Quantitative detection method of virus antigen

Abstract

The invention belongs to the technical field of biology, and particularly relates to a virus antigen detection method. The method comprises the following steps: modifying a complementary DNA chain of a virus antigen nucleic acid aptamer on the surface of an optical fiber, reacting a virus antigen in a sample with a fluorescently-labeled nucleic acid aptamer to change the conformation of the nucleic acid aptamer, and introducing a mixed solution containing the virus antigen and the fluorescently-labeled nucleic acid aptamer into the surface of a functionalized optical fiber sensing element, so as to detect the surface of the functionalized optical fiber sensing element. The fluorescence labeling nucleic acid aptamer with changed conformation is combined with complementary DNA (cDNA) on the surface of the optical fiber sensing element, the intensity of fluorescence received by the optical fiber sensing element is improved along with the increase of the combination amount, and the quantitative detection purpose is achieved by detecting the change of the intensity of the fluorescence received by the optical fiber sensing element. The method is high in accuracy, strong in specificity, small in required sample size and simple to operate, and can be used for carrying out specific detection on virus antigens.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a quantitative detection method for SARS-CoV-2 antigen. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) is highly contagious and pathogenic, spreading rapidly after its outbreak and causing a global public health problem. Timely and accurate detection of viral antigens is crucial for assisting in assessing infection status. Currently, commonly used methods for detecting the novel coronavirus (SARS-CoV-2) include: real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and flow immunoassay.

[0003] Currently, reverse transcription polymerase chain reaction (RT-PCR) has high sensitivity and specificity, making it the gold standard for SARS-CoV-2 virus detection. However, RT-PCR testing requires professionals to perform it in a biosafety level 2 laboratory, and limitations in space and personnel hinder its widespread adoption in some primary healthcare institutions. Furthermore, due to the complexity of the procedure, standard nucleic acid testing takes 4-6 hours to complete, which may lead to delays in identifying COVID-19 patients. SARS-CoV-2 antigen testing results can be used for early triage and rapid management of suspected infected individuals, serving as an effective complementary method to nucleic acid testing. Its faster testing speed and lower cost allow for large-scale rapid preliminary screening. Commonly used SARS-CoV-2 antigen detection kits, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLIA), and fluxoplasmographic immunoassay (LFIA) kits, can be used for the specific detection of SARS-CoV-2 antigens. Among the various methods, enzyme-linked immunosorbent assay (ELISA) has relatively low equipment requirements, does not require large instruments, and can achieve quantitative detection of COVID-19 antigens. However, this method is relatively cumbersome in operation and has a long detection cycle. Chemiluminescent enzyme immunoassay (CLIA) can also achieve quantitative detection of COVID-19 antigens, but this method requires expensive and complex instruments and professional operators. Fluoroassay (LFIA) can provide rapid test results, but this method can only achieve qualitative or semi-quantitative analysis of antigens and cannot accurately detect antigen concentration.

[0004] Therefore, there is a need to develop a rapid, simple, and sensitive detection technology for the clinical diagnosis of the novel coronavirus, which can effectively improve the ability to prevent and control the epidemic. Summary of the Invention

[0005] The purpose of this invention is to provide a novel coronavirus antigen detection method that enables simpler, more accurate, and minimal-volume quantitative detection of the SARS-CoV-2 spike protein, overcoming the shortcomings and limitations of existing chemiluminescent enzyme immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) methods for detecting SARS-CoV-2 antigens, such as long processing times and complex procedures. This invention offers high accuracy, strong specificity, requires a small sample volume, and is simple to operate, enabling specific detection of the SARS-CoV-2 spike protein.

[0006] The technical solution adopted in this invention is as follows:

[0007] In a first aspect, the present invention provides a method for quantitative detection of novel coronavirus antigen, comprising the following steps:

[0008] a) Add the test sample to an excess of fluorescently labeled aptamer solution for pre-incubation;

[0009] b) The incubated mixed liquid is passed into the surface of a functionalized optical fiber sensing element modified with nucleic acid aptamer complementary DNA strands to react;

[0010] c) Fluorescently labeled aptamers bind to the surface of functionalized fiber optic sensing elements, causing changes in fluorescence signals. The higher the sample concentration, the greater the fluorescence signal value. A standard curve is established between the fluorescently labeled aptamer and the SARS-CoV-2 spike protein antigen standard mixture and the fluorescence signal value of the functionalized fiber optic sensing element. The concentration of novel coronavirus antigen in the sample is calculated based on the fluorescence signal and the standard curve.

[0011] In step a) above, the sample to be tested contains the spike protein antigen of SARS-CoV-2; the fluorescently labeled aptamer is the following nucleic acid aptamer Seq No. 1, whose 5' end is modified with Cy5.5 fluorescent dye:

[0012] 5'-Cy5.5-TGGGAGCCTGGGACATAGTGGGGAAAGAGGGGAAGAGTGGGTCTT-3';

[0013] The pre-incubation time is 3–8 minutes;

[0014] In step b) above, the fabrication method of the functionalized fiber optic sensing element is as follows:

[0015] 1) Use hydrofluoric acid to etch the end of the optical fiber after the coating has been removed, and then clean and dry the optical fiber after the reaction is complete.

[0016] 2) Immerse the etched optical fiber in a piranha solution to hydroxylate the fiber surface, then clean and dry it.

[0017] 3) Place the fully dried optical fiber in an ATPES toluene solution to react and amminate the surface of the optical fiber, then wash it.

[0018] 4) Heating removes unbonded APTES from the amination-treated optical fiber;

[0019] 5) Place the optical fiber in a glutaraldehyde solution to react and then clean it;

[0020] 6) The optical fiber is placed in a solution of complementary DNA strands modified with -NH2 at the 5' end to react, thereby coupling the complementary DNA strands onto the surface of the optical fiber; the complementary DNA strands of the nucleic acid aptamer are Seq No. 2:

[0021] 5'-Biotin-CCCACTATGTCCCAGGCTCCA-3';

[0022] 7) Use BSA solution to seal unreacted sites on the surface of the optical fiber to obtain the functionalized optical fiber sensing element.

[0023] Preferably, after step c), a urea regeneration solution is introduced into the surface of the functionalized fiber optic sensing element to regenerate the functionalized fiber optic sensing element so that it can be used for the detection of the next sample.

[0024] Preferably, in step 1) above, the optical fiber has a length of 4-6 cm and a diameter of 500-600 micrometers; and is etched to a core diameter of 200-250 μm.

[0025] Preferably, in step 2) above, the piranha solution has a ratio of V(H2SO4):V(H2O2) = 3:1, and the reaction conditions are: hydroxylation time of 20-40 minutes.

[0026] Preferably, in step 3) above, the amination time is 1 to 2 hours.

[0027] Preferably, in step 4) above, the removal is completed by drying, the oven temperature is 180°C, and the reaction time is 1 to 2 hours.

[0028] Preferably, in step 5) above, the concentration of the glutaraldehyde solution is 3% to 5%, and the reaction time is 1 to 2 hours.

[0029] Preferably, in step 6) above, the temperature at which the optical fiber reacts with the complementary DNA solution is 2–6°C and the reaction time is 8–12 hours.

[0030] Preferably, in step 7) above, the concentration of the BSA solution is 1-2 mg / mL, and the sealing time is 1-2 h.

[0031] Preferably, in step c) above, the fluorescence signal acquisition time is 3 to 10 minutes;

[0032] Preferably, in step d) above, the regeneration time is 10-20 minutes;

[0033] Preferably, the concentration of the fluorescently labeled aptamer solution is 0.5–2 μM, the volume is 50–200 μL, and the volume of the sample to be tested is 100–300 μL.

[0034] In a second aspect, the present invention provides a functionalized optical fiber sensing element, which is prepared using the method described in the first aspect.

[0035] The beneficial effects achieved by this invention are as follows:

[0036] This invention modifies the surface of an optical fiber with the complementary DNA strand of a viral antigen nucleic acid aptamer. The viral antigen in the sample first reacts with the fluorescently labeled nucleic acid aptamer, causing a conformational change in the aptamer. Then, a mixed solution containing the viral antigen and the fluorescently labeled nucleic acid aptamer is passed through the surface of a functionalized optical fiber sensing element. The conformationally changed fluorescently labeled nucleic acid aptamer binds to the complementary DNA (cDNA) on the surface of the optical fiber sensing element. The fluorescence intensity received by the optical fiber sensing element increases with the amount of binding. Quantitative detection is achieved by detecting the change in fluorescence intensity received by the optical fiber sensing element. This invention overcomes the shortcomings of existing chemiluminescent enzyme immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) for detecting COVID-19 antigens, such as long processing time and complex operation. It offers high accuracy, high specificity, requires a small sample volume, and is simple to operate, enabling specific detection of viral antigens. Attached Figure Description

[0037] Figure 1 This is a typical curve diagram for the detection of the SARS-CoV-2 antigen.

[0038] Figure 2 This is a standard curve for detecting the SARS-CoV-2 antigen. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below with reference to specific implementation examples, so that those skilled in the art can better understand the invention and implement it. However, the examples given are not intended to limit the present invention.

[0040] The case cited here involves the spiked detection of novel coronavirus IgG antibodies in human serum samples.

[0041] The SARS-CoV-2 antigen (spike protein 40591-V08H) standard used in the following examples was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0042] The human serum used in the following examples was purchased from Beijing Lamborghide Biotechnology Co., Ltd.

[0043] The artificial saliva used in the following examples was purchased from Feijing Biotechnology Co., Ltd.

[0044] Example

[0045] The method for detecting the spike protein antigen of the novel coronavirus (SARS-CoV-2) provided by this invention includes the following steps:

[0046] (1) Fabrication of functional fiber optic sensing elements

[0047] a) Use hydrofluoric acid to etch the end of the optical fiber with a total length of 5.5cm until the core diameter is 220μm and the length of the etched area is 2.5cm.

[0048] b) Immerse the optical fiber in a piranha solution (H2SO4:H2O2 = 3:1) and let it stand for 30 minutes to hydroxylate the surface of the optical fiber. Then, ultrasonically clean it three times with ultrapure water until the solution is neutral and dry it.

[0049] c) The fully dried optical fiber was placed in a 2% ATPES toluene solution and reacted for 1 hour to aminate the surface of the optical fiber. Then it was cleaned three times with toluene and ethanol and dried.

[0050] d) The amination-treated optical fiber was placed in an oven at 180°C for 1 hour to remove unbound APTES by volatilization;

[0051] e) Place the optical fiber in a 4% glutaraldehyde solution and react for 1 hour, then rinse thoroughly with ultrapure water;

[0052] f) Place the optical fiber in a solution of complementary DNA (5'-Biotin-CCCACTATGTCCCAGGCTCCCA-3') with -NH2 modified at the 5' end and react overnight at 4°C to couple the complementary DNA strand onto the surface of the optical fiber.

[0053] g) Block unreacted sites on the fiber surface with 2 mg / mL BSA solution for 1 h to reduce nonspecific adsorption.

[0054] (2) Establishment of the standard curve

[0055] a) SARS-CoV-2 spike protein antigen standards with gradient concentrations of 10, 50, 100, 200, 300, 500, 1000, 1500, 5000, and 10000 ng / mL were used for detection. The specific detection process is as follows:

[0056] b) Add 200 μL of the sample to be tested to 100 μL of the 1 μM fluorescently labeled aptamer solution prepared in step (1), and pre-incubate for 5 min on a constant temperature mixer; purify the surface of the functionalized fiber optic sensing element with PBS buffer for 60 seconds to stabilize the light signal and obtain a baseline signal value 1; purify the surface of the functionalized fiber optic sensing element prepared in step (1) with 300 μL of the incubated mixture for 4 min to obtain a fluorescence signal value 2. Then purify the fiber surface with regeneration solution (30% urea solution) for 5 min to dissociate the complementary DNA strands of the fluorescently labeled aptamer from the aptamer on the fiber surface. The detection result is signal value 2 - signal value 1, and the typical real-time detection curve is shown below. Figure 1 As shown in the figure. The standard curve for SARS-CoV-2 IgG antigen detection is as follows. Figure 2 As shown.

[0057] (3) Serum sample testing

[0058] Nine different healthy individuals' serum samples were diluted 20-fold, and then novel coronavirus antigen was added at concentrations of 50, 200, and 1000 ng / mL for testing. The specific testing steps are as follows:

[0059] a) Dilute the serum to be tested 20 times with PBS buffer.

[0060] b) Add 200 μL of novel coronavirus antigen solution with concentrations of 50, 200, and 1000 ng / mL to the above serum samples, respectively, and pre-incubate for 5 min on a constant temperature mixer; purify the surface of the functionalized fiber optic sensing element with PBS buffer for 60 seconds to stabilize the optical signal and obtain a baseline signal value 1; purify the surface of the functionalized fiber optic sensing element with 300 μL of the incubated mixture for 4 min to obtain a fluorescence signal value 2. Then purify the fiber surface with regeneration solution (30% urea solution) for 5 min to dissociate the fluorescently labeled aptamer from the complementary DNA strand of the aptamer on the fiber surface.

[0061] c) The detection result is signal value 2 - signal value 1. The detection result is compared with the established SARS-CoV-2 antigen detection standard curve to obtain the concentration of SARS-CoV-2 antigen in the serum to be tested. The results are shown in Table 1 below:

[0062] Table 1. Spike recoveries of serum samples (20-fold dilution)

[0063]

[0064] The above results indicate that the recovery rate of SARS-CoV-2 antigen spiked in human serum samples was 92.32%–115.53%, with a relative standard deviation of less than 14.84%, and the detection results are reliable.

[0065] (4) Saliva sample testing

[0066] Saliva samples from nine different healthy individuals were diluted tenfold and then labeled with novel coronavirus antigen for testing. The specific testing steps are as follows:

[0067] a) Dilute the saliva sample to be tested 10 times with PBS buffer.

[0068] b) Add novel coronavirus antigen standard solution to the above saliva samples to make the concentration of novel coronavirus antigen in the mixed solution 50, 200 and 1000 ng / mL, respectively, and pre-incubate on a constant temperature mixer for 5 min; PBS buffer is passed into the surface of the functionalized fiber optic sensing element for 60 seconds to stabilize the light signal and obtain a baseline signal value 1; 300 μL of the incubated mixed liquid is passed into the surface of the functionalized fiber optic sensing element for 4 min to obtain a fluorescence signal value 2. Then, regeneration solution (30% urea solution) is passed into the fiber surface for 5 min to dissociate the fluorescently labeled aptamer from the complementary DNA strand of the aptamer on the fiber surface.

[0069] c) The detection result is signal value 2 - signal value 1. The detection result is compared with the established SARS-CoV-2 antigen detection standard curve to obtain the concentration of SARS-CoV-2 antigen in the saliva sample. The results are shown in Table 2 below:

[0070] Table 2 Spike recoveries of saliva samples (10-fold dilution)

[0071]

[0072] The above results indicate that the recovery rate of SARS-CoV-2 antigen spiked in saliva samples diluted 10 times was 92.65%–113.73%, with a relative standard deviation of less than 12.45%, and the detection results are reliable.

[0073] The detection limit is defined as the lowest concentration that can be detected. The linear range of detection is obtained from the established standard curve. The detection limit of the standard curve shown in the attached figure is 5.34 ng / mL, and the linear range is 141.49–9507.36 ng / mL.

[0074] The detection principle of this invention is as follows:

[0075] Cy5.5-labeled viral antigen aptamers are mixed with the viral antigen solution to be tested for a pre-reaction. Due to the high affinity of the nucleic acid aptamers, they bind tightly to the viral antigen, forming Cy5.5-labeled conjugates. After incubation for a certain period, the mixture is passed through the surface of a functionalized fiber optic sensing element. The fluorescently labeled nucleic acid aptamers bound to the viral antigen hybridize with the complementary DNA on the surface of the functionalized fiber optic sensing element. Then, fluorescence is induced by laser on the fluorescent molecules on the nucleic acid aptamers. The higher the concentration of viral antigen, the more Cy5.5-labeled conjugates are produced, and the easier it is for them to bind to the complementary DNA on the surface of the sensing element. Therefore, the stronger the fluorescence signal received by the sensing element, the more directly proportional the detected fluorescence signal value is to the content of SARS-CoV-2 antigen in the sample. This allows for the quantitative detection of viral antigens in serum and saliva samples.

[0076] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made using the specification and content of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for quantitative detection of novel coronavirus antigen, characterized in that, Includes the following steps: a) Add the sample to be tested to the fluorescently labeled aptamer solution for pre-incubation; b) The incubated mixed liquid is passed into the surface of a functionalized optical fiber sensing element modified with nucleic acid aptamer complementary DNA strands to react; c) Establish a standard curve between the fluorescently labeled aptamer and SARS-CoV-2 spike protein antigen standard mixture and the fluorescence signal value of the functionalized fiber optic sensing element, and calculate the concentration value of the novel coronavirus antigen in the sample based on the fluorescence signal and the standard curve. In step a) above, the fluorescently labeled aptamer is a nucleic acid aptamer Seq No. 1 whose 5' end is modified with Cy5.5 fluorescent dye: 5'-Cy5.5-TGGGAGCCTGGGACATAGTGGGGAAAGAGGGGAAGAGTGGGTCTT-3'; The pre-incubation time is 3–8 minutes; In step b) above, the fabrication method of the functionalized fiber optic sensing element is as follows: 1) Use hydrofluoric acid to etch the end of the optical fiber after the coating has been removed, and then clean and dry the optical fiber after the reaction is complete. 2) Immerse the etched optical fiber in a piranha solution to hydroxylate the fiber surface, then clean and dry it. 3) Place the fully dried optical fiber in an ATPES toluene solution to react and amminate the surface of the optical fiber, then wash it. 4) Heating removes unbonded APTES from the amination-treated optical fiber; 5) Place the optical fiber in a glutaraldehyde solution to react and then clean it; 6) The optical fiber is placed in a solution of complementary DNA strands modified with -NH2 at the 5' end to react, thereby coupling the complementary DNA strands onto the surface of the optical fiber; the complementary DNA strands of the nucleic acid aptamer are Seq No. 2: 5'-Biotin-CCCACTATGTCCCAGGCTCCA-3'; 7) Seal the unreacted sites on the surface of the optical fiber to obtain the functionalized optical fiber sensing element.

2. The method for quantitative detection of novel coronavirus antigen according to claim 1, characterized in that, In step 1) above, the optical fiber is 4-6 cm long and 500-600 μm in diameter; it is etched to a core diameter of 200-250 μm; in step 2) above, the piranha solution is V(H2SO4):V(H2O2) = 3:1, and the reaction conditions are: hydroxylation time is 20-40 minutes; in step 3) above, the amination time is 1-2 hours.

3. The method for quantitative detection of novel coronavirus antigen according to claim 1, characterized in that, In step 4) above, the removal is completed by drying, with the oven temperature at 160-200℃ and the reaction time at 1-2 hours.

4. The method for quantitative detection of novel coronavirus antigen according to claim 1, characterized in that, In step 5) above, the concentration of the glutaraldehyde solution is 3% to 5%, and the reaction time is 1 to 2 hours.

5. The method for quantitative detection of novel coronavirus antigen according to claim 1, characterized in that, In step 6) above, the temperature at which the optical fiber reacts with the complementary DNA solution is 2–6°C and the reaction time is 8–12 hours.

6. The method for quantitative detection of novel coronavirus antigen according to claim 1, characterized in that, In step 7) above, the concentration of the BSA solution is 1-2 mg / mL, and the sealing time is 1-2 h.

7. The method for quantitative detection of novel coronavirus antigen according to claim 1, characterized in that, After step c) is completed, urea regeneration solution is introduced into the surface of the functionalized fiber optic sensing element to regenerate the functionalized fiber optic sensing element so that it can be used for the detection of the next sample.

8. The method for quantitative detection of novel coronavirus antigen according to claim 1, characterized in that, In step c) above, the fluorescence signal acquisition time is 3 to 10 minutes; in step d), the regeneration time is 10 to 20 minutes.

9. The method for quantitative detection of novel coronavirus antigen according to claim 1, characterized in that, The concentration of the fluorescently labeled aptamer solution is 0.5–2 μM, the volume is 50–200 μL, and the volume of the sample to be tested is 100–300 μL.

10. A functionalized fiber optic sensing element, prepared by the method described in any one of claims 1 to 6.

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