SERS test strip for joint detection of PEDV and PoRVA and preparation method thereof

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) test strip for jointly detecting PEDV (Porcine Epidemic Diarrhea Virus) and PoRVA and a preparation method thereof, and belongs to the technical field of animal virus detection. The test strip comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially arranged on the bottom plate, a labeled antibody is fixed on the combination pad, and the labeled antibody is prepared by coupling Au (at) 4-MBA (at) Ag NPs with Anti-PEDV-Ab and / or Anti-PoRVA-Ab1; a detection line T1 formed by a PEDV monoclonal antibody, a detection line T2 formed by a PoRVA monoclonal antibody and a quality control line C formed by goat anti-mouse IgG are sequentially fixed on the nitrocellulose membrane. The test strip prepared by the invention can be simultaneously used for rapidly detecting PEDV and PoRVA on site, and has better accuracy. The test strip is prepared through the steps of Au (at) 4-MBA NPs synthesis, Au (at) 4-MBA (at) Ag NPs synthesis, antibody coupling, test paper assembly and the like. The test strip is applied to clinical sample detection, and the coincidence rate with RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reaches 93.3%; the kit has the characteristics of high sensitivity, strong specificity and high accuracy, and can be used for on-site rapid detection of PEDV and PoRVA.

Description

Technical Field

[0001] The invention belongs to the technical field of animal virus detection technology, and specifically relates to a SERS test strip for jointly detecting PEDV and PoRVA and a preparation method thereof. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) and porcine group A rotavirus (PoRVa) are two major pathogens that cause diarrhea in pigs. Their infection can cause highly contagious enteric diseases with vomiting, diarrhea and dehydration as the main symptoms in pigs, and has the characteristics of high morbidity and mortality. In recent years, SERS immunochromatography technology has been developed to detect PEDV or PoRVA alone, which is used for qualitative or quantitative determination of PEDV and PoRVA. However, since the symptoms of PEDV and PoRVA infection in pigs are similar, and PEDV and PoRVA often co-infect in many parts of the country, it is difficult to effectively determine the pathogen based on clinical experience alone, and it is difficult to formulate targeted prevention and control measures. Therefore, efficient detection of these two pathogens in the early stage of infection can provide a basis for the rapid formulation of targeted prevention and control measures, which is crucial for epidemic prevention and control.

[0003] Currently, there are methods such as RT-qPCR that can simultaneously achieve qualitative and quantitative determination of PEDV (porcine epidemic diarrhea virus) and PoRVA (porcine group A rotavirus). Although the detection results of this method are accurate, it relies heavily on equipment and professionals and is not suitable for on-site testing. At present, test strips based on the principle of immunochromatography cannot simultaneously achieve qualitative and quantitative determination of PEDV and PoRVA, and it is difficult to meet market demand. Summary of the invention

[0004] The present invention provides a SERS test strip for jointly detecting PEDV and PoRVA and a preparation method thereof, so as to solve the above technical problems.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A SERS immunochromatographic test strip for joint detection of PEDV and PoRVA, comprising:

[0007] A base plate, and a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent paper are sequentially arranged on the base plate; a labeled antibody is fixed on the conjugation pad, and the labeled antibody is prepared by coupling Au@4-MBA@Ag NPs with Anti-PEDV-Ab and / or Anti-PoRVA-Ab1; a detection line T1 formed by PEDV monoclonal antibody, a detection line T2 formed by PoRVA monoclonal antibody, and a quality control line C formed by goat anti-mouse IgG are sequentially fixed on the nitrocellulose membrane.

[0008] The method of coupling the labeled antibody is as follows:

[0009] S11, taking Au@4-MBA@Ag NPs, centrifuging, discarding the supernatant, resuspending the precipitate, and adjusting the pH of the Au@4-MBA@Ag NPs solution to alkaline;

[0010] S12, adding Anti-PEDV-Ab and / or Anti-PoRVA-Ab1, mixing, adding BSA solution and incubating;

[0011] S13, the obtained solution is centrifuged and the precipitate is redissolved to obtain the obtained product.

[0012] Furthermore, the precipitate in step S13 is redissolved using a reconstitution solution, and the reconstitution solution is prepared as follows: BSA, sucrose, and Tween-20 are dissolved in a Tris-HCl buffer and fully dissolved; the BSA concentration of the reconstitution solution is 5% (w / v), the sucrose concentration is 10% (w / v), the Tween-20 concentration is 1% (v / v), and the Tris-HCl buffer concentration is 50 mM; the pH of the reconstitution solution is 8.5.

[0013] Furthermore, the test strip is used to detect PEDV and / or PoRVA.

[0014] A method for preparing a SERS immunochromatographic test strip for joint detection of PEDV and PoRVA, comprising:

[0015] Synthesis of S21 and Au@4-MBA NPs

[0016] Take AuNPs, add 4-MBA ethanol solution, stir and mix to obtain Au@4-MBA NPs;

[0017] Synthesis of S22 and Au@4-MBA@Ag NPs

[0018] Take the Au@4-MBA NPs solution prepared by S21, centrifuge it, discard the supernatant, and dilute it 2 times with water; then heat the solution to boil, add sodium citrate solution, and then slowly drop the AgNO3 solution; after stopping heating, continue stirring until the solution cools naturally, and add water to the diluted volume;

[0019] S23, Antibody Conjugation

[0020] The Au@4-MBA@Ag NPs prepared in S22 were centrifuged, the supernatant was discarded, the precipitate was resuspended, and the pH of the Au@4-MBA@AgNPs solution was adjusted to alkaline; Anti-PEDV-Ab and / or Anti-PoRVA-Ab1 were added, mixed, and BSA solution was added for incubation; the obtained solution was centrifuged, and the precipitate was redissolved to obtain the labeled antibody;

[0021] S24, Test strip assembly

[0022] The labeled antibody prepared by S23 is fixed on the conjugate pad; the PEDV monoclonal antibody is fixed on the nitrocellulose membrane in sequence to form the detection line T1, the PoRVA monoclonal antibody is fixed to form the detection line T2, and the goat anti-mouse IgG is fixed to form the quality control line C; then the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent paper are pasted on the bottom plate and cut to obtain the product.

[0023] Furthermore, the concentration of the 4-MBA ethanol solution used in step S21 is 10 mM.

[0024] Furthermore, the concentration of the sodium citrate solution used in step S22 is 1% (w / v), and the concentration of the AgNO 3 solution is 10 mM.

[0025] Furthermore, in step S23, the pH is adjusted to 8.5 using 0.1 M K2CO3 solution.

[0026] Furthermore, before incubation in step S23, the solution was gently shaken at room temperature for 2 h, and the concentration of the BSA solution used was 10% (w / v); the precipitate was re-dissolved using a reconstitution solution, and the reconstitution solution was prepared as follows: BSA, sucrose, and Tween-20 were dissolved in Tris-HCl buffer and fully dissolved; the BSA concentration of the reconstitution solution was 5% (w / v), the sucrose concentration was 10% (w / v), the Tween-20 concentration was 1% (v / v), and the Tris-HCl buffer concentration was 50 mM; the pH of the reconstitution solution was 8.5.

[0027] Furthermore, the prepared test strip is used to detect PEDV and / or PoRVA.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The test strip is used for clinical sample testing, with a compliance rate of up to 93.3% with RT-PCR testing. It has the characteristics of high sensitivity, strong specificity and high accuracy, and can be used for on-site rapid detection of PEDV and PoRVA.

[0030] The test strip can complete the qualitative and quantitative detection of PEDV and PoRVA within 20 minutes. The visual LOD is as low as 6.25×10 2 TCID50 / mL and 7.42×10 2 copies / μL, and the LOD based on Raman signal is as low as 8.01×10 1 TCID50 / mL and 3.19×10 2 The standard curves of PEDV and PoRVA were established, and the linear relationships were good. There was no cross-reaction with other viruses with similar clinical manifestations and some common bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 The schematic diagram of the test strip structure prepared by the present invention.

[0033] Figure 2 This is a characterization diagram of 4-MBA dosage and silver shell thickness.

[0034] Figure 3 Characterization diagram of Au@4-MBA@Ag NPs prepared in the present invention.

[0035] Figure 4 The test strip prepared by the present invention is labeled with antibody concentration selection results.

[0036] Figure 5 The results are for selecting the concentration of the antibody streaking on the T line of the test strip prepared by the present invention.

[0037] Figure 6 The PEDV sensitivity test results of the test strip prepared by the present invention.

[0038] Figure 7 The test strip PoRVA sensitivity test result prepared by the present invention.

[0039] Figure 8 This is the mixed sensitivity test result of PEDV and PoRVA of the test strip prepared by the present invention.

[0040] Fig. 9 This is the specific detection result of the test strip prepared by the present invention.

[0041] Fig.10The test strips prepared by the present invention are tested for repeatability. DETAILED DESCRIPTION

[0042] To facilitate a better understanding of the present invention, the following examples are provided to illustrate the present invention in conjunction with the accompanying drawings. These examples belong to the protection scope of the present invention, but do not limit the protection scope of the present invention.

[0043] Example

[0044] A SERS immunochromatographic test strip for joint detection of PEDV and PoRVA, comprising:

[0045] A base plate, and a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent paper are sequentially arranged on the base plate; a labeled antibody is fixed on the conjugation pad, and the labeled antibody is prepared by coupling Au@4-MBA@Ag NPs with Anti-PEDV-Ab and / or Anti-PoRVA-Ab1; a detection line T1 formed by PEDV monoclonal antibody, a detection line T2 formed by PoRVA monoclonal antibody, and a quality control line C formed by goat anti-mouse IgG are sequentially fixed on the nitrocellulose membrane.

[0046] The method of labeling antibody conjugation is as follows:

[0047] S11, taking Au@4-MBA@Ag NPs, centrifuging, discarding the supernatant, resuspending the precipitate, and adjusting the pH of the Au@4-MBA@Ag NPs solution to alkaline;

[0048] S12, adding Anti-PEDV-Ab and / or Anti-PoRVA-Ab1, mixing, adding BSA solution and incubating;

[0049] S13, the obtained solution is centrifuged and the precipitate is redissolved to obtain the obtained product.

[0050] The precipitate in step S13 is redissolved using a reconstitution solution, and the reconstitution solution is prepared as follows: BSA, sucrose, and Tween-20 are dissolved in Tris-HCl buffer and fully dissolved; the BSA concentration of the reconstitution solution is 5% (w / v), the sucrose concentration is 10% (w / v), the Tween-20 concentration is 1% (v / v), and the Tris-HCl buffer concentration is 50 mM; the pH of the reconstitution solution is 8.5.

[0051] The test strips are used to detect PEDV and / or PoRVA.

[0052] A method for preparing a SERS immunochromatographic test strip for joint detection of PEDV and PoRVA, comprising:

[0053] Synthesis of S21 and Au@4-MBA NPs

[0054] Take AuNPs, add 4-MBA ethanol solution, stir and mix to obtain Au@4-MBA NPs;

[0055] Synthesis of S22 and Au@4-MBA@Ag NPs

[0056] Take the Au@4-MBA NPs solution prepared by S21, centrifuge it, discard the supernatant, and dilute it 2 times with water; then heat the solution to boil, add sodium citrate solution, and then slowly drop the AgNO3 solution; after stopping heating, continue stirring until the solution cools naturally, and add water to the diluted volume;

[0057] S23, Antibody Conjugation

[0058] The Au@4-MBA@Ag NPs prepared in S22 were centrifuged, the supernatant was discarded, the precipitate was resuspended, and the pH of the Au@4-MBA@AgNPs solution was adjusted to alkaline; Anti-PEDV-Ab and / or Anti-PoRVA-Ab1 were added, mixed, and BSA solution was added for incubation; the obtained solution was centrifuged, and the precipitate was redissolved to obtain the labeled antibody;

[0059] S24, Test strip assembly

[0060] The labeled antibody prepared by S23 is fixed on the conjugate pad; the PEDV monoclonal antibody is fixed on the nitrocellulose membrane in sequence to form the detection line T1, the PoRVA monoclonal antibody is fixed to form the detection line T2, and the goat anti-mouse IgG is fixed to form the quality control line C; then the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent paper are pasted on the bottom plate and cut to obtain the product.

[0061] The concentration of the 4-MBA ethanol solution used in step S21 is 10 mM.

[0062] The concentration of the sodium citrate solution used in step S22 is 1% (w / v), and the concentration of the AgNO3 solution is 10 mM.

[0063] In step S23, the pH is adjusted to 8.5 using 0.1 M K2CO3 solution.

[0064] Before incubation in step S23, the solution was gently shaken at room temperature for 2 h, and the concentration of the BSA solution used was 10% (w / v); the precipitate was re-dissolved using a reconstitution solution, and the reconstitution solution was prepared as follows: BSA, sucrose, and Tween-20 were dissolved in Tris-HCl buffer and fully dissolved; the BSA concentration of the reconstitution solution was 5% (w / v), the sucrose concentration was 10% (w / v), the Tween-20 concentration was 1% (v / v), and the Tris-HCl buffer concentration was 50 mM; the pH of the reconstitution solution was 8.5.

[0065] The prepared test strip is used for detecting PEDV and / or PoRVA.

[0066] In the embodiment, a labeled antibody is fixed on the conjugate pad, and the labeled antibody is prepared by coupling Au@4-MBA@Ag NPs with Anti-PEDV-Ab and / or Anti-PoRVA-Ab1; if only PEDV virus or PoRVA virus is detected, Au@4-MBA@Ag NPs can be coupled with Anti-PEDV-Ab and Anti-PoRVA-Ab1, respectively; if PEDV virus and PoRVA virus are detected at the same time, Au@4-MBA@Ag NPs are coupled with Anti-PEDV-Ab and Anti-PoRVA-Ab1.

[0067] Since different antibodies recognize different sites on the virus, using different antibodies can avoid competition between the same antibodies for the same site and improve the sensitivity of detection; therefore, the antibodies used to prepare the labeled antibody and the antibodies used to prepare the test lines T1 and T2 can be the same or different.

[0068] In the embodiment, the PEDV labeled antibody and the antibody used to prepare the detection line T1 are the same, that is, both are Anti-PEDV-Ab; the PoRVA labeled antibody and the antibody used to prepare the detection line T2 are different, namely Anti-PoRVA-Ab1 and Anti-PoRVA-Ab2.

[0069] test

[0070] 1. Test description:

[0071] Raman spectrometer, Invia Reflex, Renishaw, UK; parameter settings used: emission laser wavelength 785nm, power set to 1%, integration time set to 10s. Five points were randomly selected in the middle of each T line, and the average value of the detection signal was taken as the detection result.

[0072] TEM transmission electron microscope, Thermo Fisher Scientific, FEI-Talosf200x.

[0073] High-speed refrigerated centrifuge, Eppendorf, Germany, 5430R.

[0074] Malvern Nano Particle Size Potentiometer, Malvern Instruments, USA, ZetasizerNanoZS.

[0075] Three-dimensional film spraying gold instrument, Shanghai Jinbiao Biotechnology Co., Ltd., HM3035.

[0076] Colloidal gold reading instrument, Suzhou Hemai Precision Instrument Co., Ltd., GIC-Q1.

[0077] Sample diluent: Tris-HCl (10 mM) solution containing 1% BSA and 0.05% Tween-20, pH 8.5.

[0078] Mouse anti-PEDV monoclonal antibody (Anti-PEDV-Ab): Sangon Biotechnology Co., Ltd., mouse anti-PoRVA monoclonal antibody 1 (Anti-PoRVA-Ab1): Meridian Bioscience, USA, mouse anti-PoRVA monoclonal antibody 2: Meridian Bioscience, USA, goat anti-mouse IgG polyclonal antibody (goat anti-mouse IgG): Sigma Aldrich, USA.

[0079] PEDV, PRV and TGEV used in the present invention were isolated, identified and preserved by the Animal Infectious Disease Team of the College of Animal Science and Technology of Guangxi University; PoRVA, PoRVB, PoRVC, PDCoV and clinical samples were collected and provided by Nanning Zhufulai Animal Health Management Co., Ltd.; the sources of bacteria used in other experiments are shown in Table 1.

[0080] Table 1 Description of bacterial sources

[0081]

[0082]

[0083] 2. Select the test

[0084] 1. Preparation of AuNPs

[0085] AuNPs are mainly synthesized by Tris-assisted sodium citrate reduction method, the specific steps are as follows:

[0086] ① Take 140mL of ultrapure water, stir and heat to 100℃ under oil bath conditions, then add 10mL of sodium citrate (1%, w / v), and adjust the temperature to 137℃; ② Inject 1mL of chloroauric acid solution (1%, w / v) and 5mL of Tris solution (0.1M) into the reaction system in sequence, maintain 137℃ unchanged, and stir to react for 30min; ③ Stop heating, adjust the temperature to 100℃, and when the temperature drops to the set point, quickly inject 1mL of chloroauric acid solution, maintain 100℃ unchanged, and stir to react for 30min; ④ Repeat step ③, and finally obtain a cherry red AuNPs solution, which is stored at 4℃ away from light.

[0087] 2.4-Selection of MBA ethanol dosage

[0088] The AuNPs solution prepared above was divided into 5 groups, 10 mL of each group was placed in 5 clean glass bottles, 10, 40, 80, 160 and 320 μL of 4-MBA ethanol solution (10 mM) were added respectively, stirred and mixed, reacted at room temperature for 2 h to obtain Au@4-MBANPs, and stored at 4 °C in the dark for later use.

[0089] Figure 2 illustrate: Figure 2 -A is the Raman characteristic peak of 4-MBA at 1078cm-1 with different addition amounts -1 The Raman signal intensity at Figure 2 -B is the Raman signal intensity of different added amounts of 4-MBA.

[0090] like Figure 2 -A and Figure 2 As shown in Figure 2A, when the amount of 4-MBA added to AuNPs gradually increased, the characteristic peak at 1078 cm -1 The Raman signal at the site first increased and then decreased, and the signal was strongest when the addition amount was 40 μL.

[0091] Therefore, Au@4-MBA synthesized by adding 40 μL of 4-MBA was selected for further synthesis of Au@4-MBA@AgNPs.

[0092] 3. Selection of the dosage of AgNO3 solution

[0093] The Au@4-MBA solution synthesized by adding 40 μL of 4-MBA was used for subsequent synthesis.

[0094] Divide the Au@4-MBA NPs solution into 5 groups, and place 10 mL of each group in 5 clean glass bottles; centrifuge at 10000 rpm for 8 minutes, discard the supernatant, and dilute it 2 times with ultrapure water. Heat the solution to a boil, add 1 mL of sodium citrate solution (1%, w / v) under vigorous stirring, and slowly drip AgNO3 solution (10 mM) after 1 minute. The volumes of AgNO3 solution used are 0, 10, 25, 50, 100, 200, and 400 μL, respectively. Stop heating after 15 minutes of reaction, continue stirring until the solution cools naturally, and add ultrapure water to 20 mL.

[0095] Figure 2 illustrate: Figure 2 -C is the Raman characteristic peak 1078cm -1 The Raman signal intensity at , the inset in the upper left corner is the optical image of Au@4-MBA@Ag NPs with different silver shell thicknesses; Figure 2 -D is the Malvern particle size diagram of Au@4-MBA@Ag NPs with different silver shell thicknesses.

[0096] Figure 2 -C results show that with the increase of AgNO3 dosage, the Raman signal value gradually increases. It reaches the highest when the amount of AgNO3 added reaches 200μL, and then the signal value decreases. At the same time, the color of the solution gradually changes from the original cherry red to orange-yellow with the increase of AgNO3 dosage, which indicates that the silver shell on the surface of Au nanoparticles gradually thickens. Figure 2 -D results showed that the average hydrated particle size of the synthesized nanoparticles gradually increased from 28nm to 37nm, indicating that the thickness of the silver shell continued to increase.

[0097] Figure 3 illustrate: Figure 3 -A is the TEM image of AuNPs; Figure 3 -B and Figure 3 -C is the TEM image of Au@4-MBA@Ag NPs; Figure 3 -D to Figure 3 -F is the elemental analysis scan of Au@4-MBA@Ag NPs, red represents Au element and green represents Ag element.

[0098] Figure 3 The TEM and elemental analysis results of Au@4-MBA@Ag NPs prepared with AuNPs as the core are shown. Figure 3 -A shows that the synthesized gold nanoparticles present a round and uniform morphology. Based on this, the synthesized Au@4-MBA@AgNPs are relatively uniform and dispersed, showing a clear core-shell structure, and the thickness of the silver shell is about 4nm ( Figure 3 -B. Figure 3 -C). In addition, the elemental analysis results further proved that the core-shell nanomaterial was successfully prepared ( Figure 3 -D to Figure 3 -F). The red Au element is evenly wrapped in the green Ag element. This result is consistent with Figure 3 -B is consistent with the expected result.

[0099] Therefore, a group with an added amount of 200 μL of AgNO3 was selected for the subsequent coupling treatment.

[0100] 4. Selection of labeling antibody concentration

[0101] A group with an added amount of 200 μL of AgNO3 was selected for coupling treatment.

[0102] Au@4-MBA@Ag NPs were divided into 5 groups, each with 1 mL placed in a 1.5 mL centrifuge tube, centrifuged at 10000 rpm for 8 min, the supernatant was discarded to remove excess AgNO3, and the precipitate was resuspended to the original volume. The pH value of the Au@4-MBA@Ag NPs solution was then adjusted to 8.5 with K2CO3 solution (0.1 M). Anti-PEDV-Ab and anti-PoRVA-Ab1 were then added to the above Au@4-MBA@Ag NPs solution in amounts of 0, 2, 4, 6, 8, and 10 μg, respectively, and mixed. The mixture was gently shaken at room temperature for 2 h, and then 100 μL BSA (10%, w / v) was added and incubated for another 1 h. The resulting solution was centrifuged at 10000 rpm for 8 min, and the precipitate was redissolved with 200 μL of resolubilization solution.

[0103] The above solution was added dropwise to the conjugate pad and dried at 37°C for 2.5 h before being assembled into a test strip. 4 TCID50 / mL and 3.8×10 5 Mix the positive samples of PEDV and PoRVA at a ratio of 1:1 at 1:1, drop 3 drops on the test strip for detection, and set up a negative control. Let it stand for 20 minutes, and use a Raman spectrometer to detect it. The strongest signal peak of 4-MBA (wavelength 1078cm -1 ) to analyze the Raman signal value and determine the concentration of labeled antibody.

[0104] Figure 4 illustrate: Figure 4 -A is the result of the SERS immunochromatography test strip, 1-6 are the positive test results of the negative control and the test strip with the added amount of labeled antibody of 0, 2, 4, 6, 8 and 10 μg, T1 is the PEDV detection line, and T2 is the PoRVA detection line; Figure 4 -B is the test strip prepared under different labeled antibody concentration conditions at 1078cm -1 Raman signal intensity at the displacement.

[0105] like Figure 4 -A and Figure 4-B shows that with the increase of the amount of labeled antibody added, the Raman signal values ​​of the PEDV and PoRVA detection lines also gradually increase. When the antibody addition amount is 2, 4 and 6 μg, the Raman signal value of the PoRVA detection line increases particularly significantly. When the antibody addition amount is greater than 6 μg, the Raman signal value of the PoRVA detection line increases slowly. From the perspective of cost saving, 6 μg PoRVA antibody is added to 1 mL of Au@4-MBA@Ag NPs as the optimal antibody concentration, that is, the PoRVA antibody concentration is 6 μg / mL. Similarly, the Raman signal value of the PEDV detection line increases with the increase of the antibody addition amount. When the antibody addition amount is 10 μg, the Raman signal value of the PEDV detection line is the strongest. Therefore, 10 μg PEDV antibody is added to 1 mL of Au@4-MBA@Ag NPs as the optimal antibody concentration, that is, the PEDV antibody concentration is 10 μg / mL.

[0106] Therefore, the amount of antibody added to 1 mL of Au@4-MBA@Ag NPs solution was: 10 μg of PEDV antibody, with an antibody concentration of 10 μg / mL; 6 μg of PoRVA antibody, with an antibody concentration of 6 μg / mL.

[0107] 5. T-line antibody concentration selection

[0108] Five NC membranes were taken and coated with anti-PEDV-mAb at concentrations of 0.4, 0.6, 0.8, 1.0 and 1.2 mg / mL on the NC membrane using a three-dimensional film spray gold apparatus as the T1 line. Similarly, the T2 line was also coated with the same concentration of anti-PoRVA-mAb2 and dried at 37°C for 2.5 h. The drug loading was 1×10 4 TCID50 / mL and 3.8×10 5 The positive samples of PEDV and PoRVA were mixed at a ratio of 1:1, and 3 drops were dropped on the test strip for detection. A negative control was set at the same time. After standing for 20 minutes, the samples were detected using a Raman spectrometer. The strongest signal peak of 4-MBA (wavelength 1078 cm -1 ) were analyzed to determine the T-line membrane antibody concentration.

[0109] Figure 5 illustrate: Figure 5 -A is the result of the SERS immunochromatographic test strip, 1-6 are the positive test results of the negative control and the test strip with antibody concentrations of 0, 0.4, 0.6, 0.8, 1 and 1.2 mg / mL, T1 is the PEDV detection line, and T2 is the PoRVA detection line; Figure 5 -B is the T line of the test strip 1078cm -1 Raman signal intensity of the Raman spectrum at the shift.

[0110] like Figure 5 -A and Figure 5 -B, as the T-line film concentration increases, the color signals of the PEDV and PoRVA detection lines gradually deepen, and the Raman signal value gradually increases. When the film concentration is 0.4 and 0.6 mg / mL, the Raman signal value of the PoRVA detection line gradually increases, and when the concentration is higher than 0.8 mg / mL, although the Raman signal value of the PoRVA detection line is still increasing, the growth rate is significantly slowed down. Therefore, from the perspective of cost saving, the NC film coated with PoRVA antibody at a concentration of 0.8 mg / mL is selected as the T2 line. Similarly, the Raman signal value of the PEDV detection line increases with the increase of the film concentration. When the T-line film concentration is 1.2 mg / mL, the Raman signal value of the PEDV detection line is the strongest. Therefore, the NC film coated with PEDV antibody at a concentration of 1.2 mg / mL is selected as the T1 line.

[0111] Therefore, the membrane concentration of PEDV antibody is 1.2 mg / mL, and the membrane concentration of PoRVA antibody is 0.8 mg / mL.

[0112] 3. Test strip detection

[0113] Au@4-MBA@Ag NPs were prepared by referring to the above method, and the raw materials used were: 10 mL AuNPs, 40 μL 4-MBA (10 mM), and 200 μL AgNO3 solution (10 mM).

[0114] Labeled antibodies: 10 μg of PEDV antibody and 6 μg of PoRVA antibody were taken in 1 mL of Au@4-MBA@Ag NPs.

[0115] The concentration of the membrane was as follows: PEDV of T1 line was 1.2 mg / mL, and PoRVA of T2 line was 0.8 mg / mL.

[0116] Test strip assembly: the sample pad is made of glass fiber; the labeled antibody is fixed on the binding pad made of glass fiber using a three-dimensional film-scribing gold spraying instrument; PEDV monoclonal antibody, PoRVA monoclonal antibody, and sheep anti-mouse IgG are fixed on the NC membrane as T1, T2 and C lines respectively using a three-dimensional film-scribing gold spraying instrument, and the distance between each two lines is 5mm, and dried at 37℃ for 2.5h; finally, the NC membrane fixed with T lines and C lines, the binding pad, the sample pad and the absorbent paper are pasted to the bottom plate in turn, and cut into test strips with a width of 3mm and a length of 6.5cm using a CNC cutting machine, and stored in a dark place at 4℃ for later use; the bottom plate can be made of PVC material.

[0117] The glass fiber, NC membrane, absorbent paper, PVC material, etc. used in the test paper assembly are all existing materials and are not the innovation of this application.

[0118] 1. Sensitivity test

[0119] Use sample diluent to make the concentration of 1.0×10 4 TCID50 / mL and 3.8×10 5 PEDV and PoRVA were diluted 2 times in a gradient of 100 copies / μL. 60μL of the diluted virus solution was added to the sample pad, and the sample dilution was used as a negative control. The test results were observed with the naked eye to determine the visual LOD of the test paper, and the Raman signals of PEDV and PoRVA were measured using a Raman spectrometer. At the same time, the diluted virus solutions of PEDV and PoRVA were mixed evenly, 60μL was added to the sample pad, and the Raman signal was measured using a Raman spectrometer. The negative sample average + 3 times the standard deviation was used as the critical value. If it was lower than the critical value, it was judged as negative, and if it was higher than the critical value, it was judged as positive. Finally, the Raman signal value was used as the vertical axis, and the PEDV and PoRVA virus load was used as the horizontal axis. The Origin software was used to draw the graph and perform fitting analysis.

[0120] Figure 6 illustrate: Figure 6 -A is 1-7 test strips used to detect negative samples and the virus load is 5.0×10 3 , 2.5×10 3 , 1.25×10 3 , 6.25×10 2 , 3.13×10 2 and 1.56×10 2 PEDV-positive samples with TCID50 / mL; Figure 6 -B is the Raman signal diagram of test strips No. 1-7; Figure 6 -C is the PEDV fitting curve.

[0121] like Figure 6 As shown in Figure 1-A, when PEDV was detected alone, the color signal of the T line gradually decreased as the viral load decreased. 2 At TCID50 / mL, the T line of the test strip only showed a weak color signal. Therefore, the visualization LOD of the SERS immunochromatographic test strip for PEDV was 6.25×10 2 TCID50 / mL. Figure 6 The results of Raman spectrum detection of -B show that as the amount of PEDV loaded decreases, the Raman signal of the T line gradually weakens. Figure 6 -C, with the common logarithm of PEDV load lg (TCID50 / mL) as the horizontal axis, the Raman shift is 1078cm -1 The Raman signal value at the ordinate is used to draw a fitting curve, and the obtained linear equation is y=19742.82x-31256.31, R2 =0.99, which has a good linear relationship. The critical value 6326.25 (mean value of negative samples + 3 times standard deviation) is substituted into the linear equation to obtain the LOD of the SERS immunochromatographic test strip for PEDV, which is 8.01×10 1 TCID50 / mL.

[0122] Figure 7 illustrate: Figure 7 -A is 1-11 test strips used to detect negative samples and the virus load is 1.9×10 5 , 9.5×10 4 4.75×10 4 , 2.38×10 4 , 1.19×10 4 , 5.94×10 3 , 2.97×10 3 , 1.48×10 3 ,7.42×10 2 , 3.71×10 2 copies / μL of PoRVA-positive samples; Figure 7 -B is the Raman signal diagram of test strips No. 1-11; Figure 7 -C is the PoRVA fitting curve.

[0123] When PoRVA is measured alone, Figure 7 As shown in Figure 2A, as the sample dilution multiples increased, the intensity of the T line gradually decreased. 2 When the number of copies / μL was 2.34, the T line of the test strip showed only a weak color signal. Therefore, the visualization LOD of the SERS immunochromatographic test strip for PoRVA was 7.42×10 2 copies / μL. Figure 7 As shown in Figure 1-B, the results of Raman spectroscopy show that as the amount of PoRVA loaded decreases, the Raman signal of the T line gradually weakens. Figure 7 -C, with the common logarithm of PoRVA drug loading lg (copies / μL) as the horizontal axis, the Raman shift is 1078cm -1 The Raman signal value at the ordinate is used to draw a fitting curve, and the linear equation y = 24378.49x-55535.21 is obtained, R 2 =0.99, which has a good linear relationship. The critical value 5526.76 (mean value of negative samples + 3 times standard deviation) is substituted into the linear equation to obtain the LOD of the SERS immunochromatographic test strip for PoRVA is 3.19×10 2 copies / μL.

[0124] Figure 8Note: Test strips 1-10 are used to detect negative samples and the virus load is 2.5×10 3 , 1.25×10 3 , 6.25×10 2 , 3.13×10 2 , 1.56×10 2 ,7.81×10 1 3.90×10 1 , 1.95×10 1 and 9.75×10 0 The TCID50 / mL of PEDV positive samples and the virus load were 9.5×10 4 4.75×10 4 , 2.38×10 4 , 1.19×10 4 , 5.94×10 3 , 2.97×10 3 , 1.48×10 3 ,7.42×10 2 and 3.71×10 2 For PoRVA positive samples with 10 copies / μL, T1 is the PEDV detection line and T2 is the PoRVA detection line.

[0125] like Figure 8 As shown, when a serially diluted mixture of PEDV and PoRVA was analyzed using an immunochromatographic test strip, the LODs for PEDV and PoRVA were the same as when either PEDV or PoRVA sample was tested alone.

[0126] 2. Specificity detection

[0127] The PEDV virus liquid, PoRVA positive sample, PoRVB and PoRVC mixture (CT values ​​were 29.91 and 25.08, respectively), TGEV (1.0×10 3 TCID50 / mL), PRV (1.0×10 6 TCID50 / mL) and PDCoV (CT value was 25.04). At the same time, some common bacteria such as Escherichia coli K88, Escherichia coli K99, Escherichia coli standard strain, Salmonella Enteritidis 1, Salmonella Enteritidis 2, Salmonella Typhimurium, Salmonella Paratyphi, Staphylococcus aureus standard strain, Staphylococcus aureus resistant bacteria and Vibrio parahaemolyticus were also verified. The concentrations of the above bacterial samples have been adjusted to 1×10 7 CFU / mL. Let stand for 20 minutes and observe the test results with naked eyes. Use Raman spectrometer to detect and analyze the Raman signal value of the strongest signal peak of 4-MBA. Use Origin software to draw pictures and analyze.

[0128] Fig. 9 illustrate: Fig. 9 -A is test strips No. 1-17, which are used to detect negative samples, PEDV, PoRVA, a mixture of PoRVB and PoRVC, TGEV (transmissible gastroenteritis virus of porcine), PRV (pseudorabies virus), PDCoV (swine deltacoronavirus), Escherichia coli K88, Escherichia coli K99, Escherichia coli standard strain, Salmonella Enteritidis 1, Salmonella Enteritidis 2, Salmonella Typhimurium, Salmonella Paratyphi, Staphylococcus aureus standard strain, Staphylococcus aureus resistant bacteria and Vibrio parahaemolyticus; Fig. 9 -B is the test strip at 1078cm -1 Raman signal intensity of the Raman spectrum at the displacement; Fig. 9 -C is the Raman spectrum of the T line area of ​​the test strip.

[0129] like Fig. 9 As shown in Figure 2-A, the SERS immunochromatographic test strip can only specifically identify PEDV and PoRVA to produce red strips, and cannot cross-react with other common viruses or bacteria. Fig. 9 -B and Fig. 9 -C shows that the SERS immunochromatographic test strip has obvious Raman characteristic absorption peaks only when detecting PEDV and PoRVA. When detecting other common viruses and bacteria with similar clinical symptoms, it only shows background signals consistent with the negative control, so it has good specificity.

[0130] 3. Repeatability test

[0131] Six SERS immunochromatographic test strips were taken from the same batch and used to detect negative samples and the drug loading was 3.3×10 3 TCID50 / mL and 1.27×10 5 The positive samples of PEDV and PoRVA with a virus loading of 3.3×10 copies / μL were selected to investigate the intra-batch repeatability. Two SERS immunochromatographic test strips prepared from three different batches were taken out to detect negative samples and the virus loading was 3.3×10 3 TCID50 / mL and 1.27×10 5 Positive samples of PEDV and PoRVA with 100 copies / μL were tested to examine the reproducibility between batches. Let it stand for 20 minutes and observe the test results with the naked eye. Use Raman spectrometer to detect and analyze the Raman signal value of the strongest signal peak of 4-MBA. Use Origin software for graphical analysis. Analyze the reproducibility of the test strips by calculating the coefficient of variation (CV). The CV calculation formula is: CV = standard deviation / mean value × 100%.

[0132] Fig.10 illustrate: Fig.10 -A is the repeatability test result within the test strip batch, T1 is the PEDV test line, and T2 is the PoRVA test line; Fig.10 -B is the reproducibility test result among three batches of test strips; Fig.10 -C is the test strip within the batch at 1078cm -1 Raman signal diagram at ; Fig.10 -D is the test strip between batches at 1078cm -1 Raman signal diagram at .

[0133] like Fig.10 -As shown in A, there is no significant difference in the color signal of the test strips within the batch by naked eye observation; Fig.10 -C, the SERS test results show that the CV value of PEDV is 8.3% and the CV value of PoRVA is 3.6% when the test strip is tested within the batch, both of which are less than 10%, showing good intra-batch repeatability.

[0134] like Fig.10 -B, there is no significant difference in the strips between batches by naked eye observation; Fig.10 -D, the SERS test results show that the CV value of PEDV is 10.2% and the CV value of PoRVA is 5.6% when the test strips are tested between batches, both showing good batch-to-batch repeatability.

[0135] The above results all indicate that the SERS immunochromatographic test strip has good intra-batch and inter-batch repeatability.

[0136] 4. Clinical Sample Testing

[0137] 30 clinical stool or intestinal samples were tested using SERS immunochromatographic test strips and compared with the RT-PCR test results. The swab used for sampling was immersed in the sample diluent and mixed thoroughly. After the sample was slightly precipitated, 3 drops of supernatant were dropped on the sample pad for testing. After standing for 20 minutes, the test results were observed with the naked eye, and the Raman spectrometer was used for testing. The critical value was used to determine whether the sample was negative or positive. The results were compared with those obtained by the RT-PCR method, and the results are shown in Table 2.

[0138] As can be seen from Table 2, when the test strip of the present invention was used for analysis, a total of 16 PEDV-positive samples and 8 PoRVA samples were identified, with positive rates of 53.3% (16 / 30) and 26.6% (8 / 30), respectively. Among the positive samples, the mixed infection rate reached 13.3% (4 / 30). Among them, one sample was identified as PEDV positive by RT-PCR, but a false negative result occurred in the test strip analysis, and one sample identified as PoRVA positive by RT-PCR had a false negative result in the test strip test. The test results of the other samples by the two analysis methods were completely consistent. The above results show that the overall consistency rate of the immunochromatographic test strip of the present invention with the RT-PCR results is as high as 93.3% (28 / 30), indicating that the accuracy of the test strip based on Au@4-MBA@Ag NPs is comparable to that of RT-PCR, providing an effective option for on-site rapid diagnosis of PEDV and / or PoRVA.

[0139] Table 2 Comparison with RT-PCR clinical sample test results

[0140]

[0141] The present invention combines SERS technology with LFIA, connects 4-MBA Raman signal molecules to the surface of Tris-stabilized colloidal gold and then wraps a layer of silver shell to obtain Au@4-MBA@Ag NPs. The core-shell structure material has a nanoscale gap, retains the stability of AuNPs, and introduces silver to enhance the Raman effect. In addition, there are a large number of hot spots in the gap between the gold core and the silver shell, and more significant Raman spectra can be detected under specific excitation wavelengths, which is beneficial to improve the sensitivity of the SERS immunochromatographic test strip.

[0142] The present invention uses Au@4-MBA@Ag NPs to label antibodies, and the prepared SERS immunochromatographic test strip is used to detect PEDV and PoRVA. When the sample contains a large amount of target pathogens, the T1 and / or T2 lines of the test strip may show obvious color signals, which are used to qualitatively detect PEDV and / or PoRVA pathogens. When only a small amount of target pathogens are present in the sample, the T1 and / or T2 lines of the SERS immunochromatographic test strip have no obvious colorimetric signals, which can be further analyzed by a Raman spectrometer. The Raman shift of 4-MBA at 1078 cm -1 The Raman signals at the sites are used to quantitatively detect the samples, achieving ultra-sensitive detection of PEDV and PoRVA.

[0143] The test strip prepared by the present invention can detect trace amounts of target analytes mainly because Au@4-MBA@Ag NPs provide extremely strong colorimetric signals and SERS signals. At the same time, after optimizing the concentrations of labeled antibodies and T-line antibodies, the appropriate antigen-antibody ratio enhances the detection sensitivity.

[0144] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the statement "including an element defined by ... does not exclude the existence of other identical elements in the process, method, article or device including the element".

Claims

1. A SERS immunochromatographic test strip for joint detection of PEDV and PoRVA, characterized in that: include: A base plate, and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper sequentially arranged on the base plate; The conjugate pad is immobilized with a labeled antibody, which is prepared by coupling Au@4-MBA@Ag NPs with Anti-PEDV-Ab and / or Anti-PoRVA-Ab; The detection line T1 formed by PEDV monoclonal antibody, the detection line T2 formed by PoRVA monoclonal antibody, and the quality control line C formed by goat anti-mouse IgG are fixed on the nitrocellulose membrane in sequence.

2. A SERS immunochromatographic test strip for joint detection of PEDV and PoRVA according to claim 1, characterized in that: The method of coupling the labeled antibody is as follows: S11, taking Au@4-MBA@Ag NPs, centrifuging, discarding the supernatant, resuspending the precipitate, and adjusting the pH of the Au@4-MBA@Ag NPs solution to alkaline; S12, adding Anti-PEDV-Ab and / or Anti-PoRVA-Ab, mixing, and adding BSA solution for incubation; S13, the obtained solution is centrifuged and the precipitate is redissolved to obtain the obtained product.

3. A SERS immunochromatographic test strip for joint detection of PEDV and PoRVA according to claim 2, characterized in that: The precipitate in step S13 is redissolved using a re-solution, and the re-solution is prepared as follows: Take BSA, sucrose, and Tween-20 and dissolve them in Tris-HCl buffer until they are fully dissolved; The BSA concentration of the reconstitution solution was 5% (w / v), the sucrose concentration was 10% (w / v), the Tween-20 concentration was 1% (v / v), and the Tris-HCl buffer concentration was 50 mM; The pH of the reconstituted solution was 8.

5.

4. A SERS immunochromatographic test strip for joint detection of PEDV and PoRVA according to any one of claims 1 to 3, characterized in that: The test strip is used for detecting PEDV and / or PoRVA.

5. A method for preparing a SERS immunochromatographic test strip for joint detection of PEDV and PoRVA, characterized in that: include: Synthesis of S21 and Au@4-MBA NPs Take AuNPs, add 4-MBA ethanol solution, stir and mix to obtain Au@4-MBA NPs; Synthesis of S22 and Au@4-MBA@Ag NPs Take the Au@4-MBA NPs solution prepared by S21, centrifuge it, discard the supernatant, and dilute it 2 times with water; then heat the solution to boil, add sodium citrate solution, and then slowly drop the AgNO3 solution; after stopping heating, continue stirring until the solution cools naturally, and add water to the diluted volume; S23, Antibody Conjugation The Au@4-MBA@Ag NPs prepared in S22 were centrifuged, the supernatant was discarded, the precipitate was resuspended, and the pH of the Au@4-MBA@Ag NPs solution was adjusted to alkaline; Anti-PEDV-Ab and / or Anti-PoRVA-Ab1 were added, mixed, and BSA solution was added for incubation; the obtained solution was centrifuged, and the precipitate was redissolved to obtain the labeled antibody; S24, Test strip assembly The labeled antibody prepared by S23 is fixed on the conjugate pad; the PEDV monoclonal antibody is fixed on the nitrocellulose membrane in sequence to form the detection line T1, the PoRVA monoclonal antibody is fixed to form the detection line T2, and the goat anti-mouse IgG is fixed to form the quality control line C; then the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent paper are pasted on the bottom plate and cut to obtain the product.

6. The method for preparing a SERS immunochromatographic test strip for joint detection of PEDV and PoRVA according to claim 5, characterized in that: The concentration of the 4-MBA ethanol solution used in step S21 is 10 mM.

7. The method for preparing a SERS immunochromatographic test strip for joint detection of PEDV and PoRVA according to claim 6, characterized in that: The concentration of the sodium citrate solution used in step S22 is 1% (w / v), and the concentration of the AgNO3 solution is 10 mM.

8. The method for preparing a SERS immunochromatographic test strip for joint detection of PEDV and PoRVA according to claim 7, characterized in that: Step S23: adjusting the pH to 8.5 using 0.1 M K2CO3 solution; The precipitate was re-dissolved using a reconstitution solution prepared as follows: Take BSA, sucrose, and Tween-20 and dissolve them in Tris-HCl buffer until they are fully dissolved; The BSA concentration of the reconstitution solution was 5% (w / v), the sucrose concentration was 10% (w / v), the Tween-20 concentration was 1% (v / v), and the Tris-HCl buffer concentration was 50 mM; The pH of the reconstituted solution was 8.

5.

9. The method for preparing a SERS immunochromatographic test strip for joint detection of PEDV and PoRVA according to claim 8, characterized in that: Before incubation in step S23, the solution was gently shaken at room temperature for 2 h, and the concentration of the BSA solution used was 10% (w / v).

10. The method for preparing a SERS immunochromatographic test strip for joint detection of PEDV and PoRVA according to claim 9, characterized in that: The prepared test strip is used for detecting PEDV and / or PoRVA.

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