Cattle and sheep urea poisoning detection card and preparation method thereof

Abstract

The invention discloses a cattle and sheep urea poisoning detection card and a preparation method thereof, and relates to the technical field of urea poisoning detection. The test strip comprises a bottom plate, a nitrocellulose membrane is fixed on the upper surface of the bottom plate, a sample pad, a combination pad and a water absorption pad are sequentially overlapped on the nitrocellulose membrane, a shell is packaged outside the bottom plate, the combination pad is pre-coated with an anti-urea specific antibody-colloidal gold conjugate, and the water absorption pad is pre-coated with an anti-urea specific antibody-colloidal gold conjugate. The colloidal gold labeled anti-urease monoclonal antibody is a colloidal gold labeled anti-urease monoclonal antibody with the particle size of 30 + / -5nm, the nitrocellulose membrane is provided with a detection line T and a quality control line C, the detection line T is sprayed with an anti-urea specific antibody, and the quality control line C is sprayed with an antibody for resisting the anti-urea specific antibody. The cattle and sheep urea poisoning detection card is easy to operate, complex instruments and equipment are not needed, and farm workers can operate the detection card through simple training. The cattle and sheep urea poisoning detection card is high in detection speed, only 15-20 minutes are needed from sampling to detection result obtaining, and the requirement for rapid detection of a livestock farm can be met.

Description

Technical Field

[0001] The invention relates to the technical field of urea poisoning detection, in particular to a cattle and sheep urea poisoning detection card and a preparation method thereof. Background Art

[0002] In the process of cattle and sheep breeding, urea is widely used as a non-protein nitrogen feed additive. However, due to improper feeding and other reasons, cattle and sheep are prone to urea poisoning, which will have a serious impact on the health and breeding benefits of cattle and sheep. At present, the detection method for urea poisoning in cattle and sheep has problems such as complex operation and long detection time, which cannot meet the needs of farms for rapid and accurate detection. Therefore, we proposed a cattle and sheep urea poisoning detection card and its preparation method to solve the above problems.

[0003] The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to ordinary technicians in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a cattle and sheep urea poisoning detection card and a preparation method thereof, which can quickly and accurately detect whether cattle and sheep are urea poisoned, is simple to operate, and is suitable for on-site use in breeding farms.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a cattle and sheep urea poisoning detection card, comprising a base plate, a nitrocellulose membrane is fixed on the upper surface of the base plate, a sample pad, a binding pad and a water absorbent pad are overlapped in sequence on the nitrocellulose membrane, a shell is packaged on the outside of the base plate, an anti-urea specific antibody-colloidal gold conjugate is pre-coated on the binding pad, that is, a colloidal gold-labeled anti-urease monoclonal antibody with a particle size of 30±5nm, a detection T line and a quality control C line are provided on the nitrocellulose membrane, the detection T line is sprayed with anti-urea specific antibody, and the quality control C line is sprayed with an antibody against anti-urea specific antibody.

[0006] Preferably, the sample pad is located at one end close to the nitrocellulose membrane, and the absorbent pad is located at the other end away from the nitrocellulose membrane.

[0007] Preferably, a sample addition hole and a display area are provided on a side of the housing close to the water absorbent pad.

[0008] Preferably, the detection T-line antibody is a recombinant urease α subunit monoclonal antibody.

[0009] The preparation method of the cattle and sheep urea poisoning detection card comprises the following steps:

[0010] Step 1. Preparation of anti-urea specific antibody-colloidal gold conjugate: using colloidal gold labeling technology, coupling the anti-urea specific antibody with colloidal gold to prepare the anti-urea specific antibody-colloidal gold conjugate;

[0011] Step 2. Preparation of the conjugate pad: spray the anti-urea specific antibody-colloidal gold conjugate evenly on the glass fiber membrane, and obtain the conjugate pad after drying;

[0012] Step 3. Preparation of nitrocellulose membrane: Spray the detection T line and quality control C line on the nitrocellulose membrane using a membrane sprayer, spray the T line with anti-urea specific antibody, and spray the C line with secondary antibody, i.e., antibody against anti-urea specific antibody, and set aside after drying;

[0013] Step 4. Treatment of the sample pad and the water absorbent pad: pre-treat the sample pad and the water absorbent pad respectively to make them have good hydrophilicity and water absorbency;

[0014] Step 5. Assembly of the test card: fix the treated nitrocellulose membrane on the bottom plate, and then overlap the sample pad, conjugate pad and absorbent pad on the nitrocellulose membrane in the prescribed order, and finally seal it with a shell to obtain the cattle and sheep urea poisoning test card.

[0015] The method of using the test card is as follows:

[0016] Sampling: Use vacuum-dried negative pressure tubes or disposable needles to collect blood from cattle and sheep veins;

[0017] Sample processing: The blood sample is centrifuged to separate the serum, or left to precipitate naturally at room temperature;

[0018] Add sample: Use a dropper to drop the treated serum sample into the sample well of the test card, add 3-5 drops;

[0019] Reading the results: Place the test card at room temperature for 10-15 minutes, and observe the color of the T line and C line on the test card. If both the C line and the T line are colored, it is a positive result, indicating that the cattle and sheep may have urea poisoning; if only the C line is colored and the T line is not colored, it is a negative result, indicating that the cattle and sheep have not been poisoned by urea; if the C line is not colored, the test result is invalid and needs to be retested.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The cattle and sheep urea poisoning detection card of the present invention is easy to operate, does not require complicated instruments and equipment, and can be operated by farm staff after simple training.

[0022] (2) The cattle and sheep urea poisoning detection card of the present invention has a fast detection speed, and only takes 15-20 minutes from sampling to obtaining the test results, which can meet the needs of rapid detection in farms.

[0023] (3) The cattle and sheep urea poisoning detection card of the present invention has high detection accuracy, and the anti-urea specific antibody has a high specific binding ability with urea, which can effectively avoid the occurrence of false positive and false negative results.

[0024] (4) The cattle and sheep urea poisoning detection card of the present invention is small in size, easy to carry and store, and suitable for on-site use in breeding farms.

[0025] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the cross-sectional structure of the detection card of the present invention;

[0027] Figure 2 This is a schematic diagram of a negative result of the test card of the present invention;

[0028] Figure 3 This is a schematic diagram of a positive result of the test card of the present invention;

[0029] Figure 4 Schematic diagrams of two situations in which the test results of the test card of the present invention are invalid.

[0030] In the figure: 1, bottom plate; 2, nitrocellulose membrane; 3, sample pad; 4, binding pad; 5, absorbent pad; 6, outer shell. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] See also Figure 1 The cattle and sheep urea poisoning detection card comprises a bottom plate 1, a nitrocellulose membrane 2 is fixed on the upper surface of the bottom plate 1, a sample pad 3, a binding pad 4 and a water absorbent pad 5 are overlapped on the nitrocellulose membrane 2 in sequence, the sample pad 3 is located at one end close to the nitrocellulose membrane 2, and the water absorbent pad 5 is located at the other end away from the nitrocellulose membrane 2. The bottom plate 1 is encapsulated with a shell 6 on the outside, and a sample addition hole and a display area are opened on one side of the shell 6 close to the water absorbent pad 5. The sample addition hole is used to drip the sample, and the display area is used to observe the test results.

[0034] The conjugate pad 4 is pre-coated with an anti-urea specific antibody-colloidal gold conjugate, i.e., a colloidal gold-labeled anti-urease monoclonal antibody with a particle size of 30±5 nm. A detection T line and a quality control C line are provided on the nitrocellulose membrane 2. The detection T line is sprayed with an anti-urea specific antibody, which is a recombinant urease α subunit monoclonal antibody (SEQ ID NO: 2), and the quality control C line is sprayed with an antibody against the anti-urea specific antibody.

[0035] Example 2

[0036] The preparation method of the cattle and sheep urea poisoning detection card comprises the following steps:

[0037] Step 1. Preparation of anti-urea specific antibody-colloidal gold conjugate: using colloidal gold labeling technology, coupling the anti-urea specific antibody with colloidal gold to prepare the anti-urea specific antibody-colloidal gold conjugate;

[0038] Immunogen design: The α subunit fragment of urease (amino acid sequence: SEQ ID NO: 1) was used as the immunogen, which was purified by genetic engineering recombinant expression with a purity of > 95%; the immune adjuvant used a nanoparticle adjuvant (particle size 50 nm, chitosan carrier) to enhance the immune response;

[0039] Antibody screening: Screening of high affinity monoclonal antibodies (KD = 1.2 × 10 -9 M), cross-reaction experiments showed that it specifically binds to bovine and sheep urease, and has no cross-reaction with porcine and horse urease (ELISA verification);

[0040] Colloidal gold synthesis: trisodium citrate reduction method, controlled particle size 30 ± 5nm (dynamic light scattering verification); optimized conditions: 0.01% HAuCl 4 Solution, 100℃ for 10 minutes, pH 7.2;

[0041] Antibody labeling conditions: add 10 μg antibody per 1 mL colloidal gold solution (pH 8.2 borate buffer), centrifuge and purify, then redissolve in PBS containing 1% BSA; Characteristics: labeling efficiency > 90% (OD520 / OD280 ratio detected by UV spectrophotometry);

[0042] Step 2. Preparation of the conjugate pad: spray the anti-urea specific antibody-colloidal gold conjugate evenly on the glass fiber membrane, and obtain the conjugate pad after drying;

[0043] Step 3. Preparation of nitrocellulose membrane: Spray the detection T line and quality control C line on the nitrocellulose membrane using a membrane sprayer, spray the T line with anti-urea specific antibody, and spray the C line with secondary antibody, i.e., antibody against anti-urea specific antibody, and set aside after drying;

[0044] Step 4. Treatment of the sample pad and the water absorbent pad: pre-treat the sample pad and the water absorbent pad respectively to make them have good hydrophilicity and water absorbency;

[0045] Step 5. Assembly of the test card: fix the treated nitrocellulose membrane on the bottom plate, and then overlap the sample pad, conjugate pad and absorbent pad on the nitrocellulose membrane in the prescribed order, and finally seal it with a shell to obtain the cattle and sheep urea poisoning test card.

[0046] How to use the test card:

[0047] Sampling: Use vacuum-dried negative pressure tubes or disposable needles to collect blood from cattle and sheep veins;

[0048] Sample processing: The blood sample is centrifuged to separate the serum, or left to precipitate naturally at room temperature;

[0049] Sample addition: Use a dropper to drop the treated serum sample into the sample well of the test card, add 3-5 drops;

[0050] Reading the results: Place the test card at room temperature for 10-15 minutes, and observe the color of the T line and C line on the test card. If both the C line and the T line are colored, it is a positive result, indicating that the cattle and sheep may have urea poisoning; if only the C line is colored and the T line is not colored, it is a negative result, indicating that the cattle and sheep have not been poisoned by urea; if the C line is not colored, the test result is invalid and needs to be retested.

[0051] The detection principle of the detection card of the present invention is as follows: when the blood samples of cattle and sheep are dripped onto the sample pad 3, the samples flow toward the nitrocellulose membrane 2 under the action of capillaries. If the sample contains urea, the urea will bind to the anti-urea specific antibody-colloidal gold conjugate on the binding pad 4, and continue to flow forward to the detection T line on the nitrocellulose membrane, forming a urea-anti-urea specific antibody-colloidal gold conjugate complex, causing the T line to develop color; and the excess anti-urea specific antibody-colloidal gold conjugate will continue to flow to the quality control C line, bind to the secondary antibody on the C line, causing the C line to develop color. If the sample does not contain urea, only the C line will develop color.

[0052] The basis for judging poisoning in the detection card of the present invention is as follows: Under normal circumstances, the urea content in the blood of cattle and sheep is low, about 5-20 mg / dL (0.83-3.33 mmol / L), which is within the normal range. If cattle and sheep are poisoned by urea, the urea content in the blood is far higher than the normal level, which may exceed 50 mg / dL (8.33 mmol / L), and may be higher in severe cases. When the sample is dripped onto the sample pad 3 and moves under the capillary action, a large amount of urea will bind to the anti-urea specific antibody-colloidal gold conjugate on the binding pad 4, and the complex will be colored when it moves to the detection T line of the nitrocellulose membrane 2. If there is no poisoning, the urea content is low and not enough to make the T line color, and only the quality control C line is colored.

[0053] The technical parameters of the test card of the present invention are shown in the following table:

[0054] Serial number Components Materials / Specifications size Functional Description 1 Sample pad Glass fiber (pore size 10μm) 15×50 Pre-buried pH 7.4 buffer salt 2 Bonding pad Polyester film (containing colloidal gold conjugate) 10×50 Lyophilized antibody, reconstitution time ≤ 30 seconds 3 Nitrocellulose membrane Nitrocellulose membrane (pore size 8 μm) 25×50 T-line coated antibody concentration 1mg / mL 4 Absorbent pad Cellulose filter paper 20×50 Flow rate control: complete chromatography within 120s

[0055] The performance verification data of the test card of the present invention are as follows:

[0056] 1. Sensitivity

[0057] Minimum detection limit: 0.1 μg / mL urease (compared to 0.5 μg / mL for prior art products).

[0058] Linear range: 0.1-10μg / mL (R 2 =0.998).

[0059] 2. Specificity

[0060] The cross-reaction rate with bovine serum albumin (BSA) and hemoglobin is less than 0.1% (verified by Western Blot).

[0061] 3. Stability

[0062] 37℃ accelerated aging test: validity period ≥ 18 months (performance decrease < 5%).

[0063] Extreme environment: After being stored at 45℃ / 80% humidity for 7 days, the color intensity of the test line remains above 90%.

[0064] 4. Applicability

[0065] The test samples included Holstein cattle and goats (1-5 years old), and the coefficient of variation (CV) of the test results was <5%.

[0066] 5. Storage conditions

[0067] Sealed in aluminum foil bag with built-in desiccant, stored at 4-30℃ away from light, use within 2 hours after opening.

[0068] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0069] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, a first feature being "above", "above" or "above" a second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. A first feature being "below", "below" or "below" a second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0070] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0071] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Cattle and sheep urea poisoning detection card, characterized by: The invention comprises a bottom plate (1), a nitrocellulose membrane (2) is fixed on the upper surface of the bottom plate (1), a sample pad (3), a conjugation pad (4) and a water absorbent pad (5) are overlapped in sequence on the nitrocellulose membrane (2), a shell (6) is encapsulated outside the bottom plate (1), the conjugation pad (4) is pre-coated with an anti-urea specific antibody-colloidal gold conjugate, i.e., a colloidal gold-labeled anti-urease monoclonal antibody with a particle size of 30±5nm, and a detection T line and a quality control C line are provided on the nitrocellulose membrane (2), the detection T line is sprayed with anti-urea specific antibody, and the quality control C line is sprayed with an antibody against the anti-urea specific antibody.

2. The cattle and sheep urea poisoning detection card according to claim 1, characterized in that: The sample pad (3) is located at one end close to the nitrocellulose membrane (2), and the water absorbent pad (5) is located at the other end away from the nitrocellulose membrane (2).

3. The cattle and sheep urea poisoning detection card according to claim 1, characterized in that: A sample addition hole and a display area are provided on a side of the housing (6) close to the water absorbent pad (5).

4. The cattle and sheep urea poisoning detection card according to claim 1, characterized in that: The detection T-line antibody is a recombinant urease α subunit monoclonal antibody.

5. The method for preparing the cattle and sheep urea poisoning detection card according to any one of claims 1 to 4, characterized in that: The steps include: Step 1. Preparation of anti-urea specific antibody-colloidal gold conjugate: using colloidal gold labeling technology, coupling the anti-urea specific antibody with colloidal gold to prepare the anti-urea specific antibody-colloidal gold conjugate; Step 2. Preparation of the conjugate pad: spray the anti-urea specific antibody-colloidal gold conjugate evenly on the glass fiber membrane, and obtain the conjugate pad after drying; Step 3. Preparation of nitrocellulose membrane: Spray the detection T line and quality control C line on the nitrocellulose membrane using a membrane sprayer, spray the T line with anti-urea specific antibody, and spray the C line with secondary antibody, i.e., antibody against anti-urea specific antibody, and set aside after drying; Step 4. Treatment of the sample pad and the water absorbent pad: pre-treat the sample pad and the water absorbent pad respectively to make them have good hydrophilicity and water absorbency; Step 5. Assembly of the test card: fix the treated nitrocellulose membrane on the bottom plate, and then overlap the sample pad, conjugate pad and absorbent pad on the nitrocellulose membrane in the prescribed order, and finally seal it with a shell to obtain the cattle and sheep urea poisoning test card.

6. The method for preparing the cattle and sheep urea poisoning detection card according to claim 5, characterized in that: The method of using the test card is as follows: Sampling: Use vacuum-dried negative pressure tubes or disposable needles to collect blood from cattle and sheep veins; Sample processing: The blood sample is centrifuged to separate the serum, or left to precipitate naturally at room temperature; Add sample: Use a dropper to drop the treated serum sample into the sample well of the test card, add 3-5 drops; Reading the results: Place the test card at room temperature for 10-15 minutes, and observe the color of the T line and C line on the test card. If both the C line and the T line are colored, it is a positive result, indicating that the cattle and sheep may have urea poisoning; if only the C line is colored and the T line is not colored, it is a negative result, indicating that the cattle and sheep have not been poisoned by urea; if the C line is not colored, the test result is invalid and needs to be retested.

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