Extraction and application of salmonella pullorum lipopolysaccharide
阅读说明：本技术 鸡白痢沙门菌脂多糖的提取及其应用 (Extraction and application of salmonella pullorum lipopolysaccharide ) 是由 顾小雪 李丹 刘洋 朱国强 王传彬 刘颖昳 徐琦 刘玉良 宋晓晖 于 2020-05-13 设计创作，主要内容包括：本发明公开了鸡白痢沙门菌脂多糖的提取及其应用。本发明提供了鸡白痢沙门菌脂多糖在如下中的应用：1)制备检测鸡白痢沙门菌产品；2)制备检测鸡白痢沙门菌抗体产品。本发明提取了鸡白痢沙门菌脂多糖,以鸡白痢沙门菌脂多糖作为抗原检测抗体会提高体系的敏感性和特异性,将假阳性、假阴性的检测情况大大降低。建立并优化了鸡白痢沙门菌抗体的间接ELISA方法、荧光微球定量层析法、化学发光法,可为临床检测鸡白痢沙门菌感染鸡和种鸡群净化工作奠定基础。(The invention discloses extraction and application of salmonella pullorum lipopolysaccharide. The invention provides an application of Salmonella pullorum lipopolysaccharide in the following steps: 1) preparing a product for detecting salmonella pullorum; 2) preparing a product for detecting the salmonella pullorum antibody. The invention extracts salmonella pullorum lipopolysaccharide, improves the sensitivity and specificity of the system by taking salmonella pullorum lipopolysaccharide as an antigen detection antibody, and greatly reduces the detection conditions of false positive and false negative. The indirect ELISA method, the fluorescent microsphere quantitative chromatography method and the chemiluminescence method of the salmonella pullorum antibody are established and optimized, and a foundation can be laid for the clinical detection of the salmonella pullorum infected chickens and the purification work of breeding hens.)
1. The application of Salmonella pullorum lipopolysaccharide in at least one of the following 1) -7):
1) preparing a product for detecting salmonella pullorum;
2) preparing a product for detecting the salmonella pullorum antibody;
3) preparing a product for detecting whether a sample to be detected is infected with or contains salmonella pullorum;
4) preparing a product for detecting whether a sample to be detected is infected with or contains the salmonella pullorum antibody;
5) detecting salmonella pullorum;
6) detecting an antibody of salmonella pullorum;
7) the application of the recombinant strain in detecting the salmonella pullorum or the antigen of the antibody of the salmonella pullorum.
2. Use according to claim 1, characterized in that: the content of protein in the salmonella pullorum lipopolysaccharide is more than or equal to 200 mug/mL.
3. Use according to claim 1 or 2, characterized in that: the content of polysaccharide in the salmonella pullorum lipopolysaccharide is more than or equal to 500 mug/mL.
4. Use according to any one of claims 1 to 3, characterized in that: the Salmonella pullorum lipopolysaccharide is derived from Salmonella pullorum standard strain and Salmonella pullorum variant strain.
5. Use according to claim 4, characterized in that:
the standard strain of salmonella pullorum is CVCC 526;
the salmonella pullorum mutant is CVCC 530.
6. Use according to any one of claims 1 to 5, characterized in that: the salmonella pullorum lipopolysaccharide is prepared by the following method: extracting the mixed thallus of the standard strain of salmonella pullorum and the variant strain of salmonella pullorum by a hot phenol water method to obtain lipopolysaccharide.
7. Use according to any one of claims 1 to 6, characterized in that: the product is an indirect ELISA detection kit, a chemiluminescence detection kit or a fluorescent microsphere quantitative chromatography detection kit.
8. An indirect ELISA detection kit for detecting Salmonella pullorum or an antibody thereof, wherein the antigen is the Salmonella pullorum lipopolysaccharide of any one of claims 1-6.
9. A chemiluminescence detection kit for detecting salmonella pullorum or an antibody thereof, wherein the antigen is the salmonella pullorum lipopolysaccharide in any one of claims 1-6.
10. A fluorescent microsphere quantitative chromatography detection kit for detecting salmonella pullorum or an antibody thereof, wherein a detection line of the kit is formed by the salmonella pullorum lipopolysaccharide in any one of claims 1-6;
or, a method for preparing lipopolysaccharide, extracting the mixed thallus of the standard strain of salmonella pullorum and the variant strain of salmonella pullorum by hot phenol water method to obtain lipopolysaccharide.
Technical Field
The invention relates to the technical field of biology, in particular to extraction and application of salmonella pullorum lipopolysaccharide.
Background
Pullorum disease is a common bacterial infectious disease of chickens caused by salmonella pullorum disease, and mainly causes septicemia and pullorum disease of chickens. The infection of chicks is acute septicemia, and the morbidity and mortality are high; adult chicken infection is mostly expressed as chronic or recessive infection, the mortality rate is not high, but the adult chicken infection becomes a carrier, the yield rate is reduced, the hatchability is reduced, and serious economic loss can be caused to the poultry industry. With the rapid development of the poultry industry, salmonellosis has become one of the most important egg-borne bacterial diseases, and the purification work of poultry group salmonellosis is carried out in north america and countries of the european union, and the pullorum disease and salmonella typhi infection are successfully controlled. The national poultry improvement program was implemented in 1935 in the United states, pullorum disease in breeder birds was basically eradicated in 1967, salmonella pullorum disease was hardly detected in breeder birds and commercial flocks in 1978, and pullorum disease and typhoid disease of chickens and turkeys were extinguished in 30 states in 1985. The world animal health organization WAHIS system showed that by 2015 pullorum disease was still present and prevalent in 19 countries of asia, korea, india, brazil, argentina, uk, usa, etc., africa, south america, europe, north america, and avian typhoid was still present and prevalent in 28 developing countries of asia, africa, south america, eastern europe.
The salmonella pullorum does not have an approved effective vaccine for prevention at present, and the disease is generally controlled by adopting medicaments in poultry farms, but the phenomenon of multiple drug resistance caused by the fact that a large amount of antibacterial medicaments are used throughout the year is obvious, and a satisfactory curative effect cannot be obtained. The egg vertical transmission is also an important transmission mode of salmonella in poultry flocks, so the salmonella infection purification of the poultry flocks is the most effective prevention and control method at present, and the scheme for preventing and controlling the animal epidemic diseases for medium and long term in China (2012 and 2020) requires that all the salmonellosis in chicken farms in the whole nation in 2020 meet the purification standard. The monitoring of the infection condition of salmonella in a breeding chicken farm is the basis of purification work, and a detection method with good sensitivity and specificity is needed to identify positive breeders of salmonella, so that elimination measures are taken. The detection method used in China at present is a salmonella pullorum antibody detection method recommended by OIE (International organization for Industrial diagnostics) and vaccine handbook for terrestrial animal, namely a plate agglutination test, but the method has limited sensitivity and specificity, false negative and false positive results are easy to occur, and the difference between batches of domestic detection reagents is large, so that the identification of obsolete positive chickens and the evaluation of the purification effect are seriously influenced in practical application. Therefore, a salmonella pullorum antibody detection method with good sensitivity and specificity is urgently needed in China.
According to the difference of the salmonella O antigen spectrum, the salmonella is divided into a plurality of serogroups, the antigen formulas of the salmonella pullorum O antigen are O1, O9 and O12, the salmonella belongs to a D serogroup, and the salmonella in the D serogroup also has another clinically common salmonella, namely salmonella enteritidis. The O antigen is a heat-resistant polysaccharide antigen of a thallus cell wall, the main component of the O antigen is Lipopolysaccharide (LPS) containing lipid A, O-polysaccharide side chains and core oligosaccharide, the O antigen has species, type and specificity, and compared with a whole-bacteria antigen, the O antigen has a single antigen type and high antigen purity.
Disclosure of Invention
One object of the present invention is to provide the use of Salmonella pullorum lipopolysaccharide.
The invention provides an application of Salmonella pullorum lipopolysaccharide in at least one of the following 1) -7):
1) preparing a product for detecting salmonella pullorum;
2) preparing a product for detecting the salmonella pullorum antibody;
3) preparing a product for detecting whether a sample to be detected is infected with or contains salmonella pullorum;
4) preparing a product for detecting whether a sample to be detected is infected with or contains the salmonella pullorum antibody;
5) detecting salmonella pullorum;
6) detecting an antibody of salmonella pullorum;
7) the application of the recombinant strain in detecting the salmonella pullorum or the antigen of the antibody of the salmonella pullorum.
In the application, the content of protein in the salmonella pullorum lipopolysaccharide is more than or equal to 200 mug/mL.
In the application, the polysaccharide content in the salmonella pullorum lipopolysaccharide is more than or equal to 500 mug/mL.
In the application, the salmonella pullorum lipopolysaccharide is derived from a salmonella pullorum standard strain and a salmonella pullorum variant strain.
In the application, the standard strain of salmonella pullorum is CVCC 526; the salmonella pullorum mutant is CVCC 530.
In the application, the salmonella pullorum lipopolysaccharide is prepared according to the following method: extracting mixed thalli of a standard strain of salmonella pullorum and a variant strain of salmonella pullorum by a hot phenol water method to obtain lipopolysaccharide;
the method comprises the following specific steps:
1) culturing a standard strain CVCC526 of the salmonella pullorum and a variant strain CVCC530 of the salmonella pullorum, mixing culture solutions of the two strains, and collecting thalli;
2) extracting the thalli with phenol, and collecting a phenol phase;
3) precipitating a phenol phase by using a methanol solution containing saturated sodium acetate, and collecting the precipitate to obtain a crude lipopolysaccharide extract;
4) and then sequentially precipitating protein impurities in the crude lipopolysaccharide extract by trichloroacetic acid, removing proteins by ultracentrifugation, and dialyzing by a dialysis bag to obtain the lipopolysaccharide.
In the application, the product is an indirect ELISA detection kit, a chemiluminescence detection kit or a fluorescent microsphere quantitative chromatography detection kit.
It is another object of the present invention to provide the following kit:
the invention provides an indirect ELISA detection kit for detecting salmonella pullorum or an antibody thereof, and an antigen of the kit is the salmonella pullorum lipopolysaccharide.
The invention also provides a chemiluminescence detection kit for detecting salmonella pullorum or an antibody thereof, and the antigen of the kit is the salmonella pullorum lipopolysaccharide.
The invention also provides a fluorescent microsphere quantitative chromatography detection kit for detecting the salmonella pullorum or the antibody thereof, wherein the detection line is formed by the salmonella pullorum lipopolysaccharide.
It is still another object of the present invention to provide a method for preparing lipopolysaccharide.
The method provided by the invention is characterized in that a hot phenol water method is used for extracting mixed thalli of a salmonella pullorum standard strain and a salmonella pullorum variant strain to obtain lipopolysaccharide.
The method for extracting mixed thalli of the salmonella pullorum standard strain CVCC526 and the salmonella pullorum variant strain CVCC530 by using a hot phenol water method to obtain lipopolysaccharide comprises the following steps:
the method comprises the following specific steps:
1) culturing a standard strain CVCC526 of the salmonella pullorum and a variant strain CVCC530 of the salmonella pullorum, mixing culture solutions of the two strains, and collecting thalli;
2) extracting the thalli with phenol, and collecting a phenol phase;
3) precipitating a phenol phase by using a methanol solution containing saturated sodium acetate, and collecting the precipitate to obtain a crude lipopolysaccharide extract;
4) and then sequentially precipitating protein impurities in the crude lipopolysaccharide extract by trichloroacetic acid, removing proteins by ultracentrifugation, and dialyzing by a dialysis bag to obtain the lipopolysaccharide.
The above 4) is specifically: adding trichloroacetic acid with final concentration of 5% (volume percentage content) into the crude extractive solution obtained in step 3), stirring at room temperature for 15min, centrifuging at 10000g for 15min, discarding precipitate, dialyzing the supernatant with distilled water (the cut-off molecular weight of the dialysis bag is 3500 Dalton) overnight, changing the solution for 2 times (at least 4000mL each time), and collecting the content in the dialysis bag, namely purified LPS (hot phenol water method).
The invention extracts salmonella pullorum lipopolysaccharide, improves the sensitivity and specificity of the system by taking salmonella pullorum lipopolysaccharide as an antigen detection antibody, and greatly reduces the detection conditions of false positive and false negative. Research and development work of ELISA methods for detecting salmonella antibodies is carried out at home and abroad, but the ELISA methods mainly aim at salmonella enteritidis and salmonella typhimurium. The invention firstly adopts the hot phenol water method recommended by the world animal health organization of the terrestrial animal diagnostic test and vaccine manual to extract the salmonella pullorum lipopolysaccharide as an antigen, establishes and optimizes an indirect ELISA method, a fluorescent microsphere quantitative chromatography method and a chemiluminescence method of the salmonella pullorum antibody, and lays a foundation for the clinical detection of the salmonella pullorum infected chickens and the purification work of breeding hens.
Drawings
FIG. 1 is a lipopolysaccharide staining pattern of Salmonella pullorum.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Some of the strains used in the examples below are as follows: standard strain CVCC526 and variant CVCC530 of Salmonella pullorum are purchased from China veterinary medicine inspection institute, SPF chicken negative serum is purchased from Beijing Meiliya Winton laboratory animal technology Limited, chicken Escherichia coli O78 positive serum, Newcastle disease positive serum (ND), avian influenza H5 positive serum (AIV H5), avian influenza H7 positive serum (AIV H7), avian influenza H9 positive serum (AIV H9), Avian Encephalomyelitis (AEV) positive serum, avian adenovirus type 3 positive serum, infectious bronchitis positive blood (IBV), chicken infectious anemia virus positive serum and avian leukemia J positive serum are purchased from Holland GD company, 588 parts of clinical serum are from various chicken farms nationwide provinces.
The salmonella pullorum infection positive serum is prepared and stored in the laboratory, and the specific method is as follows: activated salmonella pullorum standard strain CVCC526 bacterial liquid (the concentration is 4 multiplied by 10)9CFU/mL) and mutant strain CVCC530 (concentration 4X 10)9CFU/mL) volume ratio of 1:1, diluting to concentration required by infection inoculation (final concentration of each bacterium is 2X 10)9CFU/mL), and storing at 4 ℃ for later use; 21 day old SPF chickens, 5 intramuscular and 5 oral, were inoculated with defined salmonella-free antibodies. Observing the mental and developmental states of the chickens, drinking water and diarrhea and other adverse reactions every day after inoculation, collecting whole blood from the veins of the wings 5, 8, 12, 15, 19, 26, 33, 40, 48, 55, 61, 89 and 103 days after inoculation, separating serum, and storing at 4 ℃ for later use.
Some of the reagents used in the following examples are as follows: the Holland BioChek D Group Salmonella (Group D) antibody detection kit was purchased from Tai Biol Ltd, Beijing Tian. Phenol, trichloroacetic acid, methanol, silver nitrate, glacial acetic acid, formaldehyde, ethanol and sodium carbonate are all from domestic analytical purity. Gelatin, Tween 20, Bovine Serum Albumin (BSA), TMB bi-component color developing solution, newborn calf serum, dialysis bag and the like are purchased from Solebao corporation. Horse Radish Peroxidase (HRP) -labeled rabbit anti-chicken IgG antibody (A9046-1ML) was purchased from SIGMA. High adsorption ELISA flat-bottom ELISA plates were purchased from Thermo.
Some of the equipment used in the following examples are as follows: the Epoch BioTek enzyme-labeling instrument is purchased from Berton instruments, Inc., the electric heating constant temperature water bath (Blue Pard) is purchased from Shanghai Blue leopard testing equipment, Inc., the desk-top high-speed refrigerated centrifuge (Legend Micro 21R) is purchased from Sammer Feishell technology, Inc., the ultra-high speed centrifuge (L-100XP) is purchased from Beckmann Kulter commercial trade, Inc. (China), and the electrophoresis apparatus (POWERP TM BASIC) is purchased from Bole Life medicine products, Inc.
Preparation of the main solutions used in the following examples:
the main reagents comprise coating solution, confining liquid, washing solution, serum antibody diluent, enzyme-labeled secondary antibody diluent, substrate buffer solution, substrate using solution, stop solution and the like, and are prepared according to reference documents.
Preparation of PBS (pH 7.4) solution: weighing 8g of NaCl, 0.2g of KCl, 0.4 g of Na2HPO41.44g of NaH2PO40.24g of NaCl and deionized water for dissolution, fixing the volume to 1L, adjusting the pH value to 7.4, and autoclaving.
Preparing a PBST solution: to 1L of PBS was added 500. mu.L of Tween-20.
Coating solution (0.05mol/L sodium carbonate-sodium bicarbonate buffer, pH 9.6): adding double distilled water into Na2CO31.5g and NaHCO32.93g, fixing the volume to 1L, adjusting the pH value to 9.6, sterilizing under high pressure, and storing at 4 ℃.
PBST with 3% gelatin as blocking solution: 3g of gelatin was weighed and added to 100mL of PBST, mixed well and stored at 4 ℃.
Serum antibody diluent: 0.5g of sucrose (0.5% sucrose in PBS) was weighed and added to 100ml of PBS.
Example 1 extraction and detection of Salmonella pullorum lipopolysaccharide
Hot phenol water method for extracting salmonella pullorum lipopolysaccharide
The method comprises the following specific steps:
1) obtaining a bacterial suspension
Activating a standard strain CVCC526 and a variant strain CVCC530 of the salmonella pullorum frozen at the temperature of-20 ℃, then streaking on a salmonella chromogenic medium (purchased from Shanghai Xin bioengineering Co., Ltd., produced by France Colma Jia company, product number SA130), and placing in a 37-DEG incubator for incubation until purple colonies appear. The purple colonies were picked, placed in 3ml of BHI medium (purchased from Beijing Leibobowder technologies Co., Ltd., product No. CM917B-05), and cultured overnight on a shaker. Respectively putting 3ml of fresh salmonella pullorum standard strain CVCC526 bacterial liquid and salmonella pullorum variant CVCC530 bacterial liquid into 300ml of BHI culture medium (the volume ratio of each bacterial liquid to the culture medium is 1: 100), and culturing overnight to obtain CVCC526 bacterial suspension and CVCC530 bacterial suspension with the bacterial contents of 4 multiplied by 10 respectively9CFU/ml bacterial solution.
2) Crushing
Sterilizing the above CVCC526 bacterial suspension and CVCC530 bacterial suspension, and mixing at a volume ratio of 1:1 (the final concentration of each bacteria after mixing is 2X 10)9CFU/mL); centrifuging at 10000g for 20min, and collecting precipitate to obtain thallus.
Weighing and calculating wet weight of the thallus, adding the wet weight of the thallus into sterilized distilled water according to the mass ratio of 1:3 (wet weight of the thallus: water mass), fully and uniformly mixing, and heating to 66 ℃; then adding equal volume of 90% (volume percentage content) phenol aqueous solution preheated at 66 ℃, continuously stirring for 15min at the temperature (66 ℃), naturally cooling at room temperature, and centrifuging for 15min at 10000g at 4 ℃; absorbing the lower layer of brownish red phenol phase by using a long tube, and filtering by using Whatman No. 1 filter paper to remove large thallus fragments; measuring the volume of a phenol phase by using a measuring cylinder, adding 3 times of volume of 2-8 ℃ precooled methanol solution (cold methanol containing 1 mass percent of saturated sodium acetate, wherein the solute is 1 mass percent of saturated sodium acetate, and the solvent is methanol) to precipitate lipopolysaccharide, incubating for 2h at 4 ℃, centrifuging for 10min at 10000g at 4 ℃, discarding supernatant, re-suspending the precipitate by using 1/2 volumes of distilled water of raw water phase, placing the precipitate in a beaker with a ceramic rotor, stirring for 18 h, and centrifuging for 10min at 10000g at 4 ℃ after stirring is finished. The supernatant solution was collected and stored at 4 ℃. Resuspending the precipitate with equal volume of sterilized distilled water, stirring at 4 deg.C for 2 hr, centrifuging to obtain supernatant, and mixing with the supernatant to obtain supernatant (crude extract of lipopolysaccharide).
3) Purification of
Subsequently, trichloroacetic acid with a final concentration of 5% (volume percentage) was added to the supernatant obtained in 2), after stirring at room temperature for 15min, 10000g was centrifuged for 15min, the precipitate was discarded, the supernatant was dialyzed with distilled water (3500 daltons cut-off molecular weight in dialysis bag) overnight, the solution was changed 2 times (at least 4000mL each time), and the contents of the dialysis bag, i.e., purified LPS (hot phenol water method), were collected.
The LPS was lyophilized and weighed to 80 mg.
Secondly, extracting salmonella pullorum lipopolysaccharide from commercial lipopolysaccharide extraction kit
A suspension (4X 10) of CVCC526 from 1) above was prepared9CFU/mL) and CVCC530 bacterial suspension (4X 10)9CFU/mL) was sterilized and mixed at a volume ratio of 1:1 (final concentration of each bacteria after mixing was 2X 10)9CFU/mL), extracting by a commercial lipopolysaccharide extraction kit according to the instruction to obtain the Salmonella pullorum LPS.
The commercial lipopolysaccharide extraction kit is a lipopolysaccharide extraction kit which is purchased from Beijing Belo Biotechnology Ltd, and has the product number of BB-31302-50T.
Third, detection
1. Lipopolysaccharide SDS-PAGE and silver staining identification
SDS-PAGE with 5% concentration gel and 12% separation gel, using 10. mu.g loading (purified LPS from the first or Salmonella pullorum LPS from the second), 40mA electrophoresis until bromophenol blue migrates to the lower end of the separation gel.
Silver staining method reference, fixing oxidized gel with 30% ethanol, 10% glacial acetic acid and 7g/L periodic acid at 22 deg.C for 20min, and fixing with ddH2Washing gel for 5min, and repeating for 3 times; staining with 1g/L silver nitrate for 30min at 30 ℃ and ddH2O washing the gel for 10 s; using 30g/L carbonDeveloping with sodium and 0.02% formaldehyde for 20 min; the color reaction was stopped with 10% glacial acetic acid, ddH2O wash gel take pictures.
The results are shown in FIG. 1, M.10-180kd marker, 1.1000. mu.g/mL commercial Salmonella enteritidis lipopolysaccharide (sigma), 2.1000. mu.g/mL commercial Salmonella typhimurium lipopolysaccharide (sigma), 3.1000. mu.g/mL of the above-mentioned one-obtained purified LPS, 4.100. mu.g/mL of the above-mentioned one-obtained purified LPS; as can be seen, the typical ladder-shaped band of the SDS-PAGE electrophoresis of the pullorum disease salmonella lipopolysaccharide is distributed between 10kd and 30kd, wherein a large amount of concentrated distribution exists between 10kd and 15kd, and partial distribution exists between 15kd and 20 kd.
2. Measurement of lipopolysaccharide concentration
The method comprises the steps of (1) measuring the lipopolysaccharide concentration of a sample to be detected by adopting an anthrone-sulfuric acid method (the specific method references are Zhangyan red, Chilobrachys lobrachys, Wuyangong, and the like, extraction and preparation of Salmonella enteritidis lipopolysaccharide [ J ] the development of animal medicine, 2001(03): 79-80.). A standard curve was drawn using glucose (purchased from Beijing Soilebao Tech. Co., Ltd., product ID0200-50mg) as a standard.
The samples to be detected are the purified LPS obtained in the first step (hot phenol water method) and the Salmonella pullorum LPS obtained in the second step (kit).
Adopting anthrone-sulfuric acid method and using glucose as standard substance to make standard curve, Y is 0.0107X +0.0703(R is20.9937), X is the glucose content (μ g/mL) and Y is the A value (OD), the average value of OD492nm for each LPS was put into the equation and calculated as follows:
the concentration of the purified LPS (hot phenol water method) polysaccharide obtained in the previous step is 834 mu g/mL; the polysaccharide concentration in the Salmonella pullorum LPS (kit) obtained in the second step is 155 mu g/mL.
3. Measurement of lipopolysaccharide protein content
The BCA method is adopted to determine the protein content in a sample to be detected (BCA protein concentration detection kit (Biyuntian) is purchased from Wuhan Bokeming biotechnology limited company, product code P0012S), and bovine serum albumin (bovine serum albumin is purchased from Beijing Solebao science and technology limited company, product code A8020-500G) is used as a standard substance to draw a concentration standard curve.
The samples to be tested are the purified LPS obtained in the first step (hot phenol water method) and the Salmonella pullorum LPS obtained in the second step (kit).
And (3) adopting a BCA method to determine, and drawing a concentration standard curve by taking BSA as a standard substance: y is 0.0002x +0.1014, R20.9946, x is the a value (OD) and y is the protein content (μ g/mL), calculated as follows:
the protein content in the purified LPS (hot phenol water method) obtained in the previous step is 328. mu.g/mL; the protein content in the Salmonella pullorum LPS (kit) obtained in the second step is 30 mug/mL.
The above results show that it is possible to obtain,
the polysaccharide concentration of the salmonella pullorum lipopolysaccharide extracted by the hot phenol water method (the purified LPS (hot phenol method)) is 834 mug/mL, and the protein concentration is 328 mug/mL; the polysaccharide concentration of the LPS (kit) of the salmonella pullorum extracted by the existing kit is 155 mug/mL, and the protein content is 30 mug/mL. As can be seen from the above, the lipopolysaccharide, the polysaccharide and the protein of the salmonella pullorum extracted by the hot phenol method are higher in content, and the lipopolysaccharide is more suitable for being used as an antigen.
Example 2 detection of Salmonella pullorum by indirect ELISA method using lipopolysaccharide of Salmonella pullorum as antigen
Method for detecting salmonella pullorum by indirect ELISA (enzyme-linked immunosorbent assay)
The indirect ELISA reaction of the salmonella pullorum lipopolysaccharide obtained in the first step and the salmonella pullorum lipopolysaccharide obtained in the second step are respectively used as antigens to carry out the indirect ELISA reaction with salmonella pullorum infection positive serum and SPF chicken serum by using 50 mu g/mL of the salmonella pullorum lipopolysaccharide obtained in the first step in the example 1, and the indirect ELISA reaction is concretely as follows:
the lipopolysaccharide of Salmonella pullorum obtained by different methods is respectively diluted to 10 mu g/mL by using antigen diluent (0.05mol/L sodium bicarbonate buffer solution, pH9.6), each well is 100 mu L, and the Salmonella pullorum is placed for 16h at 4 ℃. The liquid was discarded, washed 3 times with PBST, 3% gelatin PBST was added to each well, and blocked at 4 ℃ for 12 h. 2 parts of salmonella pullorum infection positive serum and 1 part of SPF chicken negative serum are respectively diluted according to the ratio of 1: 50. Adding 100 μ L of HRP-labeled rabbit anti-chicken IgG antibody (diluted solution: 100 μ L) into each well, reacting at 37 deg.C for 60min, discarding the liquid, washing with PBST for 3 times, and adding 100 μ L of HRP-labeled rabbit anti-chicken IgG antibody (diluted solution: 10000) into each wellPBST), reacting at 37 deg.C for 30min, discarding liquid, washing with PBST for 3 times, adding 100 μ L substrate developer (TMB bi-component developer) into each well, developing at 37 deg.C in dark for 15min, adding 50 μ L stop solution (2mol/L H) into each well2SO4Aqueous solution), OD was read at 450nm using an enzyme-linked reader.
As shown in Table 1, the reaction effect of LPS obtained by the monopyrophenol method in example 1 with the serum positive for infection with Salmonella pullorum was better than that of LPS extracted by the two kits in example 1, and was not different from that of SPF chicken serum.
Table 1 shows the comparison of the detection of Salmonella pullorum
Second, condition optimization of LPS obtained by hot phenol method as antigen for indirect ELISA reaction
1. Determination of optimal antigen coating concentration and serum dilution
A chessboard dilution method is adopted.
1) Coating: the purified LPS obtained in example 1 (the pyrogen method) was diluted to 40. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL, 2.5. mu.g/mL with an antigen diluent (0.05mol/L sodium bicarbonate buffer), and the final column was used as a blank, 100. mu.L per well, and the cells were left at 4 ℃ for 16 hours to be coated;
2) and (3) sealing: discarding the liquid, washing with PBST for 3 times, adding PBST containing 3% gelatin as blocking liquid into each well, and blocking at 4 deg.C for 12 h;
3) primary antibody reaction
The Salmonella pullorum infection positive serum and the SPF chicken negative serum are respectively diluted according to the ratio of 1:12.5 and the ratio of 1: 25. cndot. 1:800 (serum antibody diluent is diluted by PBST), added into a reaction plate according to the concentration from top to bottom, 100. mu.L of serum antibody is added into each hole, and the last line is used as a serum-free blank control. Reacting at 37 ℃ for 60 min;
4) secondary antibody reaction
Discarding the liquid, washing with PBST for 3 times, diluting with 100 μ LPBST diluent per well to obtain HRP-labeled rabbit anti-chicken IgG (horse radish peroxidase (HRP) -labeled rabbit anti-chicken IgG antibody (A9046-1ML) from SIGMA) at a ratio of 1: 10000), and reacting at 37 deg.C for 30 min;
5) color development
Discarding liquid, washing with PBST for 3 times, adding 100 μ L substrate developer (TMB bi-component developer) into each well, developing at 37 deg.C in dark for 15min, and adding 50 μ L stop solution (2mol/L H) into each well2SO4) OD was read at 450nm using an enzyme-linked reader.
The P value is the OD value of the salmonella pullorum infection positive serum, and the N value is the OD value of the SPF chicken negative serum; the condition of the maximum P/N value is determined as the optimal antigen coating concentration and the optimal antibody dilution.
The result of the square matrix experiment is shown in table 2, when the antigen coating concentration and the serum action concentration are higher, the OD value of the positive serum is maintained at a higher level; according to the condition when the P/N value is the highest, the optimal antigen coating concentration is 10 mug/mL, and the optimal serum antibody dilution is 1: 50.
Table 2 shows indirect ELISA matrix titration experiment for Salmonella pullorum
2. Determination of optimal sealing liquid and sealing conditions
The method according to 1, the different steps are as follows:
1) coating with purified LPS (Hot phenol Water) diluted to 10. mu.g/mL;
2) and (3) sealing: discarding liquid, washing with PBST for 3 times, adding 1% gelatin-PBST, 3% gelatin-PBST, 2% skimmed milk powder-PBST, 10% skimmed milk powder-PBST, 1% newborn calf serum-PBST, 10% newborn calf serum-PBST, 1% BSA-PBST, and 3% BSA-PBST (mass percentage) as blocking liquid into each well, and blocking at 4 deg.C for 1h, 2h, 12h, and 24 h;
preparation of PBS (pH 7.4) solution: weighing 8g of NaCl, 0.2g of KCl, 0.41 g of Na2HPO41, 40.24g of KH2PO40 and deionized water for dissolving, fixing the volume to 1L, adjusting the pH value to 7.4, and autoclaving.
Preparing a PBST solution: to 1L of PBS (pH 7.4) was added 500. mu.L of Tween-20.
1% gelatin-PBST, weighing 1g gelatin, adding into 100mL PBST, mixing well, and storing at 4 deg.C.
3% gelatin-PBST, 3g gelatin is weighed and added into 100mL PBST, and the mixture is uniformly mixed and stored at 4 ℃.
2% skimmed milk powder-PBST, 2g skimmed milk powder is weighed and added into 100mL PBST, and the mixture is uniformly mixed and stored at 4 ℃.
10% skimmed milk powder-PBST, weighing 10g skimmed milk powder, adding into 100mL PBST, mixing, and storing at 4 deg.C.
1% newborn bovine serum-PBST: weighing 1mL of newborn bovine serum, adding the newborn bovine serum into 99mL of BST, and uniformly mixing the newborn bovine serum and the 99mL of BST for later use at 4 ℃.
10% newborn bovine serum-PBST: weighing 10mL of newborn bovine serum, adding the newborn bovine serum into 90mLPBST, and uniformly mixing the newborn bovine serum and the 90mLPBST for later use at 4 ℃.
1% BSA-PBST: 1g BSA was weighed into 100mL PBST, mixed well and stored at 4 ℃.
3% BSA-PBST: 3g BSA was weighed into 100mL PBST, mixed well and stored at 4 ℃.
3) Primary anti-reaction: the optimal serum antibody dilution is 1: 50;
4) secondary antibody reaction;
5) developing color;
the rest of the procedure was the same as 1.
And finally, after the OD value is detected, determining the condition with the maximum P/N value as the type of the optimal sealing liquid and the optimal sealing time.
As shown in tables 3 and 4, the highest P/N value was obtained when 3% gelatin-PBST was used as a blocking solution, and the highest P/N value was obtained when blocking was carried out at 4 ℃ for 12 hours.
Table 3 shows the selection of blocking solutions
TABLE 4 selection of blocking time
3. Optimal serum antibody dilution and determination of optimal time of action of serum antibodies
The method according to 1, the different steps are as follows:
1) coating with purified LPS (Hot phenol Water) diluted to 10. mu.g/mL;
2) and (3) sealing: discarding the liquid, washing with PBST for 3 times, adding 3% gelatin-PBST as blocking liquid into each well, blocking at 4 deg.C for 12 hr to obtain the highest P/N value;
3) primary antibody reaction
Diluting salmonella pullorum infection positive serum and SPF chicken negative serum by taking 0.5% sucrose-PBST, 5% newborn bovine serum-PBST, 10% skim milk-PBST, 1% BSA-PBST, 5% BSA-PBST, 1% BSA + 5% newborn bovine serum-PBST and PBST as serum antibody diluents according to a ratio of 1:50 respectively; reacting at 37 ℃ for 0.5h, 1h, 1.5h and 2 h;
PBS (pH 7.4) solution: weighing 8g of NaCl, 0.2g of KCl, 0.41 g of Na2HPO41, 40.24g of KH2PO40 and deionized water for dissolving, fixing the volume to 1L, adjusting the pH value to 7.4, and autoclaving.
PBST solution: to 1L of PBS was added 500. mu.L of Tween-20.
0.5% sucrose-PBST: 0.5g of sucrose was weighed and added to 100ml of BST, and the mixture was uniformly mixed and stored at 4 ℃.
5% newborn bovine serum-PBST: 5mL of newborn bovine serum is measured and added into 95mLPBST, and the mixture is uniformly mixed and is reserved at 4 ℃.
10% skimmed milk powder-PBST, weighing 10g skimmed milk powder, adding into 100mL PBST, mixing, and storing at 4 deg.C.
1% BSA-PBST: 1g BSA was weighed into 100mL PBST, mixed well and stored at 4 ℃.
5% BSA-PBST: 5g BSA was weighed into 100mL PBST, mixed well and stored at 4 ℃.
1% BSA + 5% newborn bovine serum-PBST: 1g BSA and 5mL were weighed and added to 95mL PBST, mixed well and stored at 4 ℃.
4) Secondary antibody reaction;
5) and (4) developing color.
The rest of the procedure was the same as 1.
After the final detection of the OD value, the condition of the maximum P/N value is determined as the optimal serum antibody action time.
The results are shown in Table 5, with 0.5% sucrose-PBST being the optimal serum antibody dilution and the highest P/N value. As shown in Table 6, the P/N value was the highest at 30min, and the optimal serum antibody duration was 30min at 37 ℃.
TABLE 5 selection of optimal serum sample dilutions
Table 6 selection of optimal time of action of serum
4. Optimal enzyme-labeled secondary antibody diluent and determination of working concentration and time
The method according to 1, the different steps are as follows:
1) coating with purified LPS (Hot phenol Water) diluted to 10. mu.g/mL;
2) and (3) sealing: discarding the liquid, washing with PBST for 3 times, adding 3% gelatin-PBST as blocking liquid into each well, blocking at 4 deg.C for 12 hr to obtain the highest P/N value;
3) primary antibody reaction
Diluting salmonella pullorum infection positive serum and SPF chicken negative serum according to a ratio of 1:50 by taking 0.5% sucrose-PBST as serum antibody diluent; reacting for 0.5h at 37 ℃;
4) secondary antibody reaction:
5% newborn bovine serum, 10% skimmed milk and PBST are respectively used as enzyme-labeled secondary antibody diluents, and 1:2500, 1:5000, 1:10000, 1:20000 and 1:40000 are used as enzyme-labeled secondary antibody diluents for reaction for 30min, 60min and 90 min.
5) Color development
The rest of the procedure was the same as 1.
And finally determining the condition of the maximum P/N value as the action time of the optimal enzyme-labeled secondary antibody diluent and the optimal enzyme-labeled secondary antibody after detecting the OD value.
The results are shown in tables 7-9, the optimal enzyme-labeled secondary antibody diluent is PBST, the optimal enzyme-labeled secondary antibody action time is 0.5h, and the optimal enzyme-labeled secondary antibody diluent is 1: 20000.
Table 7 shows the selection of enzyme-labeled secondary antibody dilutions
Table 8 shows the selection of the optimal enzyme-labeled secondary antibody duration
Table 9 shows the selection of dilution factor of enzyme-labeled secondary antibody
5. Determination of negative and positive critical value of indirect ELISA detection method
The indirect ELISA detection method is established according to the conditions found in the 2-4 steps as follows:
1) coating: the purified LPS obtained in example 1 (hot phenol water method) was diluted to 10. mu.g/mL with an antigen diluent (0.05mol/L sodium bicarbonate buffer), and the final column was used as a blank, 100. mu.L per well, and left at 4 ℃ for 16 hours to coat;
2) and (3) sealing: discarding the liquid, washing with PBST for 3 times, adding 3% gelatin-PBST into each well as sealing liquid, and sealing at 4 deg.C for 12 h;
3) primary antibody reaction
The salmonella pullorum infection positive serum and the SPF chicken negative serum are respectively diluted according to the ratio of 1:50 (serum antibody diluent is 0.5 percent of sucrose-PBST), added into a reaction plate from top to bottom according to the concentration, 100 mu L of serum antibody is added into each hole, and the last line is used as a serum-free blank control. Reacting at 37 ℃ for 30 min;
4) secondary antibody reaction:
discarding the liquid, washing with PBST for 3 times, adding 100 μ LPBST per well to dilute 1:10000 HRP-labeled rabbit anti-chicken IgG (horse radish peroxidase (HRP) -labeled rabbit anti-chicken IgG antibody (A9046-1ML) purchased from SIGMA), and reacting at 37 deg.C for 30 min;
5) color development
Discarding the liquid, washing with PBST for 3 times, adding 100 μ L substrate developing solution (TMB) into each well, developing at 37 deg.C in dark for 15min, adding 50 μ L stop solution (2mol/L H) into each well2SO4) OD was read at 450nm using an enzyme-linked reader.
91 parts of SPF-chicken negative serum were tested by the indirect ELISA assay described above and the OD450 values were determined. The mean and Standard Deviation (SD) of the negative controls were calculated as the positive and negative cut-off values (OD450 negative control +3 SD).
The minimum value of the 91 SPF chicken negative serum OD450 values is 0.104, the maximum value is 0.466, the standard deviation is 0.065, the average value is 0.248, and 3SD is 0.195, so the critical value of the test indirect ELISA method is determined as the negative control OD450 average value +0.195, namely the OD450 of the serum sample is more than or equal to the negative control OD450 average value +0.195, and the test is judged to be positive; the negative control was judged negative when OD450 was averaged to + 0.195.
Therefore, the criteria are as follows:
if the OD450 value of the serum to be detected is more than or equal to the negative control OD450 average value +0.195, the serum to be detected is judged to be positive, the serum to be detected is infected or candidate infected salmonella pullorum, or the serum to be detected contains candidate salmonella pullorum antibodies;
if the OD450 of the serum to be detected is less than the average value of negative control OD450 and is +0.195, the serum to be detected is judged to be negative, and the serum to be detected is not infected or the candidate is not infected with the salmonella pullorum, or the serum to be detected does not contain the candidate and does not contain the antibody of the salmonella pullorum.
Second, evaluation of Indirect ELISA detection method
1. Specificity test
The optimized indirect ELISA method is used for respectively carrying out specificity detection on Escherichia coli O78 positive serum, Avian Influenza (AI) H5, H7 and H9 positive serum, avian adenovirus (EDS) positive serum, Infectious Bronchitis (IBV) positive serum, mycoplasma septicum (MG) positive serum, Avian Leukemia (AL) positive serum, Chicken Infectious Anemia (CIA) positive serum, Newcastle Disease (ND) positive serum and known positive and SPF chicken serum.
The detection results of the indirect ELISA method show that as shown in Table 10, the OD values of Escherichia coli O78 positive serum, AIV H5, H7 and H9 positive serum, EDS positive serum, IBV positive serum, MG positive serum, ALV serum, CIA positive serum and ND positive serum are all below 0.515, the OD value of pullorum disease positive serum is 1.47, and the OD value of pullorum disease negative serum is 0.319, which indicates that the indirect ELISA method has good specificity.
Table 10 shows the results of the specificity test
2. Repeatability test
5 parts of salmonella pullorum infection positive serum and 1 part of SPF chicken negative serum are selected, on the same enzyme-labeled plate, the optimized indirect ELISA method is established for detection, and each serum is repeatedly provided with 5 holes. And calculating the variation Coefficient (CV) of the OD value of each serum sample according to the detected OD value result so as to detect the repeatability of the ELISA plates of the same batch for detecting the serum samples.
Selecting 5 parts of salmonella pullorum infection positive serum and 1 part of SPF chicken negative serum, coating the enzyme label plate with 3 parts of lipopolysaccharide of different batches, and detecting by the established optimized indirect ELISA method under the same condition. And calculating the variation Coefficient (CV) of the OD value of each serum sample according to the detected OD value result so as to detect the repeatability of the ELISA plates of the same batch for detecting the serum samples.
As a result, as shown in tables 11 and 12, the maximum value of the variation coefficient in the in-lot duplicate test was 9.48%, and the maximum value of the variation coefficient in the inter-lot duplicate test was 5.79%. The method has good stability, and the operation at different time has repeatability under the condition of unchanging reaction conditions.
TABLE 11 batch repeat test for indirect ELISA
TABLE 12 replicates in indirect ELISA batches
Three, indirect ELISA detection of clinical serum
As shown in Table 13, 588 serum samples were tested by BioChek D group Salmonella ELISA kit (purchased from Tai biol, Beijing, product No. CK117) and the optimized indirect ELISA method established in the first method, respectively, and the results were as follows:
324 parts of negative serum and 264 parts of positive serum are detected by the BioChek D group salmonella ELISA kit; the method detects 312 positive serums and 276 negative serums together;
therefore, the total coincidence rate of the method and the group D salmonella ELISA kit is 89.90%, wherein the positive coincidence rate is 84.21%, and the negative coincidence rate is 90.90%.
Table 13 shows statistics of coincidence rates of salmonella indirect ELISA antibody detection method and commercial indirect kit
Example 3 establishment and optimization of chemiluminescence with Salmonella pullorum lipopolysaccharide as antigen
Establishment of chemiluminescence method of lipopolysaccharide antigen of salmonella pullorum
1. Preparing a coated plate:
the purified LPS (hot phenol water method) obtained in one of example 1 was coated with carbonate buffer (0.05mol/L sodium bicarbonate buffer) (coating concentration 1. mu.g/mL), coated at 4 ℃ for 22h, washed 2 times with 300. mu.L of wash solution (PBST) per well, and patted dry;
sealing with casein sealing solution (from Sigma Aldrich trade company, product number C7078-500G formula or selling company) at 4 deg.C for 22-24 hr, throwing off sealing solution, and drying at 37 deg.C for 3 hr.
2. Determining the chemiluminescence reaction conditions:
diluting Salmonella pullorum infection positive serum and SPF chicken negative serum with PBST diluent 1/40, adding 100 microliters per well, and incubating at 37 deg.C for 30 min; washing the plate for 5 times, wherein each hole contains 300 microliters of washing liquid, and the plate is patted dry on a patting paper;
adding PBST diluent to dilute 1:10000 HRP-labeled rabbit anti-chicken IgG (100 microliters of horseradish peroxidase (HRP) -labeled rabbit anti-chicken IgG antibody per well, incubating for 30min at 37 ℃), washing the plate for 5 times, wherein each well contains 300 microliters of washing solution, and patting the plate on a patting paper;
adding 50 microliters of luminescent substrates A and B into each hole respectively, and reading the luminescent values at room temperature in a dark place for 5 min;
preparation of luminescent substrate A: 24.23g Tris was weighed into 800ml purified water, dissolved well and adjusted to pH 8.0 with HCl. Adding 0.026g of luminol, adding 0.086g of hydroxycoumarin after the luminol is completely dissolved, and fixing the volume of purified water to 1L. And (4) performing high-pressure sterilization, subpackaging and storing at 2-8 ℃.
Preparation of luminescent substrate B: weighing 15.42g of ammonium acetate, dissolving in 800ml of purified water, fully dissolving, adjusting the pH value to 5.2 by using glacial acetic acid, adding 0.021g of vitamin C, adding 0.066g of amino acid oxidase after completely dissolving, and fixing the volume of the purified water to 1L. And (4) performing high-pressure sterilization, subpackaging and storing at 2-8 ℃.
3. Determination of the positive and negative critical values of chemiluminescence method:
detecting 100 parts of negative and positive chicken serum by an established and optimized chemiluminescence method, determining the critical value to be S/P0.55, and judging the standard as follows:
if the S/P of the serum sample is more than or equal to 0.55, the serum sample is judged to be positive, the serum to be detected is infected or the candidate is infected with the salmonella pullorum, or the serum to be detected contains the candidate antibody of the salmonella pullorum;
if the S/P of the serum sample is less than 0.55, the serum sample is judged to be negative, the serum to be detected is not infected or the candidate is not infected with the salmonella pullorum, or the serum to be detected does not contain the candidate antibody of the salmonella pullorum.
Second, clinical serum detection
As shown in Table 14, 550 serum samples were tested by BioChek D group Salmonella ELISA kit (available from Tai biol, Beijing, product No. CK117) and chemiluminescence method established in the first method, respectively, and the results were as follows:
300 parts of negative serum and 250 parts of positive serum are detected by the BioChek D group salmonella ELISA kit; 284 positive serums and 266 negative serums are detected in total by the method; therefore, the total coincidence rate of the method and the group D salmonella ELISA kit is 92.73 percent, wherein the positive coincidence rate is 90.67 percent, and the negative coincidence rate is 95.2 percent.
Table 14 shows statistics of coincidence rate of pullorum disease salmonella antibody chemiluminescence method and commercial ELISA kit
Example 4 fluorescent microsphere quantitative chromatography method established and optimized by taking Salmonella pullorum lipopolysaccharide as antigen
Establishing a fluorescent microsphere quantitative chromatography method for detecting salmonella pullorum antibody
1. Preparation of pullorum Salmonella antibody test strip (fluorescent microsphere quantitative chromatography)
Sticking a cellulose nitrate film on a PVC bottom plate, spraying 0.4mg/mL of lgG of goat anti-chicken lgY (the lgG of the goat anti-chicken lgY is purchased from Jackson, USA, and the dilution concentration is 0.4 mg/mL) diluted to 0.4mg/mL on the cellulose nitrate film by a special dot-film gold spraying machine, weighing 6.7g of boric acid and 13.4g of borax (containing 10 crystal water), dissolving in 800mL of purified water, fixing the volume to 1L, adjusting the pH value to 8.5.) to form a quality control line and the diluted purified LPS (hot phenol water method) obtained in the example 1 (the dilution concentration is 0.4 mg/mL. the formula of the diluent solution, namely a first solution (0.2mol/L of disodium hydrogen phosphate solution), weighing 2.84g of disodium hydrogen phosphate solution and purified water to 100mL, weighing a second solution (0.2mol/L of sodium dihydrogen phosphate solution), 3.12g of sodium dihydrogen phosphate (containing 2 crystal water) and 100mL of purified water, fixing the volume to 94.5 mL of the first solution and the second solution, after mixing, 25ml of purified water is added into the 25ml of purified water, and the mixture is mixed evenly. ) Forming a detection line, wherein the spraying amount is 1 mu L/cm, and then baking for 8 hours at the temperature of 37 ℃;
lanthanide fluorescent microspheres labeled with chicken lgY (fluorescent microspheres (200nm), purchased from Calcilomycetes) and lanthanide fluorescent microspheres labeled with purified LPS obtained in example 1 (same as above) were prepared. The preparation method of the lanthanide fluorescent microsphere labeled chicken lgY comprises the following steps: adding 1mL of lanthanide fluorescent microspheres into 50mg of MES (2- (N-morpholine) ethanesulfonic acid) buffer solution (0.1M, pH7.0, wherein the formula comprises the steps of weighing 0.2g of 2- (N-morpholine) ethanesulfonic acid and 100mL of purified water for dissolving, adjusting the pH to 7.0), adding 10mg of carbodiimide (EDC) and 10mg of N-hydroxysuccinimide sulfonic acid sodium salt for stirring and dissolving, carrying out centrifugal operation after reacting for 30 minutes at room temperature, redissolving the centrifugal precipitate by using 50mM boric acid buffer solution (pH8.2), adding 2mg of dialyzed chicken lgY, stirring and reacting for 24 hours at room temperature, then centrifuging, sealing, and storing in a diluent (the storage environment temperature is 2-8 ℃) to obtain the lanthanide fluorescent microspheres marked with the chicken lgY; marking lanthanide series fluorescent microspheres of salmonella pullorum lipopolysaccharide by the same method;
respectively diluting lanthanide fluorescent microspheres marked with chicken lgY and marked with salmonella pullorum lipopolysaccharide to the concentrations of 0.2 mu g/ml and 2 mu g/ml, spraying the lanthanide fluorescent microspheres on a carrier base layer by adopting a spot-film gold spraying machine to form a marking pad, wherein the spraying amount is 2.5 mu L/cm, and then drying for 8 hours at the temperature of 37 ℃;
and sequentially adhering the sample pad, the marking pad and the absorbent paper on a PVC base plate adhered with an NC film containing a detection line and a quality control line to assemble a large card, and cutting the large card into test strips with the width of 5mm by using a shearing machine to obtain the salmonella pullorum antibody detection test strip.
2. Use method of detection test strip for determining pullorum disease salmonella antibodies
Diluting chicken serum (sample to be detected) with sample diluent (PBST) at a ratio of 1:4, and mixing; sucking a sample to be detected, vertically and slowly dripping 2-3 drops (about 60 mu L) into a sample adding hole of the detection card, opening a fluorescence immunoassay analyzer, standing for 10-15 minutes, and detecting on a machine, wherein the result is invalid after more than 20 minutes; and (3) detecting by using a fluorescence immunoassay analyzer to obtain fluorescence signals of the detection line (T) and the quality control line (C), and calculating the content of the salmonella pullorum antibody in the liquid to be detected according to the contrast relation of the fluorescence signal intensity values of the quality control line (T) and the detection line (C).
Detecting 100 parts of negative and positive chicken serum by using an established and optimized fluorescent microsphere quantitative chromatography, and determining the critical value as T/C0.015; the criteria are as follows:
if the T/C of the serum sample is more than or equal to 0.015, the serum sample is judged to be positive, the serum to be detected is infected or the candidate is infected with the salmonella pullorum, or the serum to be detected contains the candidate antibody of the salmonella pullorum;
if the T/C of the serum sample is less than 0.015, the serum sample is judged to be negative, the serum to be detected does not infect or the candidate does not infect the salmonella pullorum, or the serum to be detected does not contain the candidate antibody of the salmonella pullorum.
Second, clinical serum detection
As shown in Table 15, 550 serum samples were tested by BioChek D group Salmonella ELISA kit (purchased from Tai Biol., product No. CK117, Beijing Tian) and by the fluorescent microsphere quantitative chromatography method established in the first method), respectively, and the results were as follows:
the BioChek D group salmonella ELISA kit detects 300 parts of negative serum and 250 parts of positive serum, and the method detects 283 parts of positive serum and 267 parts of negative serum; therefore, the total coincidence rate of the method and the group D salmonella ELISA kit is 91.45%, wherein the positive coincidence rate is 89.3%, and the negative coincidence rate is 94%.
Table 15 shows the statistics of the coincidence rate of the salmonella pullorum antibody detection test strip and the commercialized ELISA kit