Nano-cellulose color test strip for detecting three porcine intestinal coronaviruses

Abstract

The invention discloses a nanocellulose color test strip for simultaneously detecting porcine epidemic diarrhea virus (PEDV), porcine delta coronavirus (PDCoV) and porcine acute diarrhea syndrome coronavirus (SADS-CoV). The red nanocellulose ball labeled combination pad is coated with a red nanocellulose ball labeled anti-PEDVN protein monoclonal antibody; the green nanocellulose ball labeled combination pad is coated with an anti-PDCoVN protein monoclonal antibody labeled by green nanocellulose balls; the blue nanocellulose ball labeled combination pad is coated with an anti-SADS-CoVN protein monoclonal antibody labeled by blue nanocellulose balls; the three detection lines are respectively coated with monoclonal antibodies of N proteins of the three viruses, and the quality control line is coated with rabbit anti-mouse IgG. The test strip distinguishes target viruses through colors, is high in sensitivity and simple and convenient to operate, and can complete triple detection within 10 minutes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal virus detection, and particularly relates to a nanocellulose color test strip for detecting three porcine enteric coronaviruses, which is used for synchronously detecting porcine epidemic diarrhea virus (PEDV), porcine delta coronavirus (PDCoV) and porcine acute diarrhea syndrome coronavirus (SADS-CoV). BACKGROUND

[0002] Porcine enteric coronaviruses (PEDV, PDCoV and SADS-CoV) can cause respiratory, digestive and neurological diseases in pigs. The three viruses infect intestinal cells of pigs, cause damage to intestinal villi and malabsorption of nutrients, and cause diarrhea and severe dehydration in diseased animals, and even lead to shock and death. The harm to newborn nursing piglets is the most serious, and the main clinical manifestations are diarrhea and vomiting in piglets. In recent years, due to the gradual change of feeding mode from traditional to large-scale and intensive, the composition of pig population in China is constantly changing due to foreign introduction, which increases the possibility of diarrhea disease in pig population. Early prevention and timely detection of pathogenic factors that may cause pig diarrhea and other diseases are important contents of quarantine work. In the face of increasing international trade and cross-border animal circulation, it is important to guard the first line of defense at the port, ensure the stability and sustainable development of agricultural production, and provide a safe food and living environment for the people.

[0003] Porcine enteric coronaviruses (including PEDV, PDCoV and SADS-CoV) are important pathogens that cause respiratory, digestive and neurological diseases in pigs. These viruses infect intestinal cells of pigs, destroy the structure of intestinal villi, cause dysfunction of nutrient absorption, and further cause diarrhea, severe dehydration, and even shock and death in diseased animals. Among them, newborn nursing piglets are most seriously harmed, and the main clinical manifestations are severe diarrhea and vomiting. In recent years, the pig industry in China has changed from traditional mode to large-scale and intensive feeding, and the frequent introduction of foreign pigs has led to continuous changes in the structure of pig population. The risk of diarrhea disease in pig population has significantly increased. Therefore, early prevention and timely detection of pathogenic factors that may cause pig diarrhea have become the core content of animal quarantine work.

[0004] At present, the methods for detecting porcine enteric coronavirus include immune electron microscopy, immunofluorescence, micro serum neutralization test, enzyme-linked immunosorbent assay and RT-PCR method. The immune electron microscopy has the advantages of simplicity, directness, rapidness and qualitative correctness, but it is not suitable for large-scale diagnosis and clinical diagnosis due to the need of electron microscope equipment. The immunofluorescence method has high accuracy and specificity, but the operation is complicated and special instrument equipment is needed. The micro serum neutralization test is complicated in operation, and some human factors in the test operation have greater influence on the results. The enzyme-linked immunosorbent assay is relatively simple in operation, but it also needs instrument equipment. The RT-PCR method has good sensitivity and specificity, but it is only suitable for laboratory diagnosis at present due to the need of special instrument equipment, and is not suitable for clinical diagnosis.

[0005] The traditional colloidal gold test strip has the following defects: only single target detection, low efficiency; single colloidal gold color development, unable to distinguish co-infection; limited sensitivity (detection limit > 10 4 TCID 50 / mL). Therefore, it is urgent to develop a high-sensitivity on-site detection tool that can simultaneously distinguish the three viruses. The Li Tao research group of the Shanghai Veterinary Institute of the Chinese Academy of Agricultural Sciences successfully developed a kind of color cellulose nanoparticle with high stability and easy surface modification, and used it for multiplex lateral flow chromatography detection (Multiplex LFAs, mLFA). The preparation method of this color cellulose nanoparticle is as follows: first, spherical cellulose nanoparticles with adjustable size are synthesized, then dye molecules are loaded on the surface of the cellulose nanoparticles through mature industrial dyeing method, and then a polydopamine coating layer that can be combined with antibodies is wrapped. This material has higher color development intensity than gold nanoparticles, and also has higher salt resistance and stability against extreme pH values.

[0006] Therefore, a multiplex immunochromatographic test strip based on three-color nanocellulose spheres is proposed. SUMMARY

[0007] The purpose of the present application is to solve the defects of the traditional test strip that cannot simultaneously distinguish three viruses, low sensitivity and misjudgment of co-infection proposed in the background art, and a nanocellulose color test strip for detecting three porcine enteric coronaviruses is proposed.

[0008] Core innovation: red, green and blue three-color nanocellulose spheres (from DOI: 10.1021 / acsnano.4c15340) are used to replace colloidal gold, and multiplex detection is realized through color coding;

[0009] Structural design:

[0010] The nanocellulose sphere marker binding pad is independently coated with color marker antibodies in three areas;

[0011] The nitrocellulose membrane is provided with three detection lines (T1: PEDV, T2: PDCoV, T3: SADS-CoV) and a quality control line (C);

[0012] The detection principle is that the virus in the sample is combined with the corresponding color-labeled antibody, is captured at the detection line to form a color strip (red / green / blue), and the quality control line is colored to indicate that the system is effective.

[0013] In order to achieve the above purpose, the application adopts the following technical solutions:

[0014] A nano-cellulose color test strip for detecting three porcine enteric coronaviruses comprises a PVC base plate, a sample pad, a nano-cellulose ball marker pad, a nitrocellulose membrane and a water absorption pad, and the sample pad, the marker pad, the nitrocellulose membrane and the water absorption pad are sequentially connected to the PVC base plate.

[0015] The marker pad is divided into three independent areas: a red area, a green area and a blue area (3c), which are respectively coated with a red nano-cellulose ball marker anti-PEDV N protein monoclonal antibody, a green nano-cellulose ball marker anti-PDCoV N protein monoclonal antibody and a blue nano-cellulose ball marker anti-SADS-CoV N protein monoclonal antibody.

[0016] The nitrocellulose membrane is provided with three detection lines and a quality control line: the T1 line is coated with an anti-PEDV N protein monoclonal antibody, the T2 line is coated with an anti-PDCoV N protein monoclonal antibody, the T3 line is coated with an anti-SADS-CoV N protein monoclonal antibody, and the C line is coated with a rabbit anti-mouse IgG.

[0017] Further in the application, the nano-cellulose ball is a spherical particle with a diameter of 50-200 nm, which is modified from a cellulose nanosphere by a dyeing agent, wherein the red dyeing agent is rhodamine B, the green dyeing agent is fluorescein, and the blue dyeing agent is coomassie brilliant blue.

[0018] Further in the application, the preparation method of the nano-cellulose ball marker antibody comprises:

[0019] The nano-cellulose ball is dispersed in 0.01M pH 8.0 borate buffer;

[0020] The corresponding monoclonal antibody is added to a final concentration of 1.0 mg / mL, and the reaction is oscillated at 37°C for 1 hour;

[0021] 10% BSA is added for blocking for 30 minutes, and after centrifugation, it is resuspended in Tris-HCl buffer (pH 8.2) containing 1% sucrose.

[0022] Further in the application, the antibody spraying concentration of the T1, T2 and T3 lines is 0.8-1.2 mg / mL, and the spraying amount is 0.8 μL / cm.

[0023] The spraying concentration of rabbit anti-mouse IgG of C line is 1.5 mg / mL;

[0024] The distance between detection lines is 4 mm, and the distance between the detection line and the quality control line is 6 mm.

[0025] Further in the application, the sample pad is treated with a blocking solution, and the blocking solution comprises 0.05 M Tris-HCl (pH 8.0), 1% BSA, 0.5% PVP-40, and 0.1% Tween-20.

[0026] Further in the application, the nanocellulose spheres are purchased from the Shanghai Veterinary Institute of the Chinese Academy of Agricultural Sciences, and the preparation method is disclosed in DOI: 10.1021 / acsnano.4c15340.

[0027] Further in the application, the length of the PVC bottom plate is 60-70 mm, and the width of the sample pad is 3-4 mm.

[0028] Further in the application, the detection sensitivity of the test strip is 10 3 TCID 50 / mL, and the detection time is less than or equal to 10 minutes.

[0029] Further in the application, the test strip can simultaneously distinguish single infection or co-infection of PEDV, PDCoV, and SADS-CoV, and has no cross-reaction with TGEV, PRRSV, and PCV2.

[0030] Further in the application, the test strip is applied to rapid detection of pig diarrhea pathogens in the field.

[0031] Compared with the prior art, the application has the following beneficial effects:

[0032] 1. Triple detection: simultaneous differentiation of three viruses in a single test, with an efficiency improvement of 300%;

[0033] 2. High sensitivity: the specific surface area of nanocellulose spheres is large, and the detection limit is 10 3 TCID 50 / mL (10 times higher than that of colloidal gold);

[0034] 3. Direct results: color directly corresponds to virus species, avoiding misjudgment;

[0035] 4. Low cost: the price of nanocellulose spheres is only 1 / 5 of that of colloidal gold. DETAILED DESCRIPTION

[0036] The present application can be more easily understood and further advantages and benefits will become more apparent upon consideration of the following detailed description and the accompanying drawings, in which:

[0037] The term "comprising," "including," "carrying," "having," "containing," and variants thereof as used herein are intended to be open-ended terms that specifically permit the inclusion of unspecified elements or integers. For example, a composition, step, method, process, article, or apparatus that comprises (or includes) an element can include other elements not expressly listed or inherent to such composition, step, method, process, article, or apparatus.

[0038] The transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When the phrase "consisting of" follows the introductory transistional phrase "comprising" or "including" in a claim, it defines the scope of a claim in which the recited elements are the only elements of the claim. When the phrase "consisting of is used in a claim without the introductory transistional phrase "comprising" or "including," it defines the scope of a claim in which the recited elements are the only elements of the claim.

[0039] When equivalent or alternative values or parameters are expressed in ranges, preferred ranges, or a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pair of any upper or lower limit or preferred value, whether or not the range is expressly stated or suggested, is expressly disclosed. For example, where a range "1 to 5" is disclosed, the disclosure is intended to encompass ranges of "1 to 4," "1 to 3," "1 to 2," "1 to 2 and 4 to 5," "1 to 3 and 5," etc. When numerical ranges are disclosed herein, unless otherwise stated, the range is intended to include both the upper and lower values and all intervening values of the range, including integers and fractions thereof.

[0040] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. "Optional" or "any of" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event occurs and instances where it does not.

[0041] Approximating language in the specification and claims can be applied to modify virtually any quantitative representation that could permissibly vary without resulting in a change in the basic function to which that term is related. Accordingly, a value modified by a term or terms, such as "about" and "substantially" can not be limited to the precise value specified, in some cases. In some embodiments, the approximating language can correspond to the precision of an instrument for measuring the value. In the description and claims of the application, ranges are used as endpoints to provide embodiments. Such ranges are inclusive of the endpoints.

[0042] Also, the articles "a", "an", and "the" preceding an element or component are intended to be construed to cover both the singular and the plural, unless otherwise indicated by the context. Thus, "a" or "an" means one or at least one, and the article "the" does not exclude the presence of more than one. The use of the word "at least" followed by a list of elements means that every combination of the elements in the list is included.

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments.

[0044] The core innovation of the present application is the combination of three-color coding design and partitioning pad structure, wherein the three-color coding design:

[0045] Red nanosphere-labeled anti-PEDV antibody T1 line capture red band;

[0046] Green nanosphere-labeled anti-PDCoV antibody T2 line capture green band;

[0047] Blue nanosphere-labeled anti-SADS-CoV antibody T3 line capture blue band.

[0048] Partitioning pad structure: avoid color interference.

[0049] A nanocellulose color test strip for detecting three porcine enteric coronaviruses, comprising a PVC base plate, a sample pad, a nanocellulose ball mark combination pad, a nitrocellulose membrane, and a water absorption pad, the sample pad, the combination pad, the nitrocellulose membrane, and the water absorption pad are sequentially lapped on the PVC base plate.

[0050] The combination pad is divided into three independent areas: red area, green area, and blue area (3c), which are coated with red nanocellulose ball-labeled anti-PEDV N protein monoclonal antibody, green nanocellulose ball-labeled anti-PDCoV N protein monoclonal antibody, and blue nanocellulose ball-labeled anti-SADS-CoV N protein monoclonal antibody, respectively.

[0051] The nitrocellulose membrane is provided with three detection lines and one quality control line: the T1 line is coated with anti-PEDV N protein monoclonal antibody, the T2 line is coated with anti-PDCoV N protein monoclonal antibody, the T3 line is coated with anti-SADS-CoV N protein monoclonal antibody, and the C line is coated with rabbit anti-mouse IgG.

[0052] The nanocellulose spheres are spherical particles with a diameter of 50-200 nm, which are modified by a dyeing agent from cellulose nanospheres, wherein the red dyeing agent is rhodamine B, the green dyeing agent is fluorescein, and the blue dyeing agent is coomassie brilliant blue.

[0053] The preparation method of the nanocellulose sphere-labeled antibody comprises the following steps:

[0054] The nanocellulose spheres are dispersed in 0.01M pH 8.0 borate buffer;

[0055] The corresponding monoclonal antibody is added to a final concentration of 1.0 mg / mL, and the reaction is shaken at 37°C for 1 hour;

[0056] 10% BSA is added for blocking for 30 minutes, and after centrifugation, it is resuspended in Tris-HCl buffer (pH 8.2) containing 1% sucrose.

[0057] The antibody spraying concentration of the T1, T2 and T3 lines is 0.8-1.2 mg / mL, and the spraying amount is 0.8 μL / cm;

[0058] The rabbit anti-mouse IgG spraying concentration of the C line is 1.5 mg / mL;

[0059] The distance between the detection lines is 4 mm, and the distance between the detection lines and the quality control line is 6 mm.

[0060] The sample pad is treated with a blocking solution, and the blocking solution comprises: 0.05M Tris-HCl (pH 8.0), 1% BSA, 0.5% PVP-40, and 0.1% Tween-20.

[0061] The nanocellulose spheres are purchased from the Shanghai Veterinary Institute of the Chinese Academy of Agricultural Sciences, and the preparation method is disclosed in DOI: 10.1021 / acsnano.4c15340.

[0062] The length of the PVC base plate is 60-70 mm, and the width of the sample pad is 3-4 mm.

[0063] The detection sensitivity of the test strip is 10 3 TCID 50 / mL, and the detection time is ≤10 minutes.

[0064] The test strip can simultaneously distinguish single infection or co-infection of PEDV, PDCoV and SADS-CoV, and has no cross reaction with TGEV, PRRSV and PCV2.

[0065] Application of test strip in rapid detection of swine diarrhea pathogen in field.

[0066] Summary: The application discloses a nanocellulose color test strip for simultaneously detecting porcine epidemic diarrhea virus (PEDV), porcine delta coronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV). The test strip comprises a PVC base plate and a sample pad, a nanocellulose ball marker combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped; the nitrocellulose membrane is provided with three detection lines (T1, T2 and T3) and a quality control line (C). The red nanocellulose ball marker combination pad is coated with a red nanocellulose ball marker anti-PEDV N protein monoclonal antibody; the green nanocellulose ball marker combination pad is coated with a green nanocellulose ball marker anti-PDCoV N protein monoclonal antibody; the blue nanocellulose ball marker combination pad is coated with a blue nanocellulose ball marker anti-SADS-CoV N protein monoclonal antibody; the three detection lines are respectively coated with the anti-N protein monoclonal antibodies of the three viruses; and the quality control line is coated with a rabbit anti-mouse IgG. The nanocellulose balls are purchased from the Shanghai Veterinary Institute of the Chinese Academy of Agricultural Sciences. The test strip can distinguish the target viruses by color, has high sensitivity and is simple to operate, and the triple detection can be completed within 10 minutes.

[0067] Based on the above, the following examples are also provided

[0068] Example 1: Preparation of nanocellulose ball marker antibody

[0069] 1. Materials:

[0070] Red / green / blue nanocellulose balls (Shanghai Veterinary Institute of the Chinese Academy of Agricultural Sciences);

[0071] Monoclonal antibody: anti-PEDV / PDCoV / SADS-CoV N protein (commercially available).

[0072] 2. Steps:

[0073] ① Disperse the red / green / blue nanocellulose balls in 0.01M borate buffer (pH 8.0);

[0074] ② Add anti-PEDV / PDCoV / SADS-CoV N protein monoclonal antibodies (final concentration 1mg / mL) respectively, and oscillate at 37℃ for 1h;

[0075] ③ Add 10% BSA for blocking for 30min, and centrifuge at 12000rpm for 20min;

[0076] ④ Resuspend the precipitate in Tris-HCl buffer (pH 8.2) containing 1% sucrose, and store at 4℃.

[0077] Example 2: Test strip assembly

[0078] ① Sample pad treatment: glass fiber membrane was soaked in blocking solution (0.05M Tris-HCl, 1% BSA, 0.5% PVP-40, 0.1% Tween-20, pH 8.0) and dried at 37℃;

[0079] ② Binding pad preparation: 10 μL / cm of three-color labeled antibodies were sprayed onto the corresponding area of the binding pad and dried at 37℃;

[0080] ③ Nitrocellulose membrane coating:

[0081] T1 line: anti-PEDV N protein monoclonal antibody (1 mg / mL);

[0082] T2 line: anti-PDCoV N protein monoclonal antibody (1 mg / mL);

[0083] T3 line: anti-SADS-CoV N protein monoclonal antibody (1 mg / mL);

[0084] C line: rabbit anti-mouse IgG (1.5 mg / mL);

[0085] ④ Lamination: sample pad (15 mm), three-zone binding pad (8 mm), nitrocellulose membrane (25 mm), and water absorption pad (20 mm) were laminated in sequence on a PVC base plate with an overlap of 2 mm.

[0086] Example 3: Performance verification

[0087] ① Sensitivity test:

[0088]

[0089]

[0090] ② Specificity test:

[0091] No cross-reaction with TGEV, PRRSV, and PCV2;

[0092] No color interference in triple mixed samples (red / green / blue strips show color independently);

[0093] ③ Field application:

[0094] The coincidence rate of detection results of clinical samples (n = 120) with RT-PCR was >98%.

[0095] The examples referred to herein are merely illustrative of some features of the methods of the present application, the appended claims being intended to cover as broadly as possible the range of equivalents that can be conceived, and the embodiments presented herein are merely illustrative of selected implementations according to combinations of all possible embodiments. Accordingly, it is the applicant's intention that the appended claims not be limited to the choice of examples presented herein. Some numerical ranges are also included in the claims as sub-ranges, and the ranges in these claims are intended to cover the same as if the ranges were written out individually.

Claims

1. A nanocellulose color test strip for detecting three porcine enteric coronaviruses, comprising a PVC base plate, a sample pad, a nanocellulose spherical marker binding pad, a nitrocellulose membrane, and an absorbent pad, characterized in that, The sample pad, conjugation pad, nitrocellulose membrane, and absorbent pad are sequentially overlapped on the PVC base plate; The conjugate pad is divided into three independent regions: a red region, a green region, and a blue region (3c), which are respectively coated with anti-PEDVN protein monoclonal antibody labeled with red nanocellulose spheres, anti-PDCoVN protein monoclonal antibody labeled with green nanocellulose spheres, and anti-SADS-CoVN protein monoclonal antibody labeled with blue nanocellulose spheres. The nitrocellulose membrane has three detection lines and one control line: the T1 line is coated with anti-PEDVN protein monoclonal antibody, the T2 line is coated with anti-PDCoVN protein monoclonal antibody, the T3 line is coated with anti-SADS-CoVN protein monoclonal antibody, and the C line is coated with rabbit anti-mouse IgG.

2. The nanocellulose color test strip for detecting three porcine enteric coronaviruses according to claim 1, characterized in that, The nanocellulose spheres are spherical particles with a diameter of 50-200 nm, which are modified by dyeing agents, wherein the red dye is Rhodamine B, the green dye is fluorescein, and the blue dye is Coomassie Brilliant Blue.

3. The nanocellulose color test strip for detecting three porcine enteric coronaviruses according to claim 1, characterized in that, The method for preparing the nanocellulose sphere-labeled antibody includes: The cellulose nanospheres were dispersed in a 0.01 M pH 8.0 borate buffer solution; Add the corresponding monoclonal antibody to a final concentration of 1.0 mg / mL, and incubate at 37°C with shaking for 1 hour; Add 10% BSA to block for 30 minutes, centrifuge, and resuspend in Tris-HCl buffer (pH 8.2) containing 1% sucrose.

4. The nanocellulose color test strip for detecting three porcine enteric coronaviruses according to claim 1, characterized in that, The antibody concentration for T1, T2, and T3 lines is 0.8-1.2 mg / mL, and the spraying volume is 0.8 μL / cm. The concentration of rabbit anti-mouse IgG sprayed in line C was 1.5 mg / mL; The spacing between the test lines is 4mm, and the spacing between the test lines and the quality control lines is 6mm.

5. The nanocellulose color test strip for detecting three porcine enteric coronaviruses according to claim 1, characterized in that, The sample pad was treated with a sealing solution, which consisted of 0.05 M Tris-HCl (pH 8.0), 1% BSA, 0.5% PVP-40, and 0.1% Tween-20.

6. The nanocellulose color test strip for detecting three porcine enteric coronaviruses according to claim 1, characterized in that, The PVC base plate is 60-70mm long and the sample pad is 3-4mm wide.

7. The nanocellulose color test strip for detecting three porcine enteric coronaviruses according to claim 1, characterized in that, The detection sensitivity of the test strip is 10. 3 TCID 50 / mL, detection time ≤10 minutes.

8. The nanocellulose color test strip for detecting three porcine enteric coronaviruses according to claim 1, characterized in that, The test strip can simultaneously distinguish between single or co-infection with PEDV, PDCoV, and SADS-CoV, and has no cross-reaction with TGEV, PRRSV, and PCV2.

9. The application of the test strip according to any one of claims 1-8 in the rapid on-site detection of swine diarrhea pathogens.

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