Kit for detecting HPV mRNA based on dNTPs-CHA combined with multiple technologies and application

Abstract

The invention discloses a kit for detecting HPV (human papillomavirus) mRNA (messenger Ribonucleic Acid) based on dNTPs-CHA combined with multiple technologies and application, and belongs to the field of HPV nucleic acid detection. The invention relates to an HPV (human papillomavirus) detection probe, which comprises a probe H1, a probe H2, a probe H3, a probe H4 and a probe H5, wherein the nucleotide sequence of the probe H1 is SEQ ID NO. 2, SEQ ID NO. The nucleotide sequence of the probe H1 is SEQ ID NO.1, the nucleotide sequence of the probe H2 is SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9 or SEQ ID NO.12, and the SEQ ID NO.2, the SEQ ID NO.5, the SEQ ID NO.8 and the SEQ ID NO.11 in the probe H1 are respectively matched and corresponding. According to the present invention, through the signal amplification ability of dNTPs-CHA, the nucleic acid amplification reaction is performed under the constant temperature condition, and the fluorescent microarray detection chip and the lateral flow immunochromatography technology are combined to inspect the reaction result, such that the HPV nucleic acid can be rapidly detected with high sensitivity and specificity; and a solid foundation is hopefully laid for early screening, diagnosis and curative effect evaluation of HPV in the future.

Description

Technical Field

[0001] The present invention relates to the field of HPV nucleic acid detection, and in particular to a kit for detecting HPV mRNA based on dNTPs-CHA combined with multiple technologies and its application. Background Art

[0002] Cervical cancer is the seventh most common cancer in the world and the second most common gynecological malignancy after breast cancer. Persistent infection with high-risk human papillomavirus (HPV) is the main pathogenic factor of cervical intraepithelial neoplasia and cervical cancer. It usually takes 10 to 20 years from HPV infection to the development of invasive cervical cancer. Cervical cancer has a long and reversible precancerous lesion period. During the precancerous lesion period, treatment methods such as cervical conization can be used to prevent its malignant progression, which can prevent 99% of lesions from developing into invasive cervical cancer. Before the HPV vaccine is widely used in the population, early screening for cervical cancer is still the main means of preventing and controlling cervical cancer. The second edition of the WHO "Guidelines for Screening and Treatment of Cervical Precancer Lesions" clearly recommends HPV testing as the preferred screening method.

[0003] At present, the main methods for clinical diagnosis of HPV infection include second-generation hybrid capture technology (HC-II) and PCR methods, all of which have high sensitivity and specificity. However, in many underdeveloped areas, due to the high cost of HC-II or the lack of PCR laboratory conditions, the naked eye observation method with relatively low sensitivity and specificity but low detection cost and relatively simple operation is still the main means of early screening of cervical cancer. It can be seen that the screening method of cervical cancer cannot be simply equated with the clinical diagnosis method, and it is necessary to consider many aspects such as detection cost, medical resources (manpower and material support), the difficulty of operation of the detection method, and time consumption. Therefore, it is of great significance to develop a new HPV detection method that is easy to operate, has a short detection time, low equipment dependence, low detection cost, and can meet the needs of primary medical institutions or large-scale screening.

[0004] Based on the above background, the purpose of this application is to develop a simple, highly sensitive, and low-cost detection platform based on dNTPs-catalyzed hairpin assembly (CHA) isothermal enzyme-free nucleic acid amplification technology that reduces background signals, to achieve rapid detection of E6 / E7 mRNA of HPV16 and 18. This method is low-cost (single time <13 yuan), easy to operate, has low equipment requirements, does not require nucleic acid extraction, greatly shortens the detection time, and makes HPV self-testing at home possible. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention proposes a kit and application for detecting HPV mRNA based on dNTPs-CHA combined with multiple technologies.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] The first aspect of the present invention relates to an HPV detection probe, comprising:

[0008] Probe H1, nucleotide sequence: SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8 or SEQ ID NO.11;

[0009] And, probe H2, the nucleotide sequence is: SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9 or SEQ ID NO.12, which respectively matches SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11 in the corresponding probe H1.

[0010] Optionally, the 3' end of primer H1 is labeled (biotin when combined with a test strip, and fluorescent group FAM when combined with a fluorescence microarray detection chip), and the 3' end of H2 is labeled (digoxigenin when combined with a test strip, and quencher group BHQ1 when combined with a fluorescence microarray detection chip).

[0011] The second aspect of the present invention relates to an HPV detection reagent, comprising:

[0012] The above-mentioned HPV detection probe;

[0013] Reagents;

[0014] Target mRNA, the sequence is SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7 or SEQ ID NO.10;

[0015] And one or more of fluorescent lateral flow immunochromatography test strips (abbreviated as fluorescent LFIA), latex microsphere lateral flow immunochromatography test strips (abbreviated as latex microsphere LFIA), and fluorescent microarray detection chips.

[0016] Optionally, the fluorescent lateral flow immunochromatographic test strip is double-modified with digoxigenin and biotin, and comprises a sample pad, a conjugate pad and a NC (nitrocellulose filter membrane) membrane;

[0017] Optionally, the conjugate pad of the latex microsphere lateral flow immunochromatography test strip is sprayed with red latex microspheres labeled with streptavidin (SA).

[0018] Optionally, the fluorescent microarray detection chip mixes Streptavdin-modified magnetic beads with probe H1 and incubates them to obtain magnetic bead-bound probe H1, and mixes the probe H2 described in the claim with dNTPs, wherein the concentration of the probe H1 and probe H2 is 5 nM, and the concentration of the dNTPs is 250 nM.

[0019] Optionally, the reaction reagents include PBS buffer, polyethylene glycol 20000, polyethylene glycol 4000, bovine serum albumin, BSA-labeled biotin, casein, trehalose, anti-Digoxin monoclonal antibody, streptavidin-labeled red latex microspheres, Streptavdin-modified magnetic beads, TNaK buffer, dNTPs solution and Taqman one-step fluorescent quantitative PCR kit.

[0020] The third aspect of the present invention relates to a method for preparing an HPV detection reagent, comprising the following steps:

[0021] Designing the above-mentioned HPV detection probe;

[0022] Also includes any one or more of step A, step B or step C:

[0023] Step A: preparing a fluorescent lateral flow immunochromatography test strip, wherein one end of the NC membrane of the fluorescent lateral flow immunochromatography test strip is interlaced with a binding pad and a sample pad, and the NC membrane contains anti-Digoxin monoclonal antibodies and biotin-modified bovine serum albumin;

[0024] Step B: preparing a latex microsphere lateral flow immunochromatography test strip, wherein one end of the NC membrane of the latex microsphere lateral flow immunochromatography test strip is interlaced with a conjugate pad and a sample pad, and the conjugate pad contains red latex microspheres labeled with streptavidin.

[0025] Step C: preparing a fluorescent microarray detection chip, mixing and incubating the magnetic beads modified with Streptavdin with the probe H1 to obtain the magnetic bead-bound probe H1, and mixing the above-mentioned probe H2 with dNTPs.

[0026] Optionally, in step C, the concentrations of the probe H1 and probe H2 are 5 nM, and the concentration of dNTPs is 250 nM.

[0027] Optionally, the conjugate pad of the fluorescent lateral flow immunochromatographic test strip contains Avidin-AF647 dual-labeled PEG microspheres;

[0028] A fourth aspect of the present invention relates to a non-diagnostic HPV detection method, comprising the following steps:

[0029] Using the above-mentioned HPV detection reagent, or a reagent prepared by the above-mentioned HPV detection reagent preparation method;

[0030] Mixing the sample to be tested or the target mRNA with the HPV detection probe and performing a dNTPs-CHA reaction;

[0031] The reaction product is added dropwise to the fluorescent lateral flow immunochromatography test strip, the latex microsphere lateral flow immunochromatography test strip and the fluorescent microarray detection chip, and the result is observed.

[0032] The fifth aspect of the present invention relates to the use 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.

[0033] Reaction reagents; and target mRNA, the sequences of which are SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, and SEQ ID NO.10. Also provided are fluorescent lateral flow immunochromatography test strips, latex microsphere lateral flow immunochromatography test strips, and fluorescent microarray detection chips.

[0034] Optionally, the lateral flow immunochromatography test paper comprises:

[0035] The fluorescent immunochromatographic test strip double modified with digoxin and biotin consists of a sample pad, a conjugate release pad, a membrane and an absorption pad; the latex microsphere immunochromatographic test strip, unlike the fluorescent LFIA, has red latex microspheres labeled with streptavidin sprayed in the conjugate pad.

[0036] Optionally, the fluorescent microarray chip comprises:

[0037] The reagents also include Tris, KCl, NaCl, DEPC Treated Water, etc. The cutoff value of the blank setting is 571.

[0038] Optionally, in the nucleic acid detection step, the final concentration of the primer is 5 nM, the concentration of dNTPs is 250 nM, the concentration ratio of H1 to H2 is 1:1, the reaction time is 60 min, and the reaction temperature is 30°C.

[0039] Beneficial effects of the present invention:

[0040] The present invention proposes a scheme for detecting HPV mRNA based on dNTPs-CHA combined with multiple technologies, which is remarkable for its high accuracy, convenient operation process and short detection time. Compared with the traditional PCR technology, the test results can be obtained by direct observation with the naked eye without the assistance of complex equipment. It effectively broadens the scope of application of human papillomavirus detection and enhances the accessibility and efficiency of public health services. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will be further described below in conjunction with the accompanying drawings.

[0042] Figure 1 Schematic diagram of the human papillomavirus nucleic acid detection method using isothermal amplification of catalytic hairpin self-assembly combined with lateral flow immunochromatography. Figure 1 A in the figure represents the specific step of the isothermal amplification reaction of catalyzing the self-assembly of hairpins by adding dNTPs; Figure 1 B in the figure is a schematic diagram of a fluorescent lateral flow immunochromatographic test strip, which shows the principle of the immunoassay test strip to detect the amplification products of Dig and Bio double modifications. The immunochromatographic test strip consists of a sample pad, a conjugate pad, an NC membrane and a sticky polyvinyl chloride bottom plate. Figure 1 C in the figure is a latex microsphere immunochromatography test strip. Similarly, anti-Digoxin antibody is fixed on the T line and Biotin is fixed on the C line. Unlike fluorescent LFIA, red latex microspheres labeled with streptavidin (SA) are sprayed in the conjugate pad. Figure 1 D in it is the specific steps of the HPV typing fluorescence microarray detection chip based on dNTPs-CHA.

[0043] Figure 2 The feasibility of CHA reaction was verified by non-reducing polyacrylamide gel electrophoresis. A shows the electrophoresis of CHA reaction without using dNTPs, and B shows the electrophoresis of CHA reaction after using dNTPs.

[0044] Figure 3 Fluorescence test strips were used to detect the fluorescence values ​​of the T and C lines of HPV-negative cervical epithelial cell samples. Fluorescence LFIA was used to test the fluorescence values ​​of 20 HPV-negative cervical epithelial cell samples. 20 independent experiments showed that the fluorescence values ​​of the C line were between 8000–12000, with an average of 9940.50±872.73; the fluorescence values ​​of the T line were between 0–40, all of which did not exceed 100, with an average of 16.25±12.65. Based on this, the cutoff value was set to 100 in this study, and a positive result was reported when the T line fluorescence value was ≥100.

[0045] Figure 4is the fluorescence value of blank dNTPs-CHA coupled fluorescent test strips at different concentrations. The background leakage value of blank dNTPs-CHA reaction detected by fluorescent LFIA should be less than 100. The results of 10 independent experiments showed that high concentration of probe would increase the leakage signal. The average fluorescence values ​​of T line in 500nM and 50nM groups were 311.70±63.26 and 143.80±36.34, respectively, which were significantly different from the 0fM control group (P<0.0001). The average fluorescence values ​​of the concentration groups of 5nM and below were all lower than 100, and there was no significant difference (P=0.4571). Therefore, the optimal H1 and H2 concentrations of dNTPs-CHA coupled fluorescent LFIA are 5nM.

[0046] Figure 5 To verify the detection performance of the fluorescent test strip. (A) The fluorescence value of the fluorescent test strip detecting 1nM concentration of Biotin-Digoxin double-labeled H1-H2 DNA double strands 20 times; (B) The fluorescence test strip detecting the change of fluorescence value of the gradient diluted Biotin-Digoxin double-labeled H1-H2 DNA double strands; (C) The linear relationship between the fluorescence value detected by the fluorescent test strip and the concentration of the Biotin-Digoxin double-labeled H1-H2 DNA double strands. 1nM Biotin-H1 and Digoxin-H2 were annealed to form a double-labeled H1-H2 composite double strand, and the fluorescence LFIA was used to repeat the detection 20 times to evaluate the stability. The results showed that the average fluorescence value was 7499±284.01, the CV% was 3.79%, and the coefficient of variation was less than 5%, indicating good repeatability. The dilution gradient of Biotin-Digoxin double-labeled H1-H2 was further detected. The fluorescence value decreased with the decrease of concentration, and the logarithmic transformation of concentration and fluorescence value showed a good linear relationship (R 2 =0.9969). The results showed that 5 nM was the optimal concentration of H1 and H2 when using dNTPs-CHA coupled fluorescent LFIA, and the method had good repeatability, stability and linearity.

[0047] Figure 6The performance verification and condition optimization of latex microsphere immunochromatographic test strips. (A) Typical results of latex microsphere LFIA detection of blank dNTPs-CHA with different probe concentrations; (B) Typical results of latex microsphere LFIA detection of Biotin-Digoxin dual-labeled H1-H2 complex with different probe concentrations. Latex microsphere LFIA detects blank dNTPs-CHA background leakage signals with different probe concentrations. When the concentrations of Biotin-H1 and Digoxin-H2 are 100nM, the blank reaction produces visible red bands on the T line, while there are no obvious bands at concentrations of 10nM and below. The results of 10 repeated experiments are consistent, indicating that there is no obvious leakage signal at concentrations below 10nM. Therefore, the optimal probe concentration of dNTPs-CHA coupled latex microsphere LFIA is 10nM. The detection of Biotin-Digoxin dual-labeled H1-H2 with different concentrations shows concentration dependence, and the T line of high-concentration samples is more obvious.

[0048] Figure 7 The detection limits of dNTPs-CHA coupled fluorescent LFIA for (A) HPV16 E6, (B) HPV16 E7, (C) HPV18E6 and (D) HPV18 E7 mRNA. The present application explores the detection limits of fluorescent LFIA and latex microsphere LFIA by gradient dilution of the target sequence in HPV-negative cervical exfoliated cell samples. In the fluorescent LFIA, the concentrations of Biotin-H1 and Digoxin-H2 probes are 5nM, and the concentration of dNTPs is 250nM. After the reaction is completed, the reaction solution is added dropwise to the fluorescent LFIA for detection. With a fluorescence value of 100 as the cutoff, the detection limit (LOD) of the fluorescent LFIA for HPV16 E6 mRNA is 100aM, and the fluorescence value has a good linear relationship with the concentration (R 2 =0.9338). The detection limits of HPV16 E7, HPV18 E6, and E7 were 10aM, 100aM, and 10aM, respectively. The detection limits of the four sequences all reached the aM level, and the linear relationship was good (R 2 are all greater than 0.9).

[0049] Figure 8The detection limits of dNTPs-CHA-coupled latex microspheres LFIA for (A) HPV16 E6, (B) HPV16 E7, (C) HPV18 E6, and (D) HPV18 E7 mRNA. At concentrations of 100aM and above, there is a red band visible to the naked eye on the T line, and the red detection line on the T line tends to be less obvious as the concentration of the target sequence decreases. At concentrations of 10aM and below, there is no obvious red detection line on the T line. Similar results were obtained in 6 independent repeated experiments. Therefore, the LOD of dNTPs-CHA-coupled latex microspheres LFIA is 100aM.

[0050] Fig. 9 The results of dNTPs-CHA-coupled fluorescence LFIA for detecting HPV16 and HPV18 samples determined by RT-PCR were summarized. 87 HPV16 E6 / E7-positive, 93 HPV18 E6 / E7-positive and 120 HPV-negative cervical exfoliated cell samples were detected by dNTPs-CHA-coupled fluorescence LFIA method. The 120 negative samples were randomly divided into 4 groups, with 30 HPV16 E6, E7 and HPV18 E6, E7 probes in each group. No nucleic acid extraction was required, and the probes were directly added to the samples. HPV16 E6 mRNA was positive in 83 cases, with a sensitivity of 95.40% and a specificity of 93.33%; HPV16 E7 mRNA was positive in 86 cases, with a sensitivity of 98.85% and a specificity of 100%. HPV18E6 mRNA was positive in 89 cases, with a sensitivity of 95.70% and a specificity of 96.67%. HPV18E7 mRNA was positive in 91 cases, with a sensitivity of 97.85% and a specificity of 93.33%. The sensitivity of HPV16 / 18E6 and E7 mRNA detection was above 95%, with a total specificity of 95.83%, which was highly consistent with the RT-PCR results.

[0051] Fig.10The results of dNTPs-CHA coupled latex microspheres LFIA for detecting HPV16 and HPV18 samples determined by RT-PCR are summarized. The results of dNTPs-CHA coupled latex microspheres LFIA for clinical samples showed that 81 cases were positive for HPV16 E6 mRNA, with a sensitivity of 93.10% and a specificity of 100%; 84 cases were positive for HPV16 E7 mRNA, with a sensitivity of 96.55% and a specificity of 100%, and 79 cases were positive for both, with a total sensitivity of 90.80%. 87 cases were positive for HPV18 E6 mRNA, with a sensitivity of 93.55% and a specificity of 96.67%; 88 cases were positive for HPV18 E7 mRNA, with a sensitivity of 94.62% and a specificity of 96.67%, and 87 cases were positive for both, with a total sensitivity of 93.55%. The sensitivity of dNTPs-CHA coupled latex microspheres LFIA for detecting HPV16 and HPV18 E6 and E7 mRNA was above 90%.

[0052] Fig.11 The results of single-tube multiplex dNTPs-CHA coupled LFIA for detecting 50 HPV16 and HPV18 samples were summarized. The results showed that the detection of E6 and E7 mRNA of HPV16 and HPV18 by dNTPs-CHA-LFIA was highly consistent. In large-scale screening, only the HPV infection type can be reported without distinguishing the detection results of E6 and E7. Therefore, the detection of E6 and E7 genes can be combined to achieve single-tube multiplex dNTPs-CHA detection. To verify its feasibility, the E6 and E7 probes of HPV16 and HPV18 were mixed in one reaction tube for detection. The results of fluorescent LFIA and latex microsphere LFIA showed that the sensitivity of HPV16 and HPV18 positive samples was 96%-98%, and the specificity was 100%. This result shows that single-tube multiplex dNTPs-CHA detection does not affect the reaction efficiency, but improves the detection performance. E6 and E7 mRNA jointly trigger the reaction, which is equivalent to doubling the concentration of the target sequence, thereby improving the sensitivity and reducing the detection cost.

[0053] Fig.12 The effect of probe concentration on the signal-to-noise ratio of dNTPs-CHA fluorescence microarray. Using the CHA probe of the HPV16 E6 gene, the optimal reaction concentration was determined by comparing the signal-to-noise ratio of different concentrations of H1@Fe3O4 and H2 probes in the dNTPs-CHA reaction. The results showed that there were significant differences in the fluorescence values ​​and background signals of probes with different concentrations (P<0.0001), and the fluorescence values ​​increased with the increase of probe concentration and target sequence. In the range of 50nM to 5pM, the signal-to-noise ratio varied from 10.23±0.69 to 2.06±0.27.

[0054] Fig.13The fluorescence value of 1pM targeting sequence and negative control (TNaK buffer) was detected on the fluorescence microarray platform. At the optimal probe concentration (5nM for H1 and H2, 250nM for dNTPs), the CHA probe of the HPV16 E6 gene was used to detect 1pM targeting sequence on the fluorescence microarray platform. By repeating the experiment 10 times, the results of the fluorescence PCR instrument showed that the average fluorescence value was 8675.80±185.54, and the coefficient of variation was 2.14%, showing high stability (coefficient of variation <5%). The average fluorescence value of the blank control was 544±8.97, and the coefficient of variation was 1.65%. The cutoff value was set to 571, based on the average value of the blank dNTPs-CHA plus three times the standard deviation.

[0055] Fig.14 is the detection limit of the fluorescence microarray platform. (A) Fluorescence values ​​of different concentrations of target sequences detected by fluorescence microarray; (B) Linear relationship between fluorescence values ​​and target sequence concentration detected by fluorescence microarray. The fluorescence value of this detection method decreases as the concentration of the target sequence decreases. The minimum detection limit is 100aM, based on the cutoff value of blank dNTPs-CHA of 571. The determination coefficient R was obtained by logarithmic transformation of the fluorescence value and the concentration of the target sequence and linear fitting. 2 It is 0.9534, indicating that there is a good linear relationship between the fluorescence value and the concentration, showing that the method has strong concentration dependence.

[0056] Fig.15 The results of fluorescence microarray detection of 1pM target sequence under fluorescence microscope (10×10). Before using the fluorescence point counting method, the stability and reliability of this method need to be investigated. First, the fluorescence microscopy was used to collect the microscope image of the fluorescence microarray platform detecting 1pM target sequence. The image was adjusted by the Image J software "Best fit equalization" and other parameters. The typical result shows obvious and dense green fluorescent spots, which are fluorescent DNA-labeled magnetic beads. The distribution between the fluorescent spots is relatively uniform, without obvious aggregation.

[0057] Fig.16 The software Image J counts the fluorescent dots in the fluorescence microscopy images. Then, the software Image Jplus pro can be used to count the fluorescent dots on the collected images. The image recognition system of Image J plus pro can accurately identify each fluorescent magnetic bead in the image and count them.

[0058] Fig.17The number of fluorescent spots of 1pM target sequence was detected by the fluorescence microarray platform. To evaluate the stability of the fluorescence spot counting results, the data of 10 experiments were analyzed, and the fluorescent magnetic bead images of 6 fields of view were collected in each experiment, for a total of 60 images. The results showed that the average number of fluorescent spots was 2108±178, and the coefficient of variation was 8.46%, which was slightly higher than 5%, but lower than 10%, showing a low variability. This shows that it is feasible to use the fluorescence spot counting method to report the detection results on the fluorescence microarray platform, and the results are relatively stable.

[0059] Fig.18 Results of fluorescence microarray detection of TNaK buffer under a fluorescence microscope (10×10). Fig.10 The number of fluorescent spots in TNaK buffer was detected by the fluorescence microarray platform. For blank dNTPs-CHA, the number of fluorescent spots in each field of view was significantly less than that of 1pM target sequence. Through statistics of 60 fields of view in 10 experiments, it was found that the average number of fluorescent spots was 54.57±16.13, and the number of fluorescent spots in each field of view did not exceed 100. Based on this, 100 fluorescent spots were set as the cutoff value, and the number of fluorescent spots exceeding 100 was judged as positive, otherwise it was negative. This setting provides a clear standard to distinguish positive and negative results.

[0060] Fig.19 The fluorescence microscopy counting results of the detection gradient concentration target sequence of the fluorescence microarray platform. (A) Typical results under the microscope after contrast enhancement by Image J software; (B) Statistics of the results of 10 independent repeated experiments; (C) Linear relationship between fluorescence spot counting and detection concentration. The detection of the fluorescence microarray platform showed that the number of fluorescence spots increased with the increase of the target sequence concentration, showing obvious concentration dependence. Especially when comparing the concentrations of 1pM and 1aM, the number of fluorescence spots of 1aM was similar to that of the negative control. It was determined through 6 repeated experiments (6 images were collected each time, a total of 36 images) that the detection limit (LOD) was 10aM when 100 fluorescence spots were used as the cutoff value in each field of view. After logarithmic processing of the target sequence concentration and the number of fluorescence spots, the determination coefficient R2 of the linear relationship was 0.8296, indicating that there was a certain linear relationship between the fluorescence spots and the target sequence concentration. In addition, the relationship between the concentration of the target sequence and the amount of streptavidin-labeled Biotin-H1 on the magnetic beads is not completely linear, which may affect the accuracy of the test, but the cutoff value of 100 "lit up" magnetic beads can still effectively distinguish between positive and negative results.

[0061] Fig. 20Results of the fluorescence microarray platform for detecting HPV-negative ECC samples. The feasibility of the fluorescence microarray platform was tested using cervical exfoliated cell samples (including HPV-negative, HPV16, and HPV18 E6 / E7 mRNA-positive samples, 6 samples of each type). The fluorescence results were displayed by heat maps, and fluorescence values ​​exceeding 650 were reported as positive (green), and 650 and below were reported as negative (blue). The results showed that in the HPV-negative samples, only the internal quality control well showed positive, and the rest were negative. Under a fluorescence microscope, the internal quality control well showed obvious fluorescent spots, while the number of fluorescent spots in other detection wells was significantly less than 100.

[0062] Fig.21 The results of the fluorescence microarray platform for detecting HPV16 and 18E6 / E7 mRNA-positive ECC samples. When the fluorescence microarray platform was used to detect HPV16 or HPV18-positive samples, only the corresponding reaction wells reported positive, and the rest of the wells were negative. The results showed that there was no cross-reaction between different HPV detection CHA probes. DETAILED DESCRIPTION

[0063] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] Example 1. Design of HPV E6 / E7 mRNA detection probe and optimization and verification of dNTPs

[0065] This embodiment obtained 637 HPV16 and 146 HPV18 whole genome sequencing data from the NCBI database, and identified the mutation sites in the E6 and E7 genes through multiple sequence alignment analysis, and selected the 3'-end 30-40nt region of these genes as the target sequence to avoid the mutation sites. The secondary structure of the corresponding mRNA was predicted using NUPACK software, and the hairpin DNA probe design was optimized, in which the length of the Stem and Loop regions was 14nt / bp, and the Toehold length was adjusted to 7nt, effectively improving the CHA reaction efficiency to more than 95%. In the experiment, the catalytic efficiency of the CHA probe was close to 100% at a concentration of 1μM. The results of the fluorescent PCR instrument showed that the reaction time was prolonged, the fluorescence value increased and stabilized after 30 minutes, and the signal-to-noise ratio was 4-5. The non-reducing polyacrylamide gel electrophoresis with the addition of excess dNTPs verified the universality of the method. In addition, the optimal concentration combination of each component was also determined by the fluorescence signal-to-noise ratio calculation. All primers used were synthesized and purified by Sangon Biotechnology (Shanghai) Co., Ltd. The relevant sequences are shown in the table below (Table 1):

[0066] Table 1: Probe sequences used in this example

[0067]

[0068] Example 2. Design and preparation of fluorescent LFIA

[0069] (1) Preparation of sample pad and conjugate pad: Hydrophilic glass fiber is used as the raw material of sample pad and conjugate pad. The sample pad and conjugate pad are soaked in conjugate pad treatment solution (pH = 9.0, 0.02M PBS buffer containing 0.05wt% casein, 1wt% PEG-4000, 0.05wt% BSA, 0.01wt% trehalose) for 2 minutes for pretreatment. After soaking, take out and dry in a drying oven (25°C, 25% rh) for 5 hours. After drying, Avidin-AF647 dual-labeled PEG microspheres (5 mg / mL) are sprayed on the conjugate pad at an amount of 5 μL / cm, and then placed in a drying oven again to dry overnight. Finally, a conjugate pad containing Avidin-AF647 dual-labeled PEG microspheres is obtained.

[0070] (2) Preparation of NC membrane: Take the NC membrane with a water run-off speed of 90s and place it in a sealed box with a humidity of 50% rh for 1 hour. Dilute the anti-Digoxin monoclonal antibody to 2 mg / mL with Tris-HCl buffer at pH 6.5, and dilute the biotin-modified bovine serum albumin (Biotin-BSA) to 20 μg / mL with PBS (pH = 6.5) buffer. Streak the above two antibodies on the nitrocellulose membrane with a streaking volume of 0.8 μL / cm. After streaking, place it in a drying oven (25°C, 25% rh) to dry for 6 hours.

[0071] (3) Assembly of the test strip: The sample pad, conjugate pad and NC membrane prepared in steps (1) and (2) are adhered to the adhesive polyvinyl chloride base plate, and one end of the NC membrane is interlaced with the conjugate pad and the sample pad, with an overlapping width of 4 mm. The other end of the NC membrane is adhered to absorbent paper. Finally, the base plate is fixed and the test strip is cut into an immunochromatographic test strip of appropriate width.

[0072] Example 3. Design and preparation of latex microsphere LFIA

[0073] (1) Preparation of sample pad: The sample pad is made of glass fiber as raw material. After cutting to a suitable size, the sample pad is immersed in PBS buffer solution with a pH of 7.4. After 10 minutes, it is taken out and placed in a drying oven for vacuum drying for 6 hours.

[0074] (2) Preparation of conjugate pad: The cut glass fiber was soaked in conjugate pad buffer (PBS buffer containing 10% sucrose, 1% BSA, 0.1% PEG-20000, 2.5% trehalose and 0.02% sodium azide at pH = 7.4) for 2 minutes for pretreatment, and then placed in a drying oven for drying. The streptavidin-labeled red latex microspheres were diluted to a 0.4 w / v% dilution solution using latex buffer. The composition of the latex buffer was as follows: 0.3% PEG-20000, 0.2% PBS, 0.2% skim milk, 0.3% casein solution, 0.05% sodium azide, 0.04% potassium chloride and 0.22% glycine were added to 0.1M Tris-HCl buffer, and the pH was adjusted to 8.0 with HCl. The latex dilution was sprayed on the pretreated conjugate pad using a gold spray film scribing instrument and dried at 37°C for 4 hours.

[0075] (3) Preparation of NC membrane: Anti-digoxigenin antibody and biotin-modified albumin were diluted to 1 mg / mL with PBS buffer (pH = 7.4), and the T line and C line on the NC membrane were streaked with a streak instrument (1 μL / cm). After streaking, the membrane was dried in a vacuum oven at 30°C for 3 h.

[0076] (4) The sample pad, conjugate pad, and NC membrane obtained in the above steps are sequentially pasted on the PCV plate, and finally absorbent paper is pasted on the other end of the NC membrane to obtain a latex microsphere test strip.

[0077] Example 4. Optimization and performance verification of fluorescent immunochromatographic test strips

[0078] Before using fluorescent LFIA, conditions need to be optimized and performance needs to be verified. According to data from Nanjing Dongna Biotechnology Co., Ltd., 80 μL is the optimal sample volume, and the fluorescence reading is stable 3 minutes after sample addition, so this condition is used in subsequent experiments. First, the background fluorescence value (background fluorescence) of the fluorescent LFIA, that is, the fluorescence value generated by nonspecific binding, is evaluated. Because cervical exfoliated cell samples are detected, it is necessary to optimize for complex biological samples. Using 80 μL of sample, the T-line fluorescence value is detected after incubation at room temperature for 3 minutes, and repeated 20 times to set the cutoff value (Cutoff). To reduce background leakage, the probe concentration is optimized by blank dNTPs-CHA reaction (without adding target sequence). Different concentrations of H1-H2 probe mixture (500nM to 5pM) were tested to determine the optimal concentration by setting the Cutoff value. A 1nM mixture of Biotin-H1 and Digoxin-H2 was used to form a double-labeled H1-H2 double chain after annealing. 20 repeated tests were performed, and the coefficient of variation (CV) was calculated to evaluate repeatability. To further explore the linear relationship between fluorescence value and detection substance concentration, the double-stranded DNA was diluted 10-fold from 100 nM to 1 fM, and each concentration group was tested 10 times to verify the linear correlation between concentration and fluorescence value.

[0079] Example 5. Optimization and performance verification of latex microsphere immunochromatographic test strips

[0080] Optimization of latex microsphere LFIA Refer to Example 4. According to the optimization data of Jiangsu Yinuoweisheng Biotechnology Co., Ltd., the amount of each sample added is preferably 100 μL, and the results can be interpreted 5 minutes after adding the sample. Similarly, different concentrations (100nM–1fM) of Biotin-H1 and Digoxin-H2 were used for dNTPs-CHA reaction (without adding target sequence). After the reaction, latex microsphere LFIA was used to detect background leakage of probes of different concentrations. The probe concentration at which no obvious T line can be seen by the naked eye is the optimal concentration, and each concentration is repeated 10 times. Finally, refer to the method in Example 4 to explore the concentration dependence of latex microsphere LFIA detection.

[0081] Example 6. Detection limit of dNTPs-CHA coupled immunochromatographic test strip

[0082] Taking HPV16 E6 mRNA detection as an example, other genes are referred to the same method. The target sequence was diluted 10 times in a gradient (1nM to 1aM) with HPV-negative cervical exfoliated cell samples, and detected using dNTPs-CHA-coupled fluorescence LFIA. The concentrations of Biotin-H1 and Digoxin-H2 probes were 5nM, the concentration of dNTPs was 250nM, the reaction volume was 100μL, and the reaction was carried out at 30°C for 60 minutes. Repeat the test 6 times, with a fluorescence value of 100 as Cutoff, a T-line fluorescence value ≥100 as positive, and <100 as negative. The latex microsphere LFIA uses a Biotin-H1 and Digoxin-H2 probe concentration of 10nM and a dNTPs concentration of 500nM. After the reaction is completed, observe the T line. If a band appears, it is positive, otherwise it is negative, and repeat 6 times.

[0083] Example 7. Detection of cervical exfoliated cell samples by dNTPs-CHA coupled immunochromatographic test strips

[0084] When using dNTPs-CHA coupled immunochromatographic test strips to detect cervical exfoliated cell samples, there is no need for nucleic acid extraction. The annealed dNTPs-CHA probe is directly added to the clinical sample. After reacting at 30°C for 60 minutes, the sample is dripped on the test strip for detection. Fluorescence LFIA: Add 10μL of H1, H2 and dNTPs mixed solution (concentrations are 50nM, 50nM, and 2.5μM, respectively) to 90μL of sample. After the dNTPs-CHA reaction is completed, take 80μL and drip it on the fluorescent LFIA. T-line fluorescence values ​​≥100 are positive, and <100 are negative. Latex microsphere LFIA: Add 10μL of H1, H2 and dNTPs mixed solution (concentrations are 100nM, 100nM, and 5μM, respectively) to 90μL of sample. After the reaction is completed, take 100μL and drip it on the latex microsphere LFIA. Observe the T-line band with the naked eye. If it appears positive, it is negative, otherwise it is negative.

[0085] Example 8. Single-tube multiplex dNTPs-CHA detection

[0086] The two sets of E6 and E7 probes of HPV16 and HPV18 were mixed in a PCR tube, and a single-tube multiplex dNTPs-CHA reaction was performed to detect the mRNA of E6 and E7 at the same time. Since there are two sets of CHA probes in each reaction, the concentration of each detection probe is halved compared with Example 7. After the dNTPs-CHA reaction is completed, 50 cervical exfoliated cell samples with HPV16 E6 / E7 mRNA positive, HPV18 E6 / E7 mRNA positive, and HPV negative were detected using two test strips.

[0087] Example 9. Fluorescence microarray detection of the binding of Biotin-H1 and Streptavdin magnetic beads

[0088] According to the instructions, each milliliter of magnetic bead solution contains 10 mg of Streptavdin magnetic microspheres (about 6×10 8 ), which can bind 4nM–6nM biotin. First, mix the magnetic beads thoroughly, take 10μL of the mixed magnetic beads and mix them with 100μL of annealed Biotin-H1 probe (concentration of 1μM), and incubate them in a rotating mixer at room temperature for 10min. After the incubation, place the mixture on a magnetic stand and let it stand for 1min until the magnetic beads are absorbed to the wall of the centrifuge tube. Use a pipette to discard the liquid, wash it three times with TNaK buffer, and finally add 100μL TNaK buffer and store it at 4℃ for later use. At this time, the concentration of Biotin-H1 (referred to as H1@Fe3O4) bound to the magnetic beads in the solution is about 1μM.

[0089] Example 10. Optimization of conditions for fluorescence microarray detection chip

[0090] In order to achieve the best detection performance of the fluorescence microarray platform, the probe concentration of dNTPs-CHA (H1@Fe3O4 and FMA-BHQ1-H2) needs to be optimized. The HPV16 E6 CHA probe was used for optimization. The specific steps are as follows:

[0091] ① Dilute the H1@Fe3O4 prepared above by 10 times to 500nM, 50nM, 5nM, 500pM, and 50pM probe solutions;

[0092] ② The annealed FMA-BHQ1-H2 was diluted 10-fold to 1 μM, 100 nM, 10 nM, 1 nM, and 100 pM probe solutions, and mixed with 50 μM, 5 μM, 500 nM, 50 nM, and 5 nM dNTPs solutions at a volume ratio of 1:1 to prepare H2 probe solutions containing 50-fold concentration of dNTPs;

[0093] ③ Dilute the HPV16 E6 target sequence (T) from 62.5nM to 6.25pM in 10-fold increments, take 80μL of the test solution and add 10μL of the H1 probe solution and H2 probe solution prepared in ① and ② respectively, so that the concentrations of H1, H2 and T are the same, and perform dNTPs-CHA reaction. Use TNaK buffer as the control group and perform blank dNTPs-CHA reaction;

[0094] ④ After the reaction, use TNaK buffer for magnetic separation and washing 3 times and then adjust the volume to 100 μL. Use a fluorescent PCR instrument to measure the fluorescence value of each reaction. The fluorescence value of the blank dNTPs-CHA reaction is used as the background signal. Calculate the signal-to-noise ratio of each concentration of probe to detect the target sequence. Repeat the experiment 6 times, and the concentration with the highest signal-to-noise ratio is the optimal probe concentration.

[0095] Example 11. Automatic counting of fluorescent spots on a fluorescence microarray detection chip

[0096] Take 10 μL of the final product of the dNTPs-CHA reaction after magnetic separation and washing, drop it on a glass slide, cover it with a coverslip, and observe it under a fluorescence microscope (10×10 times). For each sample, take 6 views of evenly distributed magnetic beads. After taking pictures, use image Jpro plus 6.0 software to count the fluorescent spots in the fluorescence microscope pictures. Specifically, after importing the image to be tested, click "Best fit equalization" to automatically optimize the captured image to adjust parameters such as contrast and brightness. Then click "Count and measure objects" to count the fluorescent spots in the image. The counting method is "Automatic Bright Object", which automatically counts the number of light spots against a black background.

[0097] Example 12. Detection performance verification of fluorescence microarray detection chip

[0098] Using the optimal probe concentration explored above and the CHA probe of HPV16 E6, the detection performance of the HPV typing fluorescence microarray platform was verified. First, at the optimal probe concentration (H1 and H2 were both 5nM, dNTPs was 250nM), the target sequence at a concentration of 1pM was detected, and the results were analyzed using a fluorescent PCR instrument and a fluorescent microscope fluorescent point counting method, respectively. The test was repeated 10 times to examine the coefficient of variation of the test results. Next, the target sequence was diluted from 1pM to 1aM in a 10-fold gradient, and each concentration was repeated 6 times. The mean of the control group (TNaK buffer) plus 3 times the standard deviation was used as the cutoff value (Cutoff value), and the detection limit (LOD) of the dNTPs-CHA coupled magnetic beads was examined. The test results are as follows Figure 4 , Figure 5 shown.

[0099] Example 13. Detection of clinical samples by fluorescent microarray detection chip

[0100] Two high-risk HPV (HPV16, 18) DNA-positive (single or multiple infection) cervical exfoliated cell samples were collected, and samples with positive expression of E6 and E7 mRNA in each sample were screened by RT-PCR, and detected by fluorescence microarray platform, and compared with RT-PCR results. Specifically, 80 μL of the sample to be tested was taken, 10 μL of H1@Fe3O4 probes of E6 and E7 (the concentration of both probes was 25 nM) and 10 μL of H2 probes of E6 and E7 containing dNTPs (the concentration of both probes was 25 nM, and the concentration of dNTPs was 250 nM) were added, and after being fully mixed, placed in a 30°C water bath for reaction for 60 minutes, and the fluorescence readings in each reaction tube were read using a fluorescence PCR instrument, and images were collected using a fluorescence microscope to count the number of fluorescent spots.

[0101] Example 14. Statistical methods

[0102] Quantitative data were expressed as mean ± standard deviation. The Student-t test was used to compare the means between the two groups, and one-way ANOVA was used to compare the means between multiple groups. Enumeration data were expressed in the form of case number (rate) and compared using chi-square test. If the total sample size was less than 40 or the minimum theoretical frequency was less than 5, Fisher's exact probability method was used. P < 0.05 indicated that the difference was statistically significant. All data were plotted and statistically analyzed using GraphPad Prism 9.0.

[0103] It can be seen from the methods and experimental results of the above embodiments that the kit based on dNTPs-CHA combined with lateral flow immunochromatography technology of the present application can effectively detect HPV mRNA, and has the advantages of low detection limit, rapidity, high sensitivity and specificity. In addition, single-tube multiple dNTPs-CHA detection does not affect its reaction efficiency, but improves the detection performance.

[0104] In addition, based on the above advantages, the reagent or kit of the present application can be used in the HPV detection of biological samples, and can also be used in the detection of whether in vitro / non-biological samples or living environments contain HPV. The latter is a use for non-diagnostic purposes.

Claims

1. An HPV detection probe, characterized in that: include: Probe H1, nucleotide sequence: SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8 or SEQ ID NO.11; And, probe H2, the nucleotide sequence is: SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9 or SEQ ID NO.12, which respectively matches SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11 in the corresponding probe H1.

2. The reagent according to claim 1, characterized in that The modification scheme of the probe H1 and the probe H2 is: The 3' end of the probe H1 is labeled with biotin, and the 3' end of the probe H2 is labeled with digoxigenin; Alternatively, the 3' end of the probe H1 is labeled with a fluorescent group FAM, and the 3' end of the probe H2 is labeled with a quencher group BHQ1.

3. A HPV detection reagent, characterized in that: include: The HPV detection probe according to claim 1 or 2; Reagents; Target mRNA, the sequence is SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7 or SEQ ID NO.10; And one or more of fluorescent lateral flow immunochromatography test strips, latex microsphere lateral flow immunochromatography test strips, and fluorescent microarray detection chips.

4. The HPV detection reagent according to claim 3, characterized in that: The fluorescent lateral flow immunochromatography test strip is double-modified with digoxigenin and biotin, and comprises a sample pad, a binding pad and an NC membrane.

5. The HPV detection reagent according to claim 3, characterized in that: The conjugate pad of the latex microsphere lateral flow immunochromatography test strip is sprayed with red latex microspheres labeled with streptavidin.

6. The HPV detection reagent according to claim 3, characterized in that: The magnetic beads of the fluorescence microarray detection chip are modified with Streptavdin.

7. The HPV detection reagent according to claim 3, characterized in that: The reaction reagents include PBS buffer, polyethylene glycol 20000, polyethylene glycol 4000, bovine serum albumin, BSA-labeled biotin, casein, trehalose, anti-Digoxin monoclonal antibody, TNaK buffer and dNTPs solution.

8. 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; Also includes any one or more of step A, step B or step C: Step A: preparing a fluorescent lateral flow immunochromatography test strip, wherein one end of the NC membrane of the fluorescent lateral flow immunochromatography test strip is interlaced with a binding pad and a sample pad, and the NC membrane contains anti-Digoxin monoclonal antibodies and biotin-modified bovine serum albumin; Step B: preparing a latex microsphere lateral flow immunochromatography test strip, wherein one end of the NC membrane of the latex microsphere lateral flow immunochromatography test strip is interlaced with a conjugate pad and a sample pad, and the conjugate pad contains red latex microspheres labeled with streptavidin; Step C: preparing a fluorescent microarray detection chip, mixing and incubating the magnetic beads modified with Streptavdin with the probe H1 to obtain the magnetic bead-bound probe H1, and mixing the probe H2 with dNTPs.

9. 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 7, or the reagent prepared by the HPV detection reagent preparation method described in claim 7 or 8; Mixing the sample to be tested or the target mRNA with the HPV detection probe and performing a dNTPs-CHA reaction; The reaction product is added dropwise to the lateral flow immunochromatography test strip and the fluorescence microarray detection chip, and the result is observed.

10. Use of the HPV detection probe according to claim 1 or 2, the HPV detection reagent according to any one of claims 3 to 7, or the reagent prepared by the HPV detection reagent preparation method according to claim 7 or 8 in the preparation of a kit for detecting HPV.

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