Lateral flow immunochromatography kit based on OR logic gate combined catalysis hairpin self-assembly and application

Abstract

The invention discloses a lateral flow immunochromatography kit based on OR logic gate combined catalysis hairpin self-assembly and application, and belongs to the field of HPV nucleic acid detection. The HPV detection probe comprises a probe H1, the nucleotide sequence of which is SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9 or SEQ ID NO.13, a probe H3, a probe H4, a probe H5, a probe H6, a probe H7 and a probe H8, the nucleotide sequence of the probe H2 is as shown in SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10 or SEQ ID NO.14, and the nucleotide sequence of the probe H2 is respectively matched with the SEQ ID NO.1, the SEQ ID NO.5, the SEQ ID NO.9 and the SEQ ID NO.13 in the corresponding probe H1; the nucleotide sequence of the probe H3 is SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.11 or SEQ ID NO.15, and the nucleotide sequence of the probe H3 is SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.11 or SEQ ID NO.15 and is matched with SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9 and SEQ ID NO.13 in the corresponding probe H1 respectively.

Description

Technical Field

[0001] The present invention relates to the field of HPV nucleic acid detection, and particularly to a lateral flow immunoassay kit based on an OR logic gate combined with catalytic hairpin self-assembly and its application. Background Art

[0002] Cervical cancer is a malignant disease and the fourth leading cause of death in women. In underdeveloped regions, due to the lack of popularization of human papillomavirus (HPV) vaccination and the lack of a perfect screening program, the incidence and mortality of cervical cancer are increasing. The second edition of the WHO "Guidelines for the Screening and Treatment of Cervical Intraepithelial Neoplasia" clearly recommends HPV testing as the preferred screening method. Currently, the main HPV screening methods mainly involve the detection of HPV infection: serological testing, hybrid capture technology, qPCR technology, etc. Among them, qPCR is the most commonly used method for detecting HPV infection. However, its high cost, the necessity of professional equipment, strict detection conditions, and long detection time limit its wide application and use in various situations.

[0003] Catalytic hairpin assembly (CHA) is a series of strand displacement reactions triggered by single-stranded DNA input to achieve linear or exponential cyclic amplification, and the whole process does not require any protease assistance. At present, many CHA-based sensing methods have been developed, but they all focus on detecting a single target in a pure system or a conserved sequence in a disease. However, conserved sequences usually have complex higher-order structures, and the target sequence is likely to be masked, thus reducing the probability of the probe being triggered. In addition, a large number of inclusions in clinical samples also affect the positive detection rate indirectly. And based on the OR logic gate, a one-to-many output can be achieved, which is expected to improve the detection rate of positive samples. A logic gate is a tool for implementing Boolean logic, activated by binary-coded inputs (0 or 1), and can produce corresponding binary outputs (0 or 1).

[0004] The current mainstream kits target HPV DNA for HPV genotyping, but transient HPV infections can be cleared by the immune system. Therefore, DNA detection inevitably leads to false positives. In contrast, after persistent infection, HPV integrates into the host genome and expresses a large amount of E6 / E7 mRNA, which can reflect the integration state of HPV, thus better screening for persistent HPV infection. Compared with HPV DNA detection, E6 and E7 provide additional targets for HPV genotyping, thus improving the sensitivity of the probe for HPV genotyping. The screening method for cervical cancer cannot be simply equated with the clinical diagnosis method, and multiple aspects such as detection cost, medical resources (human and material guarantees), the ease of operation of the detection method, and the time consumption need to be considered. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a lateral flow immunoassay kit and its application based on OR logic gate combined catalytic hairpin self-assembly. The present invention is based on the OR logic gate combined catalytic hairpin self-assembly (CHA) enzyme-free nucleic acid amplification technology at room temperature, and develops a detection kit with simple operation, high sensitivity and low cost to achieve rapid detection of E6 / E7 mRNA of HPV16 and 18. This method has low cost (<20 yuan per test), simple operation, low equipment requirements, and does not require nucleic acid extraction, greatly shortening the detection time and making it possible for home self-testing of HPV.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] In the first aspect of the present invention, it relates to an HPV detection probe, including:

[0008] Probe H1, with a nucleotide sequence of SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9 or SEQ ID NO.13;

[0009] Probe H2, with a nucleotide sequence of SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10 or SEQ ID NO.14, respectively matching SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13 in the corresponding probe H1;

[0010] And probe H3, with a nucleotide sequence of SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.11 or SEQ ID NO.15, respectively matching SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13 in the corresponding probe H1.

[0011] Optionally, the 3' end of primer H1 (SEQ ID NO.1 and SEQ ID NO.5) is labeled with digoxin, the 3' end of primer H1 (SEQ ID NO.9 and SEQ ID NO.13) is labeled with fluorescein isothiocyanate, and the 3' end of all H2 and H3 is labeled with biotin.

[0012] In the second aspect of the present invention, it relates to an HPV detection reagent, including:

[0013] The above-mentioned HPV detection probe;

[0014] Reaction reagent;

[0015] And the target mRNA, with a sequence of SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12 or SEQ ID NO.16;

[0016] and a fluorescence lateral flow immunoassay strip (abbreviated as fluorescence LFIA) or a latex microsphere lateral flow immunoassay strip (abbreviated as latex microsphere LFIA).

[0017] Optionally, the fluorescence lateral flow immunoassay strip is triple-modified with Digoxin monoclonal antibody (anti-Dig), fluorescein isothiocyanate monoclonal antibody (anti-FITC), and biotin, and includes a sample pad, a conjugate pad, and an NC (nitrocellulose filter membrane) membrane;

[0018] The latex microsphere lateral flow immunoassay strip is triple-modified with Digoxin monoclonal antibody (anti-Dig), fluorescein isothiocyanate monoclonal antibody (anti-FITC), and biotin, and includes a sample pad, a conjugate pad, and an NC (nitrocellulose filter membrane) membrane;

[0019] Optionally, time-resolved fluorescence microspheres labeled with streptavidin (SA) are sprayed on the conjugate pad of the fluorescence lateral flow immunoassay strip.

[0020] Optionally, red latex microspheres labeled with streptavidin (SA) are sprayed on the conjugate pad of the latex microsphere lateral flow immunoassay strip.

[0021] Optionally, the reaction reagent includes PBS buffer, bovine serum albumin, BSA-labeled biotin, anti-Digoxin monoclonal antibody, streptavidin-labeled red latex microspheres, TNaK buffer, and a Taqman one-step fluorescence quantitative PCR kit.

[0022] The third aspect of the present invention relates to a preparation method of an HPV detection reagent, including the following steps:

[0023] Design the above-mentioned HPV detection probe;

[0024] Prepare a fluorescence lateral flow immunoassay strip, one end of the NC membrane of the fluorescence lateral flow immunoassay strip is adhesively bonded to its conjugate pad and sample pad in sequence and alternately, and the NC membrane of the fluorescence lateral flow immunoassay strip contains anti-Digoxin monoclonal antibody and biotin-modified bovine serum albumin;

[0025] Prepare a latex microsphere lateral flow immunoassay strip, one end of the NC membrane of the latex microsphere lateral flow immunoassay strip is adhesively bonded to its conjugate pad and sample pad in sequence and alternately, and the conjugate pad of the latex microsphere lateral flow immunoassay strip contains streptavidin-labeled red latex microspheres.

[0026] The fourth aspect of the present invention relates to an HPV detection method for non-diagnostic purposes, comprising the following steps:

[0027] Take the above-mentioned HPV detection reagent or the reagent prepared by the above-mentioned HPV detection reagent preparation method;

[0028] Mix the sample to be tested or the target mRNA with the above-mentioned HPV detection probe and carry out a CHA reaction;

[0029] Drop the reaction product onto the latex microsphere lateral flow immunochromatographic test strip or the fluorescence lateral flow immunochromatographic test strip and observe the result.

[0030] The above-mentioned non-diagnostic purposes include: detection of whether an HPV virus is contained in an in vitro non-biological sample, such as in the living environment.

[0031] The fifth aspect of the present invention relates to the application of the above-mentioned HPV detection probe, the above-mentioned HPV detection reagent, or the reagent prepared by the above-mentioned HPV detection reagent preparation method in the preparation of a kit for detecting HPV.

[0032] Reaction reagent; and target mRNA, with sequences of SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12 or SEQ ID NO.16. And a fluorescence lateral flow immunochromatographic test strip and a latex microsphere lateral flow immunochromatographic test strip.

[0033] Optionally, the lateral flow immunochromatographic test strip includes:

[0034] A digoxin monoclonal antibody (anti-Dig), a fluorescein isothiocyanate monoclonal antibody (anti-FITC) and a triple-modified biotin fluorescence immunochromatographic test strip, which is composed of a sample pad, a conjugate release pad, a membrane and an absorption pad, and streptavidin-labeled time-resolved microspheres are sprayed in the binding pad; the latex microsphere immunochromatographic test strip, different from the fluorescence LFIA, sprays streptavidin-labeled red latex microspheres in the binding pad.

[0035] The reagent also includes Tris, KCl, NaCl, DEPC Treated Water, etc. The cut-off values for blank settings are 204.5 (anti-Dig scribed line) and 224.5 (anti-FITC scribed line).

[0036] Optionally, in the nucleic acid detection step, the final concentration of the primer is 5 nM. The concentration ratio of H1, H2 and H3 is 1:1, the reaction time is 15 min, the reaction temperature is 30 °C, and the reaction pH is 8.0.

[0037] Advantages of the present invention:

[0038] The present invention proposes a scheme for detecting HPV mRNA using an OR logic gate-CHA based lateral flow immunoassay kit. This scheme is remarkable for its high accuracy, convenient operation process, and short detection duration. Compared with traditional PCR technology, the test results can be directly observed by the naked eye without the assistance of complex equipment. Compared with other developed kits, the detection sensitivity is enhanced by the OR logic gate, eliminating the influence of transient HPV infection. It effectively broadens the scope of application for human papillomavirus detection and enhances the accessibility and efficiency of public health services. Brief Description of the Drawings

[0039] The present invention will be further described below with reference to the accompanying drawings.

[0040] Figure 1 Schematic diagram of a method for detecting human papillomavirus nucleic acid using an OR logic gate-CHA combined with lateral flow immunoassay. Figure 1 A in it represents the specific steps of adding a clinical sample for detection using lateral flow immunoassay; Figure 1 B in it represents a schematic diagram of the catalytic hairpin assembly process; Figure 1 C in it represents a schematic diagram of a latex microsphere lateral flow immunoassay strip and the application of the OR logic gate in the lateral flow immunoassay strip. In the latex microsphere LFIA, anti-FITC antibody is immobilized on the T1 line, anti-Digoxin antibody is immobilized on the T2 line, and Biotin is immobilized on the C line. Different from the fluorescent LFIA, time-resolved microspheres labeled with streptavidin (SA) are sprayed in the conjugate pad.

[0041] Figure 2 For the feasibility and condition optimization of catalytic hairpin probe assembly. A is the verification of the reaction feasibility of CHA by non-reducing polyacrylamide gel electrophoresis; B is the verification of the feasibility of the assembly of fluorescently labeled catalytic hairpin probes; C is the optimization of the reaction time of catalytic hairpin probe assembly; D is the optimization of the reaction temperature of catalytic hairpin probe assembly; E is the optimization of the reaction pH of catalytic hairpin probe assembly; F is the optimization of the ratio of catalytic hairpin probes.

[0042] Figure 3 For the zero-background optimization of latex microsphere LFIA based on OR logic gate-CHA. When the concentration of catalytic hairpin probes is 12.5 nM or lower, no obvious bands can be observed on the T line.

[0043] Figure 4To determine the detection limits of latex microsphere LFIA based on OR logic gate-CHAs for (A) HPV18 E6 mRNA, (B) HPV18 E7 mRNA, (C) HPV18 mRNA, (D) HPV16 E6 mRNA, (E) HPV16 E7 mRNA, and (F) HPV16 mRNA in the laboratory. Among them, at concentrations of 10 pM and above, visible red bands appeared on the T lines for HPV18 E6 mRNA and HPV16 E6 mRNA. At concentrations of 1 pM and above, visible red bands appeared on the T lines for HPV18 E7 mRNA and HPV16 E7 mRNA. At concentrations of 10 fM and above, visible red bands appeared on the T lines for HPV18 E7 and HPV16 E7. Similar results were obtained from 6 independent repeated experiments. Therefore, the LOD of the OR logic gate-CHA coupled latex microsphere LFIA was 10 fM, and there was a good linear relationship between the gray value and the concentration (R 2 = 0.9507; R 2 = 0.9743). I is the verification of the specificity of the latex microsphere LFIA. The mismatched sequences, HPV 52 mRNA, HPV 52 mRNA, and HPV 33 mRNA could not trigger the latex microsphere LFIA.

[0044] Figure 5 Figure structure of the fluorescence lateral flow immunoassay strip based on OR logic gate-CHA.

[0045] Figure 6 Fluorescence values of blank OR logic gate-CHA coupled fluorescence test strips at different concentrations. The background leakage value of the fluorescence LFIA detecting the blank OR logic gate-CHA reaction should be less than 100. The results of 10 independent experiments showed that high-concentration probes would increase the leakage signal, and the average fluorescence values of the groups with concentrations of 6.25 nM and below were all less than 100, and there was no significant difference between groups. Therefore, the optimal probe concentration of the OR logic gate-CHA coupled fluorescence LFIA was 6.25 nM.

[0046] Figure 7 To determine the detection limits of fluorescence LFIA based on OR logic gate-CHAs for (A) HPV18 mRNA and (D) HPV16 mRNA in the laboratory. Under the pre-optimized experimental conditions, the linear correlation between the fluorescence values detected by TCHA-FLFIA and the target concentrations was confirmed. As shown in A, B, D, and E, the fluorescence intensity detected by FLFIA increased with the increase in mRNA concentration, and there was a good linear relationship between the fluorescence value and the logarithm of the mRNA concentration (R 2 = 96.22%; R 2= 94.66%). Using a fluorescence value of 100 as the threshold, the detection limits of FLFIA for HPV16-T and HPV18-T were both 10 aM. G is the verification of the fluorescence LFIA specificity. The mismatched sequence, HPV 52mRNA, HPV 52mRNA, and HPV 33mRNA could not trigger the latex microsphere LFIA. H and I are the fluorescence values of the fluorescence LFIA for detecting HPV 16mRNA and HPV 18mRNA multiple times, which were 1118 ± 52.02 and 918 ± 47.39 respectively, and the RSDs were 4.65% and 5.16% respectively. J is the visual output of the fluorescence LFIA under different inputs (i.e., blank, (0,0,0,0), (1,1,0,0), (0,0,1,1), and (1,1,1,1)).

[0047] Figure 8 This is a summary of the results of detecting HPV16 and HPV18 samples determined by RT-PCR using OR logic gate-CHA coupled latex microsphere LFIA / fluorescence LFIA. The OR logic gate-CHA coupled latex microsphere LFIA method was used to detect 50 cervical exfoliated cell samples positive for HPV16 E6 / E7, 50 samples positive for HPV18 E6 / E7, and 50 HPV-negative samples. Without nucleic acid extraction, the probe was directly added to the sample. The OR logic gate-CHA coupled latex microsphere LFIA detected 46 cases positive for HPV16 mRNA, with a sensitivity of 92% and a specificity of 100%; it detected 47 cases positive for HPV18 mRNA, with a sensitivity of 94% and a specificity of 100%, showing a high consistency with the RT-PCR results. The OR logic gate-CHA coupled fluorescence LFIA detected 49 cases positive for HPV16 mRNA, with a sensitivity of 98% and a specificity of 100%; it detected 48 cases positive for HPV18 mRNA, with a sensitivity of 96% and a specificity of 100%, also showing a high consistency with the RT-PCR results.

[0048] Figure 9 This is a summary of the results of detecting HPV16 and HPV18 samples determined by RT-PCR using latex microsphere LFIA / fluorescence LFIA with different probe combinations. The positive rates of the matching probes for detecting HPV16 E6 mRNA in detecting HPV16 samples were 84% / 92% (latex microsphere LFIA / fluorescence LFIA) respectively; the positive rates of the matching probes for detecting HPV16 E7 mRNA in detecting HPV16 samples were 90% / 94% (latex microsphere LFIA / fluorescence LFIA) respectively; and the positive rates of the matching probes for detecting HPV16 mRNA (E6 / E7) in detecting HPV16 samples were 92% / 98% (latex microsphere LFIA / fluorescence LFIA) respectively.

[0049] The positive rates of the matching probes for detecting HPV18 E6 mRNA in detecting HPV18 samples were 90% / 90% (latex microsphere LFIA / fluorescent LFIA) respectively; the positive rates of the matching probes for detecting HPV16 E7 mRNA in detecting HPV16 samples were 88% / 94% (latex microsphere LFIA / fluorescent LFIA) respectively; and the positive rates of the matching probes for detecting HPV16 mRNA (E6 / E7) in detecting HPV16 samples were 94% / 96% (latex microsphere LFIA / fluorescent LFIA) respectively. This result indicates that multiplex CHA detection does not affect the reaction efficiency, but instead improves the detection performance, and verifies the role of the OR logic gate in improving the detection sensitivity from clinical samples. Detailed implementation manners

[0050] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part rather than all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0051] Example 1. Design of HPV E6 / E7 mRNA detection probes and optimization and verification of reaction conditions

[0052] In this example, 637 HPV16 and 146 HPV18 whole-genome sequencing data were obtained from the NCBI database. The NUPACK software was used to predict the secondary structures of the corresponding mRNAs, and the design of hairpin DNA probes was optimized. The results of the fluorescence PCR instrument showed that as the reaction time extended, the fluorescence value increased and stabilized after 15 minutes, and the signal-to-noise ratio was 8 - 9. The universality of the method was verified by non-reducing polyacrylamide gel electrophoresis. In addition, the optimal concentration combination, reaction temperature, and pH of each component were also determined by calculating the fluorescence signal-to-noise ratio. All primers used were synthesized and purified by Shanghai Jierui Biotechnology Co., Ltd. The relevant sequences are shown in the following table (Table 1):

[0053] Table 1: Probe sequences used in this example

[0054]

[0055]

[0056] Example 2. Design and preparation of latex microsphere LFIA

[0057] I. Preparation of spray-film labeling complex:

[0058] 1. Streptavidin-microsphere labeling

[0059] 1.1 Microsphere cleaning

[0060] Take 300 nm diameter colored microspheres into a centrifuge tube, add 50 mM MES and mix well. Centrifuge to remove the supernatant, and finally ultrasonically resuspend the washed microspheres in MES buffer.

[0061] 1.2 Microsphere activation

[0062] Weigh 20 mg of NHS and EDC each, dissolve them in 50 mM MES (pH 6.5), and prepare them freshly, that is, 20 mg / mL NHS and EDC. Then, take 6 μL of 20 mg / mL NHS and 3 μL of 20 mg / mL EDC and add them to the washed microspheres respectively, mix quickly and incubate for 20 min.

[0063] 1.3 Antibody conjugation

[0064] After incubation, wash twice with 50 mM MES buffer, and finally ultrasonically resuspend the washed microspheres in 50 mM MES. Then, take the formulated amount of streptavidin into a centrifuge tube, add the activated microspheres, mix quickly, and incubate at room temperature for 2 hours.

[0065] 1.4 Microsphere blocking

[0066] After conjugation, add 60 μL of blocking solution, mix well at room temperature and block for 1 hour. Then, wash the conjugated microspheres with 600 μL of blocking solution and repeat the washing twice. Then, resuspend the microspheres in Tris buffer and store at 4°C. For long-term storage, add a preservation solution containing 0.2% BSA and preservatives.

[0067] 2. Chicken IgY-microsphere labeling

[0068] 2.1 Microsphere cleaning

[0069] Take 300 nm colored microspheres into a centrifuge tube, add 50 mM MES and mix well. Centrifuge to remove the supernatant, and finally ultrasonically resuspend the washed microspheres in MES buffer.

[0070] 2.2 Microsphere activation Weigh 20 mg of NHS and EDC each, dissolve them in 50 mM MES (pH 6.5), and prepare them freshly, that is, 20 mg / mL NHS and EDC. Then, take 6 μL of 20 mg / mL NHS and 3 μL of 20 mg / mL EDC and add them to the washed microspheres respectively, mix quickly and incubate for 20 min.

[0071] 2.3 Antibody conjugation

[0072] After the incubation, wash twice with 50 mM MES buffer, and finally resuspend the washed microspheres by ultrasound in 50 mM MES; then take the formulated amount of chicken IgY in a centrifuge tube, add the activated microspheres, mix quickly, and incubate at room temperature for 2 hours.

[0073] 2.4 Microsphere blocking

[0074] After the conjugation, add the blocking solution, mix well at room temperature and block for 1 hour; then wash the conjugated microspheres with the blocking solution, repeat the washing 2 times, and then resuspend the microspheres in Tris buffer and store at 4°C. For long-term storage, a preservation solution containing 0.2% BSA and preservatives needs to be added.

[0075] 3. Preparation of the spray film-labeled complex

[0076] Take the above-labeled streptavidin-microsphere complex and chicken IgY-microsphere labeled complex and mix them according to 15:1, and spray them evenly on the glass fiber membrane material at a spraying volume of 5 μl / cm to prepare a microsphere conjugate pad. Set the drying oven temperature at 34 ± 3°C and the humidity ≤ 40%, and dry the conjugate pad overnight for 16 - 20 h.

[0077] II. Preparation of the nitrocellulose membrane scribing

[0078] 1. Attach the nitrocellulose membrane (NC membrane) to the PVC bottom plate according to the preset position and equilibrate at room temperature for half an hour. 2. Preparation of the scribing solution:

[0079]

[0080]

[0081] 3. Then scribe the NC membrane with the liquid to be coated according to the above parameters. Set the drying oven temperature at 34 ± 3°C and the humidity ≤ 40%, and place the scribing large plate in the drying oven to dry overnight for 16 - 20 h.

[0082] III. Large plate assembly

[0083] Cut the absorbent paper and the glass cellulose membrane, and then laminate the components in the order of absorbent paper, NC membrane, conjugate pad, and sample pad.

[0084] IV. Reagent card packaging

[0085] Cut the laminated large plate into strips, then assemble the reagent strips into the card shell, add desiccant and seal and store in an aluminum foil bag.

[0086] Example 3. Design and preparation of fluorescence LFIA

[0087] I. Preparation of the spray film-labeled complex

[0088] 1. Streptavidin-Microsphere Labeling

[0089] 1.1 Microsphere Cleaning

[0090] Take time-resolved fluorescence microspheres with a particle size of 200 nm into a centrifuge tube, add 50 mM MES and mix well. Centrifuge to remove the supernatant. Finally, ultrasonically resuspend the washed microspheres in MES buffer solution.

[0091] 1.2 Microsphere Activation

[0092] Weigh 20 mg of NHS and EDC each, dissolve them with 50 mM MES (pH 6.5), and prepare them freshly, that is, 20 mg / mL NHS and EDC; then take 6 μL of 20 mg / mL NHS and 3 μL of 20 mg / mL EDC respectively and add them to the washed microspheres, mix quickly and incubate for 20 min.

[0093] 1.3 Antibody Conjugation

[0094] After the incubation, wash twice with 50 mM MES buffer solution. Finally, ultrasonically resuspend the washed microspheres in 50 mM MES; then take the formulated amount of streptavidin into a centrifuge tube, add the activated microspheres, mix quickly, and incubate at room temperature for 2 hours.

[0095] 1.4 Microsphere Blocking

[0096] After the conjugation, add 60 μL of blocking solution, mix well at room temperature and block for 1 hour; then wash the conjugated microspheres with 600 μL of blocking solution and repeat the washing twice. Then resuspend the microspheres in Tris buffer solution and store them at 4°C. For long-term storage, add a preservation solution containing 0.2% BSA and preservatives.

[0097] 2. Chicken IgY-Microsphere Labeling

[0098] 2.1 Microsphere Cleaning

[0099] Take time-resolved fluorescence microspheres with a size of 200 nm into a centrifuge tube, add 50 mM MES and mix well. Centrifuge to remove the supernatant. Finally, ultrasonically resuspend the washed microspheres in MES buffer solution.

[0100] 2.2 Microsphere Activation

[0101] Weigh 20 mg of NHS and EDC each, dissolve them with 50 mM MES (pH 6.5), and prepare them freshly, that is, 20 mg / mL NHS and EDC; then take 6 μL of 20 mg / mL NHS and 3 μL of 20 mg / mL EDC respectively and add them to the washed microspheres, mix quickly and incubate for 20 min.

[0102] 2.3 Antibody Conjugation

[0103] After the incubation, wash twice with 50 mM MES buffer, and finally resuspend the washed microspheres by ultrasonic treatment in 50 mM MES; then take the formulated amount of chicken IgY in a centrifuge tube, add the activated microspheres, mix quickly, and incubate at room temperature for 2 hours.

[0104] 2.4 Microsphere blocking

[0105] After the conjugation, add the blocking solution, mix well at room temperature and block for 1 hour; then wash the conjugated microspheres with the blocking solution, repeat the washing twice, and then resuspend the microspheres in Tris buffer, store at 4 °C, and add a preservation solution containing 0.2% BSA and preservatives for long-term storage.

[0106] 3. Preparation of the spray film-labeled complex

[0107] Take the above-mentioned labeled streptavidin-microsphere complex and chicken IgY-microsphere labeled complex and mix them in a ratio of 5:1, and spray them evenly on the glass fiber membrane material at a spraying volume of 5 μl / cm to prepare a microsphere conjugate pad. Set the drying oven temperature at 34 ± 3 °C and the humidity ≤ 40%, and dry the conjugate pad overnight for 16 - 20 h.

[0108] II. Preparation of the nitrocellulose membrane scribing

[0109] 1. Attach the nitrocellulose membrane (NC membrane) to the PVC bottom plate according to the preset position and equilibrate at room temperature for half an hour.

[0110] 2. Preparation of the scribing solution:

[0111]

[0112] 3. Then scribe the NC membrane with the liquid to be coated according to the above parameters. Set the drying oven temperature at 34 ± 3 °C and the humidity ≤ 40%, and place the scribing large plate in the drying oven to dry overnight for 16 - 20 h.

[0113] III. Large plate assembly

[0114] Cut the absorbent paper and glass cellulose membrane, and then laminate the components in the order of absorbent paper, NC membrane, conjugate pad, and sample pad.

[0115] IV. Reagent card packaging

[0116] Cut the laminated large plate into strips, then assemble the reagent strips into the card case, add desiccant and seal and store in an aluminum foil bag.

[0117] Example 4. Optimization of the latex microsphere immunochromatographic test strip.

[0118] Before using the latex microsphere LFIA, it is necessary to optimize the conditions and verify the performance. According to the data of Jiangsu Yiruoweisheng Biotechnology Co., Ltd., the sample loading volume is 120 μL, and the colorimetric result is observed 5 minutes after sample loading. Therefore, these conditions are followed in the subsequent experiments. First, evaluate the background (background) of the latex microsphere LFIA, that is, the colorimetric phenomenon caused by non-specific binding. Perform the CHA reaction (without adding the target sequence) using catalytic hairpin probes with different concentrations (100 nM–125 pM). After the reaction, use the latex microsphere LFIA to detect the background leakage of probes with different concentrations. The probe concentration at which no obvious T line can be seen with the naked eye is the optimal concentration. Further explore the linear relationship between the colorimetric gray value and the concentration of the analyte. Dilute the mRNA in a 10-fold gradient from 1 μM to 10 aM, and repeat the detection 10 times for each concentration group to verify the linear correlation between the mRNA concentration and the gray value.

[0119] Example 5. Optimization of the fluorescence immunochromatographic test strip.

[0120] Use 80 μL of the sample (cervical exfoliated cell sample), incubate at room temperature for 3 minutes, and then detect the fluorescence value of the T line. Repeat 20 times to set the cutoff value. To reduce background leakage, optimize the probe concentration through a blank CHA reaction (without adding the target sequence). Tested different concentrations of the probe mixture (50 nM to 62.5 pM) to determine the optimal concentration by setting the cutoff value. Dilute the mRNA in a 10-fold gradient from 1 μM to 10 aM, and repeat the detection 10 times for each concentration group to verify the linear correlation between the mRNA concentration and the fluorescence value.

[0121] Example 6. Detection of cervical exfoliated cell samples using the OR logic gate-CH A coupled immunochromatographic test strip

[0122] When using the OR logic gate-CH A coupled immunochromatographic test strip to detect cervical exfoliated cell samples, nucleic acid extraction is not required. Directly add the annealed CHA probe to the clinical sample, react at 30 °C for 15 minutes, and then drop the sample on the test strip for detection. Latex microsphere LFIA: Add 30 μL of the H1, H2, and H3 mixture (concentrations are 55 nM, 55 nM, and 55 nM respectively) to 90 μL of the sample. After the reaction, take 120 μL and drop it on the latex microsphere LFIA. Observe the T line strip with the naked eye. If it appears positive, otherwise it is negative. Fluorescent LFIA: Add 20 μL of the H1, H2, and H3 mixture (concentrations are 27 nM, 27 nM, and 27 nM respectively) to 90 μL of the sample. After the catalytic reaction, take 120 μL and drop it on the fluorescent LFIA. If the fluorescence value of the T line ≥ 100, it is positive; if < 100, it is negative.

[0123] Example 7. Statistical methods for the experimental data of this application

[0124] In this application, quantitative data is expressed in the form of mean ± standard deviation. The Student-t test is used for comparing means between two groups, and one-way ANOVA is used for comparing means among multiple groups. For count data, it is expressed in the form of the number of cases (rate), and the chi-square test is used for comparison. If the total sample size is less than 40 or the minimum theoretical frequency is less than 5, Fisher's exact probability method is used. P < 0.05 indicates that the difference is statistically significant. All data are plotted and statistically analyzed using GraphPad Prism 9.0.

[0125] From the methods and experimental results of the above embodiments, it can be seen that in this application, specific probe sequences are designed, and a kit based on the OR logic gate-CHA combined lateral flow immunoassay technology is prepared, which can effectively detect HPV mRNA and has the advantages of low detection limit, rapidity, high sensitivity and specificity.

Claims

1. An HPV detection probe, characterized in that: include: Probe H1, nucleotide sequence: SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9 or SEQ ID NO.13; Probe H2, nucleotide sequence: SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10 or SEQ ID NO.14, matching SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13 in the corresponding probe H1, respectively; And, probe H3, the nucleotide sequence is: SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.11 or SEQ ID NO.15, which respectively matches SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13 in the corresponding probe H1.

2. The reagent according to claim 1, characterized in that The 3' ends of the sequences SEQ ID NO.1 and SEQ ID NO.5 were labeled with digoxigenin, the 3' ends of the sequences SEQ ID NO.9 and SEQ ID NO.13 were labeled with fluorescein isothiocyanate, and the 3' ends of all probes H2 and H3 were labeled with biotin.

3. A HPV detection reagent, characterized in that: include: The HPV detection probe according to claim 1 or 2; Reagents; and a target mRNA, the sequence of which is SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12 or SEQ ID NO.16; and fluorescent lateral flow immunochromatographic test strips or latex microsphere lateral flow immunochromatographic test strips.

4. The HPV detection reagent according to claim 3, characterized in that: The fluorescent lateral flow immunochromatography test strip is modified by Digoxin monoclonal antibody, fluorescein isothiocyanate monoclonal antibody and biotin.

5. The HPV detection reagent according to claim 3, characterized in that: The latex microsphere lateral flow immunochromatography test strip comprises a sample pad, a conjugation pad and an NC membrane which are interlaced and bonded in sequence; the conjugation pad is sprayed with streptavidin-labeled latex microspheres.

6. The HPV detection reagent according to claim 3, characterized in that: The reaction reagents include BSA-labeled biotin, anti-Digoxin monoclonal antibody and TNaK buffer.

7. A method for preparing an HPV detection reagent, characterized in that: The following steps are involved: Designing the HPV detection probe according to claim 1 or 2; A fluorescent lateral flow immunochromatography test strip is prepared, wherein one end of the NC membrane of the fluorescent lateral flow immunochromatography test strip is interlacedly bonded to its binding pad and the sample pad in sequence, and the NC membrane of the fluorescent lateral flow immunochromatography test strip contains anti-Digoxin monoclonal antibody and biotin-modified bovine serum albumin; A latex microsphere lateral flow immunochromatography test strip is prepared, wherein one end of the NC membrane of the latex microsphere lateral flow immunochromatography test strip is alternately bonded to its binding pad and the sample pad in sequence, and the binding pad of the latex microsphere lateral flow immunochromatography test strip contains red latex microspheres labeled with streptavidin.

8. A method for detecting HPV for non-diagnostic purposes, characterized in that: The following steps are involved: Using the HPV detection reagent described in any one of claims 3 to 6, or the reagent prepared by the HPV detection reagent preparation method described in claim 6 or 7; Mixing the sample to be tested or the target mRNA with the HPV detection probe to perform a CHA reaction; The reaction product is added dropwise to the latex microsphere lateral flow immunochromatography test strip or the fluorescent lateral flow immunochromatography test strip, and the result is observed.

9. Use of the HPV detection probe according to claim 1 or 2, the HPV detection reagent according to any one of claims 3 to 6, or the reagent prepared by the HPV detection reagent preparation method according to claim 6 or 7 in the preparation of a kit for detecting HPV.

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