Streptococcus equi universal colloidal gold antibody detection test strip and application thereof
By developing colloidal gold antibody test strips for Streptococcus equi and utilizing colloidal gold immunochromatography, the problem of complex and time-consuming detection of Streptococcus equi in existing technologies has been solved, achieving rapid and accurate detection of Streptococcus equi antibody, which is suitable for the early diagnosis and prevention of infection in horses.
Patent Information
- Application Number
- CN202411425220.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-12
- Publication Date
- 2025-11-07
Smart Images
Figure CN120908444A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine and detection, and specifically relates to a Streptococcus equi colloidal gold antibody detection test strip and application thereof. BACKGROUND
[0002] Streptococcus equi mainly includes Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus, can infect equidae animals to cause fever, upper respiratory tract mucosa inflammation, submandibular lymph node swelling and purulence, endometritis and the like, and can cause death of the equidae animals in severe cases. Streptococcus equi can infect the upper respiratory tract of humans and various animals to cause purulent infection and meningitis. Sick horses, recovered carrier horses and contaminated stables can all cause direct or indirect transmission of Streptococcus equi, and horses, donkeys and mules can infect each other. The disease has an acute onset and a short course, is prevalent worldwide, and can seriously reduce the benefits of horse breeding farms and cause serious economic losses to the global horse breeding industry, which is a serious threat to the public health and horse and donkey breeding industry.
[0003] Streptococcus equi disease can pose a serious threat to public health and horse and donkey breeding industry, and early diagnosis and management are important for controlling infection of the bacteria. At present, clinical diagnosis and experimental diagnosis are used for diagnosis of Streptococcus equi disease. Experimental diagnosis needs to use a laboratory platform and equipment to carry out pathogenic and serological detection, including isolation and biochemical identification of Streptococcus equi, PCR, real-time fluorescent quantification, CRISPR and the like. Since the clinical symptoms are not obvious in the early stage of the disease, subclinical infection of horses only shows slight symptoms or no symptoms, and only clinical diagnosis cannot completely and accurately identify the infected horses. In addition, since both sub-species of Streptococcus equi can cause diseases in horses and cause serious losses, it is necessary to develop a rapid and instant detection technology for detection of Streptococcus equi.
[0004] A serological diagnosis method for only Streptococcus equi subsp. equi is established abroad, which is an indirect enzyme-linked immunosorbent assay (ELISA) method. The existing Streptococcus equi detection products in China are relatively single, and only PCR diagnosis kits are available. These traditional and commonly used detection methods are not only time-consuming, labor-consuming and complicated in operation, but also are prone to produce false positive results. The existing diagnosis methods usually need special equipment for diagnosis, which limits the rapid detection of samples, and the technical requirements for the detection personnel are high. Although there are test strips for detection of Streptococcus equi DNA abroad at present, the DNA amplification and detection process is complicated and time-consuming. Compared with these detection methods, colloidal gold immunochromatography test strips are a research hotspot in recent years, which are simple to operate and do not need additional detection equipment and professional personnel, and the results can be visually interpreted in 10-15 min.
[0005] The present application is based on the specific antigen of Streptococcus equi being connected to the prokaryotic expression vector peT-28a, the prokaryotic recombinant of the protein is obtained, the protein H4 is induced to express and purify by being transformed into the engineering bacteria BL-21, and the antigenicity and the diagnostic potential are preliminarily evaluated by using Western blotting and ELISA technology, so as to provide the basis for the diagnosis and prevention and control of Streptococcus equi infection. The present application has the advantages of simple and fast operation, easy judgment of results, and is suitable for early and rapid diagnosis in clinical field. SUMMARY
[0006] The present application aims at the technical defects of the prior art, and provides a colloidal gold detection test strip capable of detecting two subspecies of Streptococcus equi simultaneously, so as to solve the problems of complex process, long operation time and the like of the conventional separation culture identification and PCR detection method of Streptococcus equi in the prior art.
[0007] In order to achieve the above technical purposes, the present application adopts the following technical solutions:
[0008] The present application provides a general colloidal gold antibody detection test strip for Streptococcus equi, comprising: a supporting PVC bottom plate and a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad fixed in sequence on the supporting bottom plate.
[0009] The gold-labeled protein pad is fixed with colloidal gold-labeled Streptococcus equi surface antigen recombinant protein H4.
[0010] The chromatographic detection membrane is provided with a detection line and a quality control line, and the detection line is coated with recombinant protein H4.
[0011] The gene sequence encoding the Streptococcus equi recombinant H4 protein is from GenBank: CRU96094.
[0012] The spraying concentration of the colloidal gold-labeled Streptococcus equi recombinant H4 protein is 3 mg / mL, and the spraying amount is 5 μL / cm. The spraying concentration of the Streptococcus equi recombinant H4 protein on the nitrocellulose membrane as the detection line is 2 mg / mL, and the spraying amount is 2 μL / cm.
[0013] The polyclonal antibody of rabbit anti-Streptococcus equi H4 recombinant protein is coated on the quality control line of the nitrocellulose membrane, and the spraying concentration is
[0014] The spraying concentration is 2 mg / mL, and the spraying amount is 2 μL / cm.
[0015] The rabbit polyclonal antibody is obtained after the rabbit is immunized with the Streptococcus equi H4 recombinant protein.
[0016] The gold-labeled protein pad is coated with glass fiber, and the detection membrane is a nitrocellulose membrane.
[0017] The application further provides application of the colloidal gold immunochromatography test strip in preparation of a streptococcus equi antibody detection and infection diagnosis kit.
[0018] The detection reaction principle of the colloidal gold immunochromatography test strip provided by the application is as follows:
[0019] When the sample end of the test strip is added with a serum sample liquid to be detected, the detection solution drives the antigen and gold-labeled protein in the sample to be detected to diffuse to the nitrocellulose membrane through siphon effect, and finally penetrates into the filter paper layer. If the sample to be detected contains the streptococcus equi surface antigen recombinant protein H4 antibody, the specific antibody in the serum is combined with the gold-labeled antigen during the diffusion process, the antigen-antibody-colloidal gold complex formed is intercepted by the detection line, and a red detection line (T) is presented, and the quality control line is intercepted by the gold-labeled antigen and presents a red quality control line (C). When the sample to be detected does not contain the streptococcus equi specific antibody, the detection line does not develop color, and only the red quality control line is presented. If there is no any red mark on the detection membrane, the test strip is invalid, and the detection result is invalid.
[0020] The positive effect of the above technical solution is as follows:
[0021] The application expresses the streptococcus equi recombinant protein H4 by using an escherichia coli expression system, uses the recombinant protein H4 as a specific antigen for detecting streptococcus equi antibodies, and establishes a streptococcus equi colloidal gold antibody detection test strip. The test strip can quickly and accurately detect the streptococcus equi specific antibody in the serum sample to be detected, has strong specificity, high sensitivity, simple operation, and a minimum detection limit of 1:128. The coincidence rate of the test strip and the whole bacterial antigen ELISA in simultaneous detection of clinical serum samples is 99.52%. The results show that the detection method can accurately and quickly detect whether the streptococcus equi antibody exists in the serum to be detected, the detection time is 5-10 min, the specificity is good, the sensitivity is high, the needs of personnel at different levels and in different occasions can be met, such as disease monitoring, health and epidemic prevention, breeding, etc., and the detection method has application value and great economic and social benefits. The detection method can be applied to the diagnosis reagent and epidemiological investigation of streptococcus equi infection. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 is an expression vector schematic diagram of the recombinant antigen protein H4.
[0023] Figure 2 is an SDS-PAGE analysis result diagram of the induced H4 protein. In the diagram, M: Marker; 1: before induction; 2-3:
[0024] H4 protein whole bacteria after induction.
[0025] Figure 3Figure is the result of SDS-PAGE analysis of purified H4 protein. In the figure, M: Marker; 1: before induction; 2-3: H4 protein after purification.
[0026] Figure 4 Figure is the result of Western blot detection of H4 protein; M: protein Marker; 1: control; 2: H4.
[0027] Figure 5 Figure is the detection sensitivity result of streptococcus equi antibody test strip.
[0028] Figure 6 Figure is the specificity of streptococcus equi test strip. In the figure, 1: E. coli positive serum; 2: S. aureus positive serum; 3: no lactococcus positive serum; 4: lactococcus positive serum; 5: lactococcus positive serum; 6: Salmonella positive serum; 7: equine pneumonia positive serum; 8: equine influenza positive serum; 9: streptococcus equi positive serum; 10: streptococcus equi negative serum. DETAILED DESCRIPTION
[0029] The biological material source of the present application is:
[0030] 1. The designed recombinant sequence is synthesized by Shanghai Shengong.
[0031] The present application will be further described below in combination with specific examples, which do not constitute any limitation on the scope of the present application. The details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and these modifications and replacements all fall within the protection scope of the present application.
[0032] Example 1 Prokaryotic expression of streptococcus equi H4 protein
[0033] 1. Construction of H4 antigen vector and prokaryotic expression
[0034] According to the sequence of GenBank ID: CRU96094 obtained from GenBank, a part of the antigen fragment sequence is screened and intercepted and named as H4. According to the preference of codons of E. coli BL21 (DE3), the codons are optimized to increase the expression yield of the target protein, and BamH I and Xho I enzyme cutting sites are added at both ends, and then cloned into pET-28a plasmid, such as Figure 1 , and sent to Shanghai Shengong Biotechnology Co., Ltd. for synthesis. The ligation product is transferred into BL21 competent cells, and after overnight culture, the bacteria are picked and shaken to extract plasmid. After enzyme digestion identification, the recombinant expression plasmid is named as pET-28a-H4, such as Figure 4 , and the bacterial liquid is preserved for standby use.
[0035] 2. Induced expression of recombinant protein H4
[0036] The recombinant E. coli pET-28a-H4 / BL21 was inoculated into a liquid culture medium containing kanamycin 50 μg / mL, activated by culturing at 37°C for 12 h at 180 rpm / min, and the next day, was transferred into fresh LB (Kan+, 50 μg / mL) culture medium at 2%, and cultured at 37°C until OD600 was about equal to 0.6, and then IPTG was added to a final concentration of 0.8 mmol / L, and induced at 30°C for 10 h, and then the bacterial cells were collected by centrifugation, and lysed, and the bacterial cells were broken by ultrasonic, and the supernatant was collected by centrifugation, and the expression of the target protein in the supernatant was analyzed by SDS-PAGE, and the results are shown in Figure 2 . Then, the recombinant protein was induced for mass expression, and the recovered supernatant was column-charged by ice-bath ultrasonic, and the target protein was eluted with Wash Buffer, and the eluate was the purified target protein, and a small amount of sample loading buffer was mixed thoroughly, and the induced bacterial cells were used as a control, and the purified results were analyzed by electrophoresis on SDS-PAGE. The protein was dialyzed in a renaturation buffer, and the dialysis solution was replaced every 8 h for three times, and finally dialyzed in PBS for 12 h, and the whole process was operated in a 4°C refrigerator, and the results after electrophoresis are shown in Figure 3 , and the expected target band of H4 appeared at about 34.3 kDa.
[0037] 3. Immunogenicity verification of the recombinant protein H4
[0038] The H4 protein sample was taken for 6 μL SDS-PAGE electrophoresis, and then Western blot detection was performed to verify the immunogenicity of the protein, and the results showed that the H4 protein appeared as a single band at about 34.3 kDa, as shown in Figure 4 , and the expected size was consistent, indicating that the H4 protein was successfully expressed and had good immunogenicity.
[0039] In summary, the selected fragment was linked to a prokaryotic expression vector to construct a recombinant plasmid, and the recombinant protein H4 was expressed by induction, and the His-tag protein purification column was used to purify the protein, and the purified tagged protein H4 was obtained.
[0040] Example 2 Preparation of rabbit anti-H4 protein polyclonal antibody
[0041] The recombinant protein H4 purified in Example 1 was mixed with an equal amount of adjuvant by emulsification at a mass ratio of 1:1, and emulsified with Freund's complete adjuvant for the first immunization, and emulsified with Freund's incomplete adjuvant for the booster immunization once every 14 days. The inoculation was performed by subcutaneous injection at multiple points on the back of the rabbit, and the blood was collected from the rabbit ear vein 7 days before and after each immunization, and the antibody titer was detected by ELISA to reach 10 5 times or more. When the above was reached, the heart blood was collected. The collected blood was centrifuged at 3000 r / min for 15 min, and the supernatant was the H4 protein polyclonal antibody.
[0042] Preparation and application of immunocolloidal gold test paper of example 3
[0043] 1. Preparation of colloidal gold and gold-labeled H4 complex
[0044] Colloidal gold particles of 20 nm were prepared by sodium citrate reduction method. The pH value of the colloidal gold solution was adjusted to pH 8 with 0.2 mol / L K2CO3, and the solution was slowly shaken in a shaker under light-protected conditions. 5 μg of purified H4 recombinant protein was added dropwise to 1 mL of colloidal gold solution, and the shaking was continued for 30 min. After standing for 10 min, BSA was added as a stabilizer to a final concentration of 1% (w / w), and the shaking was continued for another 30 min. The gold-labeled antigen solution was centrifuged at 2000 r / min for 20 min at 4°C to remove the colloidal gold polymer. The supernatant was then centrifuged at 12000 r / min for 50 min at 4°C. The centrifugate was divided into three layers. The supernatant contained unbound antigen, the deepest black spot in the bottom layer was unbound colloidal gold particle polymer, and the intermediate purple-red flocculent precipitate was the well-bound gold-labeled H4 complex.
[0045] 2. Preparation of colloidal gold binding pad
[0046] The purple-red flocculent precipitate was suspended in 1% BSA in 0.01 mmol / L PB at pH 9, and stored at 4°C in the dark for later use. After labeling, 5 μL / cm was sprayed onto the glass fiber membrane and dried.
[0047] 3. Coating of detection line and quality control line
[0048] The prepared H4 protein antigen was coated on the detection line of the nitrocellulose membrane at a concentration of 1 mg / mL, and the anti-H4 protein polyclonal antibody was coated on the quality control line of the nitrocellulose membrane at a concentration of 0.5 mg / mL. The coating diluent was 0.01 mol / L PB at pH 9. The two lines were separated by 0.5 cm, and were dried for later use.
[0049] 4. Assembly of colloidal gold test strip
[0050] The sample pad, colloidal gold pad, nitrocellulose membrane, and absorbent paper were sequentially pasted on a PVC bottom plate and smoothed. The test strip was cut into 4 mm wide strips using a cutting machine, and the cut test strips were placed in a packaging bag containing a desiccant for later use.
[0051] 5. Sensitivity and repeatability test
[0052] One strain of Streptococcus equi positive serum was selected as the colloidal gold evaluation serum, which was diluted by doubling dilution. The dilution factors were 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024, respectively. Each dilution was repeated three times to evaluate the sensitivity of the Streptococcus equi colloidal gold test strip. The results are shown in Table 1.Figure 5 As shown in the figure, the detection results show that when the positive serum with a dilution ratio of 1:128 is detected, the prepared test strip detection line and the quality control line position still appear two reaction bands at the same time, which is obviously different from the negative control, and can be judged as positive.
[0053] Different batches of test strips prepared by the method of Example 3 were randomly selected and detected for 3 times, and the results showed that there was no difference in the detection results of different batches of samples, indicating that the prepared Streptococcus equi antibody detection colloidal gold immunochromatography test strip had good repeatability and stable quality.
[0054] 6. Specificity test
[0055] The prepared Streptococcus equi colloidal gold test strip was used to detect the positive serum of E. coli, S. aureus, S. agalactiae, S. dysgalactiae, S. equi, S. equi, and other pathogens, and the detection was repeated for 3 times. The results showed that the prepared test strip had no cross reaction with the positive serum of the above-mentioned pathogens, and the results are shown in Figure 6 .
[0056] 7. Conformity test
[0057] 208 horse serum samples were detected by indirect ELISA, and the detection results are shown in Table 7. Using the method for detection, 22 positive sera were detected by indirect ELISA, and the positive rate was 10.58% (22 / 208). 21 positive sera were detected by H4 test strip, and the positive rate was 10.10% (21 / 208). The coincidence rate with ELISA method was 99.52%, indicating that the established immunocolloidal gold test strip detection method had high consistency with the indirect ELISA detection method.
[0058] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all embodiments, and other embodiments can be obtained according to the present embodiments, which are within the scope of the present application.
Claims
1. A colloidal gold antibody test strip for detecting Streptococcus equi antibody, characterized in that, The test strip comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, an absorbent paper and a PVC base plate, the colloidal gold pad is a glass fiber membrane adsorbed with colloidal gold labeled H4 recombinant antigen, the nitrocellulose membrane has a rabbit anti-H4 protein polyclonal antibody coated control line and a Streptococcus equi recombinant H4 protein coated detection line, and the sample pad provides a position for adding the sample to be tested.
2. The colloidal gold immunochromatographic test strip according to claim 1, characterized in that, The gene sequence encoding the Streptococcus equi recombinant H4 protein is from GenBank: CRU96094.
3. The colloidal gold immunochromatographic test strip according to claim 1, characterized in that, The spraying concentration of the colloidal gold labeled Streptococcus equi H4 protein is 3 mg / mL, and the spraying amount is 5 μL / cm.
4. The colloidal gold immunochromatographic test strip according to claim 1, characterized in that, The spraying concentration of the Streptococcus equi H4 protein is 2 mg / mL, and the spraying amount is 2 μL / cm.
5. The colloidal gold immunochromatographic test strip according to claim 1, characterized in that, The polyclonal antibody coated on the control line is rabbit anti-Streptococcus equi H4 protein, the spraying concentration is 2 mg / mL, and the spraying amount is 2 μL / cm; the polyclonal antibody is obtained after immunizing a rabbit with the purified Streptococcus equi H4 protein.
6. The colloidal gold immunochromatographic test strip according to claim 1, characterized in that, The gold labeled protein pad is a glass fiber membrane, and the chromatographic detection membrane is a nitrocellulose membrane.
7. Use of the colloidal gold immunochromatography test strip of any one of claims 1-6 in the preparation of a Streptococcus equi infection diagnostic reagent.