results; M: Marker; 1-5: 35, 40, 45, 50, 55 cycles; Figure 8 for Bp26 (a) and VirB 12 (b) Results of 40 cycles of photothermal PCR; Figure 9Electrophoretic bands of products amplified by benchtop PCR instrument and BGGF; M: Marker; 1: Bp26 Desktop PCR instrument; 2: Bp26 Photothermal PCR instrument; 3: VirB 12 Desktop PCR instrument; 4: VirB 12 Photothermal PCR instrument; Figure 10 For detection by gel electrophoresis Bp26 (a) and VirB 12 (b) Results of photothermal PCR repeatability testing; in Figure a, M is the marker, and 1-8 represent the results of 8 positive samples. Bp26 Repeatability test results: 9 out of 1 samples were negative. Bp26 Repeatability test results; in Figure b, M is the marker, and 1-8 represent the results of 8 positive samples. VirB 12 Repeatability test results: 9 out of 1 samples were negative. VirB 12 Repeatability test results; Figure 11 For paper strip testing Bp26 (a) and VirB 12 (b) Results of photothermal PCR-LFD reaction repeatability testing; in Figure a, 1-8 represent the results of 8 positive samples. Bp26 Repeatability test results: 9 out of 1 samples were negative. Bp26 Repeatability test results; in Figure b, 1-8 represent the results of 8 positive samples. VirB 12 Repeatability test results: 9 out of 1 samples were negative. VirB 12 Repeatability test results; Figure 12 To verify the specificity of photothermal PCR using agarose gel electrophoresis results; (a) VirB 12 Genetic testing results; M: Marker; 1: VirB 12 Gene; 2: Negative control; 3: Bp26 Gene; 4: Escherichia coli; 5: Streptococcus pyogenes; 6: Enterococcus haematobium; 7: Salmonella; 8: Listeria monocytogenes; (b) Bp26 Genetic testing results; M: Marker; 1: Bp26 Gene; 2: Negative control; 3: VirB 12 Gene; 4: Escherichia coli; 5: Streptococcus pyogenes; 6: Enterococcus haematobium; 7: Salmonella; 8: Listeria monocytogenes; Figure 13 The results of the test strip were used to specifically verify the photothermal PCR-LFD assay; (a) VirB 12 Genetic testing results; 1: VirB 12 Gene; 2: Negative control; 3: Bp26Gene; 4: Escherichia coli; 5: Streptococcus pyogenes; 6: Enterococcus haematobium; 7: Salmonella; 8: Listeria monocytogenes; (b) Bp26 Genetic testing results; 1: Bp26 Gene; 2: Negative control; 3: VirB 12 Gene; 4: Escherichia coli; 5: Streptococcus pyogenes; 6: Enterococcus haematobium; 7: Salmonella; 8: Listeria monocytogenes; Figure 14 for Bp26 and VirB 12 Sensitivity test results of agarose gel electrophoresis; (a) Photothermal PCR detection Bp26 Sensitivity test, where M: Marker, 1-10 are 1.34×10⁻¹⁰. 9 1.34×10 8 1.34×10 7 1.34×10 6 1.34×10 5 1.34×10 4 1.34×10 3 1.34×10 2 1.34×10 1 and 1.34×10 0 (a) Copies / μL; 11: Negative control; (b) Photothermal PCR detection VirB 12 Sensitivity test; where M: Marker, 1-10 are 1.42×10 9 1.42×10 8 1.42×10 7 1.42×10 6 1.42×10 5 1.42×10 4 1.42×10 3 1.42×10 2 1.42×10 1 and 1.4×10 0 copies / μL, 11 is the negative control; Figure 15 for Bp26 and VirB 12 Results of photothermal PCR-LFD sensitivity assay using test strips; (a) Bp26 The results of the photothermal PCR-LFD sensitivity assay strip were 1.34 × 10⁻¹⁰ for tests 1-10. 9 1.34×10 8 1.34×10 7 1.34×10 6 1.34×10 5 1.34×104 1.34×10 3 1.34×10 2 1.34×10 1 and 1.34×10 0 copies / μL, 11: negative control; (b) VirB 12 The results of the photothermal PCR-LFD sensitivity assay strip were 1.42 × 10⁻¹⁰ for tests 1-10. 9 1.42×10 8 1.42×10 7 1.42×10 6 1.42×10 5 1.42×10 4 1.42×10 3 1.42×10 2 1.42×10 1 and 1.4×10 0 copies / μL, 11: negative control; Figure 16 The results of agarose gel electrophoresis and test strip analysis of brucellosis samples; (a) Bp26 Gene agarose gel electrophoresis results, (b) VirB12 Agarose gel electrophoresis results, where in figures a and b, M represents the marker, 1-10 are brucellosis-positive samples, and 11 is a negative control; (c) brucellosis samples. Bp26 Gene test strip results, (d) Brucellosis sample VirB12 The results of the gene test strips are shown in Figures c and d. 1-10 are brucellosis samples, and 11 is a negative control. Figure 17 Results of agarose gel electrophoresis and test strip analysis of vaccine immunization samples; (a) ∆Bp26 Agarose gel electrophoresis results of vaccine strain samples ， (b) ∆VirB12 Agarose gel electrophoresis results of vaccine strain samples. In Figures a and b, M represents the marker, 1-20 are vaccine immunization samples, and 21 is the positive control; (c) ∆Bp26 Vaccine strain sample test strip test results ， (d) ∆VirB12 The results of the vaccine strain sample test strips are shown in Figures c and d. In Figures c and d, 1-20 are vaccine immunization samples, and 21 is a positive control. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1 1. Materials and Instruments Gold chloride tetrahydrate (HAuCl4), 6-FAM polyclonal antibody, bovine serum albumin (BSA), Tris-HCl (1M, pH 8.5), DNA marker, agarose gel, and 50×TAE buffer were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China); trisodium citrate (TSC) and sucrose were purchased from Sinopharm Chemical Reagent Co., Ltd.; m-PEG-SH (MW: 5000) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; streptavidin (SA) and bovine serum albumin-biotin (BSA-Biotin) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; Takara Z-Taq™ was purchased from Takara Biotech; Triton X-100 was purchased from Sigma (St. Louis, Missouri, USA); Brucella was also present. Bp26 , VirB 12 The gene modification primers were synthesized by General Biotech (Anhui) Co., Ltd.

[0026] Glass fiber (GF-08), nitrocellulose (NC) membrane (Sartorius CN140), sample pads, conjugate pads, absorbent pads, and back pads were all purchased from Gening Biotech Co., Ltd. (Shanghai, China). K-type thermocouples were manufactured by Kapson (Jiangsu, China). PET high-temperature resistant tape, Kapton tape, elbow rinse bottles, 30 mm and 150 mm petri dishes, scissors, tweezers, steel rulers, cutting knives, and other consumables were all purchased from Taobao e-commerce platform.

[0027] Thermographic images of BGGF were recorded using an infrared thermal imager (ETS320, FLIR); the morphology of BGGF was observed using a scanning electron microscope (SEM, Zeiss Ultra Plus); the absorption spectrum of BGGF was measured using a UV-1900I (Shimadzu); the PCR amplification products were analyzed and imaged using a nucleic acid electrophoresis system (Beijing Liuyi Instrument Factory) and a gel imaging system (VILBER BIO IMAGING); the test strips were streaked using an HGS510 streaking and gold labeling machine (Hangzhou Fenghang Technology Co., Ltd.), and the test strips were cut using a microcomputer-controlled automatic cutting machine (Shanghai Jinbiao Technology Co., Ltd.).

[0028] 2. Preparation, characterization and optimization of photothermal materials 2.1 Preparation of Black Gold Glass Fiber (BGGF) Cut glass fiber (GF) into 25mm x 25mm pieces, wash with deionized water, and dry. Mix 200 µL of 1% (w / v) HAuCl4 solution with 200 µL of 100 mg / mL trisodium citrate (TSC) solution, and immerse the GF in the mixture for 100 min. Repeat this step 2-3 times, rinsing the GF thoroughly with ultrapure water and drying it before each reaction. During this process, gold ions (Au) are present. 3+ Gold nanoparticles (Au NPs) are reduced by TSC on the surface of GF. During this process, the glass fiber changes from white to black, thus obtaining the basic black gold glass fiber. The obtained basic BGGF is washed with ultrapure water and dried, and then soaked in ultrapure water for 1 h. After removing and drying, 1 mL of 10 mg / mL polyethylene glycol monomethyl ether mercapto (m-PEG-SH, MW: 5000) solution is added, and it is placed in a refrigerator overnight (>12 h). The next day, it is removed, rinsed with ultrapure water and dried, and then cut into 5 mm × 5 mm sizes for later use.

[0029] 2.2 Optimization and Characterization of BGGF To optimize the photothermal properties of BGGF, the effects of different reaction times (15, 30, 45, 60, 80, 100, and 120 min) on the loading of gold nanoparticles were investigated. After the reaction, the color change of BGGF (gradually changing from light gray to black) was observed, and its surface morphology and loading density were characterized by optical microscopy and scanning electron microscopy (SEM).

[0030] 2.3 Evaluation of the inhibitory effect of BGGF on PCR Three fiber materials with dimensions of 5 mm × 5 mm were prepared: unmodified GF, unblocked BGGF, and BGGF blocked with m-PEG-SH (MW: 5000). These materials were then immersed in a solution containing Brucella bacteria. Bp26 and VirB 12 The gene template was placed in the PCR reaction solution (refer to "3.2 Preparation of PCR Mixture"). Experimental setups included a negative control (without template), a positive control (with template but without fiber material), a GF group, a BGGF group, and a BGGF group blocked with m-PEG-SH (MW: 5000). All samples underwent PCR amplification using a standard thermal cycler. After amplification, the products were detected by agarose gel electrophoresis to evaluate the inhibitory effect of each material on nucleic acid amplification.

[0031] 2.4 Verification of Plasmon Photothermal Effect BGGF and ordinary GF were irradiated with the same white LED, and the temperature changes on the surfaces of the two materials were recorded using an infrared thermal imager. By comparing the temperature rise before and after irradiation, it was verified whether the temperature rise of BGGF originated from the plasmonic photothermal effect.

[0032] 3. Establishment of a photothermal PCR detection method for brucellosis 3.1 Primer Design and Synthesis According to Brucella published by GenBank Bp26 (Login ID: 29593322) and VirB 12 (Accession number: LT671513.1) Gene sequence, four pairs of primers were designed for each of the two gene segments using Primer Premier 5 software. To achieve... Bp26 and VirB 12 For visualization of the gene detection results, biotin and 6-carboxyfluorescein (6-FAM) were labeled at the 5' ends of the upstream and downstream primers, respectively. All primers were synthesized by General Biotech (Anhui) Co., Ltd., and the specific primer information is shown in Table 1.

[0033] 3.2 Preparation of PCR Mixture The sample loading procedure was performed according to the recommended Takara Z-Taq reaction system. The reaction system (total volume 10 μL) consisted of: 0.5 μT Takara Z-Taq (0.1 µL), 0.2 μM forward primer (0.2 µL), 0.2 μM reverse primer (0.2 µL), and 1 μL 10× Z-Taq buffer (M). 2+ , 30 mM), 0.8μL dNTP, 0.3μL pET-32a(+)- Bp26 / pET-32a(+)- VirB 12 Plasmid (1.5 ng) and 7.4 µL of enzyme-free water.

[0034] Table 1 Brucella Bp26 , VirB 12 Gene primer sequence 3.3 Optimization of the photothermal PCR reaction system 3.3.1 Primer specificity verification Will be respectively targeted Bp26 and VirB 12 The four pairs of primer powders for the gene were prepared with ultrapure water to a storage concentration of 10 μM, and the premix was prepared according to the above "3.2 Preparation of PCR Mixture".

[0035] PCR amplification was performed using Takara Z-Taq DNA polymerase. The reaction program was set according to the enzyme preparation instructions: 98℃ was used as the denaturation temperature, and the arithmetic mean of the Tm values ​​of the upstream and downstream primers used for amplification (calculated using the Nearest-Neighbor method) was used as the reaction temperature for the combined annealing and extension steps.

[0036] 3.3.2 Optimization of reaction temperature for the combined annealing and extension steps The reaction mixture was added according to the recommended reaction system in the Takara Z-Taq instruction manual. The reaction system (total volume 10 μL) consisted of: 0.5 U Takara Z-Taq (0.1 µL), 0.2 μM forward primer (0.2 µL), 0.2 μM reverse primer (0.2 µL), and 1 μL 10× Z-Taq buffer (Mg2+). 2+ 30 mM), 0.8 μL dNTP, 0.3 µL pET-32a(+)-Bp26 / pET-32a(+)-VirB12 plasmid (1.5 ng) and 7.4 µL enzyme-free water.

[0037] PCR amplification was performed using Takara Z-Taq DNA polymerase. The reaction program was set according to the enzyme preparation instructions: 98℃ was used as the denaturation temperature, and the denaturation time was 1s. The reaction times for the combined annealing and extension steps were set to 2s and 1s respectively for the Bp26 / VirB12 gene, based on the amplification efficiency of Z-Taq 100bp / s.

[0038] To optimize the reaction temperature for the annealing and extension merging steps using the selected optimal primers, a temperature gradient of 55℃-63℃ was set based on the above conditions, using the arithmetic mean of the Tm values ​​of the upstream and downstream primers (calculated using the Nearest-Neighbor method) as the baseline, and the reaction conditions were optimized in 1℃ increments. After amplification, the amplification products were analyzed by 2% agarose gel electrophoresis, and further detection using test strip chromatography was performed to eliminate interference from primer dimers and determine the optimal reaction temperature for photothermal PCR amplification.

[0039] 3.3.3 Optimization of final primer concentration The final primer concentrations were optimized under the optimal reaction temperatures for the annealing and extension merging steps. An optimization gradient of 0.1 μM to 0.3 μM was established, with gradient increments of 0.05 μM. The upstream and downstream primer concentrations remained proportionally consistent, while other reaction parameters were kept constant. After amplification, the amplification effect was analyzed by 2% agarose gel electrophoresis and test strip chromatography to determine the optimal primer concentrations for photothermal PCR amplification.

[0040] 3.3.4 Optimization of enzyme dosage Based on the recommended reaction system in the Takara Z-Taq instruction manual, an optimized gradient of polymerase dosage from 0.1 μL to 0.3 μL was set for each reaction, with a gradient increment of 0.1 μL, while keeping other reaction parameters consistent. After amplification, the amplification effect was analyzed by 2% agarose gel electrophoresis and test strip chromatography to determine the optimal enzyme dosage for photothermal PCR amplification.

[0041] 3.3.5 Optimization of Loop Count Under optimal system conditions, the number of cycles for photothermal PCR was optimized. Starting with 35 cycles, an optimized gradient of 35-55 cycles was set, with gradient increments of 5 cycles, while maintaining other reaction parameters consistently. After amplification, the amplification effect was analyzed by 2% agarose gel electrophoresis and test strip chromatography to determine the optimal number of cycles for photothermal PCR amplification.

[0042] 4. Results and Analysis 4.1 Detection principle of photothermal PCR-LFD Figure 1 This study demonstrates the operational mechanism of photothermal PCR-LFD, a system that integrates nucleic acid amplification and detection into a handheld device. The top of a test strip and a K-type thermocouple are fixed above BGGF containing pre-added PCR reaction mixture, while a white LED below provides illumination, enabling ultra-fast photothermal cycling. When the white LED is activated, photons excite plasmon electrons in the gold nanostructures on the BGGF surface, rapidly generating hot electrons. Due to the low heat capacity of these hot electrons, their temperature rises rapidly, efficiently heating the surrounding PCR solution. The device utilizes a microcontroller (MCU) to program and control the LED via pulse width modulation (PWM), precisely adjusting the light intensity, duration, and cooling cycle. Compared to laser systems, white LEDs significantly reduce power consumption, thereby reducing equipment costs, making them particularly suitable for point-of-care testing (POCT) scenarios.

[0043] After photothermal amplification, the PCR product is released onto the test strip and read visually. The liquid flows sequentially through the sample pad, conjugation pad, test line (T line), and control line (C line). The target nucleic acid is amplified using 5'-labeled primers. The amplification product binds to streptavidin-functionalized gold nanoparticles (SA-Au nanoparticles), forming an SA-Au nanoparticle-DNA complex. The T line is coated with an anti-6-FAM polyclonal antibody, which captures the 6-FAM-labeled primers, forming a visible SA-Au nanoparticle-DNA-anti-6-FAM complex. If no amplification product is present, the gold nanoparticle is only captured by the biotin labeled on the C line. Therefore, the presence or absence of the target nucleic acid can be determined by observing whether the T line shows color. The entire process (including real-time PCR amplification and visual detection) can be completed in approximately 17 minutes, facilitating rapid, visual, large-scale POCT screening during a pandemic.

[0044] 4.2 BGGF Optimization and Characterization In this invention, BGGF not only serves as a high specific surface area PCR reaction solution container but also as a highly efficient photothermal material. BGGF is prepared by in-situ reduction of Au nanoparticles on glass fibers at room temperature. During this process, HAuCl4 and TSC permeate into the voids of the glass fibers through capillary action, and over time, Au is reduced... 3+ The Au nanostructures are reduced and deposited on the GF surface.

[0045] The photothermal properties of BGGF are strongly dependent on the loading of Au nanostructures. Therefore, the reaction time of BGGF (15, 30, 45, 60, 80, 100, and 120 min) was first optimized. With increasing reaction time, the loading of Au nanostructures increased, causing BGGF to change from light gray to black. Figure 2 Optical and scanning electron microscopy images of the BGGF confirmed that the loading of gold nanostructures on the glass fiber initially increased and reached saturation at 100 min. Figure 2 The image shows that gold nanostructures of varying shapes and sizes are randomly distributed on the BGGF, which gives it strong absorption over a wide spectral range (due to the LSPR effect and mode hybridization). Taking advantage of this property, a low-cost white LED is used to achieve photothermal conversion, and the sample heats up uniformly and rapidly after illumination.

[0046] Since BGGF acts as the reaction solution container in the photothermal PCR reaction system, it is necessary to evaluate whether it inhibits nucleic acid amplification. This invention includes a negative control, a positive control, and experimental groups (GF group, BGGF group, and m-PEG-SH (MW: 5000) blocked BGGF). In the experimental groups, various fibers with dimensions of 5 mm × 5 mm were immersed in [the solution / solution]. Bp26 and VirB 12 Amplification was performed using a standard thermal cycler in the PCR solution containing the gene template. The amplification products were verified by gel electrophoresis, as shown below. Figure 2 As shown in Figure d, the electrophoretic band intensity of BGGF blocked by m-PEG-SH (MW: 5000) is comparable to that of the positive control group (band 3), indicating that BGGF blocked by m-PEG-SH (MW: 5000) has almost no inhibitory effect on PCR reaction and can be used as a container for photothermal PCR reaction.

[0047] To verify that the temperature rise mechanism of BGGF originates from the plasmon photothermal effect, the same white LED was used to illuminate both BGGF and GF, and surface temperature changes were recorded using an infrared thermal imager. Figure 2 As can be seen in Figure e, the temperature of BGGF rises rapidly after illumination, while the temperature of GF does not change significantly. This significant difference confirms that the temperature rise of BGGF after white LED illumination is due to the plasmon photothermal effect.

[0048] 4.3 Establishment of a photothermal PCR detection method for brucellosis 4.3.1 Optimization of the photothermal PCR reaction system (1) Primer specificity verification Targeting Brucella Bp26 and VirB 12 Four pairs of primers designed for gene synthesis were used for PCR amplification with Takara Z-Taq DNA polymerase. The results of 2% agarose gel electrophoresis showed that... Figure 3 ), Bp26 Primer 3 and VirB 12 Primer No. 2 amplified only the specific target band that was exactly the expected size, with no extraneous bands, no primer dimers, and no non-specific amplification signals. The primers had good specificity and could be used for subsequent optimization experiments of the photothermal PCR reaction system.

[0049] (2) Optimization of reaction temperature for the combined annealing and extension steps use Bp26 Primer 3 and VirB 12 The reaction temperature for the annealing and extension merging steps of primer 2 was optimized. Using the arithmetic mean of the primer Tm values ​​as the base, a temperature gradient of 55℃~65℃ (in 1℃ increments) was set for optimization. Agarose gel electrophoresis results showed ( Figure 4 ), Bp26 and VirB 12 When the reaction temperatures for gene annealing and extension merging steps are 61℃ and 63℃, respectively, the target band of the amplification product has the highest brightness, the band is single and free of impurities, and the amplification specificity and efficiency are optimal. Therefore, 61℃ and 63℃ are determined to be the optimal temperatures for these steps. Bp26 and VirB 12 The optimal reaction temperature for the photothermal PCR amplification, annealing, and extension merging steps.

[0050] (3) Optimization of final primer concentration At the optimal reaction temperature, the final primer concentration gradient was optimized by setting a gradient of 0.1 μM to 0.3 μM (in increments of 0.05 μM). Agarose gel electrophoresis results showed ( Figure 5 When the final concentration of the upstream and downstream primers is 0.2 μM, Bp26 and VirB 12 The target gene band exhibited the strongest gene specificity and highest brightness, with no primer dimer interference. Amplification efficiency was insufficient and band brightness was weak when primer concentration was below this value; therefore, a final primer concentration of 0.2 μM was determined to be the optimal final primer concentration for photothermal PCR amplification.

[0051] (4) Optimization of enzyme dosage Based on the baseline reaction system, a Taq DNA polymerase dosage gradient of 0.1 μL to 0.3 μL (in 0.1 μL increments) was set for optimization. Agarose gel electrophoresis results showed ( Figure 6 When the amount of polymerase used is 0.2 μL per reaction, Bp26 and VirB 12 The amplification efficiency of the target gene band can be optimized. If the polymerase dosage is too low, the amplification product is insufficient, and the band brightness is weak; if the dosage is higher than this value, there is no significant increase in amplification effect, and the detection cost is increased. Therefore, 0.2 μL was determined to be the optimal polymerase dosage for each reaction as photothermal PCR amplification.

[0052] (5) Optimization of the number of loops Based on the optimized reaction system and program parameters, a gradient of 35-55 cycles (5-cycle increments) was set for further optimization. Agarose gel electrophoresis ( Figure 7 The results showed that when the number of amplification cycles was 40 and 45, respectively... Bp26 and VirB 12 The gene yields a bright, single target band, meeting the detection sensitivity requirements. When the number of cycles is below the above value, the amplification product is insufficient, resulting in a weak signal; when the number of cycles is above this value, tailing bands may appear, and the total detection time may be prolonged. Considering both detection sensitivity and timeliness, 40 and 45 were ultimately determined to be... Bp26 and VirB 12 The optimal number of cycles for gene photothermal PCR amplification.

[0053] Example 2: Application of photothermal polymerase chain reaction in BGGF This invention utilizes 3D printing technology to fabricate a split plastic shell for a plasmonic photothermal PCR chip. The shell consists of a top cover and a bottom cover, which are tightly fastened together by plastic clips around the perimeter, forming a relatively enclosed and stable internal reaction space. The top cover integrates light source incident and result observation functions, and its surface has three types of functional openings: a central square opening for white LED excitation light incident, providing stable excitation energy for the photothermal PCR reaction; a rectangular opening as an observation window for visually interpreting the color development results of the test strips; and an opening on the bottom cover as an air inlet for a cooling fan, accelerating the PCR cooling process and improving the thermal circulation rate. The bottom cover surface is machined with a 3 mm wide positioning groove for precisely fixing nucleic acid detection test strips. The position and size of the groove are optimized to ensure that the test strips are firmly embedded and maintain a preset spatial distance from the photothermal material inside the chip, meeting the migration requirements of the amplification products.

[0054] The photothermal reaction microcavity was prepared as follows: a 5 mm × 5 mm plasmon photothermal material BGGF was taken, and 10 μL of PCR reaction mixture was dropped onto its surface; two high-temperature resistant transparent PET tapes treated with 1% (w / v) bovine serum albumin (BSA) were used to encapsulate BGGF on both sides to form a sealed PCR reaction microcavity, avoiding evaporation of the reaction solution and external contamination.

[0055] The internal components are integrated and assembled in the following order: The encapsulated BGGF reaction microcavity is fixed at the center of the square opening to ensure that the photothermal material can fully receive the LED excitation light and achieve efficient plasmon photothermal conversion. A K-type thermocouple is tightly attached to the central area of ​​the encapsulated BGGF reaction microcavity surface using polyimide (Kapton) tape for real-time monitoring of temperature changes in the reaction system (the temperature signal collected by the thermocouple is transmitted to the microcontroller unit (MCU) via a dedicated MAX31855 module; precise control of the entire thermal cycle is achieved by an STM32F407 MCU with embedded programs, which adjusts the LED irradiance and controls the fan's on / off state through its input / output ports (I / O), thereby achieving precise control of the thermal cycle); the white light excitation light is incident from the side not covered by the Kapton tape to avoid light absorption by the tape causing energy attenuation. Embed the prepared nucleic acid test strip into the positioning groove of the bottom cover, ensuring a 2 mm overlap between the sample pad of the test strip and the encapsulated BGGF reaction microcavity (non-white light irradiation side). Adjust the position so that the T-line and C-line of the test strip are aligned with the rectangular observation window of the top cover for easy visualization and result reading. After photothermal amplification, puncture the BGGF reaction microcavity with a needle and inject the running buffer. This allows the amplification products to migrate to the test strip through the overlap area under capillary force, achieving seamless integration of nucleic acid amplification and detection.

[0056] by pET-32a(+)-Bp26 / pET-32a(+)-VirB 12 The photothermal PCR reaction performance under white LED illumination was tested using plasmids as templates. Figure 8 The results of 40 stable thermal cycles are shown. The heating phase, from 64°C to 98°C, was achieved by white LED illumination, while the cooling phase was achieved by turning off the white LED and turning on the fan.

[0057] After photothermal polymerase chain reaction, the amplification products were extracted by centrifugation and analyzed by gel electrophoresis. Figure 9 Electrophoretic bands of products amplified by a benchtop PCR instrument and BGGF are shown, demonstrating matching bands and comparable amplification performance.

[0058] Example 3: Assembly of Brucellosis Photothermal PCR-LFD Test Strips 6-FAM polyclonal antibody (Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China); Catalog No.: D110013-0100) and BSA-Biotin (Solebio Technology Co., Ltd. (Beijing, China); Catalog No.: SHB063) were diluted to 1 mg / mL and immobilized on nitrocellulose membranes (NC membranes) using a gold spraying apparatus at a rate of 1 μL / cm to form detection lines (T lines) and control lines (C lines), respectively. The NC membranes were then dried at 37°C for 1 hour. Separately, streptavidin-conjugated gold nanoparticles (SA-Au NPs) were sprayed onto the conjugate pad, dried, and stored at room temperature in the dark for later use. The sample pad, conjugate pad, NC membrane, and absorbent pad were then sequentially attached to the substrate (backing plate). The assembled test strip board was cut into 3 mm wide strips using a cutter and stored in a desiccator at room temperature for later use.

[0059] Example 4: Photothermal PCR-LFD Bp26 and VirB 12 Gene duplication testing Two methods, agarose gel electrophoresis and test strip chromatography, were used to detect and compare the photothermal PCR amplification products. Bp26 and VirB 12 Using genes as the detection target, repeatability verification of photothermal PCR amplification was performed, with a total of 8 positive samples and 1 negative sample. After amplification, the products were interpreted simultaneously using 2% agarose gel electrophoresis and immunochromatographic test strips to ensure consistency between the test strip results and the agarose gel electrophoresis results.

[0060] Repeatability test results show ( Figure 10 , Figure 11 All eight positive samples achieved target gene-specific amplification, while one negative sample showed no amplification signal. There were no false positives or false negatives, indicating good reproducibility of the photothermal PCR method. The two observation methods showed good consistency and can quickly and accurately interpret photothermal PCR amplification products, providing a reliable means for rapid detection.

[0061] Example 5: Photothermal PCR-LFD Bp26 and VirB 12 Gene-specific detection Use respectively pET-32a(+)-Bp26 / pET-32a(+)-VirB 12 The plasmid and the extracted genomic DNA of Escherichia coli, Streptococcus galactiae, Enterococcus haematobacterium, Listeria monocytogenes and Salmonella were used as templates for photothermal PCR. A negative control was set up using ddH2O as a template. After the photothermal PCR was completed, the amplification products were collected by centrifugation and identified and analyzed by 2% agarose gel electrophoresis.

[0062] Bp26 and VirB 12In gene-specific experiments, agarose gel electrophoresis results showed that only the target plasmid amplified the corresponding band. Figure 12 During the test strip detection, only the target plasmid amplification product will show color at the T line, while other non-target strains will only show color at the C line. Figure 13 This indicates that the Brucella strain established in this study... Bp26 and VirB 12 Gene detection methods have good specificity.

[0063] Example 6: Photothermal PCR-LFD Bp26 and VirB 12 Sensitivity testing of genes Recombinant standard plasmids were serially diluted 10-fold. pET-32a(+)-Bp26 / pET-32a(+)-VirB 12 Using a template, amplification was performed according to the established photothermal PCR reaction system, with ddH2O set as a negative control. The amplification results were identified and analyzed by 2% agarose gel electrophoresis and test strips, respectively.

[0064] Sensitivity assay results from agarose gel electrophoresis (photothermal PCR) showed that when Bp26 The copy number of the recombinant plasmid was 1.34 × 10⁻⁶. 9 -1.34×10 3 Amplification bands were visible at copies / μL ( Figure 14 (a) The test strip detected a plasmid copy number of 1.34 × 10⁻⁶. 2 A fainter band appears at copies / μL ( Figure 15 (a) When VirB 12 The copy number of the recombinant plasmid was 1.42 × 10⁻⁶. 9 -1.42×10 3 Amplification bands were visible at copies / μL ( Figure 14 (b) The test strip detected a plasmid copy number of 1.42 × 10⁻⁶. 2 A fainter band appears at copies / μL ( Figure 15 (b); Therefore, the detection sensitivity of the test strip in this experiment is 10 times higher than that of conventional PCR.

[0065] Example 7: Application of photothermal PCR-LFD in the differential diagnosis of brucellosis First, the samples were pretreated, including brucellosis-positive samples and brucellosis vaccine immunization (gene-deleted vaccine strain). ΔBp26 Δ VirB 12 The samples were inactivated at 85℃ for 1 h. Ten samples were positive for brucellosis, and 20 samples were immunized with brucellosis vaccine. All groups underwent amplification using the BGGF prepared according to this invention and the established PCR technique. After amplification, the amplification products were verified by agarose gel electrophoresis and test strips, respectively.

[0066] The criteria for interpreting electrophoresis results are: when the sample to be tested simultaneously amplifies... Bp26 Genes and VirB12 When the gene is amplified, it is determined to be Brucella wild-type virus infection; when only the gene is amplified... VirB12 Genetic Bp26 When no amplification band is observed in the gene, it is determined to be... Bp26 Gene deletion vaccine immunization; when only the gene is amplified Bp26 Genetic VirB12 When no amplification band is observed in the gene, it is determined to be... VirB12 Immunization with gene-deleted vaccines.

[0067] The criteria for interpreting test results are as follows: when both the test line and the control line show color, it is considered a positive result for the corresponding target gene, indicating a positive natural infection; when only the control line shows color and the test line does not, it is considered a negative result for the corresponding target gene, indicating immunity to the corresponding gene-deleted vaccine strain; when the control line does not show color, the test is considered invalid.

[0068] Test results as follows Figure 16 and Figure 17 As shown, the results indicate that: brucellosis-positive samples showed a visible target band on agarose gel electrophoresis, and both the test line (T line) and control line (C line) on the test strip also developed color, indicating a positive result; the vaccine group did not show a target band on agarose gel electrophoresis, and only the C line on the test strip developed color, indicating a negative result; the positive and negative controls showed no abnormalities in band development or color development. These results demonstrate that this method can effectively distinguish brucellosis-positive samples from gene-deleted vaccine strain samples.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A kit for differentiating Brucella gene-deleted vaccines and naturally occurring strains, characterized in that, The kit includes polyethylene glycol monomethyl ether thiol-blocked black gold glass fiber photothermal material, targeting... Bp26 Genes and / or VirB12 Biotin- and 6-FAM double-labeled specific primer pairs for genes and lateral flow detection test strips; The Bp26 The nucleotide sequences of the gene-specific primer pairs are shown in SEQ ID NO. 5-6. VirB12 The nucleotide sequences of the gene-specific primer pairs are shown in SEQ ID NO.11-12.

2. The kit according to claim 1, characterized in that, The preparation method of the polyethylene glycol monomethyl ether thiol-blocked black gold glass fiber photothermal material includes the following steps: Glass fibers were immersed in a mixed solution of chloroauric acid and trisodium citrate and subjected to an in-situ reduction reaction at room temperature for 80-120 minutes to load gold nanoparticles onto the surface of the glass fibers. After cleaning, the fibers were sealed with a polyethylene glycol monomethyl ether mercapto solution for more than 12 hours, and then cleaned, dried and cut to obtain the final product.

3. The kit according to claim 1, characterized in that, The kit also includes a PCR reaction premix, a positive control, and a negative control; The PCR reaction premix includes Takara Z-Taq, 10×Z-Taq buffer, dNTPS, and enzyme-free water.

4. The use of the kit according to any one of claims 1-3 in any of the following: (1) Application in the preparation of products for differentiating Brucella gene-deleted vaccines and naturally occurring strains; (2) Application in the preparation of diagnostic products for brucellosis; (3) In the preparation and detection Bp26 Genes and VirB 12 Applications of genes in products.

5. The application as described in claim 4, characterized in that, The kit uses photothermal PCR technology and lateral flow test strips to identify Brucella gene-deleted vaccines and naturally occurring strains. The identification method includes the following steps: Using the DNA of the sample to be tested as a template, the method described above will be employed. Bp26 Gene-specific primer pairs or the aforementioned VirB12 Gene-specific primer pairs were used to construct a PCR reaction solution, which was then dropped onto the surface of a black gold glass fiber photothermal material and encapsulated to form a reaction microcavity. The top part of the lateral flow test strip and the K-type thermocouple are fixed above the black gold glass fiber optical material that has been pre-filled with PCR reaction solution, while white light LED is provided from below the black gold glass fiber optical material to perform photothermal PCR amplification. The amplification product is released onto a lateral flow test strip for chromatographic reaction. The results are interpreted by visually observing the color development of the test line and control line. Among them, when the sample to be tested is simultaneously amplified Bp26 Genes and VirB12 When the gene is amplified, it is determined to be Brucella wild-type virus infection; when only the gene is amplified... VirB12 Genetic Bp26 When no amplification band is observed in the gene, it is determined to be... Bp26 Gene deletion vaccine immunization; when only the gene is amplified Bp26 Genetic VirB12 When no amplification band is observed in the gene, it is determined to be... VirB12 Immunization with gene-deleted vaccines.

6. The application as described in claim 5, characterized in that, The PCR reaction solution comprises the following components: 0.1-0.3 μL of 0.5U Z-Taq DNA polymerase, 0.2 μL each of upstream and downstream primers with a final concentration of 0.1-0.3 μM, 1 μL of 10×Z-Taq buffer, 0.8 μL of dNTP mixture, 0.3 μL of template DNA, and enzyme-free water to a final volume of 10 μL.

7. The application as described in claim 5, characterized in that, The photothermal PCR amplification program is as follows: denaturation at 98℃ for 1 second, annealing and extension at 61~63℃, 40~45 cycles; in Bp26 The gene annealing extension conditions were 61℃ for 2 seconds, with a cycle count of 40. VirB12 The gene annealing extension conditions were 63℃ for 1s, with a cycle number of 45.

8. The application as described in claim 5, characterized in that, The method for preparing the lateral flow test strip includes the following steps: Anti-6-FAM polyclonal antibody and biotinylated bovine serum albumin were immobilized on nitrocellulose membranes to form detection lines and control lines, respectively, and dried at 37°C for 1 h. Streptavidin-conjugated gold nanoparticles were sprayed onto the conjugate pad. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad were then sequentially attached to the backing plate, and the test strips were cut to obtain the test strips.

9. The application as described in claim 5, characterized in that, The interpretation criteria for the chromatographic reaction results are as follows: when both the test line and the control line show color, it is determined that the corresponding target gene is positive, that is, the natural infection is positive; when only the control line shows color and the test line does not show color, it is determined that the corresponding target gene is negative, that is, the corresponding gene-deleted vaccine strain provides immunity; when the control line does not show color, it is determined that the test is invalid.

10. A portable detection device for identifying Brucella gene-deleted vaccines and naturally occurring strains, characterized in that, include: A white LED light source module is disposed below the black gold glass fiber optical material described in claim 1, and is used to provide photothermal excitation; Temperature sensing and feedback module, which includes a K-type thermocouple and a signal converter that are closely attached to the central region of the black gold glass fiber optical material; The microcontroller unit, which uses a chip, controls the LED irradiance intensity and fan switching through pulse width modulation, thereby achieving closed-loop temperature cycle control. And lateral chromatography test strips, which are used for the visual interpretation of amplification products.

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