CN116840472A - Double-colloidal gold test strip for detecting haemonchus contortus and preparation method thereof - Google Patents

Abstract

The invention provides a double colloidal gold test strip for detecting Haemonchus contortus and a preparation method thereof. The double colloidal gold test strip includes: a gold label pad and a nitrocellulose membrane, the gold label pad is marked with recombinant Hc-NIM1 protein labeled by colloidal gold solution and recombinant Hc-ES15 protein labeled by the colloidal gold solution, so The nitrocellulose membrane is coated with C quality control line coated with anti-Hc‑ES15/Hc‑NIM1 protein antibody. The invention provides a double colloidal gold test strip for detecting Haemonchus contortus and a preparation method thereof. The double colloidal gold test strip can be suitable for rapid, simple and cheap diagnosis of Haemonchus contortus infection, which It helps the frontline grassroots to quickly and accurately grasp the epidemic development of Haemonchus contortus in Hu sheep, and has good application prospects in the prevention and control of Haemonchus contortus in ruminants.

Description

A double colloidal gold test strip for detecting Haemonchus contortus and a preparation method thereof

Technical Field

The invention relates to the technical field of parasite detection, and more specifically to a double colloidal gold test strip for detecting Haemonchus contortus and a preparation method thereof.

Background Art

Haemonchus twister feeds on blood in sheep and can cause death of sick calves and lambs, bringing serious impacts to animal husbandry. Haemonchus twister has strong reproductive capacity and strong adaptability to the environment. It can form diapause in the host to resist external threats and survive in the host for a long time, causing severe hemorrhagic gastritis, hypoproteinemia, anemia and subcutaneous edema, leading to weight loss, decreased productivity, and reduced wool production and quality in infected sheep. Early detection and early treatment can significantly reduce the harm of Haemonchus twister to sheep, so early diagnosis of Haemonchus twister disease is particularly important.

If necessary treatment measures are not taken after Hu sheep are infected with Haemonchus twister, it is likely to lead to death. In recent years, research teams have used serological and nucleic acid molecular techniques to establish identification methods such as ELISA (Enzyme Linked Immunosorbent Assay) or PCR (Polymerase Chain Reaction), which greatly improved the accuracy of diagnosis of Haemonchus twister disease. However, these methods require professional operators and the use of expensive special instruments for operation. The operation process is cumbersome, the identification time is long, and the cost is high. Therefore, it is only suitable for small-scale laboratory diagnosis, but not for batch use at the grassroots level in clinical frontline, which makes the above two tests cannot be well promoted and applied. It can be seen that it is urgent to develop fast, simple and cheap diagnostic technology products suitable for Haemonchus twister infection.

Summary of the invention

In order to solve the above technical problems, the present invention provides a double colloidal gold test strip for detecting Haemonchus contortus and a preparation method thereof, which can be applied to the rapid, simple and inexpensive diagnosis of Haemonchus contortus infection.

On the one hand, an embodiment of the present invention provides a double colloidal gold test strip for detecting Haemonchus contortus, the double colloidal gold test strip comprising: a gold label pad and a nitrocellulose membrane, the gold label pad being labeled with recombinant Hc-NIM1 protein labeled with colloidal gold solution and recombinant Hc-ES15 protein labeled with the colloidal gold solution, the nitrocellulose membrane being coated with a C quality control line coated with anti-Hc-ES15/Hc-NIM1 protein antibodies.

Specifically, the double colloidal gold test strip further comprises a sample pad, and the sample pad comprises: Na ₂ HPO ₄ ·12H ₂ O, KH ₂ PO ₄ , Tween 20 and deionized water.

On the other hand, an embodiment of the present invention provides a method for preparing the above-mentioned double colloidal gold test strip, the preparation method comprising:

preparing the recombinant Hc-NIM1 protein and the recombinant Hc-ES15 protein;

Using colloidal gold solution to label the recombinant Hc-NIM1 protein, and spraying it on the gold label pad;

Using the colloidal gold solution to label the recombinant Hc-ES15 protein, and spraying it on the gold label pad;

The recombinant Hc-NIM1 protein is coated on the T1 detection line and sprayed on the nitrocellulose membrane, and the recombinant Hc-ES15 protein is coated on the T2 detection line and sprayed on the nitrocellulose membrane;

The mouse anti-recombinant Hc-ES15 protein polyclonal antibody is coated on the C quality control line and sprayed on the nitrocellulose membrane;

preparing a sample pad;

The nitrocellulose membrane, the gold label pad, the sample pad and the water absorbent pad are sequentially pasted onto a PVC plate to obtain the double colloidal gold test strip.

Specifically, the pH value of the labeled recombinant Hc-NIM1 protein is 6.5.

Specifically, the pH value of the labeled recombinant Hc-ES15 protein is 8.5.

Specifically, the labeled amount of the labeled recombinant Hc-NIM1 protein is 10 μg/mL.

Specifically, the labeled amount of the labeled recombinant Hc-ES15 protein is 6 μg/mL.

Specifically, the concentration of the labeled recombinant Hc-NIM1 protein is 2 mg/mL.

Specifically, the labeled amount of the labeled recombinant Hc-ES15 protein is 2 mg/mL.

Specifically, the concentration of the mouse anti-recombinant Hc-ES15 protein polyclonal antibody is 2.5 mg/mL.

The present invention provides a double colloidal gold test strip for detecting Haemonchus twister and a preparation method thereof, and the double colloidal gold test strip can be applied to the rapid, simple and inexpensive diagnosis of Haemonchus twister infection. The double colloidal gold test strip provided by the embodiment of the present invention has good sensitivity by using two detection lines respectively coated with recombinant Hc-NIM1 protein and recombinant Hc-ES15 protein. At the same time, the protein with high expression level of Haemonchus twister in L4 stage and adult stage is screened as a diagnostic antigen, and an accurate diagnosis can be made from 7 days to 120 days after infection, which is suitable for detection at all stages of the infection period. The double colloidal gold test strip provided by the embodiment of the present invention is simple to operate and has fast detection, which helps the front-line grassroots to quickly and accurately grasp the prevalence and development of Haemonchus twister disease in Hu sheep, and has a good application prospect in the prevention and control of Haemonchus twister disease in ruminants.

Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the description, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are used to provide a further understanding of the technical solution of the present invention and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present invention and do not constitute a limitation on the technical solution of the present invention.

FIG1 is a schematic diagram of the structure of a double colloidal gold test strip provided in Example 1 of the present invention, in which 1 is a sample pad, 2 is a gold label pad, 3 is a nitrocellulose membrane, 4 is a water-absorbing pad, and 5 is a PVC board.

FIG. 2 is a target band diagram of 474 bp in size provided by Example 2 of the present invention.

FIG. 3 is a target band diagram of 390 bp in size provided by Example 2 of the present invention.

FIG. 4 is a comparison diagram of the Hc-NIM1 gene sequence provided in Example 2 of the present invention.

FIG. 5 is a comparison diagram of the Hc-ES15 gene sequence provided in Example 2 of the present invention.

FIG. 6 a is a diagram showing the enzyme digestion identification of the expression vector of the target protein with a size of 36 kD provided in Example 2 of the present invention.

FIG. 6 b is a diagram showing the enzyme digestion identification of the expression vector of the target protein of 34 kD provided in Example 2 of the present invention.

FIG. 7 a shows the target band detected by the L4 sheep serum provided in Example 2 of the present invention.

FIG. 7 b shows the target band detected by the adult sheep serum provided in Example 2 of the present invention.

FIG. 7c is the detection result of the negative control group provided in Example 2 of the present invention.

FIG8a is the optimal labeling pH value of the recombinant Hc-NIM1 protein provided in Example 2 of the present invention.

FIG8b is the optimal labeling pH value of the recombinant Hc-ES15 protein provided in Example 2 of the present invention.

FIG. 9a is the optimal labeling amount of the recombinant Hc-NIM1 protein provided in Example 2 of the present invention.

FIG. 9b is the optimal labeling amount of the recombinant Hc-ES15 protein provided in Example 2 of the present invention.

FIG. 10 is an optimal concentration of polyclonal antibody coated on the C quality control line provided in Example 2 of the present invention.

FIG. 11 a shows that the concentration of the protein coated on the T1 detection line provided in Example 2 of the present invention is 1 mg/mL and the concentration of the protein coated on the T2 detection line is 1 mg/mL.

FIG. 11 b shows that the concentration of the protein coated on the T1 detection line provided in Example 2 of the present invention is 1 mg/mL, and the concentration of the protein coated on the T2 detection line is 2 mg/mL.

FIG. 11 c shows that the concentration of the protein coated on the T1 detection line provided in Example 2 of the present invention is 2 mg/mL and the concentration of the protein coated on the T2 detection line is 2 mg/mL.

FIG. 12a is a schematic diagram of negative result determination provided in Embodiment 2 of the present invention.

FIG12b is a schematic diagram of positive result determination provided in Embodiment 2 of the present invention.

FIG. 12c is a schematic diagram of determining the failure of a colloidal gold test strip provided in the second embodiment of the present invention.

FIG. 13 is a cross-reaction verification diagram provided in Example 2 of the present invention.

Figure 14 is a graph of the specific detection results of the colloidal gold test strip provided in Example 2 of the present invention, in which from left to right are the positive serum for sheep Haemonchus contortus, the positive serum for sheep Haemonchus contortus, the positive serum for sheep Fasciola hepatica, the positive serum for sheep coccidia, the sheep Echinococcus granulosus, the bovine Fasciola and the negative serum for sheep Haemonchus contortus.

Figure 15 is a diagram of the test results of serum diluted in proportion provided in Example 2 of the present invention, in which from left to right are serum dilution ratios of 1:1, 1:5, 1:10, 1:20, 1:40 and 1:80 and a negative control.

FIG. 16 a is a diagram showing the detection results of the first repeated test provided in Example 2 of the present invention.

FIG. 16 b is a diagram showing the detection results of the second repeated test provided in Example 2 of the present invention.

FIG. 16c is a diagram showing the detection results of the third repeated test provided in Example 2 of the present invention.

FIG. 17 a is a diagram showing the sensitivity test results after 30 days of storage provided in the second embodiment of the present invention.

FIG. 17 b is a diagram showing the sensitivity test results after 60 days of storage provided in the second embodiment of the present invention.

FIG. 17c is a diagram showing the sensitivity test results after 90 days of storage provided in the second embodiment of the present invention.

Figure 18a is a diagram of the serum test results on days 5, 7, 9, 11, 13 and 15 provided in Example 2 of the present invention.

Figure 18b is a graph of serum test results on days 20, 25, 30, 40, 60, 90, 120 and 125 provided in Example 2 of the present invention.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

The materials used in this embodiment are as follows:

Haemonchus contortus strain: isolated, identified and preserved by the laboratory of Zhejiang University.

L4 stage positive serum: serum of Hu sheep infected with L3 stage on the 10th day; adult positive serum: serum of Hu sheep infected with L3 stage on the 30th day; negative serum: serum of newborn calves that have not drunk breast milk; sheep hepatic fluke positive serum was donated by Yangzhou University, sheep coccidia positive serum was donated by Inner Mongolia Agricultural University, and cattle Fasciola positive serum and sheep Echinococcus granulosus positive serum were stored in this laboratory. The positive serum of Haemonchus contortus used in this study was from the same Hu sheep.

Experimental animals: 4 Hu sheep, aged 5 to 6 months, purchased from Mie Mie Sheep Farm, Huzhou City, Zhejiang Province, and kept in the animal room of Zhejiang University after purchase.

Plasmids and strains: Cloning vector pMD19-T Vector (pMD19-T vector), pET-32a Vector, Escherichia coli TOP 10, and Escherichia coli BL 21 competent strains were purchased from TaKaRa.

Enzymes and reagents: 5×HiscriptⅡq-RT Supermix reverse transcription kit (R222-01) (purchased from Nanjing Novozymes Co., Ltd.), AceQ Universal SYBR qPCR Master Mix kit (Q511-02) (purchased from Nanjing Novozymes Co., Ltd.), plasmid extraction kit (DR0201050), gel cleaning recovery kit (DR0101250) (purchased from Zhejiang Easyside Biotechnology Co., Ltd.), LA Taq enzyme, 100bp DNA marker, 250bp DNA marker, (purchased from TaKaRa Company), His-Tag protein purification agar filler, protein purification column outer column, Millipore ultrafiltration tube, 5×SDS, trehalose, bovine serum albumin BSA (purchased from Shanghai Shenggong Bioengineering Co., Ltd.), Brand Ford protein concentration determination kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.), Coomassie brilliant blue dye, HRP-labeled rabbit anti-mouse IgG, HRP-labeled rabbit anti-sheep IgG (purchased from Hangzhou Ford Biotechnology Co., Ltd.), anti-His tag mouse IgG (purchased from Sigma), color development solution (purchased from Beijing Solebow Company), protein G agarose purification resin (purchased from Shanghai Yisheng Biological Company), sodium acetate, ammonium sulfate, sodium chloride, potassium carbonate, sucrose (purchased from Sinopharm Chemical Reagent Co., Ltd.), nitrocellulose membrane (CN140), glass cellulose membrane, absorbent pad, PVC plate, and card shell (purchased from Shanghai Jie Ning Biotechnology Co., Ltd.).

Instruments: PCR instrument (Beijing Kayoudi Biotechnology Co., Ltd.), intelligent biochemical incubator (Ningbo Jiangnan Instrument Factory), shaking incubator (Shanghai Zhichu Instrument Co., Ltd.), gel imager (Shanghai Peiqing Technology Co., Ltd.), nucleic acid electrophoresis instrument (Beijing Liuyi Technology Co., Ltd.), protein electrophoresis instrument (Bio-RAD, USA), ultrasonic lysator (Ningbo Xinzhi Technology Co., Ltd.), microplate reader (Qingdao Shengjie Instrument System Co., Ltd.), gold dot membrane instrument (Bio Dot, USA).

Embodiment 1

The present embodiment provides a double colloidal gold test strip for detecting Haemonchus contortus, as shown in Figure 1, the double colloidal gold test strip includes: a gold label pad and a nitrocellulose membrane, the gold label pad includes colloidal gold labeled with recombinant Hc-NIM1 protein and recombinant Hc-ES15 protein, the nitrocellulose membrane includes: a T1 detection line coated with recombinant Hc-NIM1 protein, a T2 detection line coated with recombinant Hc-ES15 protein, and a C quality control line coated with anti-Hc-ES15/Hc-NIM1 protein antibodies.

Specifically, the double colloidal gold test strip further comprises: a sample pad, and the sample pad comprises: Na ₂ HPO ₄ ·12H ₂ O, KH ₂ PO ₄ , Tween-20 and deionized water.

Embodiment 2

This embodiment provides a method for preparing a double colloidal gold test strip, the method comprising:

Recombinant Hc-NIM1 protein and recombinant Hc-ES15 protein were obtained.

According to the sequences of NIM1 (accession number: AM040235) and ES15 (accession number: MT408359) genes published by GenBank, two pairs of specific primers were designed, and BamH I restriction enzyme site and Hind III restriction enzyme site were added to the upstream and downstream of the primers, respectively. The primer sequence design is shown in Table 1.

Table 1 shows the primer sequences

The total RNA of Haemonchus contortus was extracted according to the following steps: appropriate amount of eggs, L1 stage, L2 stage, L3 stage, L4 stage and adult stage samples of Haemonchus contortus were added to 6 centrifuge tubes of 2 mL, 1 mL of Trizol was added to each centrifuge tube and mixed by blowing; 5 steel balls with a diameter of 3 mm were added to each centrifuge tube, and the 6 centrifuge tubes were placed on a grinder respectively, the frequency of the grinder was set to 60 Hz, and the grinding time was 2 min; after grinding, each centrifuge tube was placed on ice for 5 min; 200 μL of chloroform was added to each centrifuge tube, and the mixture was allowed to stand at room temperature for 10 min after being fully shaken; centrifuged at 4°C and 10000 g for 15 min to obtain a supernatant, which was transferred to a new centrifuge tube, 500 μL of isopropanol was added to the new centrifuge tube, and the mixture was mixed by inverting and then allowed to stand at room temperature for 10 min; a 75% ethanol solution was prepared by the following method: 250 μL of DEPC (Diethyl pyrocarbonate, diethyl pyrocarbonate) water was mixed with 750 μL anhydrous ethanol; centrifuged at 10000g for 10 min at 4°C, the supernatant was discarded to obtain a first precipitate, 1 mL of 75% ethanol was added to the precipitate for resuspending; centrifuged at 10000g for 5 min at 4°C, the supernatant was discarded to obtain a second precipitate, and the centrifuge tube was inverted on a clean filter paper until dry; 10 μL of 1% DEPC water was added to the centrifuge tube to dissolve the second precipitate to obtain total RNA and stored at -80°C.

The total RNA extracted above was reverse transcribed according to the instructions of the reverse transcription kit. The reverse transcription system is shown in Table 2.

After the reverse transcription system is mixed, it is placed in a 50°C water bath for 15 minutes, then in a 85°C water bath for 5 minutes, and finally placed on ice for 5 minutes. The cDNA is obtained and stored at -80°C.

Using cDNA as a template, PCR amplification was performed using the synthesized primers NIM1-F and NIM1-R or ES15-F and ES15-R and LATaq enzyme, respectively, wherein primer NIM1-F and primer NIM1-R were used together, and primer ES15-F and primer ES15-R were used together. The PCR amplification system is shown in Table 3.

Table 3 shows the PCR amplification system

The PCR amplification program included: pre-denaturation at 95°C for 3 min; each cycle included: denaturation at 94°C for 15 s, annealing at 52°C for 30 s, extension at 72°C for 28 s, for a total of 34 cycles; extension at 72°C for 3 min; and holding at 16°C for 10 min.

The PCR amplification product was analyzed by 1% agarose gel electrophoresis, and target bands of 474 bp and 390 bp were observed, as shown in Figures 2 and 3. The PCR amplification product was recovered according to the instructions of the gel cleaning kit to obtain the PCR amplification product.

The PCR amplification product (target gene) was connected to the pMD19-T vector in a 16°C metal bath for 12 hours to obtain a connection product. The connection system is shown in Table 4.

Table 4 shows the connection system

The ligation product was transformed into Escherichia coli BL 21 competent cells and applied to ampicillin plates. After culturing at 37°C for 12 to 16 hours, a single colony was picked and placed in LB (Luria-Bertani) liquid medium containing ampicillin to obtain a bacterial solution, which was placed in a 37°C shaker for overnight culture. The plasmid in the bacterial solution was extracted according to the instructions of the plasmid extraction kit, and the positive plasmid was sequenced and compared with the reference sequence using DNAMAN software. The results are shown in Figures 4 and 5. As shown in Figure 4, the similarity between the Hc-NIM1 gene and the gene AM040235 published in the GenBank gene library is 99.79%, and as shown in Figure 5, the similarity between the Hc-ES15 gene and the gene MT408359 published in the GenBank gene library is 99.74%. The positive plasmids are pMD19-T-NIM1 and pMD19-T-ES15.

The above positive plasmids pMD19-T-NIM1 and pMD19-T-ES15 were respectively digested with the cloning vector pET-32a in a 37°C water bath for 1.5 h to obtain digestion products. The digestion system is shown in Table 5.

Table 5 shows the double restriction enzyme digestion system of recombinant cloning vector and expression vector

The digestion products were subjected to electrophoresis using 1% agarose gel, and the products were recovered using a gel cleaning recovery kit to obtain target fragment digestion recovery products and pET-32a digestion recovery products, respectively. The specific operations were performed according to the instructions of the gel cleaning recovery kit.

The target fragment digestion recovery product and the pET-32a digestion recovery product were connected in a 16°C connector for 12 hours to obtain a connection product, wherein the connection system is shown in Table 6.

Table 6 is the target fragment and expression vector fragment ligation reaction system

The ligation product was transferred into BL21 competent cells, cultured overnight after plating, and then the plasmid was extracted by picking and shaking the bacteria, and finally double enzyme digestion identification was performed. The enzyme digestion system provided in Table 5 can still be used for identification.

Take 5 mL of bacterial solution prepared from the positive plasmid and transfer it into 300 mL of LB liquid medium. After culturing at 37°C for 3-4 hours, when the OD value of the bacterial solution reaches 0.6, add 1 MIPTG (Isopropylβ-D-Thiogalactoside) inducer at a volume ratio of 1:1000, and induce expression at 37°C for 8 hours. Collect the bacterial cells, resuspend the bacterial cells with 10 mL of imidazole PBS buffer, and then centrifuge them in an ice bath for ultrasonication to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter and then slowly passed through a His Tag affinity chromatography column at a flow rate of 1 mL/min. The column was washed with 10 mM, 20 mM, 40 mM, 60 mM and 250 mM imidazole eluents, respectively, and the eluents were collected. The SDS-PAGE analysis of the eluent showed that the eluted protein concentration was the highest when the imidazole concentration was 250 mM, as shown in Figures 6a and 6b. The sizes of the target proteins were 36 kD and 34 kD, respectively.

4 μL recombinant Hc-NIM1 protein, 4 μL recombinant Hc-ES15 protein and 16 μL 5×SDS (sodium dodecyl sulfate) were mixed, boiled in boiling water for 10 min, and placed on ice to cool to complete sample preparation. The sheep serum of the adult stage of Haemonchus twister and the sheep serum of the L4 stage of Haemonchus twister were used as primary antibodies, and the HRP-labeled rabbit anti-sheep IgG was used as the secondary antibody. At the same time, the positive serum of sheep at different infection periods was used as the control group, and Western blot (protein blotting) was performed for identification. The results are shown in Figures 7a to 7c. As shown in Figures 7a to 7c, the target bands can be detected in the sheep serum of the adult stage and the sheep serum of the L4 stage of Haemonchus twister. This proves that the sheep serum of the adult stage and the L4 stage larvae contain antibodies that recognize Hc-NIM1 and Hc-ES15.

Preparation of mouse polyclonal antibodies against recombinant Hc-NIM1 and Hc-ES15 proteins

Recombinant Hc-NIM1 protein and recombinant Hc-ES15 protein were mixed with Freund's complete adjuvant at a volume ratio of 1:1 and emulsified, and injected subcutaneously at points in mice, with a dose of 100 μg protein/mouse; two weeks later, the second immunization was performed, and Freund's incomplete adjuvant was used as the emulsifier, and the immunization dose was 50 μg protein/mouse; one week later, the third immunization was performed, and the dose was the same as the second immunization; one week later, blood was collected from the tail, serum was separated, and ELISA (enzyme-linked immunosorbent assay) titer was determined, and the highest titer of both groups of mice could reach 1:8192000. The collected serum was stored at -80℃.

Measure the volume of serum, add acetate buffer (0.06M, pH=4.6), and the volume ratio of serum to acetate buffer is 1:3; under magnetic stirring, add octanoic acid (500mL serum/17mL octanoic acid) dropwise, and stir for 30 minutes; collect the supernatant after centrifugation and filter with filter paper or double-layer gauze; put the filtered supernatant into a dialysis bag and dialyze in 0.01M PB (pH=7.4) solution, change the water 3 times, and keep at 4°C overnight; take out the dialysis bag, measure the volume of serum+PBS, add an equal volume of ammonium sulfate (adjust the pH value to 7.0-7.2 with a saturated ammonium sulfate solution) dropwise, discard the supernatant after centrifugation, and obtain a precipitate, resuspend the precipitate with 0.01MPB, dialyze in 0.01M PB, and centrifuge after collecting the supernatant.

Dilute the supernatant with an appropriate amount of Binding Buffer, add it to a chromatography column filled with agarose purification resin containing Protein G, and equilibrate it at 4°C for 12 hours; adjust the valve to allow the liquid to flow out at a rate of 1 mL/min, wash the column with 30 mL of Wash Buffer, elute the target protein with 15 mL of Elution Buffer, and collect the eluate. The protein concentration can be measured using a BCA protein concentration kit (Shanghai Tongwei Biotechnology Co., Ltd.). In this example, the concentration of both polyclonal antibodies is 2 mg/mL. The purified polyclonal antibodies are stored in aliquots at -80°C.

Preparation of immunochromatographic colloidal gold test strips

Add 500 mL of ultrapure water to a clean conical flask, place it on a heatable magnetic stirrer, and boil it; weigh 0.1125 g of sodium citrate, dissolve it in 3 mL of boiling water to obtain a sodium citrate solution; add 5 mL of 1% chloroauric acid to the conical flask; quickly add the sodium citrate solution to the conical flask, react for 5 to 10 minutes, and obtain a colloidal gold solution; the colloidal gold solution can be stored at 4°C for later use.

Prepare 8 centrifuge tubes, add 1mL colloidal gold solution to each tube, then add 0μL, 1μL, 2μL, 3μL, 4μL, 5μL, 6μL and 7μL 0.2MK ₂ CO ₃ respectively, mix well; add 30μg recombinant Hc-NIM1 protein to each tube, mix well; add 100μL 10% NaCl solution to each tube, mix well, and observe the color change of the colloidal gold solution, as shown in Figure 8a. By observing the color change of the colloidal gold solution with the naked eye, it can be seen that when 2μL K ₂ CO ₃ is added, the color of the rHc-NIM1 group solution no longer changes. In actual labeling, take the last tube of the color stable tube, that is, the fourth tube, and use pH test paper to measure the pH of the solution in this tube to be 6.5, which shows that the optimal pH value for labeling recombinant Hc-NIM1 protein is 6.5.

Prepare 8 centrifuge tubes, add 1mL colloidal gold solution to each tube, then add 0μL, 1μL, 2μL, 3μL, 4μL, 5μL, 6μL and 7μL 0.2MK ₂ CO ₃ respectively, mix well; add 30μg recombinant Hc-ES15 protein to each tube, mix well; add 100μL 10% NaCl solution to each tube, mix well, and observe the color change of the colloidal gold solution, as shown in Figure 8b. By observing the color change of the colloidal gold solution with the naked eye, it can be seen that when 5μL K ₂ CO ₃ is added, the color of the rHc-ES15 group solution tends to be stable and no longer changes. When actually marking, take the last tube of the color-stable tube, that is, the 7th tube, and use pH test paper to measure the pH of the solution in this tube to be 8.5. It can be seen that the optimal pH value for marking the recombinant Hc-ES15 protein is 8.5.

Prepare 8 centrifuge tubes, add 1mL colloidal gold solution to each tube, adjust the colloidal gold solution to the optimal pH with 0.2MK ₂ CO ₃ ; add 0μg, 2μg, 4μg, 6μg, 8μg, 10μg, 12μg and 14μg recombinant Hc-NIM1 protein, mix well, and let stand for 30min; add 100μL of 10% NaCl solution to each tube, mix well, and let stand for 30min; observe the color change of the colloidal gold solution. The result is shown in Figure 9a. When 8μg/mL recombinant Hc-NIM1 protein is added, the colloidal gold solution no longer changes color, indicating that the minimum labeling amount of recombinant Hc-NIM1 protein is 8μg/mL. Generally, in practical applications, 10% to 20% more protein is added, so the optimal labeling amount of recombinant Hc-NIM1 protein is 10μg/mL.

Prepare 8 centrifuge tubes, add 1mL colloidal gold solution to each tube, adjust the colloidal gold solution to the optimal pH with 0.2MK ₂ CO ₃ ; add 0μg, 2μg, 4μg, 6μg, 8μg, 10μg, 12μg and 14μg recombinant Hc-ES15 protein, mix well, and let stand for 30min; add 100μL of 10% NaCl solution to each tube, mix well, and let stand for 30min; observe the color change of the colloidal gold solution. The results are shown in Figure 9b. When 4μg/mL recombinant Hc-ES15 protein is added, the colloidal gold solution no longer changes color, indicating that the minimum labeling amount of recombinant Hc-ES15 protein is 4μg/mL. Generally, in practical applications, 10% to 20% more protein is added, so the optimal labeling amount of recombinant Hc-ES15 protein is 6μg/mL.

Preparation of gold label pad

Take a centrifuge tube with a specification of 15 mL, add 10 mL of colloidal gold solution, add an appropriate amount of 0.2M K ₂ CO ₃ to adjust the pH value to 6.5, add purified recombinant Hc-NIM1 protein to make its final concentration 10 μg/mL, mix well and let it stand for 30 minutes, add 1 mL of 10% BSA solution, mix well, and let it stand for 1 hour; centrifuge at 13000 rpm for 30 minutes at 4°C, discard the supernatant to obtain a precipitate, resuspend the precipitate with 500 μL of gold label diluent to obtain a gold label antigen solution, spray the gold label antigen solution on the gold label pad at a dose of 6.0 μL/cm using a gold spray instrument, and place it at 37°C to dry.

Take a centrifuge tube with a specification of 15mL, add 10mL of colloidal gold solution, add appropriate amount of 0.2MK ₂ CO ₃ to adjust the pH value to 8.5, add purified recombinant Hc-ES15 protein to its final concentration of 6μg/mL, mix well and let stand for 30min, add 1mL of 10% BSA solution, mix well, let stand for 1h; centrifuge at 13000rpm for 30min at 4℃, discard the supernatant to obtain a precipitate, resuspend the precipitate with 500μL of gold label diluent to obtain a gold label antigen solution, spray the gold label antigen solution on the gold label pad at a dose of 6.0μL/cm with a gold spray instrument, and place it at 37℃ to dry.

Prepare the sample pad treatment solution according to the following formula: Na ₂ HPO ₄ ·12H ₂ O 2.87 g, KH ₂ PO ₄ 0.73 g, Tween-20 0.5 mL, deionized water 0.4 L, dilute to 0.5 L after fully dissolving, and adjust the pH value to 7.4. Soak the sample pad in the sample pad treatment solution for 2 h and place it at 37°C to dry.

Optimization of the coating concentration of the C quality control line: The purified mouse anti-Hc-ES15 protein polyclonal antibody was diluted to 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL and 3.0 mg/mL, respectively, sprayed onto the nitrocellulose (NC) membrane, placed at 37°C for drying, and negative serum was added to observe the color depth of the C quality control line. The results are shown in Figure 10, from which it can be seen that the optimal concentration of the C quality control line coated polyclonal antibody is 2.5 mg/mL.

Optimization of the concentration of the T1 test line coated with recombinant Hc-NIM1 protein and the T2 test line coated with recombinant Hc-ES15 protein: The recombinant Hc-NIM1 protein and the recombinant Hc-ES15 protein were diluted to 1.0 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL and 5.0 mg/mL, respectively, sprayed onto the NC membrane, dried at 37°C, and positive serum was added to observe the color depth of the T1 test line and the T2 test line. The results are shown in Figures 11a to 11c. The optimal concentrations of the T1 test line coating protein and the T2 test line coating protein are both 2.0 mg/mL.

Assembly of colloidal gold test strips: NC film, gold label pad, sample pad and absorbent pad were sequentially pasted onto the PVC board to obtain a colloidal gold test strip with a structure as shown in FIG1 . The strips were cut into strips with a width of 4 mm using a strip cutter and stored in an aluminum foil bag for later use.

Use and determination of colloidal gold test strips: Take a small amount of sheep positive serum and negative serum, dilute them 5 times by volume with pH 7.4, 0.1MPB solution to obtain diluted serum, and take 20μL of diluted serum as a sample for testing. Drop the diluted serum on the sample pad and let it act at room temperature for 5 to 10 minutes.

As shown in Figure 12a, negative result judgment: no red bands appear on the T1 test line and the T2 test line, and a red band appears on the C quality control line, which indicates that there is no Haemonchus contortus antibody in the sample.

As shown in FIG12 b , a positive result is judged as follows: the C quality control line and any one of the test lines (T1 test line or T2 test line) present red stripes, which indicates that antibodies to Haemonchus contortus are present in the sample.

As shown in FIG12c , if no red strip appears on the T1 test line, the T2 test line, and the C quality control line, or only the test line appears a red strip, it indicates that the colloidal gold test strip is invalid.

Testing of the performance of colloidal gold test strips

Verification of cross-reaction between ES15 and NIM1: The purified mouse anti-Hc-NIM1 protein polyclonal antibody was coated on the C quality control line at a concentration of 2.5 mg/mL, and the recombinant Hc-NIM1 protein was coated on the T1 detection line at a concentration of 2.0 mg/mL, and assembled with the gold-labeled pad sprayed with gold-labeled Hc-NIM1; the purified mouse anti-Hc-ES15 protein polyclonal antibody was coated on the C quality control line at a concentration of 2.5 mg/mL, and the recombinant Hc-ES15 protein was coated on the T2 detection line at a concentration of 2.0 mg/mL, and assembled with the gold-labeled pad sprayed with gold-labeled Hc-ES15. The positive serum of Haemonchus twistingensis was dripped on the sample pad respectively to detect whether ES15 and NIM1 had cross-reaction with each other's polyclonal antibodies. After inspection, as shown in Figure 13, ES15 and NIM had no cross-reaction with each other's polyclonal antibodies. In Figure 13, the first on the right is a negative control.

Specific detection: After the positive serum of sheep Haemonchus contortus, sheep Echinococcus granulosus, sheep coccidia, sheep Fasciola hepatica and cattle Fasciola calyx was diluted 5 times, 20 μL was taken and tested with the prepared colloidal gold test strip, and the result was determined within 10 minutes. The result is shown in Figure 14. As can be seen from Figure 14, only the T line of the colloidal gold test strip with the positive serum of sheep Haemonchus contortus added will show color.

Sensitivity test: The positive serum of Haemonchus contortus was diluted at a ratio of 1:1, 1:5, 1:10, 1:20, 1:40 and 1:80, and 20 μL of the diluted serum was tested with colloidal gold test strips, and the result was determined within 10 minutes. The results are shown in FIG15 , from which it can be seen that the minimum detection titer of the positive serum is 1:40.

Repeatability test: Three batches of colloidal gold test strips were prepared repeatedly under the same conditions, and the batch repeatability of the colloidal gold test strips was tested with positive serum and negative serum, respectively. The results are shown in Figures 16a to 16c. It can be seen from Figures 16a to 16c that the batch repeatability of the colloidal gold test strips is good.

Stability test: The colloidal gold test strips were stored at 4°C for 30 days, 60 days and 90 days, respectively, and then tested with the colloidal gold test strips. The results are shown in Figures 17a to 17c. It can be seen from Figures 17a to 17c that under stable conditions, the colloidal gold test strips can be stored at 4°C for 90 days and still be effective.

Sensitivity of determining the number of days of infection: The serum of Hu sheep infected with Haemonchus contortus was collected on the 5th, 7th, 9th, 11th, 13th, 15th, 20th, 25th, 30th, 40th, 60th, 90th, 120th and 125th days respectively, and was diluted 5 times before being dripped into the sample application area of the colloidal gold test strip to observe the color change of the T line. The results are shown in Figures 18a and 18b. As can be seen from Figures 18a and 18b, the prepared colloidal gold test strip can detect Haemonchus contortus infection as early as the 7th day, and antibodies can still be detected on the 120th day after infection.

Clinical application of diagnostic methods

Serum samples and corresponding fecal samples were collected from 114 Hu sheep, and the serum samples were tested by colloidal gold test strips, and the fecal samples were tested for fecal ova, and the two diagnostic methods were compared. The results are shown in Table 7. As can be seen from Table 7, 20 positive samples were detected by fecal ova, with a positive rate of 17.54% (20/114); 27 positive samples were detected by colloidal gold test strips, with a positive rate of 23.68% (27/114), and the fecal ova detection method had a compliance rate of 93.86% (107/114).

Table 7 is a comparison of colloidal gold detection technology and fecal examination results

It can be seen from Table 7 that, compared with fecal ova detection, the colloidal gold test strip provided in this embodiment has a higher positive rate and can be better used in clinical applications.

The present invention provides a double colloidal gold test strip for detecting Haemonchus twister and a preparation method thereof, and the double colloidal gold test strip can be applied to the rapid, simple and inexpensive diagnosis of Haemonchus twister infection. The double colloidal gold test strip provided by the embodiment of the present invention has good sensitivity by using two detection lines respectively coated with recombinant Hc-NIM1 protein and recombinant Hc-ES15 protein. At the same time, the protein with high expression level of Haemonchus twister in L4 stage and adult stage is screened as a diagnostic antigen, and an accurate diagnosis can be made from 7 days to 120 days after infection, which is suitable for detection at all stages of the infection period. The double colloidal gold test strip provided by the embodiment of the present invention is simple to operate and has fast detection, which helps the front-line grassroots to quickly and accurately grasp the prevalence and development of Haemonchus twister disease in Hu sheep, and has a good application prospect in the prevention and control of Haemonchus twister disease in ruminants.

Although the embodiments disclosed in the present invention are as above, the contents described are only embodiments adopted to facilitate understanding of the present invention and are not intended to limit the present invention. Any technician in the field to which the present invention belongs can make any modifications and changes in the form and details of implementation without departing from the spirit and scope disclosed in the present invention, but the patent protection scope of the present invention shall still be subject to the scope defined in the attached claims.

Claims (10)

1. A double-colloidal gold test strip for detecting haemonchus contortus, characterized in that the double-colloidal gold test strip comprises: the gold-labeled pad is marked with a recombinant Hc-NIM1 protein marked by a colloidal gold solution and a recombinant Hc-ES15 protein marked by the colloidal gold solution, and the nitrocellulose membrane is coated with a C quality control line coated by an anti-Hc-ES 15/Hc-NIM1 protein antibody.

2. The dual colloidal gold test strip of claim 1, further comprising a sample pad comprising: na (Na) ₂ HPO ₄ ·12H ₂ O、KH ₂ PO ₄ Tween 20 and deionized water.

3. A method for preparing the double colloidal gold test strip according to claim 1 or 2, wherein the method comprises the steps of:

preparing the recombinant Hc-NIM1 protein and the recombinant Hc-ES15 protein;

marking the recombinant Hc-NIM1 protein by using a colloidal gold solution, and spraying the recombinant Hc-NIM1 protein on the gold mark pad;

marking the recombinant Hc-ES15 protein by using the colloidal gold solution, and spraying the recombinant Hc-ES15 protein on the gold mark pad;

spraying the recombinant Hc-NIM1 protein coating the T1 detection line on the nitrocellulose membrane, and spraying the recombinant Hc-ES15 protein coating the T2 detection line on the nitrocellulose membrane;

coating the C quality control line with a polyclonal antibody of a mouse anti-recombinant Hc-ES15 protein, and spraying the C quality control line on the nitrocellulose membrane;

preparing a sample pad;

and sequentially adhering the nitrocellulose membrane, the gold label pad, the sample pad and the water absorption pad to a PVC plate respectively to obtain the double-colloidal gold test strip.

4. The method of claim 3, wherein the labeled recombinant Hc-NIM1 protein has a pH of 6.5.

5. The method of claim 3, wherein the labeled recombinant Hc-ES15 protein has a pH of 8.5.

6. The method according to claim 3, wherein the labeled recombinant Hc-NIM1 protein has a labeling amount of 10. Mu.g/mL.

7. The method according to claim 3, wherein the labeled recombinant Hc-ES15 protein is labeled in an amount of 6. Mu.g/mL.

8. The method of claim 3, wherein the concentration of the labeled recombinant Hc-NIM1 protein is 2mg/mL.

9. The method according to claim 3, wherein the labeled recombinant Hc-ES15 protein is labeled in an amount of 2mg/mL.

10. The method according to claim 3, wherein the concentration of the murine anti-recombinant Hc-ES15 protein polyclonal antibody is 2.5mg/mL.