Kit and detection method for normal-temperature nucleic acid detection of HPV (human papillomavirus)

Abstract

The invention belongs to the technical field of in-vitro detection, and particularly relates to a kit and a detection method for normal-temperature nucleic acid detection of HPV (human papillomavirus). The detection reagent is a freeze-drying reagent prepared from an RPA / CRISPR detection system, and the detection system comprises an RPA isothermal amplification reagent, a Buffer A, a front primer, a rear primer, Cas12a, gRNA, ssDNA reporter molecules, a Buffer 2.1 and a mixed freeze-drying protective additive. The composition of the mixed freeze-drying protective agent is optimized according to the composition of a detection system and a specific detection method. According to the invention, the problems of normal-temperature storage and transportation of the one-stop RPA / CRISPR detection reagent of the HPV virus are solved, the detection performance is ensured, an efficient and accurate solution is provided for large-scale screening, and the kit has a very good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology, specifically relating to a reagent kit and detection method for detecting HPV virus at room temperature using nucleic acid. Background Technology

[0002] Cervical cancer is a common malignant tumor among women worldwide. Cervical cancer screening is crucial for prevention and early diagnosis. The World Health Organization (WHO), the American Cancer Society (ACS), and Chinese expert consensus recommend human papillomavirus (HPV) testing as the preferred initial screening method. However, while current HPV PCR testing technology has good sensitivity and specificity, it is complex to operate, requires professional personnel, and has high equipment requirements, making it difficult to promote in situations with limited medical resources. Therefore, developing a convenient, rapid, and home-use HPV testing method is urgently needed.

[0003] In recent years, the combination of recombinase polymerase amplification (RPA) and the CRISPR / Cas12a system has brought new possibilities to HPV detection. It combines the rapid screening capabilities of RPA with the high efficiency and accuracy of CRISPR, providing a new approach for early screening of cervical cancer.

[0004] However, conventional RPA and CRISPR assays require cryogenic storage, and repeated freeze-thaw cycles can affect their performance stability. Therefore, this invention provides a method for preparing them as lyophilized reagents. However, RPA and CRISPR assays are complex compositions reacting within a single system, further complicating their composition. They often contain macromolecules such as DNA, RNA, and enzymes. Therefore, in the development of specific assays, improving the composition of the lyophilized reagent, particularly the composition of the lyophilization protectant, to ensure the stability of the assay and prevent performance degradation is a crucial issue. Summary of the Invention

[0005] To address the above problems, this invention provides a kit and method for detecting HPV virus using room-temperature nucleic acid testing.

[0006] A fluorescent detection reagent for detecting HPV virus at room temperature is a lyophilized reagent prepared from an RPA / CRISPR detection system, which includes the following components: One RPA isothermal amplification reagent per 28-35 μL Buffer A 36.59-45.73% v / v The pre-primer was 0.025-0.032 μM each. The subsequent primers were each at 0.025-0.032 μM. Cas12a 0.063-0.078μM gRNA 0.063-0.078 μM each ssDNA reporter molecule 0.63-0.78 μM, Buffer 2.1 2.49-3.12% v / v Mixed lyophilization protectant 18.67-23.33% v / v; The mixed lyophilization protectant comprises the following components: Glucan 40-56% w / v Maltose 30-48% w / v Mannitol 20-35%w / v Inositol 0-20% w / v.

[0007] Preferably, it is a lyophilized reagent prepared from an RPA / CRISPR detection system, wherein the RPA / CRISPR detection system comprises the following components: RPA isothermal amplification reagent 1 kit / 30μL Buffer A 39.2% v / v Each of the pre-primers was 0.027 μM. Each of the back primers was 0.027 μM. Cas12a 0.067μM 0.067 μM gRNA each ssDNA reporter molecule 0.67 μM, Buffer 2.1 2.67% v / v Mixed lyophilization protectant 20% v / v; The mixed lyophilization protectant comprises the following components: 56% w / v of dextran Maltose 48% w / v Mannitol 35%w / v Inositol 20% w / v.

[0008] This invention also provides a room-temperature nucleic acid detection strip for HPV virus, which is a lyophilized reagent made from an RPA / CRISPR detection system, the RPA / CRISPR detection system comprising the following components: One RPA isothermal amplification reagent per 28-35 μL Buffer A 36.59-45.73% v / v The pre-primer was 0.025-0.032 μM each. The subsequent primers were each at 0.025-0.032 μM. Cas12a 0.063-0.078μM gRNA 0.063-0.078 μM each ssDNA reporter molecule 0.63-0.78 μM, Buffer 2.1 2.49-3.12% v / v Mixed lyophilization protectant 18.67-23.33% v / v; The mixed lyophilization protectant comprises the following components: 15-30% w / v of dextran Lactose 0-15% w / v Monosodium glutamate 0-25% w / v Mannitol 30-40% w / v The content of lactose and monosodium glutamate is not both 0.

[0009] Preferably, it is a lyophilized reagent prepared from an RPA / CRISPR detection system, wherein the RPA / CRISPR detection system comprises the following components: RPA isothermal amplification reagent 1 kit / 30μL Buffer A 39.2% v / v Each of the pre-primers was 0.027 μM. Each of the back primers was 0.027 μM. Cas12a 0.067μM 0.067 μM gRNA each ssDNA reporter molecule 0.67 μM, Buffer 2.1 2.67% v / v Mixed lyophilization protectant 20% v / v; The mixed lyophilization protectant comprises the following components: 15-30% w / v of dextran Lactose 0-15% w / v Monosodium glutamate 0-25% w / v Mannitol 40% w / v The content of lactose and monosodium glutamate is not both 0.

[0010] Preferably, the preprime comprises at least one of the primers with nucleotide sequences as shown in SEQ ID NO. 1-SEQ ID NO. 14; The back primer includes at least one of the primers with nucleotide sequences as shown in SEQ ID NO. 15-SEQ ID NO. 28.

[0011] Preferably, the gRNA comprises at least one of the RNAs with nucleotide sequences as shown in SEQ ID NO. 29-SEQ ID NO. 42.

[0012] Preferably, the ssDNA reporter molecule is a single-stranded DNA reporter molecule modified with a fluorescent group and a quencher group.

[0013] The present invention also provides the use of the above-mentioned detection reagent in the preparation of HPV virus detection kits.

[0014] The present invention also provides a detection kit for detecting HPV virus at room temperature, comprising: Buffer B and the above-mentioned detection reagent.

[0015] The present invention also provides a method for one-stop detection of HPV virus for non-diagnostic purposes using the above-mentioned detection kit, comprising the following steps: Step 1: Redissolve the detection reagent; Step 2: Add the Buffer B and the test sample sequentially, and incubate the reaction; Step 3: Detection is performed using fluorescence detection or color development of test strips.

[0016] This invention provides an integrated RPA / CRISPR detection method for 14 high-risk HPV types, with optimized primers and gRNA, and for these specific primer and gRNA combinations, an optimal combination of lyophilization protectants. Therefore, this invention provides for the first time a lyophilized reagent and kit for integrated RPA / CRISPR detection of HPV viruses. This lyophilized reagent and kit have the following beneficial technical effects: 1. By optimizing primer and gRNA sequences and the combination of lyophilization protectants, this invention exhibits excellent sensitivity, specificity, and repeatability for HPV virus detection, meeting the needs of practical applications.

[0017] 2. This invention is the first to realize the preparation of a one-stop RPA / CRISPR detection reagent for HPV virus into a lyophilized reagent, which has the advantages of convenient storage and transportation, stable reagent performance, and ease of use.

[0018] 3. The detection method of the present invention is a one-stop detection method, which does not require the RPA amplification reaction and CRISPR / Cas12a detection to be performed in two separate reaction tubes, and there is no operation of transferring reaction solution, which can effectively avoid the problem of nucleic acid aerosol contamination.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the microfluidic chip for HPV RPA / CRISPR detection provided in Example 2.

[0022] Figure 2 The figure shows the specificity test results for Experimental Examples 3, 6 and 7. Detailed Implementation

[0023] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0024] In the following examples and experimental cases, some of the commercially available reagents were sourced from the following sources: RPA isothermal amplification reagent, Buffer A and Buffer B are from the DNA isothermal rapid amplification kit (catalog number: WLB8201KIT) of Ampu Future Biotechnology Co., Ltd.; Buffer 2.1 is from New England Biolabs and is a component of EnGen Lba Cas12a (Cpf1) (NEB #M0653).

[0025] Example 1: One-stop fluorescent detection kit and detection method for HPV virus I. Reagent Kit Composition and Preparation Method The kit in this embodiment mainly includes RPA / CRISPR system detection reagent lyophilized beads for HPV virus detection. Individual lyophilized beads are prepared for each type of HPV virus. These lyophilized beads are made by lyophilizing the RPA / CRISPR detection system shown in Table 1.

[0026] Table 1 Composition of the RPA / CRISPR detection system In other embodiments, the volume of enzyme-free water in the above system can also be adjusted as needed so that the total volume of the system is in the range of 28-35 μL.

[0027] The sequences of the front and back primers are shown in Table 2. The sequence marked with "F" is the front primer, and the sequence marked with "R" is the back primer.

[0028] Table 2. Pre- and post-primers for RPA isothermal amplification The sequence of the gRNA is shown in Table 3.

[0029] Table 3 The ssDNA reporter molecule is FAM-BHQ-labeled ssDNA: (6-FAM)TTATT(BHQ1). In other embodiments, the fluorescent group FAM can be replaced with VIC, HEX, TET, JOE, Cy5, Cy3, TAMRA, etc.

[0030] The mixed lyophilization protectant is composed of an aqueous solution containing the following components: 56% w / v dextran, 48% w / v maltose, 35% w / v mannitol, and 20% w / v inositol. The percentages are by weight / volume percentages, in g / ml.

[0031] The specific method for preparing the above RPA / CRISPR detection system into freeze-dried pellets is as follows: Using a precise dispensing system, the prepared freeze-dried mixture of the RPA / CRISPR detection system was dropped into liquid nitrogen at a volume of 30 μL. The mixture condensed into a small sphere and was allowed to sink to the bottom of the liquid nitrogen after 10 seconds. The sphere was then retrieved and transferred to a vial that had been pre-cooled with dry ice.

[0032] Once the reagent pellets have been collected, they are transferred to a freeze dryer. The freeze drying program is shown in Table 4. Table 4 II. Detection Methods Lyophilized beads prepared using the RPA / CRISPR detection system were reconstituted with 26 μL of enzyme-free water and added to the reaction tube. Then, 2 μL of Buffer B and 2 μL of the sample to be tested were added. The tube was incubated at 37°C for 50 min, while simultaneously placing it in a thermostatic fluorescence detector to collect fluorescence data every minute.

[0033] Example 2: HPV Virus One-Stop Test Strip Detection Kit and Detection Method I. Reagent Kit Composition and Preparation Method The kit in this embodiment mainly includes RPA / CRISPR system detection reagent lyophilized beads for HPV virus detection. The composition and preparation method of the lyophilized beads are basically the same as those in Example 1, except that: 1. The composition of the mixed lyophilization protectant is different. This embodiment provides two mixed lyophilization protectants suitable for one-stop HPV virus test strip detection: 1) The mixed lyophilization protectant is composed of an aqueous solution containing the following components: 15% w / v dextran, 15% w / v lactose, and 40% w / v mannitol.

[0034] 2) The mixed freeze-drying protectant is composed of an aqueous solution containing the following components: 30% w / v dextran, 25 w / v sodium glutamate, and 40% w / v mannitol.

[0035] Where the percentage is a mass-volume percentage, in g / ml.

[0036] 2. Different ssDNA reporter molecules In this embodiment, the ssDNA reporter molecule is a FAM-Biotin-labeled ssDNA: (6-FAM)TTATTATT(Biotin).

[0037] II. Detection Methods Method 1: Reconstitute lyophilized beads prepared using the RPA / CRISPR detection system with 26 μL of enzyme-free water and add them to the reaction tube. Then add 2 μL of Buffer B and 2 μL of the sample to be tested. Incubate at 37°C for 50 min to allow the reaction to proceed. Finally, take 2 μL of the reaction product, add 38 μL of 8% PEG chromatography buffer, mix well, and insert a lateral flow immunochromatographic strip (from the Milenia GenLine HybriDetect kit, purchased from Milenia Biotec). Observe the results visually within 15–22 minutes.

[0038] Method 2: Design a microfluidic chip for HPV RPA / CRISPR detection, such as Figure 1 As shown, the microfluidic chip includes a reagent area, a channel area, a reaction area, and a test strip reading area.

[0039] The testing steps are as follows: 1) Reconstitute the lyophilized bulbs prepared with the RPA / CRISPR detection system with 26 μL of enzyme-free water and add them to the reagent area.

[0040] 2) Add 2 μL of Buffer B and 2 μL of the sample to be tested to the reagent area.

[0041] 3) Pass through the channel and enter the reaction zone, where it is incubated at 37 °C for 50 min to carry out the reaction.

[0042] 4) The reaction product flows into the reading area of ​​the test strip via lateral flow immunochromatographic test strip. The results are observed visually within 15-22 minutes.

[0043] The technical solution of the present invention will be further illustrated by the following experiments.

[0044] Example 1: Optimization of Conditions for One-Stop Fluorescent Detection Method for HPV Virus I. Primer and gRNA Design This invention uses NCBI (www.ncbi.nlm.nih.gov) to search for the complete genome sequence of human papillomavirus (HPV) and selects the conserved region of the L1 gene as the target region. Based on the characteristic of Cas12a recognizing T-rich PAM sequences, target sequences are screened, and gRNAs for Cas12a of each HPV type are designed according to the cleavage characteristics of the Cas12a protein. The designed gRNAs are then optimized through experiments.

[0045] This invention utilizes Primers software for the design and optimization of specific RPA primers. All designed RPA primers were validated and analyzed using BLAST software to reduce the possibility of non-specific amplification. Furthermore, various designed RPA primers were experimentally optimized.

[0046] The optimal primers and gRNAs obtained are shown in Example 1.

[0047] II. Optimization of Detection Conditions This experimental example adjusts the detection conditions based on Example 1, comparing the fluorescence intensity of the same concentration of target gene under different conditions. The detection conditions corresponding to the fastest and highest fluorescence intensity increase within the same time period are selected as the preferred conditions. The optimal results are shown in the table below: Table 5 The composition of the optimized RPA / CRISPR detection system is shown in Example 1.

[0048] Experiment Example 2: Screening Experiment of Mixed Lyophilization Protectants for HPV One-Stop Fluorescent Detection Kit In the following experiments, reagents or conditions not specifically described were performed according to the description in Example 1. This experimental example first screened mixed lyophilization protectants using the pre-primer, post-primer, and gRNA of HPV16, and then verified that the selected mixed lyophilization protectants could be used for RPA / CRISPR system detection reagent lyophilized balls for thirteen other HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68).

[0049] 1. Screening of single protective agents This study investigated the effects of dextran, maltose, lactose, monosodium glutamate, ammonium acetate, mannitol, inositol, and dimethyl sulfoxide as lyophilization protectants in both the RPA and CRISPR detection systems on fluorescence signals.

[0050] Eight lyophilization protectants with a concentration of 20% w / v (g / ml) were added to the RPA / CRISPR detection system, respectively, to obtain RPA / CRISPR detection systems containing a single protectant (Table 6) as follows: Table 6 The fluorescence intensity of RPA / CRISPR detection systems containing different lyophilization protectants was tested. Compared with the fluorescence intensity of RPA / CRISPR detection systems without lyophilization protectants, if the fluorescence signal intensity decreased by no more than 10% within the same time period, the protectant was considered to have no inhibitory effect on the detection system. The results are shown in Table 7. Table 7 The above results indicate that dextran, maltose, lactose, mannitol, and inositol have no effect on the RPA / CRISPR detection system.

[0051] 2. Suitable concentration range of the protective agent Lyophilization protectants with a fluorescence signal intensity decrease of no more than 10% were selected and prepared at concentrations ranging from 1% to 64%. The fluorescence intensity of the RPA / CRISPR detection system was tested at different concentrations of lyophilization protectants. Compared with the fluorescence intensity of the RPA / CRISPR detection system without lyophilization protectant, if the fluorescence signal intensity decreased by no more than 10% within the same time period, the concentration was considered to have no inhibitory effect on the detection system, thus determining the suitable concentration range of the protectant. The results are shown in Table 8. Table 8 The above results indicate that the suitable concentration ranges for dextran are 8%–56%, maltose is 4%–48%, lactose is 2%–40%, mannitol is 4%–48%, and inositol is 4%–48%.

[0052] 3. Screening of lyophilization protectants for RPA / CRISPR detection system 1) The content of dextran, maltose, lactose, mannitol and inositol was optimized by a 5-factor, 4-level orthogonal experimental design, as shown in Tables 9 and 10.

[0053] Table 9 Factor Level Table Table 10 Scheme 2) Morphological examination of lyophilized bulbs using the RPA / CRISPR detection system: The morphology of the lyophilized pellets in the RPA / CRISPR testing system was observed to determine if they met the requirements. The test results are shown in Table 11. Table 11 The results above indicate that schemes 6, 11, 12, and 16 can be used for further screening.

[0054] 3) Moisture content of freeze-dried pellets in the RPA / CRISPR detection system The moisture content of the RPA / CRISPR detection system that passed the above morphological tests was determined using a cassette moisture analyzer. The results are shown in Table 12. Table 12 The results above indicate that Schemes 11 and 16 can be used for further screening.

[0055] 4) Select an RPA / CRISPR testing system with acceptable moisture content for performance testing. Following the procedure in Example 1, the fluorescence results of the above scheme were compared with the fluorescence intensity of the non-lyophilized RPA detection system and the CRISPR detection system. The results are shown in Table 13: Table 13 The above results indicate that, for the HPV virus one-stop fluorescent detection kit and detection method of the present invention, Scheme 16 is the optimal mixed lyophilization protectant formulation.

[0056] Furthermore, Scheme 16 was used to prepare lyophilized pellets of RPA / CRISPR system detection reagents for thirteen other HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68), and the obtained lyophilized pellets met the performance requirements for detection.

[0057] Example 3: Performance Validation of a One-Stop Fluorescent Detection Method for Lyophilized HPV Virus The lyophilization reagent used in this experimental example is as described in Example 1.

[0058] 1. Sensitivity test The detection method was followed as described in Example 1. The results are shown in Table 14. Table 14 2. Specificity test The detection method was followed as described in Example 1. The results are as follows: Figure 2 As shown in the figure. The results indicate that the kit in Example 1 is specific for plasmid standards of 14 high-risk HPV types.

[0059] 3. Clinical sample testing Cervical swabs were placed in saline sampling tubes, and the samples were shaken to mix. 200 μL of the sample was centrifuged at 14000 rpm for 5 min, and the supernatant was discarded. 200 μL of nucleic acid release agent was added, and the sample was incubated in a metal bath at 100℃ for 15 min. The supernatant after centrifugation was the sample to be tested. One-stop fluorescence detection was performed according to the method in Example 1. The detection results were compared with clinical detection results, and the diagnostic sensitivity and specificity were calculated, as shown in Table 15. Table 15 The above experimental results show that the HPV virus one-stop fluorescent detection kit and detection method provided in Example 1 have excellent detection performance and can meet the requirements of actual detection.

[0060] Example 4: Optimization of Conditions for One-Stop HPV Virus Test Strip Detection Method This experimental example adjusts the various detection conditions of the test strip based on Example 2. The fluorescence intensity of the same concentration of target gene detected under different conditions is compared. The detection conditions with the fastest color development speed and most obvious color development within the same time period are selected as the preferred conditions. The optimal results are shown in the table below: Table 16 Experiment Example 5: Screening Experiment of Mixed Lyophilized Protectants for HPV Virus One-Stop Test Strip Detection Kit In the following experiments, reagents or conditions not specifically described were performed according to the description in Example 2. This experimental example first screened mixed lyophilization protectants using the pre-primer, post-primer, and gRNA of HPV16, and then verified that the selected mixed lyophilization protectants could be used for RPA / CRISPR system detection reagent lyophilized balls for thirteen other HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68).

[0061] 1. Screening of single protective agents This study investigated the effects of dextran, maltose, lactose, monosodium glutamate, ammonium acetate, mannitol, inositol, and dimethyl sulfoxide as lyophilization protectants on the colorimetric signal in an RPA / CRISPR detection system.

[0062] Eight lyophilization protectants with a concentration of 20% (g / mL) were added to the RPA / CRISPR detection system respectively, resulting in RPA / CRISPR detection systems containing only one protectant (Table 17) as follows: Table 17 The colorimetric intensity of RPA / CRISPR detection systems containing different lyophilization protectants was tested and compared with the colorimetric intensity of test strips containing no lyophilization protectant. The results are shown in Table 18. Table 18 The above results indicate that dextran, lactose, monosodium glutamate, and mannitol have no effect on the color development of the test strips in the RPA / CRISPR detection system.

[0063] 2. Suitable concentration range of the protective agent The above-mentioned lyophilization protectants, which have no effect on the RPA / CRISPR detection system, were selected and prepared at concentrations ranging from 1% to 50%. The effects of different concentrations of lyophilization protectants on the color development of the RPA / CRISPR detection system were tested to determine the suitable concentration range of the protectant. The results are shown in Table 19. Table 19 The above results indicate that the suitable concentration range for dextran is 5%–45%, for lactose it is 10%–40%, for monosodium glutamate it is 5%–35%, and for mannitol it is 5%–40%.

[0064] 3. Screening of lyophilization protectants for RPA / CRISPR detection system 1) The content of dextran, lactose, monosodium glutamate and mannitol was optimized by a four-factor, four-level orthogonal experimental design, as shown in Tables 20 and 21.

[0065] Table 20 Factor Level Table Table 21 Scheme 2) Select all 16 schemes above and freeze-dry the RPA / CRISPR detection system. The freeze-drying system is shown in Table 22: Table 22 3) Preparation of freeze-dried pellets for RPA / CRISPR detection system Prepared according to the method of Example 1.

[0066] 4) Morphological examination of lyophilized bulbs using the RPA / CRISPR detection system: The morphology of the freeze-dried pellets in the RPA / CRISPR testing system was observed to determine if they met the requirements. The test results are shown in Table 23. Table 23 The results above indicate that schemes 5, 6, 9, 11, 13, and 16 can be used for further screening.

[0067] 5) Moisture content of freeze-dried pellets in the RPA / CRISPR detection system The moisture content of the freeze-dried bulbs of the RPA / CRISPR detection system that passed the above morphological tests was determined using a cassette moisture analyzer. The results are shown in Table 24. Table 24 The results above indicate that schemes 6, 9, and 11 can be used for further screening.

[0068] 6) Select RPA / CRISPR test system balls with qualified moisture content for performance testing. Following the method described in Example 2, the results of the test strips using the above scheme were compared with the color development of the test strips using the non-lyophilized RPA / CRISPR detection system, as shown in Table 25: Table 25 The above results indicate that, for the HPV virus one-stop test strip detection kit and detection method of the present invention, Scheme 6 and Scheme 9 are the optimal mixed lyophilization protectant formulations.

[0069] Furthermore, Schemes 6 and 9 were used to prepare lyophilized pellets of RPA / CRISPR system detection reagents for thirteen other HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68), and the resulting lyophilized pellets met the performance requirements for detection.

[0070] Experiment Example 6: Performance Verification of a One-Stop HPV Virus Detection Method Based on Lyophilized Protection The detection method in this experimental example is performed according to Method 1 in Example 2.

[0071] 1. Sensitivity test The detection method was performed according to Example 2. The results are shown in Table 26: Table 26 2. Specificity test The detection method was performed according to Example 2. The results are as follows. Figure 2 As shown.

[0072] 3. Clinical sample testing Cervical swabs were placed in saline sampling tubes, and the samples were shaken to mix. 200 μL of the sample was centrifuged at 14000 rpm for 5 min, and the supernatant was discarded. 200 μL of nucleic acid release agent was added, and the sample was incubated in a metal bath at 100℃ for 15 min. The supernatant after centrifugation was the test sample. The test results were compared with clinical test results, and the diagnostic sensitivity and specificity were calculated, as shown in Table 27.

[0073] Table 27 Example 7: Performance Verification of a Lyophilized One-Stop HPV Virus Test Strip Detection Method (Using a Microfluidic Chip) The detection method in this experimental example is performed according to method two in Example 2.

[0074] 1. Sensitivity test The designed concentration gradient is 10. -1 -10 6 The test sample of aM was processed according to the experimental steps of Method 2 in Example 2. The results are shown in Table 28: Table 28 2. Specificity test Select a concentration of 10 6 Fourteen high-risk HPV types from aM were used as the test samples. The experimental procedures were performed according to Method 2 in Example 2. The results are as follows: Figure 2 As shown.

[0075] 3. Clinical sample testing Cervical samples of 14 high-risk HPV types were collected and placed in saline sampling tubes. The samples were shaken to mix, and 200 μL of the sample was centrifuged at 14000 rpm for 5 min. The supernatant was discarded. 200 μL of nucleic acid release agent was added, and the sample was incubated in a metal bath at 100℃ for 15 min. The supernatant after centrifugation was the test sample. The test was performed according to Method 2 in Example 2. The test results were compared with clinical test results, and the diagnostic sensitivity and specificity were calculated. The results are shown in Table 29. Table 29 The above experimental results show that the HPV virus one-stop test strip detection kit and detection method provided in Example 2 have excellent detection performance and can meet the requirements of actual detection.

[0076] In summary, this invention solves the problems of room temperature storage and transportation of one-stop RPA / CRISPR test kits for HPV, supports POCT (point-of-care testing) and home self-testing, and ensures high sensitivity and specificity of the test. It enables early, rapid, portable and easy-to-use HPV testing, overcomes the dependence of traditional methods on expensive equipment and professional personnel, and provides an efficient and accurate solution for large-scale screening.

Claims

1. A fluorescent detection reagent for detecting HPV virus at room temperature, characterized in that: It is a lyophilized reagent made from an RPA / CRISPR detection system, which includes the following components: One RPA isothermal amplification reagent per 28-35 μL Buffer A 36.59-45.73% v / v The pre-primer was 0.025-0.032 μM each. The subsequent primers were each at 0.025-0.032 μM. Cas12a 0.063-0.078μM gRNA 0.063-0.078 μM each ssDNA reporter molecule 0.63-0.78 μM, Buffer 2.1 2.49-3.12% v / v Mixed lyophilization protectant 18.67-23.33% v / v; The mixed lyophilization protectant comprises the following components: Glucan 40-56% w / v Maltose 30-48% w / v Mannitol 20-35%w / v Inositol 0-20% w / v.

2. The detection reagent according to claim 1, characterized in that: It is a lyophilized reagent made from an RPA / CRISPR detection system, which includes the following components: RPA isothermal amplification reagent 1 kit / 30μL Buffer A 39.2% v / v Each of the pre-primers was 0.027 μM. Each of the back primers was 0.027 μM. Cas12a 0.067μM 0.067 μM gRNA each ssDNA reporter molecule 0.67 μM, Buffer 2.1 2.67% v / v Mixed lyophilization protectant 20% v / v; The mixed lyophilization protectant comprises the following components: 56% w / v of dextran Maltose 48% w / v Mannitol 35%w / v Inositol 20% w / v.

3. A test strip reagent for detecting HPV virus at room temperature, characterized in that: It is a lyophilized reagent made from an RPA / CRISPR detection system, which includes the following components: One RPA isothermal amplification reagent per 28-35 μL Buffer A 36.59-45.73% v / v The pre-primer was 0.025-0.032 μM each. The subsequent primers were each at 0.025-0.032 μM. Cas12a 0.063-0.078μM gRNA 0.063-0.078 μM each ssDNA reporter molecule 0.63-0.78 μM, Buffer 2.1 2.49-3.12% v / v Mixed lyophilization protectant 18.67-23.33% v / v; The mixed lyophilization protectant comprises the following components: 15-30% w / v of dextran Lactose 0-15% w / v Monosodium glutamate 0-25% w / v Mannitol 30-40% w / v The content of lactose and monosodium glutamate is not both 0.

4. The detection reagent according to claim 1, characterized in that: It is a lyophilized reagent made from an RPA / CRISPR detection system, which includes the following components: RPA isothermal amplification reagent 1 kit / 30μL Buffer A 39.2% v / v Each of the pre-primers was 0.027 μM. Each of the back primers was 0.027 μM. Cas12a 0.067μM 0.067 μM gRNA each ssDNA reporter molecule 0.67 μM, Buffer 2.1 2.67% v / v Mixed lyophilization protectant 20% v / v; The mixed lyophilization protectant comprises the following components: 15-30% w / v of dextran Lactose 0-15% w / v Monosodium glutamate 0-25% w / v Mannitol 40% w / v The content of lactose and monosodium glutamate is not both 0.

5. The detection reagent according to any one of claims 1-4, characterized in that: The preprime comprises at least one of the primers whose nucleotide sequences are shown in SEQ ID NO. 1-SEQ ID NO. 14; The back primer includes at least one of the primers with nucleotide sequences as shown in SEQ ID NO. 15-SEQ ID NO.

28.

6. The detection reagent according to any one of claims 1-4, characterized in that: The gRNA includes at least one of the RNAs with nucleotide sequences as shown in SEQ ID NO. 29-SEQ ID NO.

42.

7. The detection reagent according to any one of claims 1-4, characterized in that: The ssDNA reporter molecule is a single-stranded DNA reporter molecule modified with fluorescent and quenching groups.

8. Use of the detection reagent according to any one of claims 1-7 in the preparation of an HPV virus detection kit.

9. A room-temperature nucleic acid detection kit for HPV virus, characterized in that, include: Buffer B and the detection reagent according to any one of claims 1-7.

10. A method for one-stop detection of HPV virus for non-diagnostic purposes using the detection kit according to claim 9, characterized in that, Includes the following steps: Step 1: Redissolve the detection reagent; Step 2: Add the Buffer B and the test sample sequentially, and incubate the reaction; Step 3: Detection is performed using fluorescence detection or color development of test strips.

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