A bPAG detection method based on aptamer sandwich-RPA reaction and its application

Abstract

The present invention discloses a bPAG detection method based on aptamer sandwich-RPA reaction and its application. The detection method comprises an aptamer that can specifically bind to bovine pregnancy-associated glycoprotein (bPAG) as shown in SEQ ID No.1, a forward primer as shown in SEQ ID No.2, and a reverse primer as shown in SEQ ID No.3. Based on the sandwich principle, the aptamer is used as a recognition probe and an amplification template to perform a recombinase polymerase isothermal amplification reaction, and the signal is directly or indirectly amplified by a luminescent substance or biotin on the amplified product, and bPAG is qualitatively and quantitatively detected. The bPAG detection method provided by the present invention is simple to operate, has low detection cost, high detection sensitivity, and short detection time, and can be used for laboratory and field detection of early pregnancy in dairy cows.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a bPAG detection method based on aptamer sandwich-RPA reaction and application thereof. Background Art

[0002] In recent years, my country's dairy farming industry has developed rapidly, with a significant increase in the number of dairy cows, unit yield and total production. However, with the rapid development of large-scale ranches, the reproductive problems of high-yield dairy cows have seriously affected the development of the dairy farming industry. For example, due to factors such as fertilization failure and reproductive diseases, the calving interval of dairy cows has been extended, and the milk production has decreased, which has reduced the reproductive efficiency of dairy cows, increased feeding costs, and affected the economic benefits of ranch farming. Studies have reported that delaying an estrus (about 21 days) will reduce the milk production of each dairy cow by 168-315kg. Accurate early pregnancy diagnosis of livestock after mating is an important means to improve the reproductive efficiency and economic benefits of dairy cows.

[0003] At present, the main methods for diagnosing dairy cow pregnancy include rectal palpation and ultrasonic examination. Rectal palpation is the most commonly used method, but it can generally only be performed 45-60 days after mating, and it requires a high level of technical skills from the operator, which cannot meet the requirements of early pregnancy diagnosis. Although the ultrasonic diagnosis method has a high accuracy rate, the instrument is expensive and requires skilled technical operators.

[0004] Pregnancy-associated glycoprotein (PAG) is a type of glycoprotein synthesized and secreted by the placental trophoblast cells of even-toed ungulates. It can be detected in the peripheral blood of female animals 3-4 weeks after pregnancy, so PAG is often used as a biomarker for early pregnancy diagnosis in livestock. At present, the detection of bPAG in blood or milk samples based on immunoassay is the most widely used early pregnancy detection method. Its principle is antibody-antigen recognition reaction. This type of method has the advantages of being rapid, simple, and non-invasive. Currently, commercialized PAG detection kits have been applied to production, such as the cattle rapid visual detection kit based on the principle of enzyme-linked immunosorbent assay. However, the recognition molecules of PAG detection technology based on immunoassay are all antibodies, and the preparation of antibodies requires animal experiments, which has problems such as long preparation time, high cost, and poor stability.

[0005] Aptamers are ligands that can specifically bind to target substances and are screened from random single-stranded oligonucleotide libraries using the systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are essentially single-stranded oligonucleotides that fold into secondary or tertiary structures such as hairpins, stem loops, pseudoknots, and G-quadruplexes, and interact with target molecules through hydrogen bonds, van der Waals forces, etc. to form a stable complex. Compared with traditional antibodies, aptamers are not limited by immune conditions and immunogenicity, have simple preparation processes, low production costs, high stability, and are easy to label and store.

[0006] Recombinase Polymerase Amplification (RPA) is a new type of rapid isothermal nucleic acid amplification technology that uses recombinase and polymerase to rapidly amplify the target fragment under isothermal conditions of 37°C-42°C without denaturation. The product can be monitored in real time by probe fluorescence quantification, or it can be combined with lateral flow chromatography test strips, biochips, gel electrophoresis and other methods for detection.

[0007] At present, there are no reports on bPAG detection based on aptamer sandwich-recombinase polymerase isothermal amplification reaction. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides a bPAG detection method based on aptamer sandwich-RPA reaction and its application, specifically a method for detecting bovine pregnancy-associated glycoprotein based on aptamer sandwich-RPA technology and its application.

[0009] The technical solution adopted by the present invention to solve the above technical problems is:

[0010] In the first aspect, the present invention provides an aptamer-primer combination for detecting bPAG based on a ligand sandwich-RPA reaction, which comprises: an aptamer sequence, a forward primer sequence and a reverse primer sequence, the aptamer sequence is shown in SEQ ID No.1, its forward primer sequence is shown in SEQ ID No.2, and the reverse primer sequence is shown in SEQ ID No.3.

[0011] Furthermore, the forward primer or reverse primer sequence is selectively linked to a fluorescent group, biotin, streptavidin, an amino group, digoxigenin, or horseradish peroxidase.

[0012] In a second aspect, the present invention also provides a method for detecting bovine pregnancy-associated glycoprotein based on aptamer sandwich-RPA reaction, comprising the following steps:

[0013] (1) Coating anti-bPAG antibody on a 96-well ELISA plate;

[0014] (2) Blocking: Use blocking reagent to block non-specific binding sites on the surface of the ELISA plate;

[0015] (3) Sample addition: Add the bPAG sample to be tested and incubate the reaction at room temperature;

[0016] (4) Adding aptamer: adding aptamer sequence solution, the aptamer sequence binds to bPAG in the test solution to form an antibody-bPAG-aptamer sandwich complex;

[0017] (5) RPA reaction: The aptamer bound to bPAG was eluted by heating method and used as a template to construct the RPA detection system for isothermal amplification;

[0018] (6) Detection: Qualitative and quantitative detection of bPAG protein is achieved by detecting fluorescence information.

[0019] Furthermore, in step (1), the coating solution of the anti-bPAG antibody is a 50 mmol / L carbonate buffer solution with a pH of 9.6.

[0020] Furthermore, the blocking reagent is 1% BSA (w / v), and the blocking time is 2 hours.

[0021] Furthermore, in step (3), the antibody and bPAG are incubated for 0.5 h; and in step (4), bPAG and the aptamer sequence are incubated at room temperature for 0.5 h.

[0022] Furthermore, the temperature for eluting the aptamer by heating in step (5) is 95°C.

[0023] Furthermore, the RPA detection system is: preferably TwistAmp hydration buffer reconstitution buffer 29.5 μL, 5 μM upstream primer 2.5 μL, 5 μM downstream primer 2.5 μL, ultrapure water 12 μL, template 1 μL, 280 mM magnesium acetate 2.5 μL. The upstream primer is the forward primer sequence in the aptamer-primer combination, the downstream primer is the reverse primer sequence, and the template is the eluted aptamer, that is, the antibody-bPAG-aptamer complex.

[0024] Furthermore, the RPA reaction temperature is 37° C. and the reaction time is 20 min.

[0025] In a third aspect, the present invention provides the use of the aptamer-primer combination in bPAG detection and in the preparation of a kit for detecting bPAG.

[0026] Also, the method for detecting bovine pregnancy-associated glycoprotein based on aptamer sandwich-RPA reaction is used in bPAG detection, and the preparation of a bPAG detection kit.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) Low cost: Aptamers and hybrid chains can be synthesized in vitro, which overcomes the shortcomings of long antibody preparation cycle and high cost and reduces production costs;

[0029] (2) High sensitivity: After RPA reaction, the detection signal is amplified, which greatly improves the detection sensitivity;

[0030] (3) Simple operation: Compared with traditional RPA detection technology, this method does not require the extraction of DNA as a template. Instead, it uses the aptamer that specifically recognizes the target as a template for RPA amplification. It does not require expensive instruments. The entire process is carried out under constant temperature conditions. There are fewer detection steps, the detection time is short, and the operation is simple. It can be used for laboratory and on-site detection of early pregnancy in dairy cows. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A is a schematic diagram of the bPAG detection principle of Example 1 of the present invention;

[0032] Figure 1 B is a schematic diagram of the bPAG detection principle of Example 2 of the present invention;

[0033] Figure 2 This is a standard curve diagram for detecting bovine pregnancy-associated glycoprotein 9 in Example 1 of the present invention;

[0034] Figure 3 This is a standard curve diagram for detecting bovine pregnancy-associated glycoprotein 9 in Example 2 of the present invention;

[0035] Figure 4 To visualize the results of bPAG9 detection based on the method of Example 2; Figure 4 In the figure, - is the negative control, + is the positive control, 1 is the test result of pregnant cow serum, and 2 and 3 are the test results of non-pregnant cow serum. DETAILED DESCRIPTION

[0036] The technical scheme of the present invention is further described in detail below in conjunction with the accompanying drawings and specific examples. The aptamers, forward primers, and reverse primers used in the following examples are all synthesized by Shanghai Bioengineering Technology Co., Ltd. using the sequences described. The bPAG9 described in the examples is bovine pregnancy-associated glycoprotein 9, PBST is phosphate-tween buffer, PBS is phosphate buffered saline, BSA is bovine serum albumin, and EvaGreen is a green fluorescent nucleic acid dye.

[0037] An aptamer-primer combination for detecting bPAG based on a ligand sandwich-RPA reaction, comprising: an aptamer sequence, a forward primer sequence and a reverse primer sequence, wherein the aptamer sequence is shown in SEQ ID No. 1: The forward primer sequence is shown in SEQ ID No.2: The reverse primer sequence is shown in SEQ ID No.3: Based on the sandwich principle, the aptamer is used as a recognition probe and amplification template, and after adding primers, a recombinase polymerase isothermal amplification reaction is carried out. The signal is directly or indirectly amplified through the luminescent substance or biotin on the amplification product to qualitatively and quantitatively detect bPAG. Preferably, the forward primer and reverse primer sequences can be connected to fluorescent groups, biotin, avidin, amino groups, digoxigenin, horseradish peroxidase, etc.

[0038] Embodiment 1:

[0039] like Figure 1 As shown in A, the method for detecting bPAG9 based on aptamer sandwich-RPA reaction fluorescence comprises the following steps:

[0040] (1) Anti-bPAG9 antibody coated on ELISA plate: Take a 96-well ELISA plate pre-coated with bPAG antibody, wash it three times with PBST (10 mmol / L PBS, pH 7.4, 0.05% Tween-20), and pat dry;

[0041] (2) Blocking: Add 200 μL of 1% BSA (w / v) blocking solution to each well, incubate at room temperature for 2 h, wash with PBST, and pat dry;

[0042] (3) Sample addition: add 100 μL of different concentrations of bPAG9 (0, 0.05, 0.2, 1, 5, 10, 50, 100, 200 ng / mL), incubate at room temperature for 30 min, wash with PBST, and pat dry;

[0043] (4) Adding aptamer: Add 100 μL of aptamer sequence (200 nM) dissolved in buffer, incubate at room temperature for 30 min, wash with PBST, and pat dry;

[0044] (5) RPA reaction: add 100 μL ultrapure water to each well, heat at 95°C for 5 min to elute the aptamer, take 1 μL of the eluted aptamer as a template for RPA reaction, RPA reaction system: reconstitution buffer 29.5 μL, 5 μM upstream primer 2.5 μL, 5 μM downstream primer 2.5 μL, ultrapure water: 12 μL, 280 mM magnesium acetate 2.5 μL, 0.5× EvaGreen 2.5 μL; incubate at 37°C for 30 min;

[0045] (6) Fluorescence detection: The fluorescence signal of the reaction solution was measured using a full-wavelength scanning multifunctional reader at an excitation wavelength of 500 nm and an emission wavelength of 530 nm;

[0046] (7) Establishment of standard curve: Plot the standard curve using bPAG9 concentration as the horizontal axis and F-F0 as the vertical axis. Use software to draw the standard curve and establish a regression equation. F is the fluorescence intensity corresponding to the presence of bPAG9; F0 is the fluorescence intensity corresponding to the bPAG9 mass concentration of 0; Figure 2 The standard curve determined by this method is: y=245.4+175.5lgx, R=0.995, the linear range is 0.2-100ng / mL, and the detection limit is 0.09ng / mL.

[0047] Embodiment 2:

[0048] like Figure 1 As shown in B, the method for colorimetric detection of bPAG9 based on aptamer sandwich-RPA reaction comprises the following steps:

[0049] (1) Anti-bPAG antibody coated on ELISA plate: Take the ELISA plate pre-coated with bPAG antibody, wash it 3 times with PBST (same as in Example 1), and pat dry;

[0050] (2) Blocking: Add 200 μL of 1% BSA (w / v) blocking solution to each well, incubate at room temperature for 2 h, wash with PBST, and pat dry;

[0051] (3) Sample addition: add 100 μL of different concentrations of bPAG9 (0, 0.05, 0.5, 1, 5, 25, 100, 200, 400 ng / mL), incubate at room temperature for 30 min, wash with PBST, and pat dry;

[0052] (4) Adding aptamer: Add 100 μL of aptamer sequence (200 nM), incubate at room temperature for 30 min, wash with PBST, and pat dry;

[0053] (5) RPA reaction: 100 μL ultrapure water was added to each well, heated at 95°C for 5 min to elute the aptamer, and 1 μL of the aptamer was used as a template for RPA reaction. The RPA reaction system included 29.5 μL of reconstitution buffer, 2.5 μL of 5 μM biotinylated upstream primer, 2.5 μL of 5 μM downstream primer, 12 μL of ultrapure water, and 2.5 μL of 280 mM magnesium acetate. The reaction was incubated at 37°C for 20 min. After the reaction, 100 μL of 0.05 U / mL streptavidin-horseradish peroxidase (SA-HRP) was added to each well, reacted at room temperature for 10 min, washed with PBST, 100 μL of color development solution was added to each well, color was developed in the dark for 10 min, 100 μL of stop solution (2.0 mol / L H2SO4) was added, and the absorbance was measured at 450 nm using an ELISA reader.

[0054] (6) Establishment of standard curve: With bPAG9 concentration as the horizontal axis and Ai / A0 as the vertical axis, the standard curve was drawn using software and a regression equation was established, where A is the absorbance at 450 nm corresponding to each mass concentration of bPAG9, and A0 is the absorbance at 450 nm when the mass concentration of bPAG9 is 0; Figure 3 The standard curve determined by this method is: y=0.250+0181lgx, R=0.998, the linear range is 0.5-100ng / mL, and the detection limit is 0.13ng / mL.

[0055] Example 3 Early pregnancy detection in dairy cows

[0056] The method for colorimetric detection of bPAG9 based on aptamer sandwich-RPA reaction in Example 2 above was applied to early pregnancy detection in dairy cows:

[0057] (1) Preparation of test samples: 40 Holstein cows bred for 28 days were selected, and blood was collected from the vein. The blood samples were placed on ice and transported to the laboratory within 3 hours. The blood samples were centrifuged at 2000×g for 10 minutes, and the upper serum was transferred to a centrifuge tube and diluted 2-fold with PBS (0.01M, pH 7.4) for later use.

[0058] (2) Sample detection: Take several ELISA plates coated with anti-bPAG antibodies in Example 2, add 100 μL of diluted serum sample and 100 μL of aptamer solution (200 nM), incubate at room temperature for 30 min, wash with PBST, and pat dry; add 100 μL of ultrapure water to each well, heat at 95°C for 5 min, elute the aptamer, take 1 μL of the aptamer as a template for RPA reaction, the RPA reaction system: 29.5 μL of reconstitution buffer, 2.5 μL of 5 μM biotinylated upstream primer, 2.5 μL of 5 μM downstream primer, 12 μL of ultrapure water, 2.5 μL of 280 mM magnesium acetate, incubate at 37°C for 20 min; add 100 μL of ultrapure water to each well, heat at 95°C for 5 min, elute the aptamer, and take 1 μL of the aptamer as a template for RPA reaction. The RPA reaction system includes: 29.5 μL of reconstitution buffer, 2.5 μL of 5 μM biotinylated upstream primer, 2.5 μL of 5 μM downstream primer, 12 μL of ultrapure water, and 2.5 μL of 280 mM magnesium acetate. 0.05U / mL streptavidin-horseradish peroxidase (SA-HRP), react at room temperature for 10min, wash with PBST; add 100μL colorimetric solution to each well, color for 10min in the dark, add 100μL stop solution (2.0mol / L H2SO4), and measure the absorbance at 450nm with a microplate reader; add bPAG9 (5ng / mL) as a positive control, and use PBS buffer as a blank control.

[0059] (3) Test results: Pregnancy can be qualitatively determined by visually observing the color change. If the sample well is colored positive, it means pregnancy; if the sample well is colorless or the color is similar to the blank control, it means negative, which means no pregnancy. Figure 4 The colorimetric results of some samples were detected based on the method of Example 2. The dairy cows were examined by B-ultrasound detection method (gold standard method) on the 45th day after mating. The results of ultrasonic examination and aptamer sandwich-RPA colorimetric method are shown in Table 1.

[0060] Table 1 Comparison of the results of aptamer sandwich-RPA colorimetric method and B-ultrasound examination method

[0061] Diagnostic Methods Number of heads Number of positive heads Negative heads Aptamer-RPA colorimetric method 40 16 14 B-ultrasound examination 40 18 12

[0062] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various improvements and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting bovine pregnancy-associated glycoprotein 9 based on aptamer sandwich-RPA reaction, characterized in that: The specific steps include: (1) Coating the anti-bPAG9 antibody on the ELISA plate; (2) Blocking: Use blocking reagent BSA to block non-specific binding sites on the surface of the ELISA plate; (3) Sample addition: Add the bPAG sample to be tested and incubate the reaction at room temperature; (4) Adding aptamer: adding aptamer dissolved in buffer, the aptamer binds to bPAG9 in the test solution to form an antibody-bPAG-aptamer sandwich complex, the sequence of the aptamer is shown in SEQ ID No. 1; (5) RPA reaction: The aptamer bound to bPAG9 was eluted by heating method, and the eluted aptamer was used as a template to construct an RPA detection system, and isothermal amplification was performed using a forward primer with a sequence as shown in SEQ ID No. 2 and a reverse primer with a sequence as shown in SEQ ID No. 3; the sequences of the forward primer and the reverse primer were connected to a fluorescent group; the temperature of the heating method elution was 95°C; (6) Detection: Qualitative and quantitative detection of bPAG9 protein can be achieved by detecting fluorescence information.

2. The method according to claim 1, characterized in that: The coating solution of the anti-bPAG9 antibody is a 50 mmol / L, pH 9.6 carbonate buffer, the blocking reagent is 1% BSA (w / v), and the blocking time is 2 hours.

3. The method according to claim 2, characterized in that: In step (3), the antibody and the bPAG test sample are incubated for 0.5 h.

4. The method according to claim 3, characterized in that: The temperature of the RPA reaction in step (5) is 37° C. and the time is 20 min.

5. The method according to claim 4, characterized in that: The RPA detection system is: 29.5 μL of reconstitution buffer, 2.5 μL of 5 μM upstream primer, 2.5 μL of 5 μM downstream primer, 12 μL of ultrapure water, 1 μL of template, and 2.5 μL of 280 mM magnesium acetate.

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