Self-detection reagent, kit and detection method for HPV (human papillomavirus) typing

Abstract

The invention belongs to the technical field of in-vitro detection, and particularly relates to a self-detection reagent, a kit and a detection method for HPV (human papillomavirus) typing. The self-detection reagent comprises an RPA freeze-drying reagent and a CRISPR / Cas12a detection system freeze-drying reagent, wherein the RPA freeze-drying reagent comprises an RPA isothermal amplification reagent, a Buffer A, a front primer, a rear primer and a mixed freeze-drying protective additive; the CRISPR / Cas12a detection system freeze-drying reagent comprises Cas12a, gRNA (guide ribonucleic acid), ssDNA (single-stranded deoxyribonucleic acid) reporter molecules, Buffer 2.1 and a mixed freeze-drying protective agent. The composition of the mixed freeze-drying protective agent is optimized according to a detection system and a specific detection method, the problems of normal-temperature storage and transportation of the 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 mixed freeze-drying protective agent has a very good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro detection, and in particular relates to a self-test reagent, a kit and a detection method for HPV typing. Background Art

[0002] Cervical cancer is a common malignant tumor in women worldwide. Cervical cancer screening is the key to prevention and early diagnosis. The World Health Organization (WHO), the American Cancer Society (ACS) and the consensus of experts in my country recommend human papillomavirus (HPV) nucleic acid testing as the preferred initial screening method, but the currently widely used PCR detection method is complicated to operate, requires professional personnel to operate, and has high requirements for equipment, making it difficult to use in situations where medical resources are limited. In addition, patients who have been infected with HPV need to go back and forth to the hospital for reexamination many times, which brings inconvenience to patients and consumes a lot of medical resources. Therefore, there is an urgent need to develop a convenient, fast, and home-use HPV detection method.

[0003] In recent years, the CRISPR system has been widely used in gene editing and nucleic acid detection. The CRISPR / Cas12a detection method combined with RPA amplification technology not only meets the needs of rapid and highly sensitive detection, but also effectively avoids the production of non-specific products, providing a new way for early screening of cervical cancer.

[0004] However, conventional RPA and CRISPR detection reagents have harsh storage conditions and need to be stored at -20°C. In addition, repeated freezing and thawing will also affect the performance stability. The present invention intends to prepare freeze-dried detection reagents to solve the above problems. However, both RPA and CRISPR detection reagents have complex components, and during the reaction process, the RPA and CRISPR detection systems will be mixed into one system, further complicating the components. Therefore, the stability and performance of the detection reagents are difficult to guarantee. Summary of the invention

[0005] In view of the above problems, the present invention provides a self-test reagent, a kit and a detection method for HPV typing.

[0006] The present invention provides a self-test reagent for HPV typing, characterized in that: it comprises an RPA freeze-dried reagent and / or a CRISPR / Cas12a detection system freeze-dried reagent;

[0007] The RPA lyophilized reagent is a lyophilized reagent made from an RPA amplification system, and the RPA amplification system includes the following components:

[0008] RPA constant temperature amplification reagent 1 / 18-24μL,

[0009] Buffer A49.0-65.3%v / v,

[0010] Forward primers 0.033-0.044 μM each,

[0011] The latter primers were 0.033-0.044 μM each,

[0012] Mixed lyophilized protective agent 18.3-24.4% v / v;

[0013] The CRISPR / Cas12a detection system lyophilized reagent is a lyophilized reagent made from the CRISPR / Cas12a detection system, and the CRISPR / Cas12a detection system includes the following components:

[0014] Cas12a 0.167-0.250μM,

[0015] gRNA 0.167-0.250 μM each,

[0016] ssDNA reporter molecule 1.667-2.500μM,

[0017] Buffer 2.1 6.67-10.0%v / v,

[0018] Mixed lyophilized protective agent 13.3-20.0% v / v;

[0019] Wherein, the mixed lyophilization protective agent comprises the following components:

[0020] PEG 8-15% w / v,

[0021] Bovine serum albumin 4-10% w / v,

[0022] Mannitol 30-44% w / v.

[0023] Preferably, the RPA amplification system comprises the following components:

[0024] RPA constant temperature amplification reagent 1 / 22μL,

[0025] Buffer A53.4%v / v,

[0026] Forward primers 0.036 μM each,

[0027] The latter primers were 0.036 μM each,

[0028] Mixed lyophilized protective agent 20% v / v;

[0029] The CRISPR / Cas12a detection system includes the following components:

[0030] Cas12a 0.25μM,

[0031] gRNA 0.25 μM each,

[0032] ssDNA reporter molecule 2.5μM,

[0033] Buffer 2.1 10%v / v,

[0034] Mixed lyophilized protective agent 20% v / v;

[0035] Wherein, the mixed lyophilization protective agent comprises the following components:

[0036] PEG 15% w / v,

[0037] Bovine serum albumin 10% w / v,

[0038] Mannitol 44% w / v.

[0039] The present invention provides a self-test reagent for HPV typing, characterized in that: it comprises an RPA freeze-dried reagent and / or a CRISPR / Cas12a detection system freeze-dried reagent;

[0040] The RPA lyophilized reagent is a lyophilized reagent made from an RPA amplification system, and the RPA amplification system includes the following components:

[0041] RPA constant temperature amplification reagent 1 / 18-24μL,

[0042] Buffer A49.0-65.3%v / v,

[0043] Forward primers 0.033-0.044 μM each,

[0044] The latter primers were 0.033-0.044 μM each,

[0045] Mixed lyophilized protective agent 18.3-24.4% v / v;

[0046] The CRISPR / Cas12a detection system lyophilized reagent is a lyophilized reagent made from the CRISPR / Cas12a detection system, and the CRISPR / Cas12a detection system includes the following components:

[0047] Cas12a 0.167-0.250μM,

[0048] gRNA 0.167-0.250 μM each,

[0049] ssDNA reporter molecule 1.667-2.500μM,

[0050] Buffer 2.1 6.67-10.0%v / v,

[0051] Mixed lyophilized protective agent 13.3-20.0% v / v;

[0052] Wherein, the mixed lyophilization protective agent comprises the following components:

[0053] Maltose 5-20% w / v,

[0054] Bovine serum albumin 10-25% w / v,

[0055] Mannitol 20-40% w / v.

[0056] Preferably, the RPA amplification system comprises the following components:

[0057] RPA constant temperature amplification reagent 1 / 22μL,

[0058] Buffer A53.4%v / v,

[0059] Forward primers 0.036 μM each,

[0060] The latter primers were 0.036 μM each,

[0061] Mixed lyophilized protective agent 20% v / v;

[0062] The CRISPR / Cas12a detection system includes the following components:

[0063] Cas12a 0.25μM,

[0064] gRNA 0.25 μM each,

[0065] ssDNA reporter molecule 2.5μM,

[0066] Buffer 2.1 10%v / v,

[0067] Mixed lyophilized protective agent 20% v / v;

[0068] Wherein, the mixed lyophilization protective agent comprises the following components:

[0069] Maltose 20% w / v,

[0070] Bovine serum albumin 25% w / v,

[0071] Mannitol 40% w / v.

[0072] Preferably, the front primer comprises at least one of the primers having nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.14;

[0073] The rear primer includes at least one of the primers whose nucleotide sequences are shown as SEQ ID NO.15 to SEQ ID NO.28.

[0074] The gRNA includes at least one of the RNAs having nucleotide sequences as shown in SEQ ID NO.29-SEQ ID NO.42.

[0075] Preferably, the ssDNA reporter molecule is a single-stranded DNA reporter molecule modified with a fluorescent group and a quenching group.

[0076] The present invention provides use of the above-mentioned self-test reagent for HPV typing in the preparation of an HPV virus detection kit.

[0077] The present invention provides a detection kit for HPV typing, which comprises: Buffer B and the above-mentioned self-test reagent for HPV typing.

[0078] The present invention also provides a method for performing non-diagnostic HPV detection using the above-mentioned detection kit for HPV typing, which comprises the following steps:

[0079] Step 1, respectively re-dissolve the RPA lyophilized reagent and the CRISPR / Cas12a detection system lyophilized reagent;

[0080] Step 2, adding the Buffer B and the test sample to the solution of the reconstituted RPA lyophilized reagent, and incubating the reaction;

[0081] Step 3, adding a solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent to the reaction system and incubating the reaction;

[0082] Step 4, detection by fluorescence detection or color development on a test strip.

[0083] Preferably, in step 1, the solution of the reconstituted RPA lyophilized reagent is added to the reaction tube, and the solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent is added to the reaction tube cap of the reaction tube; in step 3, the solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent is added to the reaction tube by instantaneous centrifugation.

[0084] The present invention provides a self-test reagent for HPV typing, which optimizes primers and gRNA for 14 high-risk HPV viruses, and optimizes the most appropriate freeze-dried protective agent combination for these specific primer and gRNA combinations. Therefore, the present invention provides a self-test reagent for HPV typing and a kit made thereof for the first time. The self-test reagent and kit have the following beneficial technical effects:

[0085] 1. By optimizing the primer and gRNA sequences and optimizing the freeze-dried protective agent combination, the present invention has good sensitivity, specificity and repeatability for HPV virus detection and can meet the needs of practical applications.

[0086] 2. The present invention realizes for the first time the one-step RPA / CRISPR detection reagent for HPV virus to be made into a freeze-dried reagent, which has the advantages of convenient storage and transportation, stable reagent performance, and ease of use.

[0087] 3. The detection method of the present invention is a one-step detection. There is no need to separate the RPA amplification reaction and the CRISPR / Cas12a detection in two reaction tubes. There is no operation of transferring the reaction solution, which can effectively avoid the contamination problem of nucleic acid aerosol.

[0088] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0089] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 Schematic diagram of the structure of the microfluidic chip for HPV RPA / CRISPR detection provided in Example 2.

[0091] Figure 2 This is a graph showing the specificity test results in Experimental Example 3.

[0092] Figure 3 This is a graph showing the specificity test results in Experimental Example 6.

[0093] Figure 4 This is a graph showing the specificity test results in Experimental Example 7. DETAILED DESCRIPTION

[0094] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0095] In the following examples and experimental examples, the sources of some commercial reagents are as follows:

[0096] RPA constant temperature amplification reagent, Buffer A and Buffer B are from the DNA constant temperature rapid amplification kit (Cat. No.: WLB8201KIT) of Anpu Future Biotechnology Co., Ltd.; Buffer 2.1 is from New England Biolabs and is a component of EnGen Lba Cas12a (Cpf1) (NEB#M0653).

[0097] Example 1 Self-test reagent for HPV typing and its fluorescence detection kit and detection method 1. Kit composition and preparation method

[0098] The kit of this embodiment mainly includes RPA reagent freeze-dried balls for HPV virus detection and CRISPR / Cas12a detection system freeze-dried balls, and the freeze-dried balls are made by freeze-drying the RPA freeze-dried reagent in Table 1 and the CRISPR / Cas12a detection system in Table 2, respectively. The RPA freeze-dried reagent is a freeze-dried reagent made from the RPA amplification system, and the CRISPR / Cas12a detection system freeze-dried reagent is a freeze-dried reagent made from the CRISPR / Cas12a detection system. For each type of HPV virus, a separate freeze-dried ball is prepared.

[0099] Table 1 RPA lyophilized reagent composition

[0100]

[0101] In other embodiments, in the above RPA freeze-dried reagent system, the volume of enzyme-free water can also be adjusted within the range of 0.24-6.24 μL as needed.

[0102] Table 2 Composition of CRISPR / Cas12a detection system

[0103]

[0104] In other embodiments, in the above CRISPR / Cas12a detection system, the volume of enzyme-free water can also be adjusted within the range of 1.2-5.2 μL as needed.

[0105] The sequences of the front primer and the rear primer are shown in Table 3, wherein the sequence marked with "F" is the front primer, and the sequence marked with "R" is the rear primer.

[0106] Table 3 Front primer and rear primer of RPA isothermal amplification

[0107]

[0108]

[0109] The sequences of gRNAs are shown in Table 4.

[0110] Table 4

[0111]

[0112]

[0113] The ssDNA reporter molecule (Reporter) is ssDNA labeled with FAM-BHQ: (6-FAM)TTATT(BHQ1). In other embodiments, the fluorescent group FAM can also be replaced by VIC, HEX, TET, JOE, Cy5, Cy3, TAMRA, etc.

[0114] The composition of the mixed lyophilized protective agent is an aqueous solution containing the following components: 15% w / v PEG, 10% w / v bovine serum albumin, and 44% w / v mannitol, wherein the percentages are mass volume percentages, g / ml.

[0115] The specific method of preparing the above-mentioned RPA freeze-dried reagent and CRISPR detection system into freeze-dried balls is as follows:

[0116] RPA reagent freeze-dried pellets: Use a precise liquid dispensing system to drop the prepared RPA freeze-dried reagent in a volume of 22 μL into liquid nitrogen, and condense into round pellets. Wait for about 10 seconds for the pellets to sink to the bottom of the liquid nitrogen, pick them up, and transfer them to a vial that has been pre-cooled with dry ice. After the reagent pellets are collected, transfer them to the freeze dryer. The freeze-drying procedure is shown in Table 5.

[0117] CRISPR / Cas12a detection reagent freeze-dried beads: The preparation method of the RPA reagent freeze-dried beads was followed, except that the CRISPR / Cas12a detection system was dripped into liquid nitrogen in a volume of 8 μL.

[0118] Table 5

[0119]

[0120] 2. Detection Method

[0121] First, reconstitute the CRISPR / Cas12a detection reagent freeze-dried pellet with 6.8μL enzyme-free water, add it to the bottom of the reaction tube, and then add 1.2μL glycerol and mix well. Then reconstitute the RPA reagent freeze-dried pellet with 18μL enzyme-free water and add it to the reaction tube. Then add 2μL Buffer B and 2μL of the sample to be tested. Incubate at 37°C. At the same time, place the reaction tube in a constant temperature fluorescence detector and collect fluorescence data every 1 minute for 1 hour.

[0122] Example 2 Self-test reagent for HPV typing and its test strip detection kit and detection method 1. Kit composition and preparation method

[0123] The kit of this embodiment mainly includes RPA reagent freeze-dried balls for HPV virus detection and CRISPR / Cas12a detection system freeze-dried balls. The composition and preparation method of the freeze-dried balls are basically the same as those in Example 1, except that:

[0124] 1. The composition of mixed freeze-dried protective agents is different

[0125] The composition of the mixed lyophilized protective agent of this embodiment is an aqueous solution containing the following components: 25% w / v bovine serum albumin, 20% w / v maltose, and 40% w / v mannitol, wherein the percentages are mass volume percentages, g / ml.

[0126] 2. Different ssDNA reporter molecules

[0127] The ssDNA reporter molecule (Reporter) in this example is ssDNA labeled with FAM-Biotin: (6-FAM)TTATTATT (Biotin).

[0128] 2. Detection Method

[0129] Method 1:

[0130] First, reconstitute the CRISPR / Cas12a detection reagent freeze-dried ball with 6.8μL enzyme-free water, add it to the bottom of the reaction tube, and then add 1.2μL glycerol and mix well. Then reconstitute the RPA reagent freeze-dried ball with 18μL enzyme-free water, add it to the reaction tube, and then add 2μL Buffer B and 2μL of the sample to be tested. Incubate at 37°C for 60min to react. Finally, take 5μL of the reaction product, add 35μL 5% PEG chromatography buffer, mix well, insert it into the lateral flow immunochromatography test strip (from Milenia GenLineHybriDetect kit, purchased from Milenia biotec), and observe the results with the naked eye within 15 to 20 minutes.

[0131] Method 2:

[0132] 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.

[0133] The detection steps are as follows:

[0134] 1) Use 6.8 μL enzyme-free water to reconstitute the CRISPR / Cas12a detection reagent freeze-dried pellet, add it to the bottom of the reaction tube, and then add 1.2 μL glycerol. Then use 18 μL enzyme-free water to reconstitute the RPA reagent freeze-dried pellet and add it to the reagent area.

[0135] 2) Add 2 μL of Buffer B and 2 μL of the sample to be tested into the reagent area.

[0136] 3) Pass through the channel and enter the reaction zone, and incubate at 37°C for 60 minutes to carry out the reaction.

[0137] 4) The reaction product flows into the lateral flow immunochromatography test strip in the reading area of ​​the test strip, and the result is observed with the naked eye within 15 to 20 minutes.

[0138] The technical solution of the present invention is further illustrated by experiments below.

[0139] Experimental Example 1 Optimal Conditions for One-Step Fluorescence Detection of HPV Virus

[0140] 1. Primer and gRNA design

[0141] The present invention selects the conserved region of the L1 gene as the target region, and designs the gRNA of Cas12a of each HPV type according to the cleavage characteristics of the Cas12a protein. And the designed multiple gRNAs are optimized through experiments.

[0142] The present invention uses Primers software to design and optimize specific RPA primers. All designed RPA primers are verified and analyzed using BLAST software to reduce the possibility of nonspecific amplification. And the designed multiple RPA primers are optimized through experiments.

[0143] The optimal primers and gRNA are preferably as shown in Example 1.

[0144] 2. Optimization of testing conditions

[0145] This experimental example adjusted the various detection conditions based on Example 1, and compared the fluorescence intensity of the target gene at the same concentration under different conditions. In the same time period, the detection conditions corresponding to the fastest and highest increase in fluorescence intensity were selected as the preferred conditions. The preferred results are shown in the following table:

[0146] Table 6

[0147] Optimal RPA primer concentration 1~3μM Optimal RPA reaction volume 18-24 μL Optimum reaction temperature 37℃ Optimal reaction time 60~80min Optimal CRISPR reaction volume 8-12 μL Optimal crRNA and Cas12a concentration and ratio 1:1 Optimal CRISPR reaction buffer Buffer 2.1 Optimal reporter concentration 40~50μM

[0148] The composition of the optimized optimal RPA / CRISPR detection system is shown in Example 1.

[0149] Experimental Example 2 Screening experiment of mixed freeze-dried protective agent for HPV virus one-step fluorescence detection kit

[0150] In the following experiments, reagents or conditions not specifically described were performed as described in Example 1. This experimental example first screened the mixed lyophilized protective agent with the front primer, rear primer and gRNA of HPV16, and then verified that the preferred mixed lyophilized protective agent could be used for the detection reagent freeze-dried balls of other thirteen HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68).

[0151] 1. Screening of single protective agent

[0152] The effects of PEG, bovine serum albumin, lactose, ammonium acetate, maltose, mannitol, and dimethyl sulfoxide as lyoprotectants for RPA detection system and CRISPR detection system on fluorescence signals were studied.

[0153] 4.4 μL of 7 kinds of lyophilized protective agents with a concentration of 15% (mass volume percentage, g / ml) were added to the RPA reagent to obtain the RPA detection system containing a single protective agent (Table 7) as follows.

[0154] 1.6 μL of 7 kinds of lyophilized protective agents with a concentration of 15% (mass volume percentage, g / ml) were added to the CRISPR detection system to obtain a CRISPR detection system containing a single protective agent (Table 7) as follows.

[0155] Table 7

[0156]

[0157] In Table 7, the nucleic acid sequence of the front primer is SEQ ID NO.1, the nucleic acid sequence of the rear primer is SEQ ID NO.15, and the nucleic acid sequence of the gRNA is SEQ ID NO.29.

[0158] The fluorescence intensity of the RPA detection system and the CRISPR detection system containing different types of lyophilized protective agents was tested. Compared with the fluorescence intensity of the RPA detection system and the CRISPR detection system without lyophilized protective agents, if the fluorescence signal intensity decreased by no more than 10% during the same period of time, it was considered that the protective agent had no inhibitory effect on the detection system. The results are shown in Table 8:

[0159] Table 8

[0160] 15% lyophilized protective agent Fluorescence intensity PEG 93％ BSA 94％ lactose 98％ Ammonium acetate 86％ maltose 85％ Mannitol 100％ Dimethyl sulfoxide 83％

[0161] The above results show that PEG, bovine serum albumin, lactose and mannitol have no effect on CRISPR detection reagents and RPA isothermal amplification reagents.

[0162] 2. Suitable concentration range of protective agent

[0163] The above freeze-dried protective agent whose fluorescence signal intensity decreases by no more than 10% is selected, and the concentration is configured to be 1%-52%. The fluorescence intensity of the RPA detection system and the CRISPR detection system is tested for different concentrations of freeze-dried protective agent. Compared with the fluorescence intensity of the RPA detection system and the CRISPR detection system without freeze-dried protective agent, if the fluorescence signal intensity decreases by no more than 10% in the same time period, it is considered that the concentration has no inhibitory effect on the detection system, and the appropriate concentration range of the protective agent is determined. The results are shown in Table 9:

[0164] Table 9

[0165]

[0166]

[0167] The above results show that the suitable concentration range of PEG is 8%-44%, the suitable concentration range of bovine serum albumin is 4%-28%, the suitable concentration range of lactose is 2%-40%, and the suitable concentration range of mannitol is 4%-44%.

[0168] 3. Screening of RPA lyophilization protectants

[0169] 1) The contents of PEG, BSA, lactose and mannitol were optimized by orthogonal experimental design with 4 factors and 4 levels, as shown in Tables 10 and 11.

[0170] Table 10 Factor level table

[0171] factor Level 1 Level 2 Level 3 Level 4 PEG 0％ 15％ 30％ 44％ BSA 0％ 10％ 20％ 28％ lactose 0％ 15％ 30％ 40％ Mannitol 0％ 15％ 30％ 44％

[0172] Table 11 Scheme

[0173]

[0174]

[0175] 2) Morphological examination of RPA reagent freeze-dried pellets

[0176] The freeze-dried balls were prepared according to the preparation method of the RPA reagent freeze-dried balls described in Example 1, and the appearance of the RPA reagent freeze-dried balls was observed to see whether they were qualified. The test results are shown in Table 12:

[0177] Table 12

[0178]

[0179] The above results show that Scheme 5, Scheme 6, Scheme 9 and Scheme 11 can be used for the next step of screening.

[0180] 3) Moisture content of RPA reagent freeze-dried pellets

[0181] The moisture content of the RPA reagent freeze-dried pellets that passed the above morphological inspection was measured using a Karl Fischer moisture meter. The results are shown in Table 13:

[0182] Table 13

[0183] plan Moisture content in conclusion Solution 5 2.38％ qualified Solution 6 3.47％ qualified Solution 9 6.55％ Failure Solution 11 3.14％ qualified

[0184] The above results show that Scheme 5, Scheme 6 and Scheme 11 can be used for the next step of screening.

[0185] 4) Select the RPA reagent freeze-dried balls with qualified moisture content for performance inspection

[0186] The detection method described in Example 1 is different in that 8 μL of CRISPR / Cas12a detection system is directly added to the bottom of the reaction tube without re-dissolving the CRISPR / Cas12a detection reagent lyophilized pellet and adding glycerol. Negative control and positive control are also set. The fluorescence results of the above scheme are compared with the fluorescence intensity of the un-lyophilized RPA detection system and the CRISPR detection system. The results are shown in Table 14:

[0187] Table 14

[0188] plan Fluorescence intensity increase Solution 5 99％ Solution 6 91％ Solution 11 97％

[0189] The above results show that Scheme 5, Scheme 6 and Scheme 11 can be used for the next step of screening.

[0190] 4. Screening of mixed lyophilized protective agents in lyophilized protective agents for CRISPR / Cas12a detection reagents

[0191] 1) Select the RPA reagent lyophilization protectant schemes of the above schemes 5, 6, and 11, and lyophilize the CRISPR / Cas12a detection reagent. The lyophilization system is shown in Table 2.

[0192] 2) Morphological examination of freeze-dried pellets of CRISPR / Cas12a detection reagent

[0193] The freeze-dried balls were prepared according to the preparation method of the freeze-dried balls of the CRISPR / Cas12a detection reagent described in Example 1, and the appearance of the freeze-dried balls of the CRISPR / Cas12a detection reagent was observed to see whether they were qualified. The test results are shown in Table 15:

[0194] Table 15

[0195] plan Formability Surface smoothness Completeness Resolubility in conclusion Solution 5 2 2 2 1 Failure Solution 6 3 3 3 3 qualified Solution 11 3 3 3 3 qualified

[0196] The above results show that Scheme 6 and Scheme 11 can be used for the next step of screening.

[0197] 4) Moisture content of CRISPR / Cas12a detection reagent freeze-dried pellets

[0198] The moisture content of the CRISPR / Cas12a detection reagent freeze-dried pellets that passed the above morphological inspection was measured using a cardiometer, and the results are shown in Table 16:

[0199] Table 16

[0200] plan Moisture content in conclusion Solution 6 2.02％ qualified Solution 11 5.71％ Failure

[0201] The above results show that Scheme 6 can be used for the next step of screening.

[0202] 5) Select the CRISPR / Cas12a detection reagent freeze-dried pellets with qualified moisture content for performance inspection

[0203] The detection method described in Example 1 was followed, except that a negative control and a positive control were set at the same time. The fluorescence results of the above scheme were compared with the fluorescence intensity of the unfreeze-dried RPA detection system and the CRISPR detection system, and the results are shown in Table 17:

[0204] Table 17

[0205] plan Fluorescence intensity Solution 6 95％

[0206] The above results show that, for the fluorescence detection kit and detection method for HPV typing of the present invention, Scheme 6 is the optimal mixed lyophilized protective agent formula for detecting HPV16.

[0207] Scheme 6 was further used to prepare freeze-dried beads for detection reagents for other thirteen HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68), and the resulting freeze-dried beads were able to meet the performance requirements of the detection.

[0208] Experimental Example 3 Performance Verification of One-Step Fluorescence Detection Method for Freeze-dried HPV Virus

[0209] 1. Sensitivity test

[0210] The detection method described in Example 1 is used, except that the concentration range of the sample to be tested is 10 -1 ——10 5 aM, and set up negative control and positive control at the same time. The results are shown in Table 18:

[0211] Table 18

[0212]

[0213] 2. Specificity test

[0214] The detection method described in Example 1 is used, except that the samples to be tested are selected from 14 plasmid standards of high-risk HPV types. The results are as follows Figure 2 shown.

[0215] The results showed that the kit of Example 1 was specific to the plasmids of 14 high-risk HPV types.

[0216] 3. Repeatability test

[0217] The detection method described in Example 1 is used, except that the concentrations of the samples to be tested are low, medium, and high. The results are shown in Table 19:

[0218] Table 19

[0219]

[0220]

[0221] 4. Clinical sample testing

[0222] The cervical sampling cotton swab was placed in a physiological saline sampling tube, the specimen was shaken and mixed, 200uL of the sample was centrifuged at 14000rpm for 5min, and the supernatant was discarded; 200uL of nucleic acid release agent was added, and the metal bath was 100℃ for 15min. The supernatant after centrifugation was the sample to be tested, and one-step fluorescence detection was performed according to the method of Example 1. The test results were compared with the clinical test results, and the diagnostic sensitivity and specificity were calculated, as shown in Table 20:

[0223] Table 20

[0224]

[0225] The above experimental results show that the one-step fluorescence detection kit and detection method for HPV typing provided by the present invention have excellent sensitivity, specificity and repeatability, and can meet the requirements of actual detection.

[0226] Experimental Example 4 Optimal Conditions for One-Step Test Strip Detection of HPV Virus

[0227] This experimental example adjusted the various conditions of the test strip detection on the basis of Example 2, and compared the fluorescence intensity of the target gene with the same concentration under different conditions. In the same time period, the detection conditions corresponding to the fastest color development speed and the most obvious color development were selected as the preferred conditions. The preferred results are shown in the following table:

[0228] Table 21

[0229] Optimal RPA primer concentration 1~3μM Optimal RPA reaction volume 18-24 μL Optimum reaction temperature 37℃ Optimal reaction time 60~80min Optimal CRISPR reaction volume 8-12 μL Optimal crRNA and Cas12a concentration and ratio 1:1 Optimal CRISPR reaction buffer Buffer 2.1 Optimal reporter concentration 45~50μM Optimal volume of additive glycerol 1.20μL Optimal reaction product volume 2-5 μL Optimal test strip buffer concentration 5～12％ Optimal test strip buffer volume 35~40μL Optimal test strip reaction time 15~20min

[0230] Experimental Example 5 Screening experiment of mixed lyophilized protective agent for HPV virus one-step test strip detection kit

[0231] In the following experiments, reagents or conditions not specifically described were performed as described in Example 2. This experimental example first screened the mixed lyophilized protective agent with the front primer, rear primer and gRNA of HPV16, and then verified that the preferred mixed lyophilized protective agent could be used for the detection reagent lyophilized balls of other thirteen HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68).

[0232] 1. Screening of single protective agent

[0233] The method for screening a single protective agent as described in Experimental Example 2 was followed, except that the fluorescent signal was changed to a colorimetric signal.

[0234] The color intensity of the RPA detection system and the CRISPR detection system with different types of lyophilized protective agents was tested and compared with the color intensity of the RPA detection system and the CRISPR detection system without lyophilized protective agents. The results are shown in Table 22

[0235] Table 22

[0236] 15% lyophilized protective agent Color intensity BSA powerful lactose weak PEG powerful maltose powerful Ammonium acetate weak Mannitol powerful Dimethyl sulfoxide weak

[0237] The above results show that bovine serum albumin, PEG, maltose and mannitol have no effect on the color development of the test strips of CRISPR detection reagents and RPA isothermal amplification reagents.

[0238] 2. Suitable concentration range of protective agent

[0239] The above lyophilized protective agents that have no effect on CRISPR detection reagents and RPA isothermal amplification reagents were selected, and the concentration was configured to be 1%-50%. The color development effects of different concentrations of lyophilized protective agents on the RPA detection system and the CRISPR detection system were tested to determine the appropriate concentration range of the protective agent. The results are shown in Table 23:

[0240] Table 23

[0241]

[0242] The above results show that the suitable concentration range of bovine serum albumin is 10%-45%, the suitable concentration range of PEG is 10%-40%, the suitable concentration range of maltose is 5%-35%, and the suitable concentration range of mannitol is 10%-40%.

[0243] 3. Screening of freeze-dried protective agents for RPA reagents

[0244] 1) The contents of bovine serum albumin, PEG, maltose and mannitol were optimized by orthogonal experimental design with 4 factors and 3 levels, as shown in Tables 24 and 25.

[0245] Table 24 Factor level table

[0246] factor Level 1 Level 2 Level 3 BSA 0％ 25％ 45％ PEG 0％ 20％ 40％ maltose 0％ 20％ 35％ Mannitol 0％ 20％ 40％

[0247] Table 25 Scheme

[0248]

[0249] 2) All the above 9 schemes were selected, and the RPA reagent was freeze-dried. The freeze-drying system is shown in Table 1.

[0250] 3) Morphological examination of RPA reagent freeze-dried pellets

[0251] The freeze-dried balls were prepared according to the preparation method of the RPA reagent freeze-dried balls described in Example 1, and the appearance of the RPA reagent freeze-dried balls was observed to see whether they were qualified. The test results are shown in Table 26:

[0252] Table 26

[0253]

[0254]

[0255] The above results show that Schemes 3, 4, 7 and 8 can be used for further screening.

[0256] 5) Moisture content of RPA reagent freeze-dried pellets

[0257] The moisture content of the RPA reagent freeze-dried pellets that passed the above morphological inspection was measured using a Karl Fischer moisture meter. The results are shown in Table 27:

[0258] Table 27

[0259] plan Moisture content in conclusion Solution 3 2.57％ qualified Solution 4 2.26％ qualified Solution 7 2.98％ qualified Solution 8 6.97％ Failure

[0260] The above results show that Schemes 3, 4 and 7 can be used for further screening.

[0261] 6) Select the RPA reagent freeze-dried balls with qualified moisture content for performance inspection

[0262] The test method described in Example 2 is the same, except that there is no need to re-dissolve the CRISPR / Cas12a detection system freeze-dried beads, 8 μL of CRISPR / Cas12a detection system is directly added to the bottom of the reaction tube, and glycerol is added, and negative and positive controls are set at the same time. The test strip results of the above scheme are compared with the color development of the test strips of the unfreeze-dried RPA detection system and the CRISPR detection system, and the results are shown in Table 28:

[0263] Table 28

[0264] plan Color intensity Solution 3 powerful Solution 4 powerful Solution 7 powerful

[0265] The above results show that Schemes 3, 4 and 7 can be used for further screening.

[0266] 4. Screening of mixed lyophilized protective agents in lyophilized reagents for CRISPR / Cas12a detection reagents

[0267] 1) Select the RPA reagent lyophilization protectant scheme of the above scheme 3, scheme 4, and scheme 7, and lyophilize the CRISPR / Cas12a detection reagent. The lyophilization system is the lyophilization system of the CRISPR / Cas12a detection reagent described in Example 2.

[0268] 2) Morphological examination of freeze-dried pellets of CRISPR / Cas12a detection reagent

[0269] The freeze-dried balls were prepared according to the preparation method of the CRISPR / Cas12a detection reagent freeze-dried balls described in Example 2, and the appearance of the CRISPR / Cas12a detection reagent freeze-dried balls was observed to be qualified. The test results are shown in Table 29:

[0270] Table 29

[0271] plan Formability Surface smoothness Completeness Resolubility in conclusion Solution 3 3 3 2 2 Failure Solution 4 3 3 3 3 qualified Solution 7 3 3 3 3 qualified

[0272] The above results show that Scheme 4 and Scheme 7 can be used for further screening.

[0273] 3) Moisture content of CRISPR / Cas12a detection reagent freeze-dried pellets

[0274] The moisture content of the CRISPR / Cas12a detection reagent freeze-dried pellets that passed the above morphological inspection was measured using a cardiometer, and the results are shown in Table 30:

[0275] Table 30

[0276] plan Moisture content in conclusion Solution 4 2.33％ qualified Solution 7 2.71％ qualified

[0277] The above results show that Scheme 4 and Scheme 7 can be used for further screening.

[0278] 4) Select the CRISPR / Cas12a detection reagent freeze-dried pellets with qualified moisture content for performance inspection

[0279] The detection method 1 described in Example 2 is different in that a negative control and a positive control are set at the same time. The color development results of the test strips of the above scheme are compared with the color development results of the test strips of the unfreeze-dried RPA detection system and the CRISPR detection system, and the results are shown in Table 31:

[0280] Table 31

[0281] plan Color intensity Solution 4 powerful Solution 7 weak

[0282] The above results show that for the one-step test strip detection kit and detection method for HPV typing of the present invention, Scheme 4 is the optimal mixed lyophilized protective agent formula for detecting HPV16.

[0283] Scheme 4 was further used to prepare freeze-dried beads for detection reagents for other thirteen HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68), and the resulting freeze-dried beads were able to meet the performance requirements of the detection.

[0284] Experimental Example 6 Performance Verification of Freeze-dried HPV Virus One-step Test Strip Detection Method

[0285] The detection method of this experimental example is performed according to method 1 in Example 2.

[0286] 1. Sensitivity test

[0287] The detection method 1 described in Example 2 is different in that the concentration range of the sample to be tested is 10 -1 —10 6 aM, and set up negative control and positive control at the same time. The results are shown in Table 32:

[0288] Table 32

[0289]

[0290] 2. Specificity test

[0291] The detection method 1 described in Example 2 is used, except that the samples to be tested are taken from plasmid standards of 14 high-risk HPV types. The results are as follows Figure 3 shown.

[0292] 3. Repeatability test

[0293] The detection method 1 described in Example 2 was used, except that the concentrations of the samples to be tested were low, medium, and high, and negative and positive controls were set. The results are shown in Table 33:

[0294] Table 33

[0295]

[0296]

[0297] 4. Clinical sample testing

[0298] The cervical sampling cotton swab was placed in a physiological saline sampling tube, the specimen was shaken and mixed, 200 μL of the sample was taken and centrifuged at 14000 rpm for 5 min, and the supernatant was discarded; 200 μL of nucleic acid release agent was added, and the metal bath was 100°C for 15 min. The supernatant after centrifugation was the sample to be tested, and the test results were compared with the clinical test results to calculate the diagnostic sensitivity and specificity, as shown in Table 34.

[0299] Table 34

[0300]

[0301] Experimental Example 7 Performance Verification of Freeze-dried HPV Virus One-step Test Strip Detection Method (Using Microfluidic Chip)

[0302] The detection method of this experimental example is performed according to method 2 in Example 2.

[0303] 1. Sensitivity test

[0304] The designed concentration gradient is 10 -1 -10 6 The samples to be tested were aM, and the steps in the detection method 2 described in Example 2 were followed. The results are shown in Table 35:

[0305] Table 35

[0306]

[0307]

[0308] 2. Specificity test

[0309] Select a concentration of 10 6 The 14 high-risk HPV types of aM were used as samples to be tested. The experimental steps of the detection method 2 in Example 2 were followed. The results are shown in FIG. Figure 4 shown.

[0310] 3. Repeatability test

[0311] HPV high-risk types with low, medium and high concentrations were selected as samples to be tested, and the experimental steps of the detection method 2 in Example 2 were followed, with each concentration repeated 20 times. The results are shown in Table 36:

[0312] Table 36

[0313]

[0314] 4. Clinical sample testing

[0315] Collect 14 types of high-risk HPV cervical samples, place them in saline sampling tubes, shake and mix the samples, take 200μL of samples and centrifuge at 14000rpm for 5min, discard the supernatant; add 200μL of nucleic acid release agent, and heat the metal bath at 100℃ for 15min. The supernatant after centrifugation is the sample to be tested, and the experimental steps in 2 detection method 2 are followed. The test results are compared with the clinical test results, and the diagnostic sensitivity and specificity are calculated. The results are shown in Table 37:

[0316] Table 37

[0317]

[0318] The above experimental results show that the one-step test strip detection kit and detection method for HPV typing provided in Example 2 have excellent sensitivity, specificity and repeatability, and can meet the requirements of actual detection.

[0319] In summary, the present invention solves the problem of room temperature storage and transportation of one-step RPA / CRISPR detection reagents for HPV virus, supports POCT (point-of-care testing) and home self-testing; and ensures high sensitivity and specificity of detection, realizes early, rapid, portable and simple operation of HPV detection, overcomes the traditional method's dependence on expensive equipment and professionals, and provides an efficient and accurate solution for large-scale screening.

Claims

1. A self-test reagent for HPV typing, characterized in that: It includes RPA lyophilized reagent and / or CRISPR / Cas12a detection system lyophilized reagent; The RPA lyophilized reagent is a lyophilized reagent made from an RPA amplification system, and the RPA amplification system includes the following components: RPA constant temperature amplification reagent 1 / 18-24μL, Buffer A 49.0-65.3% v / v, Forward primers 0.033-0.044 μM each, The latter primers were 0.033-0.044 μM each, Mixed lyophilized protective agent 18.3-24.4% v / v; The CRISPR / Cas12a detection system lyophilized reagent is a lyophilized reagent made from the CRISPR / Cas12a detection system, and the CRISPR / Cas12a detection system includes the following components: Cas12a 0.167-0.250μM, gRNA 0.167-0.250 μM each, ssDNA reporter molecule 1.667-2.500μM, Buffer 2.1 6.67-10.0%v / v, Mixed lyophilized protective agent 13.3-20.0% v / v; Wherein, the mixed lyophilization protective agent comprises the following components: PEG 8-15% w / v, Bovine serum albumin 4-10% w / v, Mannitol 30-44% w / v.

2. The self-test reagent for HPV typing according to claim 1, characterized in that: The RPA amplification system includes the following components: RPA constant temperature amplification reagent 1 / 22μL, Buffer A 53.4% ​​v / v, Forward primers 0.036 μM each, The latter primers were 0.036 μM each, Mixed lyophilized protective agent 20% v / v; The CRISPR / Cas12a detection system includes the following components: Cas12a 0.25μM, gRNA 0.25 μM each, ssDNA reporter molecule 2.5μM, Buffer 2.1 10%v / v, Mixed lyophilized protective agent 20% v / v; Wherein, the mixed lyophilization protective agent comprises the following components: PEG 15% w / v, Bovine serum albumin 10% w / v, Mannitol 44% w / v.

3. A self-test reagent for HPV typing, characterized in that: It includes RPA lyophilized reagent and / or CRISPR / Cas12a detection system lyophilized reagent; The RPA lyophilized reagent is a lyophilized reagent made from an RPA amplification system, and the RPA amplification system includes the following components: RPA constant temperature amplification reagent 1 / 18-24μL, Buffer A49.0-65.3%v / v, Forward primers 0.033-0.044 μM each, The latter primers were 0.033-0.044 μM each, Mixed lyophilized protective agent 18.3-24.4% v / v; The CRISPR / Cas12a detection system lyophilized reagent is a lyophilized reagent made from the CRISPR / Cas12a detection system, and the CRISPR / Cas12a detection system includes the following components: Cas12a 0.167-0.250μM, gRNA 0.167-0.250 μM each, ssDNA reporter molecule 1.667-2.500μM, Buffer 2.1 6.67-10.0%v / v, Mixed lyophilized protective agent 13.3-20.0% v / v; Wherein, the mixed lyophilization protective agent comprises the following components: Maltose 5-20% w / v, Bovine serum albumin 10-25% w / v, Mannitol 20-40% w / v.

4. The self-test reagent for HPV typing according to claim 3, characterized in that: The RPA amplification system includes the following components: RPA constant temperature amplification reagent 1 / 22μL, Buffer A53.4%v / v, Forward primers 0.036 μM each, The latter primers were 0.036 μM each, Mixed lyophilized protective agent 20% v / v; The CRISPR / Cas12a detection system includes the following components: Cas12a 0.25μM, gRNA 0.25 μM each, ssDNA reporter molecule 2.5μM, Buffer 2.1 10%v / v, Mixed lyophilized protective agent 20% v / v; Wherein, the mixed lyophilization protective agent comprises the following components: Maltose 20% w / v, Bovine serum albumin 25% w / v, Mannitol 40% w / v.

5. The self-test reagent for HPV typing according to any one of claims 1 to 4, characterized in that: The front primer comprises at least one of the primers having nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.14; The rear primer includes at least one of the primers whose nucleotide sequences are shown as SEQ ID NO.15 to SEQ ID NO.

28. The gRNA includes at least one of the RNAs having nucleotide sequences as shown in SEQ ID NO.29-SEQ ID NO.

42.

6. The self-test reagent for HPV typing according to any one of claims 1 to 4, characterized in that: The ssDNA reporter molecule is a single-stranded DNA reporter molecule modified with a fluorescent group and a quenching group.

7. Use of the self-test reagent for HPV typing according to any one of claims 1 to 6 in the preparation of an HPV virus detection kit.

8. A detection kit for HPV typing, characterized in that: include: Buffer B and the self-test reagent for HPV typing according to any one of claims 1 to 6.

9. A method for performing non-diagnostic HPV detection using the detection kit for HPV typing according to claim 8, characterized in that: The steps include: Step 1, respectively re-dissolve the RPA lyophilized reagent and the CRISPR / Cas12a detection system lyophilized reagent; Step 2, adding the Buffer B and the test sample to the solution of the reconstituted RPA lyophilized reagent, and incubating the reaction; Step 3, adding a solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent to the reaction system and incubating the reaction; Step 4, detection by fluorescence detection or color development on a test strip.

10. The method for non-diagnostic HPV detection according to claim 9, characterized in that: In step 1, the solution of the reconstituted RPA lyophilized reagent is added to the reaction tube, and the solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent is added to the reaction tube cap of the reaction tube; In step 3, the solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent is added to the reaction tube by instantaneous centrifugation.

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