CN110824163A

CN110824163A - Colloidal gold test strip and reagent card for simultaneously detecting deer's disease and tuberculosis

Info

Publication number: CN110824163A
Application number: CN201911077003.4A
Authority: CN
Inventors: 杨艳玲; 张萍; 程悦宁; 郭健; 程世鹏
Original assignee: Institute Special Animal and Plant Sciences CAAS
Current assignee: Institute Special Animal and Plant Sciences CAAS
Priority date: 2019-11-06
Filing date: 2019-11-06
Publication date: 2020-02-21

Abstract

The invention discloses a colloidal gold test strip and a reagent card for simultaneously detecting deer brucellosis and tuberculosis by using sheep brucella LPS and recombinant Mycobacterium bovis MPB70 proteins, wherein the LPS and the MPB70 proteins are used as detection antigens, the established double-antigen sandwich method colloidal gold test strip is used for detecting antibodies in animal serum, and the test strip is used for detecting deer tuberculosis serum, foot-and-mouth disease serum, pasteurellosis serum and yersinia serum without cross reaction; the maximum dilution times of the positive sera for detecting the two diseases are both 1:512, bovine and deer serum samples are clinically detected, the total coincidence rate of the serum samples with the Brucella disease detection result by RBT and the total coincidence rate of the serum samples with the tuberculosis detection result by PPD is 96%, the test strip detection card is used for clinical tests in multiple areas of Jilin province, the specificity is good, the sensitivity is high, the operation is simple and convenient, the judgment is easy, and the method is suitable for clinical rapid screening of animal Brucella disease and tuberculosis.

Description

Colloidal gold test strip and reagent card for simultaneously detecting deer's disease and tuberculosis

Technical Field

The invention relates to a colloidal gold test strip and a reagent card for simultaneously detecting deer-brood and tuberculosis, belonging to the technical field of immunochromatography detection.

Background

Brucellosis (Brucella) and tuberculosis are two types of infectious diseases which seriously harm the animal husbandry, and cattle, sheep, deer, pigs and dogs are main susceptible animals. The former is caused by Brucella, and the latter is caused by mycobacterium tuberculosis (m.tuberculosis) or mycobacterium bovis (m.bovis). Brucellosis, abbreviated as brucellosis, can cause abortion and reproductive decline of female animals, and arthritis, orchitis and reduction of antler production of male animals. Tuberculosis can cause tuberculosis of multiple organs of animals, emaciation and exhaustion of organs and death. The disease distribution and tuberculosis belong to chronic infectious diseases infected by zoonosis and multi-host, which not only bring great loss to the breeding industry, but also cause serious harm to the health of the public, and are one of the important public health problems in the world.

With the rapid development of the deer industry, the disease distribution and the tuberculosis have seriously influenced the healthy breeding of the deer industry and the quality safety of deer products, and also bring serious hidden troubles to public health safety.

At present, the prevention and treatment measures for animal diseases and tuberculosis in various countries in the world are mainly quarantine-culling, so that accurate diagnosis is very important. At present, the diagnosis methods of animal brucellosis mainly comprise a tiger red plate agglutination test (RBT) and a test tube agglutination test (SAT), and have the defects of not strict judgment standard and poor specificity. Tuberculosis uses traditional intradermal allergy (PPD) and blood-gamma interferon release test (IGRA) to screen infected animals, and the two technologies are complex to operate and have the defects of poor specificity and low sensitivity. The imperfection of the detection technology brings many difficulties to the diagnosis and prevention of the disease distribution and tuberculosis, thereby causing the two diseases to be widely popularized in the cultured animal groups. Therefore, it is imperative to establish a rapid and simple diagnostic method with high sensitivity, high specificity and low cost.

Disclosure of Invention

In view of the above, the colloidal gold antibody test strip developed by the invention by using the Brucella melitensis LPS and the recombinant Mycobacterium bovis MPB70 protein as detection antigens is used for clinical rapid diagnosis of deer's disease and tuberculosis.

In order to achieve the purpose, the invention adopts the following technical scheme:

the utility model provides a detect deer's cloth disease and tuberculosis colloidal gold test paper strip simultaneously, including the PVC bottom plate, be fixed with the cellulose nitrate membrane on the PVC bottom plate in proper order, the gold mark pad, sample pad and absorption pad, the gold mark pad of gold mark antigen is adsorbed to the gold mark pad, be equipped with detection line T line and quality control line C line on the cellulose nitrate membrane, the C line sets up the one end near the absorption pad, detection line T line department is wrapped by the antigen that has LPS and MPB70 protein, quality control line C line department is wrapped by LPS and MPB70 protein polyclonal antibody, sample pad overlap joint is close to the top of sample pad one end on the gold mark pad, the other end of gold mark pad and the top of the one end difference overlap joint at the both ends of cellulose nitrate membrane of absorption pad.

The invention uses LPS and MPB70 protein as detection antigen, and the established double-antigen sandwich colloidal gold test strip is used for detecting antibody in animal serum. The test paper strip detects no cross reaction of deer tuberculosis serum (brucellosis serum), tuberculosis serum, foot-and-mouth disease serum, pasteurellosis serum and yersinia serosa; the maximum dilution times of positive serum of the two diseases are both 1: 512. The bovine and deer derived serum samples are clinically detected, the positive coincidence rate of the result with the RBT detection brucellosis result is 100%, the negative coincidence rate is 96%, and the total coincidence rate is 96%. The positive coincidence rate of the result of the detection of the tuberculosis by PPD is 94 percent, the negative coincidence rate is 97 percent, and the total coincidence rate is 96 percent. The test strip detection card is used for clinical tests in multiple areas of Jilin province, has good specificity, high sensitivity, simple and convenient operation and easy interpretation, and is suitable for clinical rapid screening of animal brucellosis and tuberculosis.

Further, the preparation method of the gold-labeled pad comprises the following steps:

(1) with K₂CO₃Adjusting the pH value of the colloidal gold to 7.3-7.7, adding LPS antigen with the concentration of 30-36 mug/mL and MPB70 protein antigen with the concentration of 20-24 mug/mL, fully and uniformly mixing, standing for 30min, adding BSA (bovine serum albumin) to the final concentration of 1%, continuously stirring for 20min, and standing for 15min to obtain gold-labeled antigen for later use;

(2) and (3) placing the combined pad in a sealing solution, sealing for 30min at 37 ℃, taking gold-labeled antigen, mixing with 0.2% sucrose, fully dissolving, adding to the combined pad until saturation, and naturally airing to obtain the gold-labeled pad, wherein the pH value of the sealing solution is 7.4, and each 0.01M of the sealing solution contains 1% BSA, 0.2% Tween-20 and 5mM NaCl.

The method for arranging the detection line T line and the quality control line C line on the nitrocellulose membrane comprises the following steps:

immersing the nitrocellulose membrane in a methanol solution for sensitization for 10min, placing the nitrocellulose membrane in a treatment solution, soaking at 37 ℃ for 30min, washing with PBS for 2 times, naturally airing, coating antigens of LPS and MPB70 protein and polyclonal antibodies of LPS and MPB70 protein on the nitrocellulose membrane, sealing with 1% BSA for 30min, and drying at 4 ℃, wherein the pH value of the treatment solution is 7.4, and each 0.01M of the treatment solution contains 0.2% Tween-20, 5mM NaCl and 5mM sucrose.

The sample pad was soaked in 0.01M PBS (pH 7.4) containing 3% BSA and 1% Tween-20 and then air dried.

Further, the preparation method of the LPS antigen comprises the following steps:

inoculating Brucella melitensis 16M into a meat soup culture medium, carrying out shake culture at 37 ℃ and 200rpm until OD600 is 2.0, inactivating the bacterial liquid at 100 ℃ for 60min, centrifuging for 15min to collect bacterial mud of the inactivated Brucella melitensis 16M, washing the bacterial mud with PBS for three times, weighing the bacterial mud, weighing saturated phenol according to the ratio of the saturated phenol to the bacterial mud of 4: 1, adding equal amount of sterilized water into the saturated phenol, preheating for 30min at 72 ℃, slowly adding the preheated bacterial mud into the bacterial mud, fully suspending the bacterial mud, continuing to act for 20min in 72 ℃ water bath, fully suspending once at an interval of 5min, centrifuging for 8h at 4 ℃, collecting a phenol water layer, adding the collected phenol water layer into a dialysis bag, dialyzing for 36h in pure water, subpackaging the dialyzed product into 2ml ampoules, freezing for 2h at-80 ℃, freeze-drying for 24h on a freeze dryer, adding 1% saturated sodium cold methanol with volume being 3 times of the freeze-dried product, slowly stirring for acting for 1h at 4 ℃, centrifuging for 15min, removing supernatant, collecting precipitate, adding 500ul of sterilized pure water, dissolving precipitate sufficiently, adding 50 μ g/mL of DNase and RNase respectively, performing enzymolysis at 37 deg.C for 2h, adding 15 μ g/mL of proteinase K, performing enzymolysis at 37 deg.C for 1h, cooling in ice bath, centrifuging for 20min at 1500rmp, adding 2 times volume of anhydrous ethanol into supernatant, and precipitating to obtain LPS.

The invention uses an improved method to extract 16M LPS of Brucella melitensis, selects four inactivation modes of 60 ℃ inactivation for 60min, 75 ℃ inactivation for 40min, 80 ℃ inactivation for 30min and 100 ℃ inactivation for 60min respectively according to the prior research method, cultures bacterial liquid on a TSA culture medium at 37 ℃ for 72h after inactivation, checks whether the inactivation is complete, and selects the optimal inactivation condition, and a great amount of experiments show that the Brucella melitensis can be completely inactivated at 100 ℃ for 60min, the traditional 67 ℃ water bath is changed in the operation of extracting LPS by saturated phenol, the action time of the water bath is changed from 10min to 20min, and simultaneously, one key step is grasped, namely, bacterial sludge is fully suspended by 72 ℃ sterilized water and then preheated saturated phenol is slowly added, the step has important significance to the extraction effect, not only saves the extraction time, but also obtains high-purity LPS, has important value for developing commercial products.

Further, the MPB70 protein antigen is prepared by the following method:

(1) taking 1-2 mul of activated bacterial liquid to 20ml of LB liquid culture medium, culturing at 37 ℃, culturing at 200rpm, transferring the cultured bacterial liquid to 1000ml of LB liquid culture medium, culturing at 37 ℃, 200rpm until OD is 0.6-0.8, inducing at 16 ℃ for overnight at 0.1mM IPTG, centrifuging at 6000rpm for 5min, removing supernatant to collect bacteria, and blowing the bacteria by using 20-30ml of 10mM Tris-HCl solution with the pH value of 8.0;

(2) and (2) washing the nickel column with deionized water until the pH value is 7.0, hanging nickel until the pH value is 2.0-3.0, washing the column with deionized water until the pH value is 7.0, balancing the nickel column with 10mM Tris-HCl solution with the pH value of 8.0, adding the thalli treated in the step (1), washing the column with 10mM Tris-HCl solution with 0.5M sodium chloride and the pH value of 8.0 after finishing, and eluting with 10mM Tris-HCl solution with the pH value of 8.0 and the pH value of 0.5M sodium chloride to obtain MPB70 protein.

Further, the preparation method of the polyclonal antibody of the LPS and MPB70 protein comprises the following steps:

the method comprises the steps of using purified LPS and MPB70 protein as antigens to respectively immunize New Zealand white rabbits, adopting a back subcutaneous multipoint injection mode, enabling the immunization time to be 0, 2, 4 and 6 weeks for 4 times, fully mixing the antigens with Freund's complete adjuvant according to the proportion of 1:1 before the 1 st immunization, fully mixing the antigens with Freund's incomplete adjuvant according to the proportion of 1:1 before the later 3 times of immunization, enabling the quantity of the antigens to be 400 mu g/mouse each time of immunization, taking blood from hearts at 7 weeks, placing at 37 ℃ to separate out serum, namely LPS and MPB70 protein polyclonal antibodies, and storing at-80 ℃ for later use.

The invention also provides a colloidal gold test card for simultaneously detecting the deer antler disease and the tuberculosis, which comprises a shell, wherein the colloidal gold test strip for simultaneously detecting the deer antler disease and the tuberculosis is arranged in the shell, a sample adding hole is arranged at the position, corresponding to the sample pad, on the shell, and an observation window is arranged at the position, corresponding to the nitrocellulose membrane, of the shell.

The colloidal gold detection card is firstly developed and used for simultaneously detecting deer brucellosis and tuberculosis, the selected antigens are LPS of brucella and MPB70 of mycobacterium bovis respectively, the existing antigen extraction and purification technology is optimized, the specific antigen LPS of brucella and the specific antigen MPB70 of mycobacterium bovis are obtained, a sensitive, specific and convenient-to-use rapid colloidal gold detection test strip is developed, and the colloidal gold detection card is constructed and can be simultaneously used for detecting deer brucellosis and tuberculosis. The current detection products aiming at the deer's disease and tuberculosis are not available, the deer is a special economic animal and has different physiological characteristics and disease incidence rules with other animals, the research is to develop the colloidal gold rapid antibody detection test strip product of the deer's disease and tuberculosis for the first time after research and analysis are carried out according to the disease incidence characteristics and epidemic rules of the deer's disease and tuberculosis, and the accurate diagnosis and purification of the deer's disease can be met. Meanwhile, products for simultaneously detecting the diseases of the deer and the tuberculosis are developed for the first time, after the research is deeply considered and researched by improving the existing method, the rapid colloidal gold antibody detection test strip capable of simultaneously detecting the diseases of the deer and the tuberculosis is obtained, and the detection card is assembled. Provides technical support for diagnosis and purification of deer diseases and tuberculosis in China.

Drawings

FIG. 1 is a graph showing the result of SDS-PAGE silver staining of Brucella LPS according to example 1 of the present invention;

FIG. 2 is a graph showing the result of the agarose immunodiffusion reaction between LPS and Brucella positive serum of example 1 of the present invention;

FIG. 3 is a graph showing the results of the purification of MPB70 protein in example 1 of the present invention;

FIG. 4 is a graph showing the results of Western blotting of MPB70 in example 1 of the present invention;

FIG. 5 is a graph showing the results of pH measurement of the antigen best labeled in example 3 of the present invention;

FIG. 6 is a graph showing the results of measurement of the concentration of the antigen-optimizing marker in example 3 of the present invention;

FIG. 7 is a diagram of a colloidal gold test strip according to embodiment 3 of the present invention;

FIG. 8 is a graph showing the test strip determination results in example 3 of the present invention;

FIG. 9 is a graph showing the results of clinical sample testing in example 4 of the present invention.

In the drawings, the structures represented by the respective reference numerals are as follows:

1. nitrocellulose membrane, 2, gold label pad, 3, sample pad, 4, absorption pad, 5, PVC base plate.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Antigen and antibody preparation:

1. the method comprises the following steps of (1) extracting and purifying the LPS of the Brucella melitensis 16M strain:

(1) the Brucella melitensis 16M is inoculated into a meat soup culture medium and is subjected to shake culture at 37 ℃ and 200rpm until the OD600 is about 2.0. The bacterial liquid is inactivated in a hot water bath at 100 ℃ for 60 min.

(2) Extraction of LPS from saturated phenol

10000g, centrifuging for 15min, collecting inactivated brucella 16M bacterial mud, washing the bacterial mud with PBS for three times, weighing after collecting the bacterial mud, and marking. Weighing saturated phenol according to the ratio of saturated phenol to bacterial sludge of 4: 1, preheating sterilized water and saturated phenol at 72 ℃ for 30min, slowly adding the sterilized water with the same amount as the saturated phenol into the bacterial sludge, fully suspending the bacterial sludge, slowly adding the saturated phenol, fully suspending, continuously acting in a water bath at 72 ℃ for 20min, and fully suspending once at intervals of 5 min.

(3)5000g, centrifuging at 4 ℃ for 8h, and collecting a phenol water layer.

(4) Adding the collected phenol water layer into dialysis bag, and dialyzing in pure water for 36 hr until there is no phenol smell.

(5) The dialyzed product was dispensed into 2ml ampoules, frozen at-80 ℃ for 2h and then lyophilized on a lyophilizer for 24 h.

(6) The lyophilized product was added to 3 volumes of 1% saturated sodium acetate in cold methanol and slowly stirred at 4 ℃ for 1 h.

(7)8000g are centrifuged for 15min, the supernatant is removed and the precipitate is collected, this time crude LPS.

(8) Adding 500ul of sterilized pure water to dissolve the precipitate sufficiently, and the amount of water added can be determined according to the amount of the precipitate.

(9) Adding 50. mu.g/mL of each of DNase and RNase, and carrying out enzymolysis at 37 ℃ for 2 h. Then adding 15 mu g/mL proteinase K, and carrying out enzymolysis for 1h at 37 ℃. Cooling in ice bath, centrifuging at 1500rmp for 20min, adding 2 times volume of anhydrous ethanol into the supernatant, and precipitating to obtain LPS.

(10) The concentration was determined with LPS quantification kit. And (3) separating the purified LPS by SDS-PAGE, and detecting after silver staining.

Extracting LPS of Brucella melitensis virulent strain 16M by using an improved hot phenol method, and detecting the concentration of the LPS by using an LPS quantitative kit after purification to be 0.23 mg/mL. SDS-PAGE separation and silver staining detection show that the extracted LPS is uniform smear, and the result is shown in figure 1, wherein M: pre-staining a Marker with protein; LPS-1, LPS-2: brucella 16M LPS extract.

2. Immunogenicity analysis of LPS

LPS is respectively used as an antigen, clinically collected deer-brood positive serum is used as an antibody to carry out agarose diffusion reaction to detect the reactionogenicity of the LPS, obvious precipitation lines appear around LPS holes under the condition that the LPS is diluted to be 3 times and 6 times, the extracted LPS is proved to have stronger immunogenicity, and the result is shown in figure 2, and the middle hole is the LPS; the 1-6 holes are all deer Brucella positive serum.

3. Expression of Mycobacterium bovis MPB70 protein

(1) 1-2. mu.l of the activated bacterial suspension was inoculated into 20ml of LB liquid medium and cultured at 37 ℃ at 200 rpm.

(2) The cultured bacterial suspension was transferred to 1000ml of LB liquid medium, cultured at 37 ℃ and 200rpm until OD becomes 0.6-0.8, and induced overnight with IPTG (0.1mM) at 16 ℃.

(3) Collecting bacteria: centrifuge at 6000rpm for 5 min. The supernatant was discarded.

(4) Ultrasonic bacteria breaking: the cells were lysed with 20-30ml 10mM Tris-HCl (pH 8.0) (resuspended in lysate for GST soluble protein purification) and sonicated (500W, 60 times 10s each, 15s apart).

(5) Electrophoresis to determine the expression pattern: mu.l of the sonicated bacterial suspension was centrifuged at 12000rpm for 10min, 50. mu.l of the supernatant was transferred to another EP tube, and after the supernatant was removed, the pellet was blown off with 50. mu.l of 10mM Tris-HCl (pH 8.0). And (4) carrying out electrophoresis detection.

The improved prokaryotic expression technology and the optimized nickel column purification method are utilized to express and purify the Mycobacterium bovis MPB70 protein, MPB70 protein with high expression abundance and purity is obtained, and the result is shown in figure 3. Meanwhile, MPB70 protein can generate specific immunoreaction with deer tuberculosis positive serum, and the result is shown in the result of immunoblot analysis in FIG. 4.

4. Purification of Mycobacterium bovis MPB70 protein

(1) The nickel column (Ni Sepharose 6Fast Flow, GE Healthcare) was washed with deionized water to pH 7.0.

(2) Hanging nickel until the pH value is 2-3.

(3) The column was washed with deionized water to pH 7.0.

(4)10mM Tris-HCI (pH 8.0) solution equilibrated the nickel column, about 100 ml.

(5) The nickel column was equilibrated with a 10mM Tris-HCI (pH 8.0) solution containing 0.5M sodium chloride, and about 50 ml.

(6) And (4) diluting the sample and loading. The sample contained sodium chloride at a final concentration of 0.5M.

(7) After the completion of the loading, the column was washed with a 10mM Tris-HCI solution (pH 8.0) containing 0.5M sodium chloride.

(8) The protein peaks were collected by eluting with 10mM Tris-HCI (pH 8.0) containing 15mM imidazole, 60mM imidazole, and 300mM imidazole, respectively, containing 0.5M sodium chloride.

(9) And (5) detecting the protein purification effect by electrophoresis.

5. MPB70 immunogenicity analysis

MPB70 is used as an antigen, deer tuberculosis positive serum is used as an antibody, the reactogenicity of MPB70 protein is detected by a WesternBlotting method, and the result shows that MPB70 protein can generate specific immunoreaction with the deer brood positive serum.

Example 2

Preparation of Rabbit anti-LPS, MPB70 polyclonal antibody

New Zealand white rabbits were immunized with purified LPS and MPB70 protein, respectively, as antigens. The immunization time is 4 times of

week

0, 2, 4 and 6. The immunization mode adopts subcutaneous multi-point injection on the back, the antigen is fully mixed with Freund's complete adjuvant according to the proportion of 1:1 before the 1 st immunization, and the antigen is fully mixed with Freund's incomplete adjuvant according to the proportion of 1:1 before the 3 last immunization. The amount of antigen per immunization was 400. mu.g/mouse. Blood was collected from the heart at week 7. The serum is deposited at 37 ℃ and stored at-80 ℃ for later use.

Example 3

Preparation of test paper strip

1. Determination of optimal pH value of gold-labeled antigen

20 centrifuge tubes were taken, 10 tubes each. Adding 1mL of colloidal gold solution into each tube, and adding 3% K₂CO₃The solution adjusted the pH of the colloidal gold solution (table 1). Adding 80 μ g LPS and MPB70 protein into the two groups of centrifuge tubes, mixing, and standing for 30 min. Then, 100. mu.L of 10% NaCl solution was added to each tube, and the mixture was mixed well and allowed to stand for 1 hour. By OD₅₅₀The value is detected and combined with the visual observation to determine the optimum pH value. OD₅₅₀The maximum value corresponds to the best pH value, and the minimum pH value of the solution red is maintained to be the best.

TABLE 1 pH of colloidal gold solution per tube

K₂CO₃Solution (mu L)	1	2	4	6	8	10	12	14	16	20
											pH value	6.8	7.1	7.3	7.5	7.7	7.9	8.1	8.4	8.6	8.8

The results are shown in FIG. 5, in which LPS was bound to colloidal goldAdding 3% K into colloidal gold solution with optimal pH value of 1mL₂CO₃Solution 6uL, pH 7.5. The optimal pH value for combining MPB70 protein and colloidal gold is 1mL of colloidal gold solution, and 3 percent K is added into the colloidal gold solution₂CO₃Solution 8uL, pH 7.5.

2. Determination of optimal concentration of gold-labeled antigen

Adjusting the pH value of the colloidal gold solution to an optimal value, adding 1mL of the colloidal gold solution into 10 centrifugal tubes of each group, respectively adding LPS and MPB70 proteins into the centrifugal tubes according to antigen amounts of 5 mug, 10 mug, 15 mug, 20 mug, 25 mug, 30 mug, 35 mug, 40 mug, 45 mug and 50 mug, fully and uniformly mixing, standing for 30min, adding 100 mug 10% NaCl solution, fully and uniformly mixing, standing for 1h, and performing OD (optical density) passing through₅₅₀The value is measured and combined with visual inspection to determine the antigen minimum label concentration. OD₅₅₀Values are plotted on the ordinate and antigen concentration on the abscissa, with the curve corresponding to the lowest marker concentration (lowest concentration that maintains the solution red) at the inflection point of the curve equilibrium. The optimum marker concentration is the minimum concentration × 120%.

As a result, as shown in FIG. 6, the lowest labeling concentration of LPS was 30. mu.g/mL, and the optimal labeling concentration was 36. mu.g/mL instead of 30X 120%. The lowest marker concentration of MPB70 protein is 20ug/mL, and the optimal protein concentration is 20 × 120% ═ 24 μ g/mL.

3. Preparation of gold label pad

3％K₂CO₃Adjusting the pH value of the colloidal gold to be 7.5(LPS group) and 7.7(MPB70 group) respectively; adding antigen according to the optimal concentration, mixing well, and standing for 30 min. Adding BSA to the final concentration of 1%, continuing stirring for 20min, and standing for 15min to allow the antigen to be stably combined with the colloidal gold particles. The conjugate pad was placed in blocking solution (pH7.4, 1% BSA in 0.01M PBS, 0.2% Tween-20 and 5mM NaCl) and blocked at 37 ℃ for 30 min. The treated bond pads were cut into strips of 0.40cm width. Adding 0.2% of sucrose into the gold-labeled antigen, fully dissolving, uniformly adding the gold-labeled antigen on the gold-labeled binding pad until the gold-labeled antigen is saturated, and drying the gold-labeled antigen at room temperature to obtain the gold-labeled pad.

4. Film scribing for detection line (T line) and quality control line (C line)

The nitrocellulose membrane (with backing) was sensitized by dipping in a methanol solution for 10 min. The membrane was placed in a treatment solution (pH7.4, 0.01M PBS containing 0.2% Tween-20, 5mM NaCl and 5mM sucrose), soaked at 37 ℃ for 30min, washed with PBS for 2 times, and air-dried at room temperature. The antigen membrane-spraying concentrations of LPS and MPB70 protein T lines were 0.25mg/mL and 0.5mg/mL, respectively. The lineation of the LPS and MPB70 protein polyclonal antibody on the C line is 1uL/cm and 2uL/cm respectively. Coating antigen antibody on nitrocellulose membrane with JY-EQ03 continuous membrane-drawing instrument, blocking with 1% BSA for 30min after coating, and drying at 4 ℃.

5. Sample pad treatment

The sample pad was soaked with 0.01M PBS (containing 3% BSA and 1% Tween-20) at pH7.4 and allowed to air dry at room temperature.

6. Test strip assembly and result determination

And sequentially adhering the nitrocellulose membrane, the gold label pad, the sample pad and the absorption pad to the PVC base plate. The width of the slit test strip is 4mm, as shown in fig. 7. During detection, 100uL (fresh serum diluted by 3-5 times) is added to a sample hole, and the result is judged after 10 min. The positive and negative of the sample or the failure of the test strip (the C line does not develop color) is judged according to the color development strips of the T line and the C line, the judgment result is shown in figure 8, brucellosis, tuberculosis +, brucellosis and tuberculosis are sequentially detected from left to right, the brucellosis and the tuberculosis of the detected animal can be simultaneously detected, the strips displayed by the test strip are clear, the color development is stable, no dragging is caused, and the negative and positive of the brucellosis and the tuberculosis in clinic can be better distinguished. .

Example 4

Test strip detection

1. Specificity test

The prepared test paper strips are used for respectively detecting deer brucellosis serum, tuberculosis serum, foot-and-mouth disease serum, pasteurellosis serum and yersinia serosa, and the results are shown in table 2.

TABLE 2 test strip specificity detection

Therefore, the prepared test strip is used for respectively detecting the deer brucellosis serum, the tuberculosis serum, the foot-and-mouth disease serum, the pasteurellosis serum and the yersinia serum, the result shows that no cross reaction exists, and the test strip is proved to have good specificity.

2. Sensitivity test

After the deer brucellosis positive serum (RBT and SAT detection both strong positive) and the tuberculosis serum (PPD and IGRA detection both strong positive) are diluted in a multiple ratio, a test strip is used for detection, the maximum dilution times of positive display are recorded, and the maximum dilution times of the recorded results are all 1: 512.

3. Coincidence rate detection

206 parts of cattle serum and 387 parts of deer serum collected clinically are detected. And (3) comparing the coincidence rate of the detection results of the tiger red plate agglutination test (RBT) and the colloidal gold test strip detection (GICA), the intradermal allergic reaction (PPD) and the colloidal gold test strip detection. The results are shown in Table 3.

TABLE 3 GICA, RBT and PPD test results

The total positive rate of Brucella detected by RBT is 6.57%, and the total positive rate of Brucella detected by GICA is 10.3%; compared with the RBT method, the GICA has the positive coincidence rate of 100 percent, the negative coincidence rate of 96 percent and the total coincidence rate of 96 percent. The total positive rate of the PPD detection tuberculosis is 21.2 percent, and the total positive rate of the GICA detection tuberculosis is 22.4 percent; compared with the RBT method, the GICA has a positive coincidence rate of 94 percent, a negative coincidence rate of 97 percent and a total coincidence rate of 96 percent. The two groups of methods have better conformity.

4. Clinical sample testing

3000 detection cards are prepared, popularization tests are carried out in areas of 29682Chun, people, Tunhua, Shulan, Changling, Changbai, Siping and the like, field detection is carried out in a basic farm, and the results are shown in figure 9, and as can be seen from figure 9, the results are clear, and judgment is simple.

Claims

1. The utility model provides a detect deer's cloth disease and tuberculosis colloidal gold test paper strip simultaneously, includes the PVC bottom plate, be fixed with cellulose nitrate membrane, gold mark pad, sample pad and absorption pad in proper order on the PVC bottom plate, its characterized in that, the gold mark pad of gold mark antigen is adsorbed to the gold mark pad, be equipped with detection line T line and quality control line C line on the cellulose nitrate membrane, the C line sets up and is being close to the one end of absorption pad, detection line T line department is wrapped by the antigen that has LPS and MPB70 protein, quality control line C line department is wrapped by LPS and MPB70 protein polyclonal antibody.

2. The colloidal gold test strip for simultaneously detecting deer's disease and tuberculosis as claimed in claim 1, wherein said sample pad is lapped on said gold-labeled pad above one end close to said sample pad, and the other end of said gold-labeled pad and one end of said absorption pad close to said nitrocellulose membrane are lapped on both ends of said nitrocellulose membrane respectively.

3. The colloidal gold test strip for simultaneously detecting deer Buse disease and tuberculosis as claimed in claim 1, wherein the preparation method of the gold-labeled pad comprises the following steps:

(1) with K₂CO₃Adjusting the pH value of the colloidal gold to 7.3-7.7, adding LPS antigen and MPB70 protein antigen, mixing well, standing for 30min, adding BSA to a final concentration of 1%, stirring for 20min, standing for 15min to obtain gold-labeled antigen for later use;

(2) and (3) sealing the bonding pad in a sealing solution at 37 ℃ for 30min, taking the gold-labeled antigen, mixing with 0.2% sucrose, fully dissolving, adding the gold-labeled antigen onto the bonding pad until the gold-labeled antigen is saturated, and naturally drying to obtain the gold-labeled pad.

4. The colloidal gold test strip for simultaneously detecting deer Buse disease and tuberculosis as claimed in claim 3, wherein the concentration of LPS antigen in step (1) is 30-36 μ g/mL;

the concentration of the MPB70 protein antigen is 20ug/mL-24 ug/mL.

5. The colloidal gold test strip for simultaneously detecting deer Buse disease and tuberculosis as claimed in claim 3, wherein the pH value of the blocking solution in step (2) is 7.4, and each 0.01M of the blocking solution contains 1% BSA, 0.2% Tween-20 and 5mM NaCl.

6. The colloidal gold test strip for simultaneously detecting deer's disease and tuberculosis as claimed in claim 1, wherein the method for setting detection line T and quality control line C on the nitrocellulose membrane comprises the following steps:

immersing the nitrocellulose membrane in a methanol solution for sensitization for 10min, placing the nitrocellulose membrane in a treatment solution, soaking for 30min at 37 ℃, washing for 2 times by PBS, naturally airing, coating antigens of LPS and MPB70 protein and polyclonal antibodies of LPS and MPB70 protein on the nitrocellulose membrane, sealing for 30min by 1% BSA, and drying at 4 ℃.

7. The colloidal gold test strip for simultaneously detecting deer Buse disease and tuberculosis as claimed in claim 6, wherein the pH value of the treatment solution is 7.4, and each 0.01M treatment solution contains 0.2% Tween-20, 5mM NaCl and 5mM sucrose;

the preparation method of the polyclonal antibody of the LPS and MPB70 protein comprises the following steps:

respectively immunizing New Zealand white rabbits with purified LPS and MPB70 protein as antigens, injecting subcutaneously at multiple points on the back for 4 times in 0, 2, 4 and 6 weeks, mixing the antigen with Freund's complete adjuvant at a ratio of 1:1 before the 1 st immunization, mixing the antigen with Freund's incomplete adjuvant at a ratio of 1:1 before the 3 later immunizations, taking blood from heart at 7 weeks with an amount of 400 μ g of antigen per immunization, and standing at 37 deg.C to separate out serum, i.e. LPS and MPB70 protein polyclonal antibody, and preserving at-80 deg.C for later use.

8. The colloidal gold test strip for simultaneously detecting deer antler disease and tuberculosis as claimed in claim 3 or 6, wherein the preparation method of the LPS antigen comprises the following steps:

inoculating 16M of Brucella melitensis into a meat soup culture medium, carrying out shake culture at 37 ℃ and 200rpm until OD600 = 2.0, inactivating the bacterial liquid at 100 ℃ for 60min, centrifuging for 15min, collecting bacterial sludge of the inactivated Brucella melitensis 16M, washing the bacterial sludge with PBS for three times, collecting the bacterial sludge, weighing, and mixing according to saturated phenol: bacterial sludge = 4: 1, adding equivalent sterilized water into saturated phenol, preheating at 72 ℃ for 30min, slowly adding into bacterial mud, after the bacterial mud is fully suspended, continuing to act for 20min in 72 ℃ water bath, fully suspending once at intervals of 5min, centrifuging at 4 ℃ for 8h, collecting a phenol water layer, adding the collected phenol water layer into a dialysis bag, dialyzing for 36h in pure water, subpackaging the dialyzed product into 2mL ampoules, freezing at-80 ℃ for 2h, freeze-drying for 24h in a freeze dryer, adding 1% saturated sodium acetate cold methanol in a volume of 3 times, slowly stirring at 4 ℃ for 1h, centrifuging for 15min, removing supernatant, collecting precipitate, adding 500ul sterilized pure water, fully dissolving precipitate, adding 50 mu g/mL each of DNase and RNAse, carrying out enzymolysis for 2h at 37 ℃, adding 15 mu g/mL protease K, carrying out enzymolysis for 1h at 37 ℃, cooling, centrifuging at 1500rmp for 20min, adding 2 times volume of absolute ethyl alcohol into the supernatant, and precipitating to obtain LPS;

the MPB70 protein antigen is prepared by the following method:

(1) taking 1-2 mul of activated bacterial liquid to 20ml of LB liquid culture medium, culturing at 37 ℃, culturing at 200rpm, transferring the cultured bacterial liquid to 1000ml of LB liquid culture medium, culturing at 37 ℃, 200rpm until OD =0.6-0.8, inducing at 0.1mM IPTG 16 ℃ overnight, centrifuging at 6000rpm for 5min, discarding supernatant to collect bacteria, and blowing the bacteria by using 20-30ml of 10mM Tris-HCl solution with the pH value of 8.0;

(2) and (2) washing the nickel column with deionized water until the pH value is 7.0, hanging nickel until the pH value is 2.0-3.0, washing the column with deionized water until the pH value is 7.0, balancing the nickel column with 10mM Tris-HCl solution with the pH value of 8.0, adding the thalli treated in the step (1), washing the column with 10mM Tris-HCl solution with 0.5M sodium chloride and the pH value of 8.0 after finishing, and eluting with 10mM Tris-HCl solution with the pH value of 8.0 and containing imidazole and 0.5M sodium chloride to obtain MPB70 protein.

9. The colloidal gold test strip for simultaneously detecting deer Buse disease and tuberculosis as claimed in claim 1, wherein the sample pad is a sample pad naturally dried after being soaked in 0.01M PBS with pH7.4, and the PBS contains 3% BSA and 1% Tween-20.

10. The colloidal gold test card for simultaneously detecting the deer's disease and the tuberculosis comprises a shell, and is characterized in that the colloidal gold test strip for simultaneously detecting the deer's disease and the tuberculosis as claimed in any one of claims 1 to 9 is arranged in the shell, a sample adding hole is formed in the position, corresponding to the sample pad, of the shell, and an observation window is formed in the position, corresponding to the nitrocellulose membrane, of the shell.