WO2022110733A1

WO2022110733A1 - Fluorescent immunochromatographic test strip capable of detecting early infection of swine trichinella spiralis and preparation method therefor

Info

Publication number: WO2022110733A1
Application number: PCT/CN2021/097205
Authority: WO
Inventors: 刘明远; 刘晓雷; 杨勇; 王鑫宇; 王学林; 白雪; 唐斌; 丁静
Original assignee: 吉林大学
Priority date: 2020-11-30
Filing date: 2021-05-31
Publication date: 2022-06-02
Also published as: CN112526125A

Abstract

A fluorescent immunochromatographic test strip capable of detecting the early infection of swine trichinella spiralis and a preparation method therefor, which belong to the technical field of serological detection. In order to detect the infection of trichinella spiralis more quickly, conveniently and sensitively, the test strip of the present invention comprises a sample pad, a binding pad, a chromatography film, a water-absorbing pad and a bottom plate. The binding pad is spray-coated with a fluorescent probe, wherein the fluorescent probe comprises a time-resolved fluorescent microsphere coupled goat anti-rabbit IgG as an indicator probe and a time-resolved fluorescent microsphere coupled trichinella spiralis ES mixed antigen as a capture probe, and the trichinella spiralis ES mixed antigen is composed of a trichinella spiralis muscle larvae excretion secretion ML-ESP antigen and a trichinella spiralis intestinal tract muscle larvae excretion secretion iML-ESP antigen. The chromatography film is provided with a detection line and a quality control line, wherein the detection line is spray-coated with a mouse anti-swine IgG, and the quality control line is spray-coated with a rabbit anti-goat IgG. The present invention can be used for on-site rapid detection of trichinella spiralis.

Description

A fluorescent immunochromatographic test strip for detecting early infection of porcine Trichinella spiralis and preparation method thereof

technical field

The invention belongs to the technical field of serological detection, and in particular relates to a fluorescent immunochromatographic test strip capable of detecting early infection of Trichinella swine and a preparation method thereof.

Background technique

Trichinosis caused by Trichinella spiralis is an important zoonotic parasitic disease, which can infect more than 150 kinds of carnivores and omnivores. Humans mainly eat raw or semi-raw meat and meat products containing Trichinella muscle larvae. The infection, while only human infection shows obvious clinical symptoms. According to the International Commission on Trichinella, between 1986 and 2009, a total of 65,818 Trichinella infections occurred worldwide. At the same time, according to relevant literature reports, during the period from 1964 to 2011, a total of 38,794 cases of Trichinella infection occurred in my country, of which 336 patients died unfortunately. Therefore, my country has become one of the main countries affected by trichinosis. In view of the above-mentioned serious situation, my country spends 2.2 billion yuan every year to prevent and control trichinosis. Since ancient times in my country, there has been a custom of eating pork, and pigs, as the intermediate host of trichinella, have played a certain role in the spread of trichinosis.

At present, the detection methods of Trichinella are divided into direct detection method and indirect detection method. Direct detection, artificial digestion, is also the gold standard detection method for trichinosis. The indirect detection method is based on ELISA method and colloidal gold immunochromatography method based on muscle larval excretion secretion product (ML-ES) antigen. However, the above method has the disadvantages of time-consuming and labor-intensive, expensive detection instruments and low sensitivity. Therefore, in order to ensure food safety and stabilize the economic growth of animal husbandry, it is urgent to develop an on-site rapid detection method for trichinosis.

Fluorescent microsphere immunochromatographic test strip is a rapid and sensitive detection method emerging in recent years. The advantages of long Stokes shift and long fluorescence quenching time of fluorescent microspheres have greatly improved its sensitivity compared with colloidal gold immunochromatography, and this technology has been successfully applied in environmental hygiene, food safety, chemical Drug residue detection work. As the patent with the application number of 201910604044.8 in the prior art, a fluorescent immunochromatographic detection test strip for trichinosis is provided, and the test strip includes a sample pad, a binding pad, a chromatography membrane, a water-absorbing pad and a bottom plate. The binding pad is marked with time-resolved fluorescent microspheres coupled with goat anti-pig IgG; the chromatographic membrane is provided with a detection line and a quality control line, wherein the detection line is sprayed with ML-ESP antigen and ML-ESP antigen excreted by Trichinella muscle larvae. Trichinella spiralis muscle larvae ES mixed antigen composed of iMP-ESP antigen from muscle larvae excreta secretion in intestinal stage of Trichinella spiralis; rabbit anti-goat IgG is sprayed on the quality control line. This test strip is compatible with the ES ELISA method specified by OIE. Compared with Trichinella, it can accurately detect Trichinella antibody positive at an earlier stage after Trichinella infection, shortening the "window" period of detection, but the earliest time point when the test strip can detect Trichinella antibody positive is before the development of Trichinella. It is still some time before the infective larvae are capable of infecting, so the technology needs to be further improved.

SUMMARY OF THE INVENTION

In order to solve the problem of on-site detection of trichinosis and to improve the sensitivity and specificity of the detection of Trichinella test strips, the present invention provides a fluorescent immunochromatographic test strip that can detect early infection of Trichinella swine and a preparation method thereof. The test strip includes a sample pad, a binding pad, a chromatographic film, a water-absorbing pad and a bottom plate, the sample pad is provided with a sample hole; the binding pad is sprayed with fluorescent probes, and the fluorescent probes are included as indicator probes The time-resolved fluorescent microspheres coupled with goat anti-rabbit IgG and the time-resolved fluorescent microspheres coupled with Trichinella sp. ES mixed antigen as capture probes; the chromatographic membrane is provided with a detection line and a quality control line, wherein the detection line Mouse anti-pig IgG was sprayed on the top, and rabbit anti-goat IgG was sprayed on the quality control line.

To be further defined, the Trichinella ES mixed antigen consists of the ML-ESP antigen of the excretory secretion of Trichinella muscle larvae and the iML-ESP antigen of the excretory secretion of the muscle larvae of the intestinal stage of Trichinella spiralis.

Further limited, the time-resolved fluorescent microspheres are Eu nanoparticles; the material of the sample pad is glass cellulose membrane XQ-Y8; the material of the binding pad is glass cellulose membrane SB-06; the material of the chromatography membrane Millipore135 for nitrocellulose membrane.

The present invention also provides a preparation method of a fluorescent immunochromatographic test strip capable of early detection of Trichinella swine infection, the method comprising the following steps:

1) Preparation of Trichinella ES mixed antigens: The ML-ESP antigen of the excretory secretion of Trichinella spiralis muscle larvae and the excretory secretion iML-ESP antigen of the muscle larvae of Trichinella spiralis in the intestinal stage are mixed according to the mass ratio of 1:1-3:1, Obtain Trichinella ES mixed antigen;

2) Preparation of the binding pad: the time-resolved fluorescent microspheres coupled with the Trichinella sp. ES mixed antigen fluorescent probe and the time-resolved fluorescent microspheres coupled with the goat anti-rabbit IgG fluorescent probe were sprayed in a mass ratio of 10:1-5:1 On the binding pad, vacuum dry and store it for later use;

3) Preparation of detection line and quality control line: The mouse anti-pig IgG antibody and the rabbit anti-goat IgG antibody were sprayed on the chromatographic membrane in a mass ratio of 2:1 to form a detection line and a quality control line, and vacuum dried for 2 -3h, sealed and preserved for later use;

4) Assembly of test strips: On the bottom plate, the sample pad, the binding pad, the chromatography pad, and the water-absorbing pad are pasted on the bottom plate in sequence along the chromatographic direction during sample detection to form a test strip.

To further define, the preparation method of the ML-ESP antigen of the excretory secretion of the Trichinella muscle larvae described in step 1) is as follows: the Trichinella muscle larvae are washed with a culture solution containing penicillin and streptomycin and then naturally precipitated for 20-30min, and then It was put into a culture medium containing a final concentration of 250U/mL penicillin and a final concentration of 250U/mL streptomycin, the density was 3000-5000/mL, 37°C, 10% carbon dioxide, and the supernatant was collected after culturing for 18-24h. The collected supernatant was dialyzed and concentrated with a 3KDa ultrafiltration tube in turn to obtain the ML-ESP antigen in the excretory secretion of Trichinella muscle larvae.

Further limit, step 1) the preparation method of described Trichinella intestinalis muscle larvae excretory secretion iML-ESP antigen is as follows: Trichinella muscle larvae are orally infected with rats, the infection dose is 8000/only, and put to death after 6h , take out the rat small intestine, cut it into pieces, place it on the screen of the petri dish containing the culture medium, incubate at 37°C for 2-3 hours, collect the worms at the bottom of the petri dish, wash with the culture medium containing penicillin and streptomycin, and then precipitate naturally 20-30min, then put it into the culture medium containing the final concentration of 250U/mL penicillin and the final concentration of 250U/mL streptomycin, the density is 3000-5000 strips/mL, 37 ℃, 10% carbon dioxide, culture 18- After 24 hours, the supernatant was collected, and the collected supernatant was dialyzed and concentrated with a 3KDa ultrafiltration tube in turn, which was the iML-ESP antigen that was excreted by the muscle larvae of the intestinal stage of Trichinella spiralis.

It is further defined that the preparation method of the time-resolved fluorescent microspheres coupled with Trichinella sp. ES mixed antigen fluorescent probes in step 2) is as follows: 1% (w/v) time-resolved fluorescent microspheres per 14000g are centrifuged and discarded for storage. Glycerol and phosphate in the liquid; add 100 μL of 2-morpholineethanesulfonic acid MES solution with a concentration of 0.025 mol/L, 1.5 μL of N-hydroxysuccinimide NHS with a concentration of 10 mg/mL, and 10 mg/mL of NHS with a concentration of 10 mg/mL. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide EDC 1 μL and 10 μL time-resolved fluorescent microspheres with a concentration of 10 mg/ml were mixed well, stirred and incubated for 45 min; after the reaction was over, centrifuged and discarded The supernatant was washed with phosphate buffer with a concentration of 0.05mol/L, and the precipitate was resuspended by adding 200 μL of phosphate buffer; then, 5 μL of goat anti-rabbit IgG antibody with a concentration of 1 mg/mL was added, and incubated at room temperature for 2-3 h; After the reaction is over, discard the supernatant after centrifugation, then add 200 μL of BSA solution with a mass fraction of 5%, block at room temperature for 2 hours, discard the supernatant after centrifugation, and add 200 μL of the storage solution to the pellet to resuspend; the coupling storage solution is 0.05 mol/L phosphate buffer solution, each liter of coupling stock solution contains 0.5mL Proclin300, BSA 10g and polyethylene glycol 400 20g.

To further define, the preparation method of the time-resolved fluorescent microspheres-coupled goat anti-rabbit IgG fluorescent probe in step 2) is as follows: each 1% (w/v) time-resolved fluorescent microspheres are centrifuged at 14,000 g/min and discarded. Glycerol and phosphate in the preservation solution; add 100 μL of 2-morpholineethanesulfonic acid MES solution with a concentration of 0.025 mol/L at pH=6.1, and 1.5 μL of N-hydroxysuccinimide NHS with a concentration of 10 mg/mL. 10mg/mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC 1μL and 10mg/ml time-resolved fluorescent microspheres 10μL were mixed evenly, stirred and incubated for 45min; , after centrifugation, discard the supernatant, rinse the precipitate with 0.05 mol/L phosphate buffer at pH=7, add 200 μL of phosphate buffer to resuspend; then, add 5 μL of 1 mg/mL goat anti-rabbit IgG antibody, Incubate for 2-3 hours at room temperature; after the reaction is over, discard the supernatant after centrifugation, then add 200 μL of BSA solution with a mass fraction of 5%, block at room temperature for 2 hours, discard the supernatant after centrifugation, and add 200 μL of storage solution to the pellet to resuspend; The coupling storage solution is 0.05mol/L phosphate buffer solution, and each liter of the coupling storage solution contains 0.5 mL of Proclin300, 10 g of BSA and 20 g of polyethylene glycol 400.

It is further defined that the spotting speed of the sprayed fluorescent probe in step 2) is 10 μL/cm.

It is further defined that in step 3), the spotting speed of mouse anti-pig IgG sprayed on the detection line and the spotting speed of rabbit anti-goat IgG sprayed on the quality control line are both 0.8 μL/cm.

The schematic diagram of the detection principle of the immunochromatographic detection test strip for trichinosis is shown in B in Figure 1. When the test strip detects positive serum, the fluorescent microsphere-ES probe on the binding pad will interact with the Trichinella antibody in the serum. Binding, and further under the action of chromatography, the probe conjugate will bind to the mouse anti-pig IgG antibody on the detection line; and the fluorescent microsphere-goat anti-rabbit IgG probe will react with the rabbit anti-goat IgG antibody on the quality control line. Fluorescent signals can be seen in the final detection line and quality control line. When the test strip detects negative serum, only the fluorescent microsphere-goat anti-rabbit IgG probe reacts with the rabbit anti-goat antibody on the quality control line, and only the quality control line can see the fluorescent signal.

beneficial effect

The fluorescent immunochromatographic test strip which can detect the early infection of Trichinella suis prepared by the present invention is mainly composed of five basic structural units: a sample pad, a binding pad, a chromatography pad, a water absorption pad and a bottom plate. The ends are sequentially superimposed on the support base plate. The sample pad is a treated glass cellulose membrane XQ-Y8, which is used to quickly absorb the sample solution to be tested and make it flow laterally to the binding pad by siphoning; the binding pad is a glass cellulose membrane SB-06, which adsorbs labeled Biologically active materials: time-resolved fluorescent microspheres coupled with Trichinella sp. ES mixed antigen fluorescent probes and time-resolved fluorescent microspheres coupled with goat anti-rabbit IgG fluorescent probes, which can be combined with the detection target in the sample solution to be detected to form visible to the naked eye. When the test strip detects positive serum, the fluorescent microsphere-conjugated Trichinella ES mixed antigen fluorescent probe acts as a capture probe and binds to the Trichinella positive antibody in pig serum, while the pig non-antigen in the serum The specific antibody will not be bound to the capture probe, thus completing the screening of positive antibodies in the serum at the beginning, reducing the background of the test strip detection, which in turn helps to improve the sensitivity and specificity of the test strip detection, Therefore, compared with the test strip provided in the patent with the application number of 201910604044.8, it is possible to accurately detect Trichinella antibody positive at an earlier stage when pigs are infected with Trichinella; the chromatographic membrane is a nitrocellulose membrane Millipore135, on which two kinds of Antibodies to form "detection line" (T line) and "control line" (C line) blots to intercept labeled immune complexes; absorbent pads are absorbent cardboard for absorbing test samples that flow through the chromatographic membrane The sample solution is used to maintain the pressure difference between the two ends of the chromatographic membrane, so that more sample solution to be tested flows in a certain direction under the action of capillary, which can speed up the flow rate of the entire immunochromatography system and prevent the random flow of the sample. In addition to sample pads, binding pads, chromatography membranes, absorbent pads and bottom plates, the test strips of the present invention can also add some auxiliary structures, such as outer plastic films or plastic shells, etc., to be assembled into different types of immunochromatographic test paper products .

The test strip of the present invention has the characteristics of simple operation, time-saving and labor-saving, easy interpretation of results, etc., and has no restrictions on the professional skills of users. The high detection specificity and sensitivity can be widely used in on-site rapid detection of Trichinella spiralis, and has market development value and prospects.

Description of drawings

Figure 1.A is a schematic diagram of the structure of the fluorescent immunochromatographic detection test strip: wherein, 1 is a sample pad; 2 is a binding pad; 3 is a detection line; 4 is a quality control line; 5 is a nitrocellulose membrane; 6 is an absorbent pad; B is the schematic diagram of the detection principle of the fluorescent immunochromatographic detection test strip;

Figure 2. Schematic diagram for determination of test results of fluorescent immunochromatographic test strips: 1 is the sample addition hole; 2 is the detection line; 3 is the quality control line; a) is a schematic diagram of a negative result; b) is a schematic diagram of a positive result;

Fig. 3 Fluorescence immunochromatographic detection test strip sensitivity test results, wherein, A is the test result of the test strip in swine serum (0-120 days) infected with 100 Trichinella spiralis; B is the pig serum (0-120 days) infected with 1000 Trichinella spiralis 120 days) test strip test result; C is the test result of 10000 Trichinella swine serum (0-120 days) test strip test; 1 is the quality control line; 2 is the detection line;

Figure 4 Fluorescence immunochromatography detection test strip specific detection results, wherein 1 is the quality control line; 2 is the detection line.

Detailed ways

The reagents or instruments of the present invention can be purchased through commercial channels.

Embodiment 1, Trichinosis Fluorescence Immunochromatography Detection Test Strip

The fluorescent immunochromatographic test strip for early detection of Trichinella swine infection described in this embodiment includes a sample pad, a binding pad, a chromatography membrane, a water-absorbing pad and a bottom plate, and the sample pad is provided with a sample addition hole; the binding pad The fluorescent probes are sprayed with fluorescent probes, and the fluorescent probes include time-resolved fluorescent microspheres coupled with goat anti-rabbit IgG as indicator probes and time-resolved fluorescent microspheres coupled with Trichinella sp. ES mixed antigens as capture probes. The Trichinella ES mixed antigen is composed of ML-ESP antigen of Trichinella muscle larvae excretory secretion and iML-ESP antigen of Trichinella intestinalis muscle larvae excretory secretion; Among them, mouse anti-pig IgG was sprayed on the detection line, and rabbit anti-goat IgG was sprayed on the quality control line. The time-resolved fluorescent microspheres are Eu nanoparticles; the sample pad is made of glass cellulose membrane XQ-Y8; the binding pad is made of glass cellulose membrane SB-06; the chromatography membrane is made of nitrocellulose Membrane Millipore135, the schematic diagram of the structure of the test strip is shown in A in Figure 1.

Embodiment 2. The preparation method of the test strip for the detection of trichinosis by fluorescence immunochromatography.

1) Preparation of Trichinella ES mixed antigens: Mix the ML-ESP antigen of the excretory secretion of Trichinella spiralis muscle larvae and the iML-ESP antigen of the excretory secretion of Trichinella spiralis intestinal stage muscle larvae according to the mass ratio of 1:1 to obtain Trichinella spiralis ES Mixed antigens; specific methods are:

① Preparation of ML-ESP antigen from the excretory secretion of Trichinella muscle larvae:

The collected muscle larvae of Trichinella spiralis were washed three times with the culture medium containing penicillin (500U/mL) and streptomycin (500U/mL) (natural precipitation, 20min each time), and then put into penicillin-containing (250U/mL) and streptomycin (250U/mL) in the culture medium (density is 5000/mL), 37°C, 10% carbon dioxide, and collect the supernatant after culturing for 18-24h. After filtering the remaining muscle larvae and debris with a disposable syringe filter, the collected culture supernatant was dialyzed overnight with PBS and concentrated to 2-3 mg/mL with a 3KDa ultrafiltration tube to obtain Trichinella ML-ESP antigen and stored. Reserve at -70°C. During the culturing process, it is necessary to ensure that the medium is free from bacterial, fungal and other contamination, and the contaminated antigen cannot be used.

②Preparation of iML-ESP antigen from excretory secretion of muscle larvae in intestinal stage of Trichinella spiralis:

The collected Trichinella muscle larvae were orally infected with female Wistar rats, 6-8 weeks old, purchased from the Experimental Animal Center of Jilin University, the infective dose of the Wistar rats was 8000/rat, killed after 6 hours, and taken out immediately Rat small intestine, use a syringe to wash the small intestine with phosphate buffered saline (PBS), cut the small intestine into pieces of 1-2 cm vertically and horizontally, place them on the screen of a petri dish containing culture medium, incubate at 37 °C for 3 hours, and place the bottom of the petri dish. The worms were collected in a 15mL centrifuge tube, washed three times with the culture solution containing penicillin (500U/mL) and streptomycin (500U/mL) (natural precipitation, 20min each time), and then put them into penicillin (500U/mL) and streptomycin (500U/mL) 250U/mL) and streptomycin (250U/mL) in the culture medium (density is 5000/mL), at 37°C, under 10% carbon dioxide conditions, collect the supernatant after culturing for 18-24h. After filtering the residual muscle larvae and debris with a disposable syringe filter, the collected culture supernatant was dialyzed overnight with PBS and concentrated to 2-3 mg/mL with a 3KDa ultrafiltration tube to obtain Trichinella iML-ESP antigen and stored. Reserve at -70°C. During the culturing process, it is necessary to ensure that the medium is free from bacterial, fungal and other contamination, and the contaminated antigen cannot be used.

③ Preparation of Trichinella ES mixed antigen:

Trichinella ML-ESP antigen and Trichinella ML-ESP antigen were respectively diluted with 0.01mol/L PBS buffer to dilute the iML-ESP antigen of Trichinella muscle larvae excretory secretion to a concentration of 2mg/mL, and the two antigens were 1:1 The mass ratio is fully mixed (working concentration is 1mg/L).

2) Preparation of the binding pad: The time-resolved fluorescent microspheres coupled with the Trichinella sp. ES mixed antigen fluorescent probe and the time-resolved fluorescent microspheres coupled with the goat anti-rabbit IgG fluorescent probe were sprayed on the binding pad at a ratio of 10:1, and the mixture was vacuumized. After draining, dry it and save it for later use; the specific method is:

① Preparation of time-resolved fluorescent microspheres coupled to Trichinella sp. ES mixed antigen fluorescent probe:

Centrifuge 14000g 1% (w/v) time-resolved fluorescent microspheres and discard the glycerol and phosphate in the preservation solution; add 100 μL of 2-morpholineethanesulfonic acid MES solution with a pH of 6.1 and a concentration of 0.025mol/L , 1.5 μL of N-hydroxysuccinimide NHS at a concentration of 10 mg/mL, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC at a concentration of 10 mg/mL 1 μL and a concentration of 10 mg Mix 10 μL of time-resolved fluorescent microspheres per ml, and incubate with stirring for 1-2 h; when the reaction is over, discard the supernatant after centrifugation, rinse the precipitate with phosphate buffer with pH=7 and a concentration of 0.05 mol/L, and add 200 μL of phosphoric acid. Resuspend in salt buffer; then, add 5 μL of Trichinella ES mixed antigen with a concentration of 1 mg/mL, and incubate at room temperature for 2-3 h; after the reaction is over, discard the supernatant after centrifugation, and then add 200 μL of 5% BSA solution , closed at room temperature for 2 h, discarded the supernatant after centrifugation, and added 200 μL of storage solution to the pellet to resuspend; the coupling storage solution was 0.05 mol/L phosphate buffer solution, and each liter of the coupling storage solution contained 0.5 mL of Proclin300, BSA 10g and polyethylene glycol 400 20g.

②Preparation of time-resolved fluorescent microsphere-conjugated goat anti-rabbit IgG antibody fluorescent probe

The preparation method of the time-resolved fluorescent microsphere-conjugated goat anti-rabbit IgG antibody fluorescent probe is the same as that of the time-resolved fluorescent microsphere-conjugated Trichinella ES mixed antigen fluorescent probe.

③ Preparation of binding pads

The fluorescent microspheres containing the conjugated Trichinella sp. ES mixed antigen prepared above and the fluorescent microspheres containing the conjugated goat anti-rabbit IgG antibody were sprayed onto a 300 × 5 mm glass cellulose membrane SB-06 with a test paper sprayer. The sample speed was 10 μL/cm, vacuum-dried at 4°C, and dried at 4°C for later use.

3) Preparation of detection line and quality control line: The mouse anti-pig IgG antibody and the rabbit anti-goat IgG antibody were sprayed on the chromatographic membrane according to the mass ratio of 2:1 to form the detection line and the quality control line, and vacuumed at 37°C Dry for 2h, and store it at 4℃ for later use; the specific method is as follows:

The concentration of 1 μg/mL mouse anti-pig IgG antibody and 0.5 μg/mL rabbit anti-goat IgG antibody (diluted in 0.01mol/L PBS buffer) were used as the quality control reagents of the detection line box respectively. The two reagents were respectively spotted on the nitrocellulose membrane Millipore135 with a test paper sprayer at a speed of 0.8 μL/cm. Dry at 37°C for 2h. The dried nitrocellulose membrane Millipore135 was soaked in a blocking agent (phosphate buffered saline PBS containing 2% BSA) for 1-2 hours, then dried at 37°C for 1-2 hours, and sealed at 4°C for later use.

4) Assembly of test strips: Paste the sample pad, binding pad, chromatography pad and water-absorbing pad on the bottom plate in sequence along the chromatographic direction during sample detection on the bottom plate to make test strips. The specific method is as follows: as shown in the figure As shown in 1, glue the black bottom plate for fluorescence, sample pad XQ-Y8, binding pad SB-06, absorbent pad H5072 and nitrocellulose membrane Millipore135 together, and cut them into 3.5mm wide test pieces with a test strip cutter. Put the test strip into the test paper card, put the test strip into a sealed bag containing desiccant, and store it at 4°C after sealing.

5) Trichinosis Fluorescence immunochromatographic detection test strips are used to detect trichinosis.

Sample dilution: Dilute the samples of Trichinella antibody positive serum (such as swine infected Trichinella sera), negative serum (such as normal swine serum) and the serum to be tested (such as porcine serum) by sample diluent 100 times, that is, to 99 μL of diluent. Add 1 μL of porcine serum to the mixture and mix with a pipette. Place the test strip on the operating table with the sample hole facing up, add 100 μL of the diluted serum sample to the sample hole, let it stand for 10 minutes at room temperature, and irradiate the test strip with an ultraviolet lamp at 360-370nm to observe the results. The dilution consisted of 0.9% NaCl, 1.5% BSA and 0.05% Tween-20.

The result judgment should be carried out within 20min after adding the sample. The sample to be tested is judged to be positive if both the quality control line and the test line develop color; if only the quality control line develops color, but the test line does not develop color, it is judged to be negative; if the quality control line does not develop color, the test is invalid , the test strip should be replaced and re-tested. Figure 2 shows a schematic diagram of the determination of the detection results of fluorescent immunochromatographic test strips.

6) Investigate the sensitivity and specificity of the test strips for the detection of trichinosis by fluorescence immunochromatography.

①Sensitivity detection:

The swine serum of Trichinella muscle larvae with infection doses of 100, 1000 and 10000 respectively (serum collection time points: 0, 7, 9, 11, 13, 15, 17, 19, 21, 25, 30, 35, 45, 60, 90 and 120 days) using test strips and a commercial Qiagen ELISA, respectively. Infected dose of 10,000 Trichinella muscle larvae in pig serum, ELISA detected Trichinella antibody positive at 21-25 days, while test strips could detect Trichinella infection at 17-19 days. The infection dose was 1,000 Trichinella muscle larvae, in which ELISA detected Trichinella antibody positive at 25-30 days, while test strips could detect Trichinella infection at 21-25 days. The infection dose was 100 muscle larvae, of which ELISA detected Trichinella antibody positive in 30-45 days, and test strips could detect Trichinella antibody in 25-35 days. The results show that compared with Qiagen ELISA, the sensitivity of the test strip is significantly higher, and the positive antibody can be detected earlier, and it can be used for the screening of Trichinella spiralis infection serum.

②Specific detection:

Test strips were used to detect pigs infected with Clonorchis sinensis, cysticercosis, Cryptosporidium, subtropical tapeworm, Toxoplasma, etc. 10 sera (two sera for each species), and set up a positive control. After testing: the test strip results showed no positive bands, indicating that the test strip has good specificity, and the test results are shown in Figure 4.

③ Fluorescent microsphere immunochromatographic test strip for clinical sample detection

Test strips, digestion method and Qiagen ELISA were used to test 15,000 pigs in Yunnan and Inner Mongolia. The test results showed that 19 pigs were positive for antibodies by test strips and were diagnosed as trichinosis. Seventeen pigs were diagnosed with trichinosis by digestion and Qiagen ELISA. The test strip has an accuracy rate of over 99%.

④ Fluorescent microsphere immunochromatographic test strip repeatability and stability test

Three batches of test strips from different batches were used for detection, and the sensitivity and specificity of 30 negative sera and 30 positive sera from Trichinella infection were 100% after ELISA detection. Experiments show that the prepared fluorescent microsphere immunochromatographic test strip has good repeatability and stability.

⑤Comparison with the sensitivity of Trichinella immunofluorescence test strips labeled with time-resolved fluorescent microspheres conjugated with goat anti-pig IgG on the binding pad.

The swine serum of Trichinella muscle larvae with infection doses of 100, 1000 and 10000 were respectively used for immunofluorescence of Trichinella spiralis labeled with time-resolved fluorescent microspheres coupled with goat anti-pig IgG on the test strip of the present invention and the binding pad. Test strips (test strips described in the patent application with application publication number CN110221006A) are detected. The infection dose is the pig serum of 10,000 Trichinella muscle larvae, wherein the test strip of the present invention can detect Trichinella antibody positive in 17 days at the earliest, and the Trichinella immunofluorescence test strip that distinguishes fluorescent microspheres coupled with goat anti-pig IgG Trichinella infection can be detected as early as 21 days. The infection dose is 1000 Trichinella muscle larvae, wherein the test strip test strip of the present invention detects Trichinella infection in 21-25 days, and the Trichinella immunofluorescence test strip that distinguishes fluorescent microspheres coupled with goat anti-pig IgG Trichinella infection can be detected within 25-35 days. The infection dose is 100 muscle larvae, wherein the test strip test strip of the present invention detects Trichinella infection at 25-35 days, and the Trichinella immunofluorescence test strip with fluorescent microspheres coupled with goat anti-pig IgG is detected at 35 days. Trichinella infection can be detected within 40 days. The results show that the test strip of the present invention is more sensitive than the Trichinella immunofluorescence test strip with fluorescent microspheres coupled with goat anti-pig IgG, and can be used for the detection of Trichinella spiralis infected serum. Screening.

Claims

A test strip for detection of trichinosis by fluorescence immunochromatography, comprising a sample pad, a binding pad, a chromatographic film, a water-absorbing pad and a bottom plate, wherein the sample pad is provided with a sample addition hole; the binding pad is sprayed with Fluorescent probes, the fluorescent probes include time-resolved fluorescent microspheres coupled with goat anti-rabbit IgG as indicator probes and time-resolved fluorescent microspheres coupled with Trichinella sp. ES mixed antigens as capture probes; the chromatographic membrane There is a detection line and a quality control line on it, among which, the detection line is sprayed with mouse anti-pig IgG, and the quality control line is sprayed with rabbit anti-goat IgG.
The trichinellosis fluorescence immunochromatographic detection test strip according to claim 1, wherein the Trichinella ES mixed antigen is composed of the Trichinella muscle larvae excretory secretion ML-ESP antigen and the Trichinella intestinalis muscle larvae Excretory secretion iML-ESP antigen composition.
The trichinosis fluorescence immunochromatographic detection test strip according to claim 1, wherein the time-resolved fluorescent microspheres are Eu nanoparticles; the material of the sample pad is glass cellulose film XQ-Y8; The material of the binding pad is glass cellulose membrane SB-06; the material of the chromatography membrane is nitrocellulose membrane Millipore135.
The preparation method of the trichinosis fluorescence immunochromatography detection test strip according to claim 1, is characterized in that, comprises the steps:

1) Preparation of Trichinella ES mixed antigens: The ML-ESP antigen of the excretory secretion of Trichinella spiralis muscle larvae and the excretory secretion iML-ESP antigen of the muscle larvae of Trichinella spiralis in the intestinal stage are mixed according to the mass ratio of 1:1-3:1, Obtain Trichinella ES mixed antigen;

2) Preparation of the binding pad: the time-resolved fluorescent microspheres coupled with the Trichinella sp. ES mixed antigen fluorescent probe and the time-resolved fluorescent microspheres coupled with the goat anti-rabbit IgG fluorescent probe were sprayed in a mass ratio of 10:1-5:1 On the binding pad, vacuum dry and store it for later use;

3) Preparation of detection line and quality control line: The mouse anti-pig IgG antibody and the rabbit anti-goat IgG antibody were sprayed on the chromatographic membrane in a mass ratio of 2:1 to form a detection line and a quality control line, and vacuum dried for 2 -3h, sealed and preserved for later use;

4) Assembly of test strips: On the bottom plate, the sample pad, the binding pad, the chromatography pad, and the water-absorbing pad are pasted on the bottom plate in sequence along the chromatographic direction during sample detection to form a test strip.
The preparation method according to claim 4, wherein the preparation method of the ML-ESP antigen of the excretory secretion of the Trichinella muscle larvae described in step 1) is as follows: the Trichinella muscle larvae are treated with a culture solution containing penicillin and streptomycin After washing, it is naturally precipitated for 20-30min, and then placed in a culture medium containing a final concentration of 250U/mL penicillin and a final concentration of 250U/mL streptomycin, at a density of 3000-5000 strips/mL, 37°C, 10% carbon dioxide After culturing for 18-24h, collect the supernatant, and the collected supernatant is ML-ESP antigen in the excretory secretion of Trichinella muscle larvae after being concentrated by dialysis and 3KDa ultrafiltration tube in turn.
The preparation method according to claim 4, characterized in that, in step 1), the preparation method of the excretory secretion iML-ESP antigen of Trichinella intestinalis muscle larvae is as follows: The dose was 8,000 rods/rat. After 6 hours, the rats were killed. The small intestines of the rats were taken out, cut into pieces, placed on the screen of a petri dish containing culture medium, incubated at 37 °C for 2-3 hours, and the worms at the bottom of the petri dish were collected. After washing, the culture solution of streptomycin is naturally precipitated for 20-30min, and then put into the culture solution containing the final concentration of 250U/mL penicillin and the final concentration of 250U/mL streptomycin, the density is 3000-5000/mL, The supernatant was collected after culturing for 18-24 hours at 37°C in 10% carbon dioxide. The collected supernatant was dialyzed and concentrated with a 3KDa ultrafiltration tube in turn to obtain iML-ESP antigens excreted by muscle larvae in the intestinal stage of Trichinella spiralis.
The preparation method according to claim 4, wherein the preparation method of the time-resolved fluorescent microsphere-coupled Trichinella ES mixed antigen fluorescent probe in step 2) is as follows: 1% (w/v) time per 14000g Discard the glycerol and phosphate in the preservation solution after centrifuging the resolution fluorescent microspheres; add 100 μL of 2-morpholineethanesulfonic acid MES solution with a pH of 6.1 and a concentration of 0.025 mol/L, and N-hydroxyl with a concentration of 10 mg/mL. Succinimide NHS 1.5 μL, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC 1 μL at a concentration of 10 mg/mL and 10 μL of time-resolved fluorescent microspheres at a concentration of 10 mg/mL were mixed After the reaction is over, discard the supernatant after centrifugation, rinse the precipitate with 0.05 mol/L phosphate buffer at pH=7, and add 200 μL of phosphate buffer to resuspend; then, add 5 μL of concentration Mix antigens with 1 mg/mL Trichinella sp. ES, incubate at room temperature for 2-3 hours; after the reaction is over, discard the supernatant after centrifugation, then add 200 μL of 5% BSA solution, block at room temperature for 2 hours, and discard the supernatant after centrifugation. Add 200 μL of storage solution to the precipitation to resuspend; the coupling storage solution is 0.05mol/L phosphate buffer solution, and each liter of the coupling storage solution contains 0.5 mL of Proclin300, 10 g of BSA and 20 g of polyethylene glycol 400.
The preparation method according to claim 4, wherein the preparation method of the time-resolved fluorescent microsphere-coupled goat anti-rabbit IgG fluorescent probe in step 2) is as follows: 1% (w/v) time-resolved per 14,000 g After centrifuging the fluorescent microspheres, discard the glycerol and phosphate in the preservation solution; add 100 μL of 2-morpholineethanesulfonic acid MES solution with a concentration of 0.025 mol/L, and N-hydroxysuccinimide NHS with a concentration of 10 mg/mL. 1.5 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC at a concentration of 10 mg/mL and 1 μL of time-resolved fluorescent microspheres at a concentration of 10 mg/ml were mixed well, and incubated with stirring. 45min; after the reaction is over, discard the supernatant after centrifugation, rinse the precipitate with phosphate buffer with a concentration of 0.05mol/L, and add 200 μL of phosphate buffer to resuspend; then, add 5 μL of goat anti-rabbit IgG with a concentration of 1 mg/mL Antibody, incubate for 2-3h at room temperature; after the reaction is over, discard the supernatant after centrifugation, then add 200μL of 5% BSA solution, block at room temperature for 2h, discard the supernatant after centrifugation, and add 200μL of storage solution to the pellet to resuspend ; Described coupling storage solution is 0.05mol/L phosphate buffered solution, and every liter of coupling storage solution contains 0.5mL Proclin300, BSA 10g and polyethylene glycol 400 20g.
The preparation method according to claim 4, wherein the spotting speed of the sprayed fluorescent probe in step 2) is 10 μL/cm.
The preparation method according to claim 4, characterized in that, in step 3), the spotting speed of spraying mouse anti-pig IgG on the detection line and the spotting speed of spraying rabbit anti-goat IgG on the quality control line are both 0.8 μL/cm.