Colloidal gold immunochromatography test strip for detecting antigens of orientia tsutsugamushi

Abstract

The invention discloses a colloidal gold immunochromatography test strip for detecting antigens of orientia tsutsugamushi, the test strip comprises a nitrocellulose membrane, one end of the nitrocellulose membrane is provided with a sample pad and a gold-labeled pad, the other end of the nitrocellulose membrane is provided with a water absorption pad, the gold-labeled pad is arranged on the nitrocellulose membrane, and the water absorption pad is arranged on the nitrocellulose membrane. The sample pad, the water absorption pad and the nitrocellulose membrane are all arranged on the bottom plate, the gold mark pad contains an anti-oriental body 56kDa rabbit polyclonal antibody marked by colloidal gold, the nitrocellulose membrane is provided with a detection line and a control line, the detection line contains an orientia tsutsugamushi disease 56kDa protein monoclonal antibody, and the quality control line contains a goat anti-rabbit polyclonal antibody. A colloidal gold immunochromatography method is established by adopting a double-antibody sandwich method principle to detect the orientia tsutsugamushi antigen, and the kit has the advantages of low cost of a detection instrument, simplicity and rapidness in operation, good stability, good specificity, high sensitivity, relatively strong practicability, easiness in storage and market development value.

Description

Technical Field

[0001] The present invention relates to the technical field of colloidal gold immunoassay, and particularly relates to a colloidal gold immunochromatographic test strip for detecting Orientia tsutsugamushi antigen by a double antibody sandwich method, a preparation method thereof, and an application thereof. Background Art

[0002] The information provided in this part is only background information related to the present disclosure, and it is not necessarily prior art.

[0003] Scrub typhus is a natural zoonotic disease caused by Orientia tsutsugamushi (Ot). The larva of chigger mite is the only vector of scrub typhus, and the larva of chigger mite forms an infection cycle in nature by biting rodents such as mice. Orientia tsutsugamushi enters the human body following the bite of chigger mite, first multiplies at the skin bite site, then enters the blood, initially attacks the myeloid cells at the damaged site, and then attacks the vascular endothelial cells. Subsequently, Orientia tsutsugamushi attaches itself to the target cells by using the surface proteoglycan existing on the host cells and the surface protein of the bacterial cell. Scrub typhus is widely distributed in China and is greatly affected by geographical features, climate, and wild animals. The antigenicity and virulence of Orientia tsutsugamushi in different strains and different regions are different, and the pathogenic mechanism of scrub typhus has not been fully clarified yet, and the auxiliary examination has no specificity, resulting in great difficulties in the prevention and diagnosis of this disease.

[0004] There are mainly two methods for detecting Orientia tsutsugamushi, one is serological detection and the other is molecular biological detection. Serological detection includes the Weil-Felix reaction, indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA). The Weil-Felix test is a mature agglutination method for detecting Rickettsia infection in patients' serum samples. Although it is simple to operate, it is prone to non-specific reactions, resulting in inaccurate results and unable to meet clinical requirements. The IFA method has certain limitations: complex operation, high cost, the need for experienced operators, and the risk of missed detection when only one strain of Orientia is used during the detection process. Therefore, the application of IFA in epidemic areas is limited. ELISA has higher specificity and sensitivity, but ELISA also has deficiencies: high cost, the need for trained personnel, and the need for a fluorescence microscope, etc. Molecular biological detection has high specificity and sensitivity and can even detect bacterial infections at an early stage. It mainly includes polymerase chain reaction (PCR), loop-mediated isothermal amplification technology, qPCR, and recombinase polymerase amplification technology, etc. The 56 kDa type-specific antigen gene, GroEL gene, 16S RNA gene, TraD conjugal transfer protein gene, and 47 kDa membrane protease gene are often used as target genes of Orientia for specific nucleic acid detection of target organisms. Generally speaking, the diagnostic accuracy of the PCR technique is superior to other methods for the early diagnosis of scrub typhus. Although nucleic acid detection methods have many advantages, they all require expensive instruments and professional personnel for operation, which also limits their application.

[0005] Immunochromatographic technology is a solid-phase membrane immunoassay technology that combines chromatographic separation technology, immunological technology, and immunolabeling technology. Its diagnostic principle is as follows: The sample solution migrates on a nitrocellulose (NC) membrane by capillary action. During chromatography, the complex of the labeled substance and the analyte to be detected is captured by the corresponding ligand and concentrated to show color. Qualitative or quantitative analysis is performed based on the presence or amount of the colored band on the NC membrane. ICA detection has good sensitivity, specificity, and a rapid reaction time (10 - 15 minutes), does not require complex instrument equipment, and has broad application prospects in the diagnosis of scrub typhus.

[0006] Colloidal gold was first observed under an electron microscope as a tracer marker. It has excellent properties such as good stability, simple preparation, high charge density, the ability to adsorb proteins without affecting the protein structure, which has gradually made it be applied to the markers in immunoassays. Currently, the most widely used method in rapid disease diagnosis is colloidal gold immunochromatography. The basic principle of colloidal gold immunochromatography is that antigens and antibodies flow directionally on a nitrocellulose membrane and undergo specific binding reactions, and gold particles aggregate at fixed positions, generating a precipitate visible to the naked eye, thereby achieving the detection of the target. It has the advantages of low cost, convenient detection, strong specificity, intuitive results, safety and environmental protection, etc.

[0007] In the research on the diagnosis of scrub typhus, almost all related studies on colloidal gold immunochromatography are for the detection of Orientia-specific antibodies. However, in the early stage of the disease, detecting antigens has more advantages. Therefore, constructing a colloidal gold immunochromatographic test strip for detecting Orientia tsutsugamushi antigens is of great significance for providing rapid and simple diagnosis in the early stage of the disease. In the present invention, monoclonal antibodies are prepared against the conserved region of the 56 kDa outer membrane protein of Orientia, combined with polyclonal antibodies, and a colloidal gold immunochromatographic method is established using the principle of sandwich ELISA to detect Orientia tsutsugamushi, and a standard curve is constructed using qPCR to determine the detection limit of the colloidal gold immunochromatographic test strip. The test strip is simple to operate, sensitive and specific, and can provide visible results to the naked eye within 15 minutes, and has good application prospects in primary hospitals, remote towns and epidemic outbreak areas. Summary of the Invention

[0008] Object of the Invention: The technical problem to be solved by the present invention is to provide a colloidal gold immunochromatographic test strip for detecting Orientia tsutsugamushi antigens in view of the deficiencies of the prior art.

[0009] The present invention also provides a test card or kit including the above-mentioned colloidal gold immunochromatographic test strip for detecting Orientia tsutsugamushi antigens.

[0010] The present invention also provides the application of the above-mentioned colloidal gold immunochromatographic test strip, test card or kit for detecting Orientia tsutsugamushi antigens.

[0011] To solve the above first technical problem, the present invention discloses a colloidal gold immunochromatographic test strip for detecting Orientia tsutsugamushi antigens, including a nitrocellulose membrane, one end of the nitrocellulose membrane is provided with a sample pad and a gold-labeled pad, the other end is provided with an absorbent pad, the gold-labeled pad is placed on the nitrocellulose membrane, the sample pad, the absorbent pad and the nitrocellulose membrane are all placed on a bottom plate, the sample pad is placed on both the bottom plate and the gold-labeled pad at the same time, the absorbent pad is placed on both the nitrocellulose membrane and the bottom plate at the same time, and the nitrocellulose membrane is provided with a detection line 5 and a control line 6.

[0012] Among them, the sample pad is provided with a sample addition hole.

[0013] Among them, the gold conjugate pad contains a rabbit polyclonal antibody against Orientia tsutsugamushi 56 kDa prepared by colloidal gold labeling; preferably, the colloidal gold-polyantibody is sprayed on the gold conjugate pad.

[0014] Among them, the test line contains a monoclonal antibody against Orientia tsutsugamushi 56 kDa protein; preferably, the monoclonal antibody against Orientia tsutsugamushi 56 kDa is sprayed on the test line. The monoclonal antibody against Orientia tsutsugamushi 56 kDa protein is secreted by a hybridoma cell line with the strain preservation number of CCTCC NO: C2022244 preserved in the China Center for Type Culture Collection.

[0015] Among them, the control line contains a goat anti-rabbit polyclonal antibody; preferably, the goat anti-rabbit polyclonal antibody is sprayed on the control line.

[0016] Among them, the preparation method of the gold conjugate pad includes adjusting the pH of the colloidal gold solution, stirring, adding a rabbit polyclonal antibody against Orientia tsutsugamushi 56 kDa to the colloidal gold solution, stirring, adding a polyethylene glycol solution, stirring, adding a bovine serum albumin solution, stirring; the obtained solution is stored at low temperature, centrifuged at low temperature to obtain a gold conjugate precipitate; the gold conjugate precipitate is dissolved, and the obtained gold conjugate solution is sprayed on glass fiber and dried. Among them, the colloidal gold is synthesized by the sodium citrate reduction of chloroauric acid method. Chloroauric acid is added to ultrapure water to make the final concentration of chloroauric acid 0.01% wt. The chloroauric acid solution is boiled, the bottle mouth is covered with tin foil to prevent water vapor from escaping, sodium citrate is added, and heated for 30 min. The color will gradually turn into wine red, and then cooled to room temperature and stored at 4°C for standby. Observe the prepared colloidal gold, determine the maximum absorption peak wavelength of the colloidal gold in the wavelength range of 450 - 600 nm with a spectrophotometer, and observe the size of the colloidal gold particles with a transmission electron microscope to see if the particles are uniform and dispersed.

[0017] Among them, the pH is 8 - 9, preferably 8.5.

[0018] Among them, when adding a rabbit polyclonal antibody against Orientia tsutsugamushi 56 kDa to the colloidal gold solution, the concentration of the rabbit polyclonal antibody against Orientia tsutsugamushi 56 kDa is 18 - 22 μg / mL, preferably 20 μg / mL.

[0019] Among them, the addition amounts of the polyethylene glycol solution and the bovine serum albumin solution are independently selected from 0.5 - 1.5 mL, such as 1 mL; the mass percentage of polyethylene glycol in the polyethylene glycol solution is 0.5% - 1.5%, such as 1%; the mass percentage of bovine serum albumin in the bovine serum albumin solution is 8% - 12%, such as 10%.

[0020] Among them, the gold-labeled precipitate is dissolved with a complex solution; the complex solution is a mixed solution of polyethylene glycol 20000, sucrose, BSA, Tween and 0.01M Tris solution. In the complex solution, the concentration of polyethylene glycol 20000 is 0.01 g / mL, the concentration of sucrose is 0.1 g / mL, the concentration of BSA is 0.01 g / mL, and the dosage of Tween 20 is 100 μL / 10 mL Tris solution.

[0021] Among them, the membrane coating concentration of the 56 kDa protein monoclonal antibody of Orientia tsutsugamushi in the test line is 0.4 - 0.6 mg / mL, preferably 0.5 mg / mL.

[0022] Among them, the membrane coating concentration of the goat anti-rabbit polyclonal antibody in the quality control line is 0.4 - 0.6 mg / mL, preferably 0.5 mg / mL.

[0023] To solve the above-mentioned second technical problem, the present invention discloses a test strip card, which includes the above-mentioned test strip.

[0024] The present invention also discloses a kit, which also includes the above-mentioned test strip.

[0025] To solve the above-mentioned third technical problem, the present invention discloses the application of the test strip described in the first aspect, the test strip card described in the second aspect and the test kit in the detection of Orientia tsutsugamushi antibody.

[0026] Among them, the application includes the following steps:

[0027] (1) Add 30 μL of the sample to the sample application hole of the sample pad of the test strip, and let it stand at room temperature for 10 min, then observe.

[0028] (2) When both the quality control line and the test line show color, it indicates that the sample is positive and the test result is valid, and it is determined as positive; when there is no band in the test line and there is a band in the quality control line, it indicates that the sample is negative and the test result is valid; when there is no band in the quality control line and there is or is not a band in the test line, the test is invalid.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0030] The present invention uses the double antibody sandwich method to detect the antigen of Orientia tsutsugamushi, which has the advantages of low cost of detection instruments, simple operation, rapidity, good stability, good specificity, high sensitivity, strong practicability, easy storage and market development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0032] Figure 1 It is a schematic diagram of the structure of a fluorescence immunochromatographic test strip; among them, 1 is the sample pad; 2 is the bottom plate; 3 is the nitrocellulose membrane; 4 is the gold-labeled pad; 5 is the test line; 6 is the quality control line; 8 is the absorbent pad.

[0033] Figure 2 It is a schematic diagram for judging the test results of a colloidal gold immunochromatographic test strip; among them, T is the test line; C is the quality control line; a) is a schematic diagram of a positive result; b) is a schematic diagram of a negative result; c) and d) are schematic diagrams of invalid results.

[0034] Figure 3 It is a schematic diagram of the color change of colloidal gold solution at different pH values; 1) is the control; 2) is 6.5; 3) is 7.0; 4) is 7.5; 5) is 8.0; 6) is 8.5; 7) is 9.0; 8) is 9.5.

[0035] Figure 4 It is a schematic diagram of the color change of colloidal gold solution at different amounts of protein; 1) is 40 μg / mL; 2) is 35 μg / mL; 3) is 30 μg / mL; 4) is 25 μg / mL; 5) is 20 μg / mL; 6) is 15 μg / mL; 7) is 10 μg / mL; 8) is 5 μg / mL.

[0036] Figure 5 It is a schematic diagram for determining the sensitivity of a colloidal gold immunochromatographic test strip at different recombinant protein concentrations; 1) is 37.00 μg / mL; 2) is 18.75 μg / mL; 3) is 9.38 μg / mL; 4) is 4.69 μg / mL; 5) is 2.35 μg / mL; 6) is 1.18 μg / mL; 7) is the blank control.

[0037] Figure 6 It is a schematic diagram of the qPCR amplification curve; A) is the qPCR amplification curve graph; B) is the qPCR melting curve graph; C) is the qPCR standard curve graph.

[0038] Figure 7 It is a schematic diagram for determining the sensitivity of a colloidal gold immunochromatographic test strip at different Orientia copy numbers; 1) is 1×10 8 copies / μL; 2) is 1×10 7 copies / μL; 3) is 1×10 6 copies / μL; 4) is 1×10 5 copies / μL; 5) is the blank control.

[0039] Figure 8Schematic diagram for the specificity determination of colloidal gold immunochromatographic test strips when detecting different pathogenic bacteria; 1) Escherichia coli; 2) Salmonella; 3) Staphylococcus aureus; 4) Listeria monocytogenes; 5) blank control; 6) positive control. Specific implementation mode

[0040] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0041] In the following embodiments, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0042] In the present invention, the concentrations are mass concentrations unless otherwise specified.

[0043] The monoclonal antibody against Orientia tsutsugamushi 56 kDa protein in the following embodiments was prepared by the research group in the early stage. Specifically, it can refer to Chinese invention patent ZL202210943095.5, a hybridoma cell line 5B3, a monoclonal antibody against Orientia tsutsugamushi 56 kDa protein, and its preparation method and application.

[0044] The mixed recombinant antigen of Orientia tsutsugamushi in the following embodiments was prepared by the research group in the early stage. Specifically, it can refer to Chinese invention patent ZL202110854578.3, a fluorescence immunochromatographic test strip for detecting antibodies against Orientia tsutsugamushi, and its preparation method and application.

[0045] The polyclonal antibody against Orientia tsutsugamushi 56 kDa rabbit in the following embodiments was prepared by the research group in the early stage. The specific method was to immunize animals with the mixed recombinant antigen of Orientia tsutsugamushi (rabbits for experiments were selected as animals), immunize once every 2 weeks, and immunize a total of 4 times throughout the process. Whole blood was collected by carotid artery intubation, antiserum was separated and prepared, and the rabbit polyclonal antibody against Orientia tsutsugamushi was further purified by affinity chromatography.

[0046] The polyclonal antibody against rabbit IgG labeled with HRP goat anti-rabbit in the following embodiments was purchased from Beijing Boao Sen Biotechnology Co., Ltd.

[0047] Example 1: Determination of the optimal conditions for the test strip for detecting Orientia tsutsugamushi antigen by the antibody sandwich method

[0048] 1. Preparation of colloidal gold

[0049] Colloidal gold was prepared by reducing chloroauric acid with trisodium citrate. Measure 99 mL of ultrapure water, and then add 1 mL of an aqueous chloroauric acid solution with a mass percentage of 1% so that the final mass percentage of chloroauric acid is 0.01%. Boil the chloroauric acid solution, put the rotor soaked in aqua regia into the conical flask, stir the liquid evenly, cover the mouth of the flask with tin foil to prevent the escape of water vapor, quickly add 2 mL of 1% trisodium citrate aqueous solution, heat for 30 min, the color will gradually turn into wine red, then cool to room temperature and store at 4 °C for standby. Observe the prepared colloidal gold. The synthesized gold particles have uniform particle size, and the colloidal gold particles have good morphology, are spherical, and there is no overlapping and aggregation phenomenon.

[0050] 2. Determination of the Optimal pH of the Colloidal Gold Solution

[0051] Take 1 mL of the prepared colloidal gold solution into 8 1.5-mL centrifuge tubes, numbered 1-8, and add 0.1 M K 2 CO 3 to adjust the pH to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0. Set another colloidal gold solution without any treatment as a blank control, add an excessive amount of anti-Oriental body 56 kDa rabbit polyclonal antibody, shake well and let stand at room temperature for 15 min. Add 100 μL of a 10% NaCl solution by mass, and observe the color after standing at room temperature for 2 h. Select the smallest pH when the solution color is closest to the original solution color and there is no aggregation and precipitation as the optimal pH. As Figure 3 shown: When the pH of the colloidal gold is 8.5, its color is closest to the color of the colloidal gold stock solution, always showing wine red and being relatively stable. Therefore, the optimal pH for colloidal gold to label polyclonal antibody is 8.5.

[0052] 3. Determination of the Optimal Protein Dosage in Colloidal Gold Labeling of Anti-Oriental Body 56 kDa Rabbit Polyclonal Antibody

[0053] Take 1 mL of the prepared colloidal gold solution into 8 1.5-mL centrifuge tubes, numbered 1-8, and add 0.1 M K 2 CO 3 solution to make the pH of the colloidal gold solution 8.5. Add 40, 35, 30, 25, 20, 15, 10, 5 μg of anti-Oriental body 56 kDa rabbit polyclonal antibody to tubes 1-8 respectively. After mixing and standing for 15 min, add 100 μL of a 10% NaCl solution by mass and let stand for 2 h. When the protein amount is too much, the solution color remains unchanged. When the protein amount is too little, the solution color will turn blue and precipitate, as Figure 4As shown; in the first 6 tubes, since the amount of protein in the colloidal gold solution is sufficient, it appears as a clear and transparent wine-red color, indicating that the amount of protein is sufficient. The color of the 7th tube is bluish, and the 8th tube is completely blue-black, indicating that the amount of protein in these 2 tubes is too small. Therefore, it is determined that the amount of protein in the 6th tube is the critical value, and its stable minimum concentration is 15 μg / mL. In order to make the solution more stable and in the principle of cost saving, the optimal protein dosage is selected as 20 μg / mL.

[0054] 4. Determination of the optimal T-line and C-line antibody concentrations

[0055] The monoclonal antibody against Orientia tsutsugamushi 56 kDa protein and the goat anti-rabbit polyclonal antibody were respectively diluted to 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. The diluted monoclonal antibody and secondary antibody were respectively drawn on the NC membrane as the T-line and C-line using a membrane spraying instrument. The test sample was dropped on the sample pad, and the optimal coating amounts of the monoclonal antibody and secondary antibody were determined according to their color development. The monoclonal antibody was drawn on the NC membrane and then verified with the recombinant protein. The results showed that when the drawing concentration was 0.5 mg / mL, the band brightness was approximately close to that at 1 mg / mL, and there was no background color interference. Therefore, 0.5 mg / mL was selected as the optimal membrane-drawing concentration for the T-line. The goat anti-rabbit IgG was drawn on the NC membrane and verified with the recombinant protein. The results showed that when the drawing concentration was 0.5 mg / mL, the band brightness was approximately close to that at 1 mg / mL, and there was no false positive situation. Therefore, 0.5 mg / mL was selected as the optimal membrane-drawing concentration for the C-line.

[0056] Example 2: Preparation method and detection method of a colloidal gold immunochromatographic test strip for detecting Orientia tsutsugamushi antigen by the double antibody sandwich method

[0057] In this example, based on Example 1, the preparation of the gold-labeled pad and the assembly of the test strip were studied (if not otherwise specified, other steps were the same as in Example 1).

[0058] 1. Preparation of the gold-labeled pad

[0059] Adjust the colloidal gold solution obtained in step 1 of Example 1 according to the optimal pH obtained in step 2 of Example 1, stir it with a magnetic stirrer, add the anti-Oriental body 56 kDa rabbit polyclonal antibody with the optimal protein concentration obtained in step 3 of Example 1 to the colloidal gold solution, stir with a magnetic stirrer for 30 min, then add 1 mL of an aqueous solution of polyethylene glycol 20000 with a mass percentage of 1%, stir with a magnetic stirrer again for 30 min, add 1 mL of a BSA solution with a mass percentage of 10%, continue to stir for 30 min, then transfer the solution to a centrifuge tube and place it in a 4°C refrigerator overnight. Take it out the next day, centrifuge it at 12000 rpm for 20 min at 4°C with a refrigerated centrifuge. After centrifugation, carefully aspirate and discard the supernatant. Dissolve 0.1 g of polyethylene glycol 20000, 1 g of sucrose, 0.1 g of BSA and 100 μL of Tween 20 in 10 mL of 0.01 M Tris solution to obtain a reconstitution solution, and then dissolve the gold-labeled precipitate with one-tenth volume of the reconstitution solution and place it at 4°C. Spray the gold-labeled solution evenly on the glass fiber and dry it at 37°C for 4 - 6 h.

[0060] 2. Preparation of nitrocellulose membrane

[0061] Set the parameters of the membrane spraying instrument, and evenly spray the optimal membrane scribing concentration obtained in step 4 of Example 1, that is, 0.5 mg / mL of the monoclonal antibody against Orientia tsutsugamushi 56 kDa protein and 0.5 mg / mL of the goat anti-rabbit polyclonal antibody on the NC membrane, respectively, as the test line and the quality control line, and the distance between adjacent two lines is 4 mm.

[0062] 3. Assembly of the test strip

[0063] As Figure 1 shown, along the chromatographic direction during sample detection on the bottom plate, sequentially paste the sample pad 1, the gold-labeled pad 4, the nitrocellulose membrane 3, and the absorbent pad 8 on the bottom plate 2. Paste the absorbent pad 8 at one end of the PVC bottom plate close to the quality control line 6, press it on the NC membrane 3, with an overlap of 2 mm. Paste the gold-labeled pad 4 at one end of the NC membrane close to the test line 5, and the gold-labeled pad 4 is pasted under the NC membrane with an overlap of 2 mm. Paste the sample pad 1 under the gold-labeled pad 4 with an overlap of 2 mm, and also press it on the NC membrane 3; finally, use a tablet press to compact the pasted PVC bottom plate, cut it into test strips 4 mm wide with a strip cutting machine, place them in a sealed bag containing a desiccant, and store them at 4°C.

[0064] 4. The detection method is as follows:

[0065] Drop 30 μL of the sample onto the sample addition hole of the sample pad of the test strip, let it stand at room temperature for 10 min, observe the experimental results. As Figure 2 shown, if there are bands on both the T line and the C line, it indicates that the sample is positive and the detection result is valid. As Figure 2-a, it is determined as positive; if there is no band in the T line and there is a band in the C line, it indicates that the sample is negative and the test result is valid, as Figure 2 -b; if there is no band in the C line and there is or is not a band in the T line, the test is invalid, as Figure 2 -c and 2-d.

[0066] Example 3: Performance Evaluation of the Sandwich ELISA for Detecting Orientia tsutsugamushi Antigen

[0067] In this example, the optimal parameters in Example 1 were selected, and the test strip and detection method were based on those in Example 2.

[0068] 1. Determination of the sensitivity of the test strip

[0069] The mixed recombinant antigen of Orientia tsutsugamushi was serially diluted 2-fold with sterile PBS, and sterile PBS was used as a blank control. 30 μL of each dilution was dropped onto the sample pad. After reacting for 10 min, the sensitivity of the method was judged by observing the results of the test strip. As Figure 5 shown: when the recombinant protein was 1.18 μg / mL, there was no band in the T line of the test strip. Therefore, the lowest detection limit of the test strip was 2.35 μg / mL. Since the sample loading volume was 30 μL, the actual lowest detection amount was 70.5 ng.

[0070] The infected cells were cultured in a T75 cell culture flask until confluent, digested with Trypsin-EDTA, rinsed with cell culture medium, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of PBS. The cell suspension was diluted and counted using a hemocytometer, and the counting was repeated 3 times to calculate the number of infected cells. The counted cells were serially diluted 10-fold with PBS for 8 gradients (10 -1 -10 -8 ) to extract the DNA of Orientia tsutsugamushi. The extracted DNA was subjected to qPCR, and the relationship between the cell number and the copy number of Orientia tsutsugamushi was constructed based on the standard curve, as Figure 6 shown: among them, the amplification curve was S-shaped, the Ct value increased with the decrease of the template concentration, the gradient was obvious, the data within the group had good parallelism, the qPCR melting curve showed that the qPCR amplification product was correct, the data was reliable, and it belonged to specific amplification. A standard curve was established with the copy number of the standard as the X-axis and the cycle number (Ct) value as the Y-axis. The qPCR standard curve showed that its linear correlation coefficient was 0.99743, and the linear correlation between the copy number and the Ct value was relatively high.

[0071] The cells infected with Orientia tsutsugamushi were diluted and counted, and the DNA of the diluted cells was extracted for qPCR. Based on the established qPCR standard curve, the relationship between the cell number and the copy number was further determined.

[0072] Collect the cell line infected with Orientia tsutsugamushi, concentrate the Orientia tsutsugamushi cell precipitate into a 15 mL centrifuge tube, add 10 mL of PBS to it, centrifuge at 1500 rpm for 5 min, discard the supernatant, then add 1 mL of PBS, mix the cells and transfer them to a 2 mL centrifuge tube. Take 20 μL of the cell suspension into a 1.5 mL centrifuge tube for later cell counting. Add glass beads with a specification of 2.5 mm to the centrifuge tube to make the glass bead column equal in height to the upper liquid column. Place it on an oscillator and oscillate at a gentle rate for 30 s to break the cells, then place it in an ice bath on ice for 30 s and repeat 15 times to achieve the purpose of breaking the cells and releasing Orientia tsutsugamushi. After breaking the cells, use a pipette to aspirate the suspension in the centrifuge tube into a 2 mL centrifuge tube, centrifuge at 2000 rpm for 10 min to centrifuge down the cell debris, take the supernatant, use PBS as a blank control, add 30 μL dropwise onto the sample pad, react for 10 min, and observe the result of the test strip. As Figure 7 shown: When the test strip detects the Orientia tsutsugamushi antigen, when the copy number of Orientia tsutsugamushi corresponding to the cell number is 1×10 5 copies / μL, there is no band in the T line of the test strip. When the copy number of Orientia tsutsugamushi is 1×10 6 copies / μL, there is a band in the T line of the test strip. Therefore, the lowest detection limit for detecting the antigen is 1×10 6 copies / μL.

[0073] 2. Specificity determination of the test strip

[0074] Use Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes as control strains, and the Orientia tsutsugamushi antigen as a positive control. Use a sterile THB culture medium solution as a blank control, add 30 μL dropwise onto the sample pad respectively, and after reacting for 10 min, observe the result of the test strip. As Figure 8 shown: The results of repeated experiments many times all show that the detection results of the 4 pathogenic bacteria are all negative, indicating that the method has good specificity.

[0075] 3. Stability determination of the test strip

[0076] After thoroughly drying the prepared colloidal gold immunochromatographic test strip, put it into an aluminum foil bag with a desiccant and store it sealed at 4℃. Take the prepared test strip out of the 4℃ refrigerator, balance it at room temperature for 30 min, detect the test strips at 7 days, 1 month, 2 months, and 3 months respectively. Drop the sample onto the sample pad, and the results show that they are consistent with the previous experimental results, indicating that the test strip has good stability and can be stored for 3 months at 4℃.

[0077] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

Claims

1. A test strip for detecting Orientia tsutsugamushi antigen, characterized in that: The invention comprises a nitrocellulose membrane (3), wherein one end of the nitrocellulose membrane (3) is provided with a sample pad (1) and a gold label pad (4), and the other end is provided with a water absorbent pad (8), wherein the gold label pad (4) is placed on the nitrocellulose membrane (3), and the sample pad (1), the water absorbent pad (8) and the nitrocellulose membrane (3) are all placed on a bottom plate (2), wherein the gold label pad (4) contains a colloidal gold-labeled anti-Orientia tsutsugamushi 56 kDa rabbit polyclonal antibody, and the nitrocellulose membrane (3) is provided with a detection line (5) and a control line (6), wherein the detection line (5) contains a monoclonal antibody against the 56 kDa protein of Orientia tsutsugamushi, and the quality control line (6) contains a goat anti-rabbit polyclonal antibody.

2. The test strip according to claim 1, characterized in that: The monoclonal antibody against the 56kDa protein of Orientia tsutsugamushi is secreted and produced by a hybridoma cell line which is deposited in the China Center for Type Culture Collection and has a strain collection number of CCTCC NO: C2022244.

3. The test strip according to claim 1 or 2, characterized in that: The preparation method of the gold label pad comprises adjusting the pH of a colloidal gold solution, stirring, adding an anti-Orientia 56kDa rabbit polyclonal antibody to the colloidal gold solution, stirring, adding a polyethylene glycol solution, stirring, adding a bovine serum albumin solution, and stirring; The obtained solution was stored at low temperature and centrifuged at low temperature to obtain a gold-labeled precipitate; Dissolve the gold label precipitate, spray the gold label solution evenly on the glass fiber, and dry it.

4. The test strip according to claim 3, characterized in that: The pH is 8-9, preferably 8.

5.

5. The test strip according to claim 3, characterized in that: The anti-Orientia 56 kDa rabbit polyclonal antibody is added to the colloidal gold solution, so that the concentration of the anti-Orientia 56 kDa rabbit polyclonal antibody is 18-22 μg / mL, preferably 20 μg / mL.

6. The test strip according to claim 3, characterized in that: The added amounts of the polyethylene glycol solution and the bovine serum albumin solution are independently selected from 0.5-1.5 mL; the mass percentage of polyethylene glycol in the polyethylene glycol solution is 0.5%-1.5%; and the mass percentage of the bovine serum albumin solution is 8%-12%.

7. The test strip according to claim 1, characterized in that: The stripping concentration of the monoclonal antibody against the 56 kDa protein of Orientia tsutsugamushi in the test line (5) is 0.4-0.6 mg / mL, preferably 0.5 mg / mL.

8. The test strip according to claim 1, characterized in that: The stripping concentration of the goat anti-rabbit polyclonal antibody in the quality control line (6) is 0.4-0.6 mg / mL, preferably 0.5 mg / mL.

9. A test paper card or a test paper box, characterized in that: The invention comprises the test strip according to any one of claims 1 to 8.

10. Use of the test strip according to any one of claims 1 to 8, the test card according to claim 9, or the test box according to claim 9 in detecting antibodies against Orientia tsutsugamushi.

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