Salmonella pullorum high-throughput detection method and kit

Abstract

The invention relates to the technical field of bacterial detection, in particular to a Salmonella pullorum high-throughput detection method which comprises the following steps: inoculating Salmonella pullorum to a TSB culture medium, culturing to obtain a bacterial liquid, inactivating the bacterial liquid, centrifuging to remove supernatant, dyeing by using a crystal violet ethanol solution, adjusting the concentration of the Salmonella pullorum to 5 * 10 < 7 > CFU / mL to 5 * 10 < 9 > CFU / mL, and carrying out high-throughput detection on the Salmonella pullorum. And preparing to obtain a salmonella gallinarum antigen solution. The method comprises the following steps: diluting serum to be detected, adding into a porous plate, drying and fixing, sealing by using a PBS (Phosphate Buffer Solution), adding a salmonella gallinarum antigen solution, incubating, washing, and observing by naked eyes or measuring by using a microplate reader so as to judge the result. According to the invention, high-flux, low-cost and rapid detection of pullorum disease can be realized, the problem of insufficient accuracy of traditional manual interpretation is solved, and a technical method is provided for identification and diagnosis of pullorum disease.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial detection, and in particular to a high-throughput detection method and kit for Salmonella pullorum. Background Art

[0002] Salmonella is a common and important zoonotic pathogen. Based on their invasiveness in different hosts, Salmonella can be divided into broadly tropic and host-specific Salmonella. Chickens are the primary host of Salmonella. Pullorum, caused by Salmonella pullorum, is a bacterial infectious disease that seriously harms the poultry industry. The disease manifests as acute septicemia with high mortality in chicks, while in adult chickens, it often presents as a chronic or latent infection, leading to decreased egg production and vertical transmission. The most effective measure to control the spread of the Salmonella pullorum pathogen is to achieve its elimination. Therefore, rapid identification and culling of chickens infected with Salmonella pullorum is crucial. Timely and accurate detection and diagnosis of the pathogen are crucial for the effective prevention and control of Salmonella in poultry farms.

[0003] Currently, traditional serum testing methods, such as plate agglutination tests and tube agglutination tests, suffer from low throughput, subjective interpretation, and susceptibility to interference from sample impurities, often leading to false-positive and false-negative results. While molecular biology methods, such as PCR, offer high specificity, they are expensive and complex to perform, making them difficult to meet the large-scale screening needs of large-scale farms.

[0004] However, as the poultry industry develops towards intensive and large-scale development, the industry has put forward higher requirements for disease monitoring technology. There is an urgent need to establish a Salmonella pullorum detection method that can simultaneously meet the requirements of high-throughput detection, high sensitivity, and be suitable for different detection scenarios. Summary of the Invention

[0005] To solve the above problems, the present invention provides a high-throughput detection method and kit for Salmonella pullorum.

[0006] The present invention is achieved through the following technical solutions: A high-throughput detection method for Salmonella pullorum comprises the following steps: Salmonella enterica was inoculated into TSB medium. After the culture was harvested, the culture was inactivated and the supernatant was removed by centrifugation. The culture was stained with crystal violet ethanol solution and the concentration of Salmonella enterica was adjusted to 5×10 7 CFU / mL~5×10 9 CFU / mL, and the Salmonella Gallinarum antigen solution was prepared.

[0007] The serum to be tested is diluted and added to a multi-well plate, dried and fixed, blocked with PBS blocking solution, and then incubated with a Salmonella enterica antigen solution. The plate is washed and the results are determined by visual observation or microplate reader.

[0008] In the result determination, the formation of purple granular precipitation at the bottom of the well by naked eye was determined to be positive; the absorbance at 620 nm was measured by a microplate reader, and the OD value > 0.8 was determined to be positive.

[0009] Preferably, the serum is diluted with PBS buffer; the volume ratio of the serum to PBS buffer is 1:30-50.

[0010] Preferably, the PBS buffer further comprises Tween-20, and the volume fraction of Tween-20 in the PBS buffer is 0.01% to 0.05%.

[0011] Preferably, the inactivation method is to use formaldehyde with a volume fraction of 0.4% to 0.6%.

[0012] Preferably, the mass volume concentration of crystal violet in the crystal violet ethanol solution is 0.05% to 0.15%, and the volume fraction of ethanol is 70%.

[0013] Preferably, the drying temperature is 36°C to 38°C.

[0014] Preferably, the incubation temperature is 36° C. to 38° C., and the incubation time is 29 minutes to 31 minutes.

[0015] Preferably, the PBS blocking solution contains bovine serum albumin and gelatin; the mass concentration of the bovine serum albumin in the PBS blocking solution is 0.5% to 1.5%; the mass concentration of the gelatin in the PBS blocking solution is 0.05% to 0.15%.

[0016] A high-throughput serum detection kit for pullorum disease, comprising the Salmonella antigen solution according to claim 1 and PBS blocking solution.

[0017] The principle of the high-throughput detection method for Salmonella pullorum is based on the specific antigen-antibody binding reaction and indirect agglutination reaction. First, Salmonella pullorum is cultured, inactivated, and stained with crystal violet to produce particulate antigen. During the test, specific antibodies in the serum to be tested bind to the antigen coated on a multi-well plate. The stained Salmonella antigen solution is then added. If the corresponding antibodies are present in the serum, they bind to the antigen to form a visible purple granular precipitate, indicating a positive reaction. If the antibodies are absent, no visible precipitate forms, indicating a negative reaction. This method also supports quantitative detection using a microplate reader, measuring absorbance at a wavelength of 620 nm (OD values ​​> 0.8 are considered positive), enabling objective interpretation. The entire process combines the advantages of high-throughput multi-well plate detection with standardized antigen preparation and colorimetric determination procedures. This allows for rapid screening of hundreds to thousands of samples at once, making it suitable for serological monitoring and epidemiological surveys in large-scale livestock farms.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a high-throughput detection method for Salmonella pullorum, comprising the following steps: inoculating Salmonella Gallinarum in TSB culture medium, culturing and harvesting bacterial liquid, inactivating the bacterial liquid, centrifuging and removing the supernatant, staining with crystal violet ethanol solution, adjusting the Salmonella Gallinarum concentration to 5×10 7 CFU / mL~5×10 9 CFU / mL, prepare a Salmonella Gallinarum antigen solution; dilute the test serum and add it to the multi-well plate, dry and fix it, use PBS blocking solution to block, add the Salmonella Gallinarum antigen solution to incubate, wash, and observe by naked eye or measure with a microplate reader to determine the result; in the determination of the result, the formation of purple granular precipitate at the bottom of the well by naked eye is judged as positive; measure the absorbance at 620nm with a microplate reader, and the OD value>0.8 is judged as positive; This method utilizes a 96-well plate design, allowing for simultaneous testing of 92 samples (with 4 wells reserved for controls). This method increases throughput by over 10 times compared to traditional plate agglutination tests, and the entire process takes only 3-4 hours to complete, significantly improving detection efficiency. This method innovatively integrates dual criteria: visual observation (purple granule precipitation) and quantitative detection by a microplate reader. This method not only meets the precise quantitative needs of laboratories but is also adaptable to rapid on-site screening at the grassroots level. The interpretation consistency is 100%, addressing the subjectivity inherent in traditional manual interpretation. Regarding cost control, this method uses crystal violet staining instead of fluorescent labeling, reducing reagent costs by over 60%. Furthermore, the antigen preparation process is simplified, resulting in a single test cost of only one-fifth that of an ELISA method, and it can be performed at the grassroots level without specialized equipment. Furthermore, experiments have demonstrated high accuracy, excellent reproducibility, and reliability. This method can be used for large-scale screening for cleanroom monitoring of breeder farms and provides an early diagnostic tool for vertical transmission prevention and control. Furthermore, the digital recording of test data facilitates the establishment of a comprehensive disease traceability system. The present invention also possesses excellent detection sensitivity, effectively identifying samples with low antibody concentrations, significantly improving the ability to detect early-stage infections and weakly positive samples. Through the innovative combination of high-throughput design, dual-mode interpretation, and low-cost processes, it effectively addresses key technical bottlenecks such as low throughput, high cost, and highly subjective interpretation of traditional detection methods. It provides a highly effective solution for population monitoring and precise prevention and control of Salmonella pullorum, and is particularly suitable for critical applications such as regular screening of breeding poultry farms, health monitoring of commercial chicken flocks, and import and export quarantine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 This is a 96-well plate test diagram of the present invention. Figure 1 In the test wells, A1 and B1 are positive controls, C1 and D1 are negative controls, and the remaining wells are sample wells to be tested. DETAILED DESCRIPTION

[0021] To facilitate understanding of the present invention, the present invention will be described more fully below, along with preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0023] The beneficial effects of the present invention are described below through specific examples.

[0024] The Salmonella Gallinarum used in the present invention is a standard Salmonella Gallinarum purchased from the China Microbiological Culture Collection Center.

[0025] Example 1: A high-throughput serum detection method for pullorum disease 1. Sample preprocessing Chicken peripheral blood was collected and centrifuged at 3000 rpm for 10 minutes. After obtaining serum, it was diluted to 1:30 with PBS buffer containing 0.01% Tween-20.

[0026] 2. Preparation of antigen solution Antigen preparation: Standardized Salmonella enterica strains were inoculated into TSB medium and cultured with shaking at 37°C for 18 h. The bacterial suspension was harvested and inactivated with 0.3% formaldehyde for 24 h, centrifuged and the supernatant removed. The suspension was stained with 70% ethanol containing 0.05% crystal violet for 30 min. The stained bacteria were resuspended in PBS containing 2% glycerol, pH 7.4, and the concentration was adjusted to 5 × 10 7 CFU / mL, and a standard Salmonella Gallinarum antigen solution was prepared.

[0027] 3. Sample coating Add the diluted serum sample to the sample test well, 50 μL / well; add the positive serum sample to the positive control well, and add the negative serum sample to the negative control well, both at 50 μL / well, and place them at 36°C for drying and fixation.

[0028] 4. Sealing and washing A PBS blocking solution containing 1% BSA and 0.1% gelatin was added to each well of a 96-well plate and incubated at 37°C for 1 hour. 200 μL of 1× PBS solution was then added to each well for washing, and the washing was repeated three times.

[0029] 5. Antigen-antibody reaction Add 50 μL of the prepared antigen solution to each well of a 96-well plate and incubate at 36°C with gentle shaking for 29 minutes. Discard the liquid in the wells and add 200 μL of wash buffer (1× PBS) to each well. Let it stand for 1 minute and then discard. Repeat the wash three times. Finally, add another 200 μL of wash buffer and keep it.

[0030] 5. Result determination Interpretation by naked eye: If a purple granular precipitate is formed in the well (adsorbed at the bottom of the well), it is judged as positive; if there is no precipitate in the well, it is judged as negative.

[0031] Instrumental testing: Use a microplate reader to measure the absorbance (OD value) at 620 nm. The negative control OD reading is less than or equal to 0.2, the positive control OD reading is greater than 0.8, and the test sample OD reading greater than 1.5 is considered strongly positive. The test sample OD reading is greater than 0.2 and less than 0.8 is considered weakly positive. The test sample OD reading is less than 0.2 is considered negative.

[0032] Example 2: A high-throughput serum detection method for pullorum disease 1. Sample preprocessing Chicken peripheral blood was collected and centrifuged at 3000 rpm for 10 minutes. After obtaining serum, it was diluted to 1:35 with PBS buffer containing 0.03% Tween-20.

[0033] 2. Preparation of antigen solution Antigen preparation: Standardized Salmonella enterica strains were inoculated into TSB medium and cultured at 37°C for 18 h. The bacterial suspension was harvested and inactivated with 0.5% formaldehyde for 24 h, centrifuged and the supernatant removed. The suspension was stained with 70% ethanol containing 0.1% crystal violet for 30 min. The stained bacteria were resuspended in PBS containing 2% glycerol, pH 7.4, and the concentration was adjusted to 5 × 10 8 CFU / mL, and a standard Salmonella Gallinarum antigen solution was prepared.

[0034] 3. Sample coating Add the diluted serum sample to the sample test well, 50 μL / well; add the positive serum sample to the positive control well, and add the negative serum sample to the negative control well, both at 50 μL / well, and place them at 37°C for drying and fixation.

[0035] 4. Sealing and washing A PBS blocking solution containing 1% BSA and 0.1% gelatin was added to each well of a 96-well plate and incubated at 37°C for 1 hour. 200 μL of 1× PBS solution was then added to each well for washing, and the washing was repeated three times.

[0036] 5. Antigen-antibody reaction Add 50 μL of the prepared antigen solution to each well of a 96-well plate and incubate at 37°C with gentle shaking for 30 minutes. Discard the liquid in the wells and add 200 μL of wash buffer (1× PBS) to each well. Let it stand for 1 minute and then discard. Repeat the wash three times. Finally, add another 200 μL of wash buffer and keep it.

[0037] 5. Result determination Interpretation by naked eye: If a purple granular precipitate is formed in the well (adsorbed at the bottom of the well), it is judged as positive; if there is no precipitate in the well, it is judged as negative.

[0038] Instrumental testing: Use a microplate reader to measure the absorbance (OD value) at 620 nm. The negative control OD reading is less than or equal to 0.2, the positive control OD reading is greater than 0.8, and the test sample OD reading greater than 1.5 is considered strongly positive. The test sample OD reading is greater than 0.2 and less than 0.8 is considered weakly positive. The test sample OD reading is less than 0.2 is considered negative.

[0039] Example 3: A high-throughput serum detection method for pullorum disease 1. Sample preprocessing Chicken peripheral blood was collected and centrifuged at 3000 rpm for 10 minutes. After obtaining serum, it was diluted to 1:40 with PBS buffer containing 0.05% Tween-20.

[0040] 2. Preparation of antigen solution Antigen preparation: Standardized Salmonella enterica strains were inoculated into TSB medium and cultured with shaking at 37°C for 18 h. The bacterial suspension was harvested and inactivated with 0.6% formaldehyde for 24 h, centrifuged and the supernatant removed. The suspension was stained with 70% ethanol containing 0.15% crystal violet for 30 min. The stained bacteria were resuspended in PBS containing 2% glycerol, pH 7.4, and the concentration was adjusted to 5 × 10 9 CFU / mL, and a standard Salmonella Gallinarum antigen solution was prepared.

[0041] 3. Sample coating Add the diluted serum sample to the sample test well, 50 μL / well; add the positive serum sample to the positive control well, and add the negative serum sample to the negative control well, both at 50 μL / well, and place them in a 38°C oven-dried container for fixation.

[0042] 4. Sealing and washing A PBS blocking solution containing 1.5% BSA and 0.15% gelatin was added to each well of a 96-well plate and incubated at 37°C for 1 hour. 200 μL of 1× PBS solution was then added to each well for washing, and the washing was repeated three times.

[0043] 5. Antigen-antibody reaction Add 50 μL of the prepared antigen solution to each well of a 96-well plate and incubate at 38°C with gentle shaking for 30 minutes. Discard the liquid in the wells and add 200 μL of wash buffer (1× PBS) to each well. Let it stand for 1 minute and then discard. Repeat the wash three times. Finally, add another 200 μL of wash buffer and keep it.

[0044] 5. Result determination Interpretation by naked eye: If a purple granular precipitate is formed in the well (adsorbed at the bottom of the well), it is judged as positive; if there is no precipitate in the well, it is judged as negative.

[0045] Instrumental testing: Use a microplate reader to measure the absorbance (OD value) at 620 nm. The negative control OD reading is less than or equal to 0.2, the positive control OD reading is greater than 0.8, and the test sample OD reading greater than 1.5 is considered strongly positive. The test sample OD reading is greater than 0.2 and less than 0.8 is considered weakly positive. The test sample OD reading is less than 0.2 is considered negative.

[0046] Example 4 From 2022 to 2024, a total of 1,200 blood samples were collected from breeder chickens in Gansu and Ningxia provinces and regions. The high-throughput detection method for Salmonella pullorum described in Example 1 was used for the detection and diagnosis of Salmonella pullorum. Visual interpretation revealed 248 positive samples, while microplate reader analysis revealed 248 strongly positive samples and 52 weakly positive samples.

[0047] At the same time, the 300 positive samples tested by the traditional whole blood plate agglutination test with the microplate reader showed that there were 186 strongly positive samples and 14 weakly positive samples. The above results show that the patented technology of the present invention has a high sensitivity in detecting pullorum disease and its accuracy is much higher than the traditional whole blood plate agglutination test results.

[0048] It should be noted that the present invention also uses the high-throughput detection method for detecting Salmonella pullorum of Examples 2 and 3 to detect and diagnose Salmonella pullorum. The results are the same as those of Example 1, and have high sensitivity in detecting pullorum disease. The accuracy is also much higher than the detection results of the traditional whole blood plate agglutination test.

[0049] Example 5: A high-throughput serum detection kit for pullorum disease Kit Composition: Sample diluent: PBS buffer containing 0.05% Tween-20 Positive control: serum from known infected chickens Negative control: SPF chicken serum Antigen solution: Standard Salmonella Gallinarum antigen solution Washing buffer: 10× PBS concentrate, diluted 10 times with deionized water before use Blocking solution: PBS blocking solution (containing 1% BSA, 0.1% gelatin) Instructions for use of the kit: 1. Fix the sample: dilute chicken serum to 1:40 with PBS (containing 0.05% Tween-20), then dispense 50 μL into the sample wells to be tested. Add positive serum sample to the positive control wells and negative serum sample to the negative control wells, both at 50 μL / well. Dry and fix at 37°C.

[0050] 2. Blocking and washing: Add 50 μL of PBS blocking solution to each well of a 96-well plate, incubate at 37°C for 1 hour, then add 200 μL of washing buffer (1× PBS) to each well for washing, and repeat the washing process three times.

[0051] 3. Antigen-antibody reaction: Add 50 μL of the prepared standard Salmonella Gallinarum antigen solution to each well and incubate at 37°C with slow shaking for 30 minutes. Discard the liquid in the wells and add 200 μL of washing solution (1× PBS). Let it stand for 1 minute and then discard. Repeat the washing process three times. Finally, add 200 μL of washing buffer and keep it.

[0052] Determination method of using this kit: 1. Visual interpretation: If a purple granular precipitate forms in the well (adsorbed at the bottom of the well), it is judged as positive; if there is no precipitate in the well, it is judged as negative.

[0053] 2. Instrumental Detection: Use a microplate reader to measure the absorbance (OD value) at 620 nm. The negative control OD reading should be less than or equal to 0.2, the positive control OD reading should be greater than 0.8, and the test sample OD reading greater than 1.5 is considered strongly positive. The test sample OD reading greater than 0.2 and less than 0.8 is considered weakly positive, and the test sample OD reading less than 0.2 is considered negative.

[0054] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0055] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. A person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims.

Claims

1. A high-throughput detection method for Salmonella pullorum, characterized in that: The following steps are involved: Salmonella enterica was inoculated into TSB medium. After the culture was harvested, the culture was inactivated and the supernatant was removed by centrifugation. The culture was stained with crystal violet ethanol solution and the concentration of Salmonella enterica was adjusted to 5×10 7 CFU / mL~5×10 9 CFU / mL, and the Salmonella Gallinarum antigen solution was prepared; The serum to be tested is diluted and added to a multi-well plate, dried and fixed, blocked with PBS blocking solution, and then incubated with a Salmonella enterica antigen solution, washed, and the results are determined by visual observation or microplate reader. In the result determination, the formation of purple granular precipitation at the bottom of the well by naked eye was determined to be positive; the absorbance at 620 nm was measured by a microplate reader, and the OD value > 0.8 was determined to be positive.

2. The method according to claim 1, characterized in that Serum was diluted with PBS buffer; The volume ratio of the serum to PBS buffer is 1:30-50.

3. The method according to claim 2, characterized in that The PBS buffer further comprises Tween-20, and the volume fraction of Tween-20 in the PBS buffer is 0.01% to 0.05%.

4. The method according to claim 1, wherein The inactivation method is to use formaldehyde with a volume fraction of 0.4% to 0.6% for inactivation.

5. The method according to claim 1, characterized in that The mass concentration of crystal violet in the crystal violet ethanol solution is 0.05%-0.15%, and the volume fraction of ethanol is 70%.

6. The method according to claim 1, characterized in that The fixed temperature of the drying is 36°C to 38°C.

7. The method according to claim 1, characterized in that The incubation temperature is 36° C. to 38° C., and the incubation time is 29 minutes to 31 minutes.

8. The method according to claim 1, characterized in that The PBS blocking solution contains bovine serum albumin and gelatin; the mass concentration of the bovine serum albumin in the PBS blocking solution is 0.5% to 1.5%; the mass concentration of the gelatin in the PBS blocking solution is 0.05% to 0.15%.

9. A high-throughput serum detection kit for pullorum disease, characterized in that: The kit comprises the Salmonella antigen solution and PBS blocking solution as claimed in claim 1.

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