Colloidal gold test strip for detecting adenovirus as well as preparation method and application of colloidal gold test strip

Abstract

The invention relates to a colloidal gold test strip for detecting adenovirus as well as a preparation method and application of the colloidal gold test strip, and belongs to the technical field of adenovirus detection. The invention provides a preparation method of a colloidal gold test strip, the colloidal gold test strip is used for detecting adenovirus, the colloidal gold test strip is composed of a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the colloidal gold test strip is fixed on a bottom plate, colloidal gold is used for marking an adenovirus detection antibody, the colloidal gold test strip is sprayed on the surface of the combination pad, and an adenovirus capture antibody is diluted. The lowest detection limit of the prepared colloidal gold test strip on the adenovirus is 2ng / mL, and the colloidal gold test strip is simple to prepare, convenient to use, capable of rapidly obtaining a detection result, free of professional complex operation and suitable for rapid clinical screening of the adenovirus.

Description

Technical Field

[0001] The present invention relates to the technical field of adenovirus detection, and in particular to a colloidal gold test strip for detecting adenovirus, a preparation method thereof and an application thereof. Background Art

[0002] Adenovirus is a widely existing non-enveloped double-stranded DNA virus. Its icosahedral symmetric structure is composed of 252 capsomeres and can be divided into 7 subgenera and 114 genotypes from A to G. Different types can cause multi-system infections such as the respiratory tract, eyes, and gastrointestinal tract. The virus is mainly transmitted through droplets, contact with contaminated objects, and the fecal-oral route, and is prone to outbreaks in enclosed places (such as schools, hospitals, swimming pools). Its harmfulness is manifested as follows: about 60%-70% of infected people have respiratory symptoms such as high fever (>39°C) and intractable cough, and severe cases can develop into pneumonia or even respiratory failure (the case fatality rate of severe pneumonia in children reaches 6.8%); keratoconjunctivitis caused by type D adenovirus can cause corneal infiltration in 41% of patients, and 8.7% of them have permanent vision damage; about 15% of infants and young children in gastroenteritis cases need hospitalization for dehydration caused by watery diarrhea; the mortality rate of hemorrhagic cystitis or meningitis in immunocompromised groups after infection is 6 times higher than that in the healthy population. It should be noted that the virus is stable in the environment (surviving at room temperature for 10 days), and there is no specific antiviral drug yet. Severe cases rely on supportive therapy for treatment, and prevention mainly depends on basic protective measures such as frequent handwashing and wearing masks.

[0003] Adenovirus detection is not only the core link of disease diagnosis and treatment, but also a key public health measure to control community transmission and reduce the severe case rate. Early and accurate detection can significantly improve clinical decision-making. Molecular detection techniques (such as PCR) can detect viral DNA in the early stage of infection for 3-5 days, shortening the antibody detection window period by 7-10 days, which is crucial for the timing selection of antiviral treatment for immunosuppressed patients. However, the PCR technique has its insurmountable defects. Traditional PCR operations are easily contaminated by aerosols, especially during open-tube detection. Extremely trace nucleic acid residues can lead to false positive results; it requires professional personnel to operate and strict environmental control (such as an environment free of nuclease contamination). Improper sample handling may lead to template loss or degradation; in addition, real-time fluorescence quantitative PCR (qPCR) relies on expensive instruments (unit price exceeding 250,000 yuan) and cold-chain transportation reagents, which are limited in popularization in primary medical institutions. Moreover, the sensitivity of qPCR to early infection samples with low viral loads drops sharply, and the false negative rate can reach 38.9%; due to differences in primer design and instrument calibration among different laboratories, the comparability of results is poor, affecting the consistency of clinical decision-making. Considering the above disadvantages, there is an urgent need for a simple, fast, and low-operation-difficulty adenovirus detection method to achieve early, efficient, and rapid screening of adenoviruses. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a method for preparing a colloidal gold test strip for detecting adenovirus. The colloidal gold test strip is composed of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, and is fixed on a bottom plate. An adenovirus detection antibody is labeled with colloidal gold and sprayed on the surface of the conjugate pad. The adenovirus capture antibody is diluted and sprayed on the nitrocellulose membrane to form a test line.

[0005] The first object of the present invention is to provide a method for preparing a colloidal gold test strip for detecting adenovirus, comprising the following steps:

[0006] (1) Prepare a sample pad, a conjugate pad, an absorbent pad, a nitrocellulose membrane, and a bottom plate;

[0007] (2) Label an adenovirus detection antibody with colloidal gold and spray it on the surface of the conjugate pad. The concentration of the adenovirus detection antibody is 10 - 20 μg / mL, the spraying amount is 3 - 5 μL / cm, and the pH value of the colloidal gold-labeled adenovirus detection antibody solution is 8 - 8.5;

[0008] (3) Dilute the adenovirus capture antibody and spray it on the nitrocellulose membrane to form a test line. The concentration of the adenovirus capture antibody is 0.7 - 1.5 mg / mL, and the spraying amount is 1 - 2 μL / cm;

[0009] (4) Assemble the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad on the bottom plate in sequence. The two ends of the nitrocellulose membrane overlap and are bonded to the conjugate pad and the absorbent pad respectively.

[0010] Further, the sample pad and the conjugate pad are pretreated. The sample pad pretreatment solution includes a buffer solution, casein, polyvinylpyrrolidone K30, surfactant S9, Tween 20, a preservative, and a heterophilic antibody blocker HBR. The conjugate pad pretreatment solution includes a buffer solution, bovine serum albumin, Tween 20, and a preservative.

[0011] Further, soak the sample pad in 0.05 - 0.1 mL / cm 2 of the sample pad treatment solution, and soak the conjugate pad in 0.05 - 0.1 mL / cm 2 of the conjugate pad treatment solution.

[0012] Further, the step of labeling an adenovirus detection antibody with colloidal gold includes preparing a colloidal gold solution and a colloidal gold labeling complex solution, adjusting the pH value of the colloidal gold solution, adding the adenovirus detection antibody, mixing well and then adding bovine serum albumin, centrifuging to obtain a precipitate, and adding the colloidal gold labeling complex solution to the precipitate. The colloidal gold solution includes chloroauric acid and sodium citrate, and the colloidal gold labeling complex solution includes a buffer solution, casein, sucrose, trehalose, and a preservative.

[0013] Further, the final concentration of the bovine serum albumin is 0.5 - 1% w / w.

[0014] Further, the adenovirus capture antibody is diluted with a coating diluent, which includes a buffer and sucrose, and the pH value of the coating diluent is 7 - 7.5.

[0015] The second object of the present invention is to provide a colloidal gold test strip prepared by the above method.

[0016] Further, the colloidal gold test strip further includes a sample diluent, which includes a buffer, casein, a surfactant, and a preservative.

[0017] The third object of the present invention is to provide the application of the above colloidal gold test strip in the detection of adenovirus antigen.

[0018] The fourth object of the present invention is to provide a method for detecting adenovirus antigen, using the above colloidal gold test strip to detect a sample to be tested.

[0019] Advantages of the present invention:

[0020] The colloidal gold test strip provided by the present invention combines the advantages of high sensitivity, rapid detection, convenient operation, and strong stability. It is especially suitable for the early screening and primary diagnosis of adenovirus infection, providing an efficient and reliable on-site detection tool for clinical use. The lowest detection limit for adenovirus is 2 ng / mL, and the detection result can be obtained in 10 - 15 minutes without professional and complex operations, reducing the detection and screening costs of adenovirus. Description of the Drawings

[0021] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention and in combination with the drawings, wherein:

[0022] Figure 1 is a schematic diagram of the overall structure of the colloidal gold test strip provided by the present invention, where 1 is the sample pad, 2 is the conjugate pad, 3 is the nitrocellulose membrane, 31 is the test line, 32 is the control line, 4 is the absorbent pad, and 5 is the bottom plate;

[0023] Figure 2 is the test result diagram of the colloidal gold test strip provided by the present invention. Detailed Embodiments

[0024] The following further illustrates the present invention in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.

[0025] Example 1: Preparation of the colloidal gold test strip

[0026] 1. Pretreatment of the sample pad

[0027] Prepare the sample pad pretreatment solution according to the following ratio: Tris-HCl buffer 40 - 80 mM, casein 0.4 - 0.8% (w / w), polyvinylpyrrolidone K30 0.8 - 1.5% (w / w), surfactant S9 0.8 - 1.5% (w / w), Tween 20 0.04 - 0.1% (w / w), preservative ProClin300 0.02 - 0.06% (w / w), heterophilic antibody blocker HBR 0.002 - 0.006% (w / w). After preparation, stabilize the pH of the solution in the range of 8.0 - 9.0.

[0028] Transfer the prepared sample pad pretreatment solution to an impregnation container and inject it according to the liquid volume standard of 0.05 - 0.1 mL / cm2. Completely immerse the sample pad to be treated in the solution, ensuring that the continuous soaking time is more than 5 minutes. After soaking, remove the excess liquid by centrifugation. Transfer the dehydrated sample pad to a constant temperature oven and continuously dry it for more than 7 hours within the temperature range of 33 - 50 °C. Immediately after drying, pack the sample pad in a moisture-proof package and seal it in an environment with a relative humidity ≤ 30%.

[0029] 2. Pretreat the conjugate pad

[0030] Prepare the conjugate pad pretreatment solution according to the following ratio: Tris-HCl buffer 40 - 80 mM, bovine serum albumin 0.4 - 0.8% (w / w), Tween 20 0.04 - 0.08% (w / w), ProClin300 0.02 - 0.06% (w / w). After preparation, stabilize the pH of the solution in the range of 8.0 - 9.0.

[0031] Treat the conjugate pad according to the above method, with the difference that the amount of the conjugate pad pretreatment solution is 0.05 - 0.1 mL / cm 2 .

[0032] 3. Prepare the colloidal gold solution

[0033] Prepare the colloidal gold solution, which mainly contains two components, chloroauric acid and sodium citrate. The concentration of chloroauric acid is 0.01 - 0.04% (w / w), and the mass ratio of chloroauric acid to sodium citrate is 1:1 to 1:1.5.

[0034] Prepare the colloidal gold labeling complex solution, whose components include Tris-HCl buffer 35 - 60 mM, casein 0.3 - 0.7% (w / w), sucrose 10 - 30% (w / w), trehalose 2 - 7% (w / w), ProClin300 0.02 - 0.06% (w / w). After preparation, stabilize the pH of the solution in the range of 8.0 - 9.0.

[0035] 4. Label detection antibody and quality control antibody

[0036] Measure 1 mL of colloidal gold solution, adjust the pH with 0.1 M potassium carbonate, and control the final pH value in the range of 8.0 - 8.5. Add the adenovirus detection antibody at a concentration of 10 - 20 μg / mL, and shake and mix for more than 10 minutes. Then supplement bovine serum albumin (BSA) to 0.5 - 1% (w / v), and continue mixing for no less than 10 minutes. Transfer the mixed system to a centrifuge, and centrifuge at a speed of 7000 - 14000 rpm at a temperature of 0 - 8 °C for no less than 5 minutes. After removing the supernatant, quantitatively add 20 - 30 μL of the colloidal gold labeling complex solution prepared above to the precipitate, and resuspend to form a homogeneous labeling system to obtain the colloidal gold labeling complex solution of the adenovirus detection antibody. Use a spectrophotometer to measure the optical density OD value.

[0037] Measure 1 mL of colloidal gold solution, adjust the pH with 0.1 M potassium carbonate, and control the final pH value in the range of 8.5 - 9.0. Add the chicken IgY antibody at a concentration of 10 - 20 μg / mL, and shake and mix for more than 10 minutes. Then supplement bovine serum albumin (BSA) to 0.6 - 1% (w / v), and continue mixing for no less than 10 minutes. Transfer the mixed system to a centrifuge, and centrifuge at a speed of 9000 - 14000 rpm at a temperature of 0 - 8 °C for no less than 5 minutes. After removing the supernatant, quantitatively add 30 - 40 μL of the colloidal gold labeling complex solution prepared above to the precipitate, and resuspend to form a homogeneous labeling system to obtain the colloidal gold labeling complex solution of the chicken IgY antibody. Use a spectrophotometer to measure the optical density OD value.

[0038] 5. Coating capture antibody and quality control capture antibody

[0039] Prepare the coating dilution solution, and its composition and ratio are: 8 - 15 mM PBS, 1.5 - 3 wt% sucrose, and the pH value of the solution is 7 - 7.5.

[0040] Dilute the adenovirus capture antibody with the coating dilution solution to 0.7 - 1.5 mg / mL to obtain the adenovirus capture antibody coating solution.

[0041] Dilute the goat anti-chicken IgY antibody with the coating dilution solution to 0.8 - 1.2 mg / mL to obtain the goat anti-chicken IgY antibody coating solution.

[0042] 6. Prepare the sample dilution solution

[0043] Prepare the sample diluent according to the following ratio: Tris-HCl buffer 50 - 150 mM, casein 0.5 - 1.2% (w / w), non-ionic surfactant 0.2 - 0.6% (w / w), ProClin300 0.02 - 0.06% (w / w). After preparation, stabilize the pH of the solution in the range of 7.0 - 7.5.

[0044] 7. Assembly of the test strip

[0045] Adjust the concentration of the colloidal gold complex solution of the adenovirus detection antibody to OD50 - 70, and the concentration of the colloidal gold-labeled complex solution of the chicken IgY antibody to OD25 - 35. After adjustment, mix the two thoroughly, and uniformly spray the mixture onto the surface of the pretreated conjugate pad at a rate of 3 - 5 μL / cm. After spraying, transfer the conjugate pad to an environment of 30 - 50 °C and bake for at least 6 hours. After complete drying, store it in a sealed container, and the storage environment needs to strictly control the humidity ≤ 30%.

[0046] Spray the adenovirus capture antibody coating solution and the goat anti-chicken IgY antibody coating solution onto the test line and the quality control line of the nitrocellulose membrane at a spraying rate of 1 - 2 μL / cm respectively. Then, place the treated nitrocellulose membrane in an environment of 30 - 50 °C and bake for at least 6 hours. After complete drying, store it in a sealed container, and the storage environment needs to strictly control the humidity ≤ 30%.

[0047] Assemble the components of the test strip onto the bottom plate in the following order: Using the nitrocellulose membrane as the central positioning point, lay it flat in the middle section of the bottom plate, and lap and bond its two ends to the absorbent pad and the conjugate pad respectively. The lap overlap width is strictly controlled within the range of 1 - 3 mm. Among them, the upper end (test end) of the nitrocellulose membrane overlaps and connects with the end of the conjugate pad, and the lower end (sample migration terminal) overlaps and connects with the starting end of the absorbent pad. The other end of the conjugate pad is connected to the sample pad with the same overlap specification, finally forming a chromatographic path of "sample pad - conjugate pad - nitrocellulose membrane - absorbent pad". The bottom plate serves as a support base to carry all components and ensure the precise alignment of each functional layer.

[0048] The schematic structural diagram of the prepared colloidal gold test strip is as Figure 1 shown, where 1 is the sample pad, 2 is the conjugate pad, 3 is the nitrocellulose membrane, 31 is the test line, 32 is the quality control line, 4 is the absorbent pad, and 5 is the bottom plate.

[0049] Example 2: Application of the colloidal gold test strip

[0050] The sample diluent prepared in Example 1 was used to dilute the adenovirus antigen standard solution to concentrations of 1.4 μg / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, and 1 ng / mL, respectively. 80 μL of the adenovirus antigen standard solution of the five concentrations was taken and applied to the colloidal gold test strip prepared in Example 1. Due to capillary action, the sample moved along the test strip to the binding pad and the nitrocellulose membrane until it flowed to the absorbent pad. The result was observed for 10 minutes, and the color development was invalid after 15 minutes.

[0051] (c) Observation of results: The test results are as follows: Figure 2 As shown, the quality control lines of all test strips are clearly and obviously displayed, indicating that the test strips can normally play the role of indicating antigens. When the adenovirus antigen concentration is ≥2ng / mL, the detection line is colored (due to problems such as shooting angle, contrast, pixels, etc. Figure 2 The visual effect of the detection line is not obvious. By comparing the framed part, it can be seen that the test line does exist when the antigen concentration is 2ng / mL). Therefore, the minimum detection limit of adenovirus antigen in this test strip is 2ng / mL. When the adenovirus antigen concentration is ≥2ng / mL, the detection line is colored, indicating that the adenovirus test result is positive; when the adenovirus antigen concentration is <2ng / mL, the detection line is not colored, indicating that the adenovirus test result is negative.

[0052] Comparative Example 1: Effect of sample dilution adjustment on detection performance

[0053] The colloidal gold test strips were prepared according to the method described in Example 1, except that the sample diluent was adjusted.

[0054] Prepare the sample diluent according to the following ratios: sodium chloride 2-3% (w / w), Tris 0.5-1% (w / w), bovine serum albumin 0.5-1% (w / w), Tween 20 2-3% (w / w), ProClin300 0.02-0.06% (w / w). After preparation, stabilize the solution pH in the range of 7.0-7.5.

[0055] The adenovirus antigen standard solution was tested according to the method described in Example 2, and the minimum detection limit was adenovirus antigen concentration value ≥ 5ng / mL for a positive test result, and < 5ng / mL for a negative test result. The detection limit was significantly increased compared with the colloidal gold test strip prepared in Example 1, and the detection effect was significantly reduced. The detection performance of the colloidal gold test strip was affected.

[0056] Comparative Example 2: Effect of pH adjustment of colloidal gold solution on detection performance

[0057] The test strip was prepared according to the method described in Example 1, except that in step 4, the pH value of the colloidal gold solution was adjusted to 7 / 7.5 / 9 / 9.5 with a 0.1M potassium carbonate solution, and then the adenovirus antigen standard solution was detected according to the method described in Example 2. The detection results are shown in Table 1.

[0058] Table 1 Influence of pH value adjustment on the lowest detection limit

[0059] pH value 7 7.5 9 9.5 Minimum detectable limit 5 ng / mL 3 ng / mL 4 ng / mL 6 ng / mL

[0060] As can be seen from the results in Table 1, adjusting the pH value of the colloidal gold solution will affect the detection performance of the colloidal gold test strip, resulting in an increase in the detection limit and a decrease in the detection effect, which greatly affects the sensitivity of the test strip.

[0061] Comparative Example 3: Influence of the adjustment of the detection antibody and capture antibody loading on the detection performance

[0062] 1. Adjustment of the detection antibody loading

[0063] The test strip was prepared according to the method described in Example 1, except that in step 7, the mixed solution of the adenovirus detection antibody colloidal gold labeling complex solution and the chicken IgY antibody colloidal gold labeling complex solution was sprayed on the treated conjugate pad at spraying amounts of 1 μL / cm, 2 μL / cm, 6 μL / cm, and 7 μL / cm respectively, and then the adenovirus antigen standard solution was detected according to the method described in Example 2. The detection results are shown in Table 2.

[0064] Table 2 Influence of the adjustment of the detection antibody loading on the lowest detection limit

[0065] Spraying amount 1 μL / cm 2 μL / cm 6 μL / cm 7 μL / cm Minimum detectable limit 5 ng / mL 4 ng / mL 2 ng / mL 2 ng / mL

[0066] As can be seen from the results in Table 2, adjusting the loading of the detection antibody will affect the detection performance of the colloidal gold test strip. A decrease in the loading of the detection antibody will increase the lowest detection limit of the colloidal gold test strip, and when the loading of the detection antibody increases to a certain amount, the lowest detection limit remains stable and the detection sensitivity will not be further improved.

[0067] 2. Adjustment of the capture antibody loading

[0068] The test strip was prepared according to the method described in Example 1, except that in step 7, the adenovirus capture antibody coating solution was sprayed on the nitrocellulose membrane at spraying amounts of 0.5 μL / cm, 2.5 μL / cm, and 3 μL / cm respectively, and then the adenovirus antigen standard solution was detected according to the method described in Example 2. The detection results are shown in Table 3.

[0069] Table 3 Influence of the adjustment of the capture antibody loading on the lowest detection limit

[0070] Spraying amount 0.5 μL / cm 2.5 μL / cm 3 μL / cm Minimum detectable limit 4 ng / mL 2 ng / mL 2 ng / mL

[0071] As can be seen from the results in Table 2, adjusting the loading amount of the capture antibody will affect the detection performance of the colloidal gold test strip. A decrease in the loading amount of the capture antibody will increase the minimum detection limit of the colloidal gold test strip. When the loading amount of the capture antibody increases to a certain level, the minimum detection limit remains stable and the detection sensitivity will not be further improved.

[0072] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing a colloidal gold test strip for detecting adenovirus, characterized in that: The following steps are involved: (1) Prepare the sample pad, conjugate pad, absorbent pad, nitrocellulose membrane and base plate; (2) using colloidal gold to label adenovirus detection antibodies and spraying them on the surface of the conjugate pad, wherein the concentration of the adenovirus detection antibodies is 10-20 μg / mL, the spraying amount is 3-5 μL / cm, and the pH value of the colloidal gold labeled adenovirus detection antibody solution is 8-8.5; (3) diluting adenovirus capture antibody and spraying it on the nitrocellulose membrane to form a detection line, wherein the concentration of the adenovirus capture antibody is 0.7-1.5 mg / mL and the spraying amount is 1-2 μL / cm; (4) Assembling a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad in sequence on a bottom plate, wherein two ends of the nitrocellulose membrane overlap and bond with the conjugation pad and the water absorbent pad respectively.

2. The preparation method according to claim 1, characterized in that: The sample pad and the conjugate pad are pretreated, the sample pad pretreatment solution comprises a buffer, casein, polyvinyl pyrrolidone K30, a surfactant S9, Tween 20, a preservative and a heterophilic antibody blocking agent HBR, and the conjugate pad pretreatment solution comprises a buffer, bovine serum albumin, Tween 20 and a preservative.

3. The preparation method according to claim 2, characterized in that: Soak the sample pad in 0.05-0.1mL / cm 2 The conjugate pad is immersed in the sample pad treatment solution of 0.05-0.1mL / cm 2 of the conjugate pad treatment solution.

4. The preparation method according to claim 1, characterized in that: The steps of using colloidal gold to label adenovirus detection antibodies include preparing a colloidal gold solution and a colloidal gold labeling re-solution, adjusting the pH value of the colloidal gold solution, adding the adenovirus detection antibody, adding bovine serum albumin after mixing, centrifuging to obtain a precipitate, and adding the colloidal gold labeling re-solution to the precipitate, wherein the colloidal gold solution includes chloroauric acid and sodium citrate, and the colloidal gold labeling re-solution includes a buffer, casein, sucrose, trehalose and a preservative.

5. The preparation method according to claim 4, characterized in that: The final concentration of the bovine serum albumin is 0.5-1% w / w.

6. The preparation method according to claim 1, characterized in that: The adenovirus capture antibody is diluted with a coating diluent, wherein the coating diluent includes a buffer and sucrose, and the pH value of the coating diluent is 7-7.

5.

7. The colloidal gold test strip prepared by the method according to any one of claims 1 to 6.

8. The colloidal gold test strip according to claim 7, characterized in that: The colloidal gold test strip also includes a sample diluent, which includes a buffer, casein, a surfactant and a preservative.

9. Use of the colloidal gold test strip according to claim 7 or 8 in adenovirus antigen detection.

10. A method for detecting adenovirus antigens, characterized in that: The colloidal gold test strip according to claim 7 or 8 is used to detect the sample to be tested.

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