A one-pot high-risk HPV detection freeze-dried reagent, a kit and its use

Abstract

The present invention belongs to the field of in vitro detection technology, and specifically relates to a one-pot detection freeze-dried reagent, a kit and its use for high-risk HPV. The freeze-dried reagent of the present invention includes an RPA freeze-dried reagent and a CRISPR / Cas12a detection system freeze-dried reagent; the RPA freeze-dried reagent includes: RPA constant temperature amplification reagent, Buffer A, front primer, rear primer, mixed freeze-dried protective agent; the CRISPR / Cas12a detection system freeze-dried reagent includes: Cas12a, gRNA, ssDNA reporter molecule, Buffer 2.1, mixed freeze-dried protective agent. The composition of the mixed freeze-dried protective agent is optimized according to the detection system and the specific method of the detection, which solves the problem of room temperature storage and transportation of the RPA / CRISPR detection reagent of the HPV virus, and ensures the detection performance, providing an efficient and accurate solution for large-scale screening, and has a 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 one-pot high-risk HPV detection freeze-dried reagent, a kit and uses thereof. Background Art

[0002] Cervical cancer is the fourth most common malignant tumor faced by women worldwide, and it is imperative to eliminate cervical cancer. It is now clear that high-risk HPV infection is the cause of external genital cancer, cervical cancer, and high-grade cervical intraepithelial neoplasia. However, it usually takes 8 to 10 years for HPV to persist in infection and develop into cervical precancerous lesions and eventually cervical cancer. Therefore, improving screening coverage and adopting high-quality screening methods are key means to eliminate cervical cancer.

[0003] There are 14 high-risk HPV types, and nucleic acid detection typing is the WHO recommended method for initial screening of cervical cancer. However, the widely used PCR method requires expensive equipment, special conditions and skilled personnel, and is not suitable for areas with limited conditions. In addition, HPV-infected people need to continuously monitor their treatment status, and repeated testing of 14 types wastes a lot of medical resources, so there is an urgent need to develop a new convenient, home-based, and targeted HPV detection method. RPA is an efficient constant temperature amplification method that is suitable for large-scale screening and resource-poor areas, but non-specific products affect its application. CRISPR / Cas12a can enhance RPA results through precise pairing and efficient cutting. The combination of the two is expected to replace PCR and become a fast, home-based, and efficient means of initial screening for cervical cancer.

[0004] However, conventional RPA and CRISPR detection reagents need to be stored and transported at low temperatures, and repeated freezing and thawing will affect the stability of performance. Therefore, it is crucial to develop reagents that can be stably stored at room temperature and easy to transport. Preparing the detection reagents as freeze-dried reagents is a feasible idea to solve the above problems. However, RPA and CRISPR detection reagents are a complex composition, especially the freeze-dried RPA reagents and freeze-dried CRISPR reagents will be mixed into a system during the reaction process, and its composition is further complicated, including DNA, RNA and enzymes, freeze-dried ingredients and other substances. Therefore, in the process of developing specific detection reagents, how to improve the composition of the freeze-dried reagents, especially how to set the composition of the freeze-dried protectant, so as to ensure the stability of the detection reagents and avoid their performance degradation, is an important issue. Summary of the invention

[0005] In view of the above problems, the present invention provides a one-pot high-risk HPV detection freeze-dried reagent, a kit and uses thereof.

[0006] A one-pot high-risk HPV detection freeze-dried reagent, comprising 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 / 22-25 μL,

[0009] Buffer A 47.1-53.5% v / v,

[0010] Forward primers 0.032-0.036 μM each,

[0011] The latter primers were 0.032-0.036 μM each,

[0012] Mixed lyophilized protective agent 22-25% 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 0.20-0.29μM,

[0015] gRNA 0.20-0.29 μM each,

[0016] ssDNA reporter molecule 2.0-2.9μM,

[0017] Buffer 2.1 8-11.4% v / v,

[0018] Mixed lyophilized protective agent 20-29% v / v;

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

[0020] Sorbitol 8-20% w / v,

[0021] Dimethyl sulfoxide 0-15% w / v,

[0022] Bovine serum albumin 0-15% w / v,

[0023] Trehalose 40-56% w / v;

[0024] The RPA constant temperature amplification reagent and Buffer A are reagents in the DNA constant temperature rapid amplification kit of Anpu Future Biotechnology Co., Ltd.; the Buffer 2.1 is a reagent in the EnGen Lba Cas12a kit of New England Biolabs.

[0025] Preferably, the RPA lyophilized reagent is a lyophilized reagent made from an RPA amplification system, and the RPA amplification system includes the following components:

[0026] RPA constant temperature amplification reagent 1 / 22 μL,

[0027] Buffer A 53.5% v / v,

[0028] Forward primers 0.036 μM each,

[0029] The latter primers were 0.036 μM each,

[0030] Mixed lyophilized protective agent 25% v / v;

[0031] 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:

[0032] Cas12a 0.25μM,

[0033] gRNA 0.25 μM each,

[0034] ssDNA reporter molecule 2.5μM,

[0035] Buffer 2.1 10% v / v,

[0036] Mixed lyophilized protective agent 25% v / v;

[0037] Wherein, the mixed lyophilization protective agent comprises the following components:

[0038] Sorbitol 20% w / v,

[0039] Dimethyl sulfoxide 0-15% w / v,

[0040] Bovine serum albumin 0-15 %w / v

[0041] Trehalose 40-56 %w / v.

[0042] Preferably, it includes RPA lyophilized reagent and / or CRISPR / Cas12a detection system lyophilized reagent;

[0043] The RPA lyophilized reagent is a lyophilized reagent made from an RPA amplification system, and the RPA amplification system includes the following components:

[0044] RPA constant temperature amplification reagent 1 / 22-25 μL,

[0045] Buffer A 47.1-53.5% v / v,

[0046] Forward primers 0.032-0.036 μM each,

[0047] The latter primers were 0.032-0.036 μM each,

[0048] Mixed lyophilized protective agent 22-25% v / v;

[0049] 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:

[0050] Cas12a 0.20-0.29μM,

[0051] gRNA 0.20-0.29 μM each,

[0052] ssDNA reporter molecule 2.0-2.9μM,

[0053] Buffer 2.1 8-11.4% v / v,

[0054] Mixed lyophilized protective agent 20-29% v / v;

[0055] Wherein, the mixed lyophilization protective agent comprises the following components:

[0056] PEG 20-40% w / v,

[0057] Bovine serum albumin 15-30% w / v,

[0058] Trehalose 20-40% w / v,

[0059] Mannitol 16-32% w / v;

[0060] The RPA constant temperature amplification reagent and Buffer A are reagents in the DNA constant temperature rapid amplification kit of Anpu Future Biotechnology Co., Ltd.; the Buffer 2.1 is a reagent in the EnGen Lba Cas12a kit of New England Biolabs.

[0061] Preferably, the RPA lyophilized reagent is a lyophilized reagent made from an RPA amplification system, and the RPA amplification system includes the following components:

[0062] RPA constant temperature amplification reagent 1 / 22 μL,

[0063] Buffer A 53.5% v / v,

[0064] Forward primers 0.036 μM each,

[0065] The latter primers were 0.036 μM each,

[0066] Mixed lyophilized protective agent 25% v / v;

[0067] 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:

[0068] Cas12a 0.25μM,

[0069] gRNA 0.25 μM each,

[0070] ssDNA reporter molecule 2.5μM,

[0071] Buffer 2.1 10% v / v,

[0072] Mixed lyophilized protective agent 25% v / v;

[0073] Wherein, the mixed lyophilization protective agent comprises the following components:

[0074] PEG 40% w / v,

[0075] Bovine serum albumin 30% w / v,

[0076] Trehalose 40% w / v,

[0077] Mannitol 32% w / v.

[0078] 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;

[0079] The rear primer includes at least one of the primers having nucleotide sequences as shown in SEQ ID NO. 15 to SEQ ID NO. 28;

[0080] The gRNA includes at least one of the RNAs having nucleotide sequences as shown in SEQ ID NO. 29-SEQ ID NO. 42.

[0081] Preferably, the ssDNA reporter molecule is a single-stranded DNA reporter molecule modified with a fluorescent group and a quenching group.

[0082] The present invention also provides use of the above-mentioned freeze-dried reagent for HPV one-pot detection in preparing an HPV virus detection kit.

[0083] The present invention also provides a high-risk HPV one-pot detection kit, comprising: Buffer B and the above-mentioned freeze-dried reagent for HPV one-pot detection; the Buffer B is a reagent in the DNA constant temperature rapid amplification kit of Anpu Future Biotechnology Co., Ltd.

[0084] The present invention also provides a method for performing non-diagnostic HPV detection using the high-risk HPV one-pot detection kit, comprising the following steps:

[0085] Step 1, respectively re-dissolve the RPA lyophilized reagent and the CRISPR / Cas12a detection system lyophilized reagent;

[0086] Step 2, adding the Buffer B and the test sample to the solution of the reconstituted RPA lyophilized reagent, and incubating the reaction;

[0087] Step 3, adding a solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent to the reaction system and incubating the reaction;

[0088] Step 4, detection by fluorescence detection or color development on a test strip.

[0089] 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.

[0090] The present invention optimizes primers and gRNA for one-pot RPA / CRISPR detection of 14 high-risk HPV viruses, and optimizes the most appropriate combination of lyophilized protective agents for these specific primer and gRNA combinations. Therefore, the present invention provides for the first time a lyophilized reagent capable of performing a two-step reaction in one reaction vessel (one-pot method) and a kit made thereof. The lyophilized reagent and kit have the following beneficial technical effects:

[0091] 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.

[0092] 2. The present invention realizes for the first time the one-pot 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.

[0093] 3. The present invention realizes a one-pot two-step detection method, and can realize the detection in one reaction container. There is no operation of transferring the reaction liquid, which can effectively avoid the contamination problem of nucleic acid aerosol.

[0094] 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.

[0095] 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

[0096] Figure 1 Schematic diagram of the structure of the microfluidic chip for HPV RPA / CRISPR detection provided in Example 2.

[0097] Figure 2 These are the specificity test result diagrams of Experimental Examples 3, 6 and 7. DETAILED DESCRIPTION

[0098] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0099] In the following examples and experimental examples, the sources of some commercial reagents are as follows:

[0100] 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 NewEngland Biolabs and is a component of EnGen Lba Cas12a (Cpf1) (NEB #M0653).

[0101] Example 1 HPV virus one-pot fluorescence detection kit and detection method

[0102] 1. Kit composition and preparation method

[0103] The kit of this embodiment mainly includes RPA freeze-dried reagent and CRISPR / Cas12a detection system freeze-dried reagent. For each type of HPV virus, a separate freeze-dried ball is prepared.

[0104] The RPA lyophilized reagent is prepared by lyophilizing the RPA amplification system in Table 1.

[0105] Table 1

[0106]

[0107] In other embodiments, in the above system, the volume of enzyme-free water can also be adjusted within the range of 3.14-6.14 as needed.

[0108] The CRISPR / Cas12a detection system freeze-dried reagent is prepared by freeze-drying the CRISPR / Cas12a detection system in Table 2.

[0109] Table 2

[0110]

[0111] In other embodiments, in the above system, the volume of enzyme-free water can also be adjusted within the range of 0.7-1.0 as needed.

[0112] 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.

[0113] Table 3 Front primer and back primer of RPA isothermal amplification

[0114]

[0115]

[0116] The sequences of gRNAs are shown in Table 4.

[0117] Table 4

[0118]

[0119]

[0120] 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.

[0121] The composition of the mixed lyophilization protective agent is an aqueous solution containing the following components: 1) 20% w / v sorbitol, 15% w / v bovine serum albumin, 40% w / v trehalose; or, 2) 20% w / v sorbitol, 15% w / v dimethyl sulfoxide, 56% w / v trehalose. The percentages are mass volume percentages, g / ml.

[0122] The preparation method of RPA lyophilized reagent is as follows:

[0123] Using a precise liquid dispensing system, drop the prepared RPA amplification system solution into liquid nitrogen at a volume of 22 μL to condense into round balls. Wait for 10 seconds for the balls to sink to the bottom of the liquid nitrogen. Pick up the balls and transfer them to a vial that has been pre-cooled with dry ice.

[0124] After the reagent pellets are collected, they are transferred to the freeze dryer. The freeze drying procedure is shown in Table 5:

[0125] Table 5

[0126]

[0127] The preparation method of the CRISPR / Cas12a detection system lyophilized reagent is the same, except that the volume of the CRISPR / Cas12a detection system solution condensed into round beads is 8 μl.

[0128] 2. Detection Method

[0129] Reconstitute the lyophilized pellet made of RPA lyophilized reagent with 18.86 μL enzyme-free water and add it to the reaction tube. Then add 1.14 μL Buffer B and 2 μL of the sample to be tested. Then reconstitute the CRISPR / Cas12a detection system lyophilized reagent with 8 μL enzyme-free water, add it to the reaction tube cap, and gently cover it. Incubate at 37°C for 50 minutes for amplification. After the amplification is completed, centrifuge instantly to centrifuge the reconstituted CRISPR / Cas12a detection system on the cap to the bottom of the tube. Finally, place the reaction tube in a constant temperature fluorescence detector and collect fluorescence data every 1 minute for 50 minutes.

[0130] Example 2 HPV virus one-pot test strip detection kit and detection method

[0131] 1. Kit composition and preparation method

[0132] The kit of this embodiment mainly includes RPA lyophilized reagent and CRISPR / Cas12a detection system lyophilized reagent. The composition and preparation method of the two lyophilized reagents are basically the same as those in Example 1, except that:

[0133] 1. The composition of mixed freeze-dried protective agents is different

[0134] The composition of the mixed lyophilized protective agent in this example is: polyethylene glycol 6000 (PEG, purchased from BioFroxx) 40% w / v, bovine serum albumin 30% w / v, trehalose 40% w / v, and mannitol 32% w / v, wherein the percentages are mass volume percentages, g / ml.

[0135] 2. Different ssDNA reporter molecules

[0136] The ssDNA reporter molecule (Reporter) in this example is ssDNA labeled with FAM-Biotin: (6-FAM)TTATTATT(Biotin).

[0137] 2. Detection Method

[0138] Method 1:

[0139] Reconstitute the lyophilized pellets made of RPA lyophilized reagent with 18.86 μL enzyme-free water and add them to the reaction tube. Then add 1.14 μL Buffer B and 2 μL of the sample to be tested. Then reconstitute the CRISPR / Cas12a detection system lyophilized reagent with 8 μL enzyme-free water, add it to the lid of the reaction tube, and gently cover it. Incubate at 37°C for 50 minutes for amplification. After the amplification is completed, centrifuge instantly to centrifuge the reconstituted CRISPR / Cas12a detection system on the lid to the bottom of the tube. Incubate at 37°C for 50 min. Finally, take 3 μL of the reaction product, add 37 μL 5% PEG chromatography buffer, mix well, insert into the lateral flow immunochromatography test strip (from Milenia GenLine HybriDetect kit, purchased from Milenia biotec), and observe the results with the naked eye within 10-15 minutes.

[0140] Method 2:

[0141] Design a microfluidic chip for HPV RPA / CRISPR detection, such as Figure 1 As shown, the chip includes an RPA area, a CRISPR area, a mixing channel area, a CRISPR shearing area, and a test strip reading area.

[0142] The detection steps are as follows:

[0143] 1) RPA lyophilized reagent was coated on the RPA area, and 18.86 μL enzyme-free water was added to reconstitute it. Then 1.14 μL Buffer B and 2 μL test sample were added to the RPA area and incubated at 37°C for 50 min for amplification.

[0144] 2) The CRISPR / Cas12a detection system freeze-dried reagent is coated in the CRISPR area, and 8 μL of enzyme-free water is added to reconstitute it. The sample product after RPA amplification and the reconstituted CRISPR / Cas12a detection system freeze-dried reagent enter the mixing channel area together, and the two liquids in the mixing channel are fully micro-mixed.

[0145] 3) The mixed solution enters the CRISPR cutting area and incubates at 37°C for 50 min.

[0146] 4) The reaction products of the CRISPR cutting area flow into the lateral flow immunochromatography test strip in the reading area of ​​the test strip, and the results are observed with the naked eye within 15 to 20 minutes.

[0147] The technical solution of the present invention is further illustrated by experiments below.

[0148] Experimental Example 1 Optimal Conditions for One-Pot Fluorescence Detection of HPV Virus

[0149] 1. Primer and gRNA design

[0150] The present invention searches for the full gene sequence of human papillomavirus (HPV) through NCBI (www.ncbi.nlm.nih.gov) and selects the conserved region of the L1 gene as the target region. According to the characteristics of Cas12a recognizing T-rich PAM sequences, the target sequence is screened and detected, and the gRNA of Cas12a for each HPV type is designed according to the cleavage characteristics of Cas12a protein. And the various designed gRNAs are optimized through experiments.

[0151] 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. The designed multiple RPA primers are optimized through experiments.

[0152] The optimal primers and gRNA are preferably as shown in Example 1.

[0153] 2. Optimization of testing conditions

[0154] 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:

[0155] Table 6

[0156]

[0157] The composition of the optimized optimal RPA / CRISPR detection system is shown in Example 1.

[0158] Experimental Example 2 Screening experiment of mixed lyophilized protective agent for one-pot fluorescence detection kit of HPV virus

[0159] In the following experiments, reagents or conditions not specifically described were performed as described in Example 1. This experimental example first screened mixed lyophilized protectants using HPV16 front primers, back primers and gRNA, and then verified that the preferred mixed lyophilized protectants could be used for RPA / CRISPR system detection reagent freeze-dried balls of other thirteen HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68).

[0160] 1. Screening of mixed lyophilization protectants in RPA lyophilization reagents

[0161] 1. Screening of single protective agent

[0162] The effects of sorbitol, dimethyl sulfoxide, dextran, PEG, bovine serum albumin, trehalose, alanine, xylitol, mannitol, and inositol as lyoprotectants for RPA detection system and CRISPR detection system on the fluorescence signal were studied.

[0163] 5.5 μl and 2.0 μl of 10 lyophilized protective agents with a concentration of 15% (mass volume percentage, g / ml) were added to the RPA reagent and the CRISPR reagent, respectively, to obtain the RPA detection system (Table 7) and CRISPR detection system (Table 8) containing a single protective agent as follows.

[0164] Table 7

[0165]

[0166] Table 8

[0167]

[0168] 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 decreases by no more than 10% during the same period of time, it is considered that the protective agent has no inhibitory effect on the detection system. The results are shown in Table 9:

[0169] Table 9

[0170]

[0171] The results showed that sorbitol, dimethyl sulfoxide, bovine serum albumin and trehalose had no effect on CRISPR detection reagents and RPA isothermal amplification reagents.

[0172] 2. Suitable concentration range of protective agent

[0173] The above freeze-dried protective agents whose fluorescence signal intensity decreases by no more than 10% are selected, and the concentration is configured to be 1%-64%. The fluorescence intensity of the RPA detection system and the CRISPR detection system is tested for different concentrations of freeze-dried protective agents. Compared with the fluorescence intensity of the RPA detection system and the CRISPR detection system without freeze-dried protective agents, if the fluorescence signal intensity decreases by no more than 10% within 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 10:

[0174] Table 10

[0175]

[0176] The results showed that the suitable concentration range of sorbitol was 8%-56%, the suitable concentration range of dimethyl sulfoxide was 4%-40%, the suitable concentration range of bovine serum albumin was 2%-40%, and the suitable concentration range of trehalose was 4%-56%.

[0177] 3. Screening of freeze-dried protective agents for RPA isothermal amplification reagents

[0178] 1) The contents of sorbitol, dimethyl sulfoxide, bovine serum albumin, and trehalose were optimized by orthogonal experimental design with 4 factors and 4 levels, as shown in Tables 11 and 12.

[0179] Table 11 Factor level table

[0180]

[0181] Table 12 Scheme

[0182]

[0183] 2) Select all 16 schemes above and freeze-dry the RPA isothermal amplification reagent. The freeze-drying system is shown in Table 13:

[0184] Table 13

[0185]

[0186] 3) Preparation of RPA isothermal amplification reagent freeze-dried beads

[0187] Prepared according to the method of Example 1.

[0188] 4) Morphological examination of freeze-dried pellets of RPA isothermal amplification reagent:

[0189] Observe whether the appearance of the RPA constant temperature amplification reagent freeze-dried pellet is qualified. The test results are shown in Table 14:

[0190] Table 14

[0191]

[0192] Based on the above results, Scheme 5, Scheme 6, and Scheme 9 were selected for the next step of screening.

[0193] 5) Moisture content of RPA constant temperature amplification reagent freeze-dried pellet

[0194] The moisture content of the RPA constant temperature amplification reagent freeze-dried balls that passed the above morphological inspection was measured using a card type moisture meter. The results are shown in Table 15:

[0195] Table 15

[0196]

[0197] Based on the above results, Scheme 5 and Scheme 6 were selected for the next step of screening.

[0198] 6) Select the RPA freeze-dried reagent balls with qualified moisture content for performance inspection

[0199] First, the RPA lyophilized reagent was placed in a reaction tube and 18.86 μL of enzyme-free water was added to reconstitute it, followed by 1.14 μL of Buffer B and 2 μL of the target to be tested, and negative and positive controls were set up at the same time. Then, 8 μL of the CRISPR / Cas12a detection system was added to the lid of the reaction tube and the lid was gently closed. Incubate at 37°C for 50 min for amplification. After the amplification is completed, centrifuge instantly to centrifuge the CRISPR / Cas12a detection system on the lid to the bottom of the tube. Finally, the reaction tube was placed in a constant temperature fluorescence detector, and fluorescence data was collected every 1 minute for 50 minutes. 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. The results are shown in Table 16:

[0200] Table 16

[0201]

[0202] The results show that both Scheme 5 and Scheme 6 can achieve ideal detection results. Scheme 5 and Scheme 6 can be used as the preferred mixed lyophilization protective agent schemes.

[0203] 2. Screening of mixed lyophilized protective agents in lyophilized reagents for CRISPR / Cas12a detection system

[0204] 1) Select the RPA constant temperature amplification reagent lyophilization protectant scheme of the above scheme 5 and scheme 6, and lyophilize the CRISPR / Cas12a detection reagent. The lyophilization system is shown in Table 17:

[0205] Table 17

[0206]

[0207] 2) Preparation of CRISPR / Cas12a detection reagent freeze-dried pellets

[0208] Prepared according to the method of Example 1.

[0209] 3) Morphological examination of freeze-dried pellets of CRISPR / Cas12a detection reagent:

[0210] Observe whether the appearance of the RPA isothermal amplification reagent freeze-dried pellet is qualified. The test results are shown in Table 18:

[0211] Table 18

[0212]

[0213] The results showed that the CRISPR / Cas12a detection reagent lyophilized protectant screening schemes 5 and 6 were qualified.

[0214] 4) Moisture content of CRISPR / Cas12a detection reagent freeze-dried pellets

[0215] 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 19:

[0216] Table 19

[0217]

[0218] The results showed that the CRISPR / Cas12a detection reagent lyophilized protectant screening schemes 5 and 6 were qualified.

[0219] 5) Select the CRISPR / Cas12a detection reagent freeze-dried pellets with qualified moisture content for performance inspection

[0220] The detection method of Example 1 was used for the operation. 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. The results are shown in Table 20:

[0221] Table 20

[0222]

[0223] The results showed that Scheme 5 had the best effect and could be used as a preferred mixed lyophilization protectant scheme.

[0224] Scheme 5 was further used to prepare freeze-dried beads of RPA / CRISPR system 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.

[0225] Experimental Example 3 Performance Verification of the One-Pot Fluorescence Detection Method for Freeze-dried HPV Virus

[0226] The RPA lyophilized reagent and CRISPR / Cas12a detection lyophilized reagent used in this experimental example are as described in Example 1.

[0227] 1. Sensitivity test

[0228] The detection method of Example 1 was used. The results are shown in Table 21:

[0229] Table 21

[0230]

[0231]

[0232] 2. Specificity test

[0233] The detection method of Example 1 was used to operate. The results were as follows Figure 2 shown.

[0234] 3. Clinical sample testing

[0235] The cervical sampling cotton swab was placed in a physiological saline sampling tube, the specimen was shaken and mixed, 200 uL of the sample was centrifuged at 14000 rpm for 5 min, and the supernatant was discarded; 200 uL of nucleic acid release agent was added, and the metal bath was 100℃ for 15 min. The supernatant after centrifugation was the sample to be tested, and the test results of the one-pot fluorescence detection method were compared with the clinical test results, and the diagnostic sensitivity and specificity were calculated, as shown in Table 22.

[0236] Table 22

[0237]

[0238] The above results show that the HPV virus one-pot fluorescence detection kit and detection method of the present invention have good sensitivity and specificity and can meet application requirements.

[0239] Experimental Example 4 Optimal Conditions for One-Pot Test Strip Detection of HPV Virus

[0240] 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:

[0241] Table 23

[0242]

[0243] Experimental Example 5 Screening experiment of mixed lyophilized protective agent for HPV virus one-pot test strip detection kit

[0244] In the following experiments, reagents or conditions not specifically described were performed as described in Example 2. This experimental example first screened mixed lyophilized protectants using HPV16 front primers, back primers and gRNA, and then verified that the preferred mixed lyophilized protectants could be used for RPA / CRISPR system detection reagent freeze-dried balls of other thirteen HPV viruses (HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68).

[0245] 1. Screening of mixed lyophilization protectants in RPA lyophilization reagents

[0246] 1. Screening of single protective agent

[0247] The effects of sorbitol, dimethyl sulfoxide, dextran, PEG, bovine serum albumin, trehalose, alanine, xylitol, mannitol, and inositol as lyoprotectants for RPA detection system and CRISPR detection system on the colorimetric signal were studied.

[0248] 5.5 μL and 2.0 μL of 10 lyophilized protective agents with a concentration of 15% (mass volume percentage, g / mL) were added to the RPA reagent and the CRISPR reagent, respectively, to obtain the RPA detection system (Table 24) and CRISPR detection system (Table 25) containing a single protective agent as follows:

[0249] Table 24

[0250]

[0251] Table 25

[0252]

[0253] The color intensity of the RPA detection system and the CRISPR detection system containing 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 26:

[0254] Table 26

[0255]

[0256] The results showed that dimethyl sulfoxide, PEG, bovine serum albumin, trehalose and mannitol had no effect on CRISPR detection reagents and RPA isothermal amplification reagents.

[0257] 2. Suitable concentration range of protective agent

[0258] 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%-64%. 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 27:

[0259] Table 27

[0260]

[0261] The above results show that the suitable concentration range of dimethyl sulfoxide is 4%-48%, the suitable concentration range of PEG is 8%-56%, the suitable concentration range of bovine serum albumin is 4%-40%, the suitable concentration range of trehalose is 2%-56%, and the suitable concentration range of mannitol is 4%-48%.

[0262] 3. Screening of freeze-dried protective agents for RPA isothermal amplification reagents

[0263] 1) The contents of PEG, sucrose, trehalose and mannitol were optimized by orthogonal experimental design with 5 factors and 3 levels, as shown in Tables 28 and 29.

[0264] Table 28 Factor level table

[0265]

[0266] Table 29 Scheme

[0267]

[0268] 2) Select all 16 schemes above and freeze-dry the RPA isothermal amplification reagent. The freeze-drying system is shown in Table 30:

[0269] Table 30

[0270]

[0271] 3) Preparation of RPA isothermal amplification reagent freeze-dried beads

[0272] Prepared according to the method of Example 1.

[0273] 4) Morphological examination of freeze-dried pellets of RPA isothermal amplification reagent:

[0274] Observe whether the appearance of the RPA constant temperature amplification reagent freeze-dried pellet is qualified. The test results are shown in Table 31:

[0275] Table 31

[0276]

[0277] Based on the above results, Scheme 2, Scheme 3, Scheme 6, and Scheme 11 were selected for further screening.

[0278] 5) Moisture content of RPA constant temperature amplification reagent freeze-dried pellet

[0279] The moisture content of the RPA constant temperature amplification reagent freeze-dried balls that passed the above morphological inspection was measured using a card type moisture meter. The results are shown in Table 32:

[0280] Table 32

[0281]

[0282] Based on the above results, Scheme 2, Scheme 3, and Scheme 11 were selected for further screening.

[0283] 6) Select the RPA freeze-dried reagent balls with qualified moisture content for performance inspection

[0284] First, the RPA lyophilized reagent was placed in a reaction tube and 18.86 μL of enzyme-free water was added to reconstitute it, followed by 1.14 μL of Buffer B and 2 μL of the target to be tested, and negative and positive controls were set up at the same time. Then, 8 μL of the CRISPR / Cas12a detection system was added to the lid of the reaction tube and the lid was gently closed. Incubate at 37°C for 50 min for amplification. After the amplification is completed, centrifuge instantly to centrifuge the CRISPR / Cas12a detection system on the lid to the bottom of the tube. Incubate at 37°C for 50 min. Finally, take 3 μL of the reaction product, add 37 μL of 5% PEG chromatography buffer, mix well, insert the lateral flow immunochromatography test strip, and observe the results with the naked eye within 10 to 15 minutes. The results of the above test strips were 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 33:

[0285] Table 33

[0286]

[0287] The above results show that Scheme 2, Scheme 3 and Scheme 11 are better choices.

[0288] 2. Screening of mixed lyophilized protective agents in lyophilized reagents for CRISPR / Cas12a detection system

[0289] 1) Select Scheme 2, Scheme 3, or Scheme 11, and freeze-dry the CRISPR / Cas12a detection reagent. The freeze-drying system is shown in Table 34:

[0290] Table 34

[0291]

[0292] 2) Preparation of CRISPR / Cas12a detection reagent freeze-dried pellets

[0293] Prepared according to the method of Example 1.

[0294] 3) Morphological examination of freeze-dried pellets of CRISPR / Cas12a detection reagent:

[0295] Observe whether the appearance of the mixed RPA constant temperature amplification reagent freeze-dried ball is qualified. The test results are shown in Table 36:

[0296] Table 36

[0297]

[0298] Therefore, Scheme 3 and Scheme 11 were selected for further screening.

[0299] 4) Moisture content of CRISPR / Cas12a detection reagent freeze-dried pellets

[0300] 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 37:

[0301] Table 37

[0302]

[0303] Therefore, option 3 was selected for further verification.

[0304] 5) Select the CRISPR / Cas12a detection reagent freeze-dried pellets with qualified moisture content for performance inspection

[0305] The detection method 1 of Example 2 was followed. The color development results of the test strips of the above scheme were 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 38:

[0306] Table 38

[0307]

[0308] The above results show that Scheme 3 is the preferred mixed lyophilization protectant scheme.

[0309] Scheme 3 was further used to prepare freeze-dried beads of RPA / CRISPR system 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.

[0310] Experimental Example 6 Performance Verification of the One-Pot Test Strip Detection Method for Freeze-Dried HPV Virus

[0311] The detection method of this experimental example is performed according to method 1 in Example 2.

[0312] 1. Sensitivity test

[0313] The test was performed according to the detection method 1 of Example 2. The results are shown in Table 39:

[0314] Table 39

[0315]

[0316]

[0317] 2. Specificity test

[0318] The results were as follows: Figure 2 shown.

[0319] 3. Clinical sample testing

[0320] 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; 200uL of nucleic acid release agent was added, and the metal bath was 100℃ 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 40.

[0321] Table 40

[0322]

[0323] Experimental Example 7 Performance Verification of the One-Pot Test Strip Detection Method for Freeze-dried HPV Virus (Using Microfluidic Chip)

[0324] The detection method of this experimental example is performed according to method 2 in Example 2.

[0325] 1. Sensitivity test

[0326] The designed concentration gradient is 10 -1 —10 5 The sample to be tested was aM, and the operation was performed according to the method 2 in Example 2. The results are shown in Table 41:

[0327] Table 41

[0328]

[0329]

[0330] 2. Specificity test

[0331] Select a concentration of 10 5 The 14 high-risk HPV types of aM were used as samples to be tested. The operation was performed according to the method 2 in Example 2. The results are shown in FIG. Figure 2 shown.

[0332] 3. Clinical sample testing

[0333] Cervical samples of 14 high-risk HPV types were collected respectively, 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 metal bath was 100°C for 15 min. The supernatant after centrifugation was the sample to be tested, and the operation was performed according to method 2 in Example 2. The test results were compared with the clinical test results, and the diagnostic sensitivity and specificity were calculated. The results are shown in Table 42:

[0334] Table 42

[0335]

[0336] The above experimental results show that the HPV virus one-pot test strip detection kit and detection method provided in Example 2 have excellent detection performance and can meet the requirements of actual detection.

[0337] In summary, the present invention solves the problem of room temperature storage and transportation of 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 one-pot high-risk HPV detection freeze-dried reagent, characterized in that: It includes RPA lyophilized reagents and CRISPR / Cas12a detection system lyophilized reagents; 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, Buffer A 47.1-53.5% v / v, Forward primers 0.032-0.036 μM each, The latter primers were 0.032-0.036 μM each, Mixed lyophilized protective agent 22-25% v / v; The volume of the RPA amplification system is 22-25 μL; 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 0.20-0.29μM, gRNA 0.20-0.29 μM each, ssDNA reporter molecule 2.0-2.9μM, Buffer 2.1 8-11.4% v / v, Mixed lyophilized protective agent 20-29% v / v; Wherein, the mixed lyophilization protective agent consists of the following components: Sorbitol 8-20% w / v, Dimethyl sulfoxide 15% w / v, Trehalose 40-56% w / v; or, Sorbitol 8-20% w / v, Bovine serum albumin 15% w / v, Trehalose 40-56% w / v; The RPA constant temperature amplification reagent and Buffer A are reagents in the DNA constant temperature rapid amplification kit of Anpu Future Biotechnology Co., Ltd.; the Buffer 2.1 is a reagent in the EnGen Lba Cas12a kit of New England Biolabs.

2. The one-pot high-risk HPV detection freeze-dried reagent according to claim 1, characterized in that: 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, Buffer A 53.5% v / v, Forward primers 0.036 μM each, The latter primers were 0.036 μM each, Mixed lyophilized protective agent 25% v / v; The volume of the RPA amplification system is 22 μL; 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 0.25μM, gRNA 0.25 μM each, ssDNA reporter molecule 2.5μM, Buffer 2.1 10% v / v, Mixed lyophilized protective agent 25% v / v; Wherein, the mixed lyophilization protective agent consists of the following components: Sorbitol 20% w / v, Dimethyl sulfoxide 15% w / v, Trehalose 40-56% w / v; or, Sorbitol 20% w / v, Bovine serum albumin 15% w / v, Trehalose 40-56 %w / v.

3. A one-pot high-risk HPV detection freeze-dried reagent, characterized in that: It includes RPA lyophilized reagents and CRISPR / Cas12a detection system lyophilized reagents; 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, Buffer A 47.1-53.5% v / v, Forward primers 0.032-0.036 μM each, The latter primers were 0.032-0.036 μM each, Mixed lyophilized protective agent 22-25% v / v; The volume of the RPA amplification system is 22-25 μL; 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 0.20-0.29μM, gRNA 0.20-0.29 μM each, ssDNA reporter molecule 2.0-2.9μM, Buffer 2.1 8-11.4% v / v, Mixed lyophilized protective agent 20-29% v / v; Wherein, the mixed lyophilization protective agent consists of the following components: PEG 20-40% w / v, Bovine serum albumin 15-30% w / v, Trehalose 20-40% w / v, Mannitol 16-32% w / v; The RPA constant temperature amplification reagent and Buffer A are reagents in the DNA constant temperature rapid amplification kit of Anpu Future Biotechnology Co., Ltd.; the Buffer 2.1 is a reagent in the EnGen Lba Cas12a kit of New England Biolabs.

4. The one-pot high-risk HPV detection freeze-dried reagent according to claim 3, characterized in that: 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, Buffer A 53.5% v / v, Forward primers 0.036 μM each, The latter primers were 0.036 μM each, Mixed lyophilized protective agent 25% v / v; The volume of the RPA amplification system is 22 μL; 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 0.25μM, gRNA 0.25 μM each, ssDNA reporter molecule 2.5μM, Buffer 2.1 10% v / v, Mixed lyophilized protective agent 25% v / v; Wherein, the mixed lyophilization protective agent consists of the following components: PEG 40% w / v, Bovine serum albumin 30% w / v, Trehalose 40% w / v, Mannitol 32% w / v.

5. The one-pot high-risk HPV detection freeze-dried reagent 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 having nucleotide sequences as shown in 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 one-pot high-risk HPV detection freeze-dried reagent 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 one-pot high-risk HPV detection freeze-dried reagent according to any one of claims 1 to 6 in the preparation of an HPV virus detection kit.

8. A one-pot detection kit for high-risk HPV, characterized in that: include: Buffer B and the lyophilized reagent for one-pot detection of high-risk HPV according to any one of claims 1 to 6; The Buffer B is a reagent in the DNA isothermal rapid amplification kit of Anpu Future Biotechnology Co., Ltd.

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