阅读说明：本技术 登革热病毒抗原、检测试剂、检测试纸、检测试剂盒及其使用方法 (Dengue virus antigen, detection reagent, detection test paper, detection kit and use method thereof ) 是由 朱天川 何建安 李微 龙军 叶颖 赵纯中 顾大勇 谭选平 于 2019-08-30 设计创作，主要内容包括：本发明涉及一种登革热病毒抗原、检测试剂、检测试纸、检测试剂盒及其使用方法。该登革热病毒抗原包括氨基酸序列如SEQ ID NO.1所示的多肽及氨基酸序列如SEQ ID NO.2所示的多肽中的至少一种。上述登革热病毒抗原能够与登革热病毒抗体特异性结合,灵敏度和特异性均较高,并且在检测登革热病毒时,样本使用量较少、检测周期短且成本低。(The invention relates to a dengue virus antigen, a detection reagent, detection test paper, a detection kit and a use method thereof. The dengue virus antigen comprises at least one of polypeptide with an amino acid sequence shown as SEQ ID NO.1 and polypeptide with an amino acid sequence shown as SEQ ID NO. 2. The dengue virus antigen can be specifically combined with a dengue virus antibody, the sensitivity and the specificity are high, and when detecting dengue virus, the sample usage amount is small, the detection period is short and the cost is low.)

1. A dengue virus antigen, characterized by comprising at least one of a polypeptide having an amino acid sequence as shown in SEQ ID No.1 and a polypeptide having an amino acid sequence as shown in SEQ ID No. 2.

2. Use of the dengue virus antigen of claim 1 in the preparation of a dengue virus detection reagent, in the preparation of a dengue virus test strip or in the preparation of a dengue virus detection kit.

3. A reagent for detecting dengue virus, comprising the dengue virus antigen of claim 1.

4. The dengue virus detection reagent according to claim 3, wherein the detection reagent is a magnetic bead reagent, the dengue virus antigen comprises a polypeptide with an amino acid sequence shown as SEQ ID No.1 and a polypeptide with an amino acid sequence shown as SEQ ID No.2, and the dengue virus antigen is coated on the magnetic bead.

5. A test strip for detecting dengue virus, which is characterized by comprising a substrate and dengue virus antigens arranged on the substrate, wherein the dengue virus antigens are the dengue virus antigens of claim 1.

6. The test strip for dengue virus according to claim 5, wherein the envelope is coated with an antigen having an amino acid sequence as shown in SEQ ID No.1 and an antigen having an amino acid sequence as shown in SEQ ID No. 2.

7. A detection kit for dengue viruses, comprising:

The dengue virus antigen of claim 1; or

The dengue virus detection reagent of any one of claims 3 to 4; or

a test strip for dengue virus of any one of claims 5 to 6.

8. The dengue virus detection kit of claim 7, further comprising at least one of a sample diluent, a coating solution, a blocking solution, a washing solution, and a developing solution.

9. The dengue virus detection kit of claim 7, wherein the sample diluent is selected from one of carbonate buffer, PBS buffer and TBS buffer.

10. the use method of the dengue virus detection kit is characterized by comprising the following steps:

Mixing a sample to be tested with a dengue virus antigen coated on a solid phase carrier, then incubating, adding a detection antibody, and continuing the incubation, wherein the dengue virus antigen is the dengue virus antigen of claim 1, and the detection antibody can be combined with the dengue virus antibody and is provided with a detection label; and

And detecting to obtain a detection result.

Technical Field

the invention relates to the field of molecular biology detection, in particular to a dengue virus antigen, a detection reagent, detection test paper, a detection kit and a use method thereof.

Background

Dengue Virus (DENV) is a mosquito-borne infectious Virus widely spread in the world, mainly spread by aedes aegypti and aedes albopictus, can cause Dengue fever, Dengue hemorrhagic fever and Dengue shock syndrome, and has a great threat to human health.

At present, the main method for clinically detecting DENV infection is a serum method, namely, DENV recombinant protein is used as a coating antigen to detect anti-dengue virus antibodies in serum, but the problems of large sample usage amount and long detection time exist in the DENV detection by the serum method. In addition, the preparation process of the DENV recombinant protein is complex, the detection cost is high, and the use of the DENV recombinant protein in regions with poor economic conditions is limited.

Disclosure of Invention

Accordingly, there is a need for a dengue virus antigen that can reduce the amount of sample used, shorten the detection time, and reduce the cost.

In addition, the detection reagent, the detection test paper, the detection kit and the use method for the dengue fever virus are provided, wherein the use amount of a sample is small, the detection time is short, and the cost is low.

A dengue virus antigen comprises at least one of a polypeptide with an amino acid sequence shown as SEQ ID NO.1 and a polypeptide with an amino acid sequence shown as SEQ ID NO. 2.

The dengue virus antigen comprises at least one of polypeptide with an amino acid sequence shown as SEQ ID NO.1 and polypeptide with an amino acid sequence shown as SEQ ID NO. 2. Through research, specific epitopes of DENV and its subtypes are mainly located on domain I and domain III of envelope proteins. The polypeptide with the amino acid sequence shown as SEQ ID NO.1 is designed aiming at the structural domain I of the DENV envelope protein, and the polypeptide with the amino acid sequence shown as SEQ ID NO.2 is designed aiming at the structural domain III of the DENV envelope protein.

proved by experiments, the dengue virus antigen can be specifically combined with a dengue virus antibody, the sensitivity and the specificity are both higher, and when detecting dengue virus, the sample usage amount is less, and the detection period is short. In addition, the dengue fever virus antigen can be artificially synthesized, the process is mature and simple, and the production cost is low.

The dengue virus antigen is applied to preparation of a dengue virus detection reagent, preparation of dengue virus detection test paper or preparation of a dengue virus detection kit.

A reagent for detecting dengue virus comprises the dengue virus antigen.

in one embodiment, the detection reagent is a magnetic bead reagent, the dengue virus antigen comprises a polypeptide with an amino acid sequence shown as SEQ ID No.1 and a polypeptide with an amino acid sequence shown as SEQ ID No.2, and the dengue virus antigen is coated on the magnetic bead.

A test strip for detecting dengue viruses comprises a substrate and dengue virus antigens arranged on the substrate, wherein the dengue virus antigens are the dengue virus antigens.

In one embodiment, the coating film is coated with an antigen with an amino acid sequence shown as SEQ ID NO.1 and an antigen with an amino acid sequence shown as SEQ ID NO. 2.

A detection kit for dengue virus comprising:

The above dengue virus antigen; or

a detection reagent for the dengue virus; or

The test paper for detecting the dengue fever virus.

In one embodiment, the kit further comprises at least one of a sample diluent, a coating solution, a sealing solution, a washing solution and a developing solution.

In one embodiment, the sample diluent is selected from one of carbonate buffer, PBS buffer, and TBS buffer.

a method for using a dengue virus detection kit comprises the following steps:

Mixing a sample to be tested with a dengue virus antigen coated on a solid phase carrier, then incubating, adding a detection antibody, and continuing the incubation, wherein the dengue virus antigen is the dengue virus antigen, and the detection antibody can be combined with the dengue virus antibody and is provided with a detection label; and

And detecting to obtain a detection result.

Drawings

FIG. 1 is a schematic diagram showing the positions of envelope proteins of dengue viruses against which E1 to E10 of example 1 are directed;

FIG. 2 is a graph showing the results of indirect CLEIA experiments from E1 to E10;

FIG. 3 is a ROC curve for detection of anti-DENV IgM as a coating antigen by E1 in example 3;

FIG. 4 is a scatter plot of the levels of anti-DENV IgM detected as coating antigen by E1 in example 3;

FIG. 5 is a ROC curve for detection of anti-DENV IgM as a coating antigen by E7 in example 3;

FIG. 6 is a scatter plot of the levels of anti-DENV IgM detected as coating antigen by E7 in example 3;

FIG. 7 is a ROC curve for detection of anti-DENV IgM as coating antigen by E1/E7 in example 3;

FIG. 8 is a scatter plot of the levels of anti-DENV IgM detected as coating antigen by E1/E7 in example 3.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

One embodiment of the present invention provides a dengue virus antigen including at least one of a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and a polypeptide having an amino acid sequence as shown in SEQ ID NO. 2. Specifically, the amino acid sequence shown as SEQ ID NO.1 is: TMAKDKPTLDIELLKTEVTNPAVLRKLCIE are provided. The amino acid sequence shown as SEQ ID NO.1 is as follows: FKLEKEVAETQHGTVLVQVKYEGTDAPCKI are provided.

in one embodiment, the dengue virus antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and a polypeptide having an amino acid sequence as shown in SEQ ID NO. 2. Further, the mass ratio of the polypeptide with the amino acid sequence shown as SEQ ID NO.1 to the polypeptide with the amino acid sequence shown as SEQ ID NO.2 is 1: 10-10: 1.

Through research, specific epitopes of dengue virus (DENV) and its subtypes are mainly located on domain I and domain III of envelope proteins. The polypeptide with the amino acid sequence shown as SEQ ID NO.1 is designed aiming at the structural domain I of the DENV envelope protein, and the polypeptide with the amino acid sequence shown as SEQ ID NO.2 is designed aiming at the structural domain III of the DENV envelope protein.

Proved by experiments, the dengue virus antigen can be specifically combined with a dengue virus antibody, the sensitivity and the specificity are high, and in the process of detecting dengue virus, the sample usage amount is small and the detection is rapid. In addition, the dengue virus antigen can be artificially synthesized, and compared with the synthesis of DENV recombinant protein, the process is mature and simple, and the production cost is low.

The dengue virus antigen is applied to preparation of a dengue virus detection reagent, preparation of dengue virus detection test paper or preparation of a dengue virus detection kit.

The invention also provides a detection reagent for dengue viruses, and the detection reagent for dengue viruses comprises the dengue virus antigen.

Specifically, the dengue virus antigen can be used as a coating antigen for binding with dengue virus antibodies in a sample to be tested. Of course, the dengue virus antigen can also be used as a detection antibody for detecting the binding of the dengue virus antibody and the coating antigen in the sample to be detected.

In one embodiment, the dengue virus detection reagent is a magnetic bead reagent, and the dengue virus antigen is coated on the magnetic bead.

Furthermore, the magnetic beads are coated with an antigen with an amino acid sequence shown as SEQ ID NO.1 or an antigen with an amino acid sequence shown as SEQ ID NO. 2.

Furthermore, the magnetic beads are coated with an antigen with an amino acid sequence shown as SEQ ID NO.1 and an antigen with an amino acid sequence shown as SEQ ID NO. 2. The magnetic beads coated with two antigens are more convenient to use.

The detection reagent for the dengue virus comprises the dengue virus antigen, can be specifically combined with a dengue virus antibody when detecting the dengue virus, and has the advantages of high sensitivity, good specificity, less sample usage, rapid detection and low cost.

the invention also provides test paper for detecting the dengue fever virus. The test paper for detecting the dengue virus comprises a substrate and the dengue virus antigen arranged on the substrate.

In one embodiment, the substrate is in the form of a porous plate, and the dengue virus antigen is coated in the pores.

In one embodiment, the test strip for detecting dengue viruses comprises a substrate and a coating film arranged on the substrate, wherein the coating film is provided with the dengue virus antigen. Further, the coating film is a nitrocellulose film.

The test strip for detecting the dengue virus comprises the dengue virus antigen, can be specifically combined with a dengue virus antibody when detecting the dengue virus, and has the advantages of high sensitivity, good specificity, less sample usage, rapid detection and low cost.

An embodiment of the present invention also provides a detection kit for dengue virus, including: the above dengue virus antigen; or a reagent for detecting dengue virus as described above; or test strips for dengue virus antigens.

In one embodiment, the reagent for detecting dengue viruses further comprises at least one of a sample diluent, a coating solution, a confining solution, a washing solution and a color developing solution.

Specifically, the sample diluent is at least one selected from the group consisting of a carbonate buffer, a PBS buffer, and a TBS buffer. Further, the sample diluent is one of a carbonate buffer and a PBS buffer. Of course, it will be appreciated that in other embodiments, the sample diluent may be other sample diluents as are common in the art.

Specifically, the coating solution is at least one selected from carbonate buffer, PBS buffer and TBS buffer. Further, the coating solution is selected from one of carbonate buffer solution and PBS. Of course, it is understood that in other embodiments, the coating solution may be other coating solutions commonly found in the art.

Specifically, the blocking solution is at least one selected from a PBS solution containing 1-3% (v/v) BSA, a Casein solution containing 1-3% (v/v) BSA and a 10% (v/v) horse serum solution. Further, the blocking solution is selected from one of a PBS solution containing 3% (v/v) BSA and a Casein solution containing 3% (v/v) BSA. Of course, it is understood that in other embodiments, the confining liquid may be other confining liquids commonly found in the art.

Specifically, the washing solution is selected from at least one of PBST containing 0.05-0.2% (v/v) Tween-20, TBS containing 0.05-0.2% (v/v) Tween-20, and 10% Triton. Further, the washing solution is PBST (phosphate Tween buffer) containing 0.05% -0.1% (v/v) Tween-20. Of course, it is understood that in other embodiments, the washing solution may be other washing solutions commonly found in the art.

In particular, the detection antibody is capable of binding to an antibody against dengue virus and carries a detection label. The detection label may be any one of an enzyme label, a fluorescein label, a biotin label, and a colloidal gold label. Further, the detection antibody is goat anti-human IgM-mu chain labeled with horseradish peroxidase (HRP).

Specifically, the developing solution is selected according to the type of the label of the detection antibody.

The method for using the dengue virus detection kit comprises the steps S110 to S150. Specifically, the method comprises the following steps:

Step S110, coating the dengue virus antigen on a solid phase carrier.

Specifically, the dengue virus antigen is diluted by a diluent and then mixed with a solid phase carrier, or the diluted dengue virus antigen is added to the solid phase carrier, and then a coating solution is added, and the temperature is 2-4 ℃ for 8-16 h. Then discarding the redundant coating solution, washing with washing solution, adding confining solution, incubating for 1-3 h at 37 ℃, discarding the redundant confining solution, and washing with washing solution to obtain the coated dengue virus antigen. Further, the solid phase carrier can be magnetic beads, latex microspheres, polystyrene plates or nitrocellulose membranes. Of course, the dengue virus antigen must be capable of being coated onto a solid support. For example, a solid carrier having a group capable of binding to the dengue virus antigen.

In one embodiment, the dengue virus antigen is coated at a concentration of 5 μ g/mL to 20 μ g/mL. Further, the coating concentration of the dengue virus antigen is 8 to 15 μ g/mL.

in one embodiment, the solid support is a magnetic bead. Further, the solid phase carrier is immunomagnetic beads. And mixing the dengue virus antigen and magnetic beads, and coating the dengue virus antigen on the magnetic beads to obtain the coated dengue virus antigen.

In one embodiment, the solid support is a polystyrene plate with a plurality of holes spaced apart. The wells are surface treated to coat the dengue virus antigens into the wells to obtain the coated dengue virus antigens.

Of course, in some embodiments, the step of coating the dengue virus antigen is already performed when preparing the dengue virus detection kit, and in this case, the use of the detection kit may be omitted.

And step S130, mixing the sample to be tested with the dengue virus antigen coated on the solid phase carrier, then incubating, adding the detection antibody, and continuing the incubation.

Specifically, the sample to be tested is serum. Diluting the sample to be tested by 200-800 times, mixing the diluted sample with the coated dengue fever virus antigen, and incubating the mixture at 37 ℃ for 20-45 min. Further, the incubation time of the sample to be tested and the dengue virus antigen is 25 min-35 min. Unbound material is then discarded and washed with a wash solution, followed by addition of the detection antibody and incubation at 37 ℃ for 20-45 min. Further, the incubation time for detecting the antibody is 25min to 35 min. Furthermore, the dilution multiple of the sample to be detected is 300-400 times. The dilution factor of the detection antibody is 8-32 ten thousand times, namely the concentration of the detection antibody is 3.125-12.5 ng/mL.

And S150, detecting to obtain a detection result.

Specifically, unbound detection antibody is discarded and washed with a wash solution. Then, a developing solution corresponding to the label for detecting the antibody is added for development. And after the color development is finished, detecting to obtain a detection result.

The application method of the dengue virus detection kit is simple and convenient.