A nucleic acid combination, kit and method for detecting monkeypox virus based on CRISPR technology

Abstract

The present invention belongs to the field of in vitro detection technology, and specifically relates to a nucleic acid combination, a kit and a method for detecting monkeypox virus based on CRISPR technology. The detection reagents of the present invention include RPA freeze-dried reagents and CRISPR / Cas12a detection system freeze-dried reagents; RPA freeze-dried reagents include: RPA constant temperature amplification reagents, Buffer A, front primers, back primers, mixed freeze-dried protective agents; CRISPR / Cas12a detection system freeze-dried reagents include: Cas12a, gRNA, ssDNA reporter molecules, Buffer 2.1, mixed freeze-dried protective agents. The composition of the mixed freeze-dried protective agent is optimized according to the composition of the detection system and the specific method of detection. The present invention solves the problem of room temperature storage and transportation of RPA / CRISPR detection reagents for monkeypox virus, has the characteristics of simple operation, rapid detection, high sensitivity, and suitability for on-site and large-scale screening, provides an efficient and accurate solution for large-scale screening, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro detection technology, and specifically relates to a nucleic acid combination, a kit and a method for detecting monkeypox virus based on CRISPR technology. Background Art

[0002] Monkeypox virus (MPXV) is a double-stranded DNA virus belonging to the genus Orthopoxvirus in the Poxviridae family, along with smallpox and cowpox viruses. It can be transmitted through contact with body fluids, cutaneous or mucous membrane wounds, respiratory droplets, and contaminated objects. Consuming undercooked meat products from infected animals also increases the risk of infection. Although MPXV patients often present with skin lesions, herpes simplex virus, molluscum contagiosum virus, and various bacterial skin infections can also cause similar skin lesions. Therefore, reliable laboratory diagnostic methods are needed to differentiate between monkeypox infection. Existing immunological, molecular biological, and microbiological methods are of limited value in distinguishing MPXV from other orthopoxviruses and diagnosing acute monkeypox infection, and cannot be used independently for MPXV diagnosis.

[0003] Recombinase polymerase amplification (RPA) plays an important role in point-of-care testing (POCT). Although RPA has been used to detect MPXV with good sensitivity, the problem of nonspecific products cannot be ignored. By leveraging the characteristics of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), guide RNA (gRNA) tightly binds to the Cas protein, thereby activating the Cas protein to precisely cleave the target DNA or RNA sequence. Therefore, the combination of RPA and CRISPR ensures a fast, efficient, and accurate detection process.

[0004] However, existing methods require opening the lid and transferring some of the amplified product, which carries the risk of aerosol contamination. Furthermore, nucleic acid detection reagents must be stored at -20°C, and repeated freeze-thaw cycles during transportation or use can degrade their detection performance. Therefore, developing a detection reagent and method that is stable at room temperature and does not require opening the lid is crucial. Summary of the Invention

[0005] To address the above problems, the present invention provides a nucleic acid combination, kit and method for detecting monkeypox virus based on CRISPR technology.

[0006] A one-pot fluorescence detection reagent for monkeypox virus, comprising a lyophilized RPA reagent and / or a lyophilized CRISPR / Cas12a detection system reagent;

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

[0008] RPA constant temperature amplification reagent 1 / 20-25μL,

[0009] Buffer A48-60% v / v,

[0010] Forward primer 0.04-0.05 μM,

[0011] Rear primer 0.04-0.05μM,

[0012] Mixed lyophilized protective agent 20-25% 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 0.2-0.33μM,

[0015] gRNA 0.2-0.33 μM,

[0016] ssDNA reporter molecule 1.2-3μM,

[0017] Buffer 2.18.0-13.3% v / v,

[0018] Mixed lyophilized protective agent 16.0-26.7% v / v;

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

[0020] Polyethylene glycol 10-20% w / v,

[0021] Trehalose 4-15% w / v,

[0022] Sucrose 20-32% w / v,

[0023] Mannitol 15-30% w / v.

[0024] Preferably, the RPA isothermal amplification system includes the following components:

[0025] RPA constant temperature amplification reagent 1 / 25μL,

[0026] Buffer A 60% v / v,

[0027] Forward primer 0.04 μM,

[0028] The rear primer 0.04 μM,

[0029] Mixed lyophilized protective agent 20.0% v / v;

[0030] The CRISPR / Cas12a detection system includes the following components:

[0031] Cas12a 0.25μM,

[0032] gRNA 0.25 μM,

[0033] ssDNA reporter molecule 1.5μM,

[0034] Buffer 2.110% v / v,

[0035] Mixed lyophilized protective agent 20% v / v;

[0036] Wherein, the mixed lyophilization protective agent includes the following components:

[0037] Polyethylene glycol 20% w / v,

[0038] Trehalose 15% w / v,

[0039] Sucrose 32% w / v,

[0040] Mannitol 30% w / v.

[0041] A one-pot test strip for monkeypox virus, comprising a lyophilized RPA reagent and / or a lyophilized CRISPR / Cas12a detection system reagent;

[0042] The RPA lyophilized reagent is a lyophilized reagent made from an RPA constant temperature amplification system, and the RPA constant temperature amplification system includes the following components:

[0043] RPA constant temperature amplification reagent 1 / 20-25μL,

[0044] Buffer A48-60% v / v,

[0045] Forward primer 0.04-0.05 μM,

[0046] Rear primer 0.04-0.05μM,

[0047] Mixed lyophilized protective agent 20-25% v / v;

[0048] 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:

[0049] Cas12a 0.2-0.33μM,

[0050] gRNA 0.2-0.33 μM,

[0051] ssDNA reporter molecule 1.2-3μM,

[0052] Buffer 2.18.0-13.3% v / v,

[0053] Mixed lyophilized protective agent 16.0-26.7% v / v;

[0054] Wherein, the mixed lyophilization protective agent includes the following components:

[0055] Polyethylene glycol 4-16% w / v,

[0056] Trehalose 2-20% w / v,

[0057] Mannitol 2-20% w / v.

[0058] Preferably, the RPA isothermal amplification system includes the following components:

[0059] RPA constant temperature amplification reagent 1 / 25μL,

[0060] Buffer A 60% v / v,

[0061] Forward primer 0.04 μM,

[0062] The rear primer 0.04 μM,

[0063] Mixed lyophilized protective agent 20% v / v;

[0064] The CRISPR / Cas12a detection system includes the following components:

[0065] Cas12a 0.25μM,

[0066] gRNA 0.25 μM,

[0067] ssDNA reporter molecule 1.5μM,

[0068] Buffer 2.110% v / v,

[0069] Mixed lyophilized protective agent 20% v / v;

[0070] Wherein, the mixed lyophilization protective agent includes the following components:

[0071] Polyethylene glycol 16% w / v,

[0072] Trehalose 20% w / v,

[0073] Mannitol 20% w / v.

[0074] Preferably, the front primer comprises at least one of the primers having nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2;

[0075] The rear primer includes at least one of the primers having nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4;

[0076] The nucleotide sequence of the gRNA is shown in SEQ ID NO.5.

[0077] Preferably, the ssDNA reporter molecule is a single-stranded DNA reporter molecule modified with a fluorescent group and a quencher group.

[0078] The present invention also provides use of the above-mentioned detection reagent in preparing a monkeypox virus detection kit.

[0079] The present invention also provides a monkeypox virus detection kit, characterized in that it comprises: Buffer B and the above-mentioned detection reagent.

[0080] The present invention also provides a method for detecting monkeypox virus for non-diagnostic purposes using the monkeypox virus detection kit, comprising the following steps:

[0081] Step 1: Redissolve the RPA lyophilized reagent and the CRISPR / Cas12a detection system lyophilized reagent separately;

[0082] Step 2, adding the Buffer B and the test sample to the solution of the reconstituted RPA freeze-dried reagent, and incubating the reaction;

[0083] Step 3: Add the solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent to the reaction system and incubate the reaction;

[0084] Step 4: Detection by fluorescence detection or color development on a test strip.

[0085] 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.

[0086] The present invention optimizes primers and gRNA for one-pot RPA / CRISPR detection of monkeypox virus, and also optimizes the most appropriate lyoprotectant combination for these specific primer and gRNA combinations. Thus, the present invention provides, for the first time, a lyophilized reagent and a kit for use thereof that can perform one-pot RPA / CRISPR detection of monkeypox virus. The lyophilized reagent and kit have the following beneficial technical effects:

[0087] 1. By designing primers and gRNA sequences and optimizing the freeze-dried protective agent combination, the present invention has good sensitivity, specificity and repeatability for monkeypox virus detection, which can meet the needs of practical applications.

[0088] 2. This invention realizes for the first time the conversion of a one-pot RPA / CRISPR detection reagent for monkeypox virus into a freeze-dried reagent, which has the advantages of convenient storage and transportation, stable reagent performance, and ease of use.

[0089] 3. The detection method of the present invention is a one-pot detection. There is no need to separate the RPA amplification reaction and 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.

[0090] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0091] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Schematic diagram of the structure of the microfluidic chip for MPXV RPA / CRISPR detection provided in Example 2.

[0093] Figure 2 This is a graph showing the sensitivity experimental results of the one-pot fluorescence detection method for freeze-dried MPXV virus.

[0094] Figure 3 This is a graph showing the specificity experimental results of the one-pot fluorescence detection method for freeze-dried MPXV virus.

[0095] Figure 4 This is a graph showing the reproducible experimental results of the one-pot fluorescence detection method for freeze-dried MPXV virus.

[0096] Figure 5This is the clinical sample test results of the one-pot fluorescence detection method for freeze-dried MPXV virus.

[0097] Figure 6 This is a graph showing the sensitivity and specificity experimental results of the one-pot test strip detection method for freeze-dried MPXV virus.

[0098] Figure 7 This is a graph showing the sensitivity and specificity experimental results of the one-pot test strip detection method for freeze-dried MPXV virus (using a microfluidic chip).

[0099] Figure 8 This is a diagram of the clinical sample test results of the freeze-dried MPXV virus one-pot test strip detection method (using a microfluidic chip). DETAILED DESCRIPTION

[0100] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available.

[0101] In the following examples and experimental examples, the sources of some commercial reagents are as follows:

[0102] RPA constant temperature amplification reagent, Buffer A and Buffer B were from the DNA constant temperature rapid amplification kit (Cat. No.: WLB8201KIT) of Anpu Future Biotechnology Co., Ltd.; Buffer 2.1 was from New England Biolabs and is a component of EnGenLba Cas12a (Cpf1) (NEB#M0653).

[0103] Example 1 MPXV virus one-pot fluorescence detection kit and detection method

[0104] 1. Kit composition and preparation method

[0105] The kit of this embodiment mainly includes RPA / CRISPR system detection reagent freeze-dried balls for monkeypox virus (MPXV) detection, which include RPA freeze-dried reagent balls made by freeze-drying the RPA constant temperature amplification system in Table 1 and CRISPR detection reagent freeze-dried balls made by freeze-drying the CRISPR / Cas12a detection system in Table 2.

[0106] Table 1

[0107]

[0108] In other embodiments, in the above system, the volume of enzyme-free water can also be adjusted within the range of 1 μL-6 μL as needed.

[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 0.9 μL-3.2 μL as needed.

[0112] The sequences of the front primer and the back primer are shown in Table 3, wherein the sequence marked with "F" is the front primer, and the sequence marked with "R" is the back primer; the preferred front primer in this embodiment is RPA-A27-F2, and the preferred back primer is RPA-A27-R2.

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

[0114]

[0115] gRNA: The crRNA of Cas12a was designed based on the cleavage characteristics of Cas12a protein, and its sequence number is SEQ ID NO.5, and the sequence is: UAAUUUCUACUAAGUGUAGAUUCAUUAUUAGGUGGGGGAUG.

[0116] The ssDNA reporter molecule is FAM-BHQ-labeled ssDNA: (6-FAM)TTATT(BHQ1). In other embodiments, the fluorescent group FAM can also be replaced by VIC, HEX, TET, JOE, Cy5, Cy3, TAMRA, etc.

[0117] The composition of the mixed lyophilization protectant is an aqueous solution containing the following components: 20% w / v polyethylene glycol 6000 (PEG, purchased from BioFroxx), 15% w / v trehalose, 32% w / v sucrose, and 30% w / v mannitol, wherein the percentages are weight-volume percentages, g / ml.

[0118] The specific method for preparing the above-mentioned RPA / CRISPR detection system into freeze-dried pellets is as follows:

[0119] 1. Prepare RPA lyophilized reagent pellets: Using a precise liquid dispensing system, add 25 μL of the prepared lyophilized mixed solution of the RPA constant temperature amplification system into liquid nitrogen to condense into round pellets. Wait for about 10 seconds for the pellets to sink to the bottom of the liquid nitrogen. Pick up the pellets and transfer them to a vial that has been pre-cooled with dry ice.

[0120] 2. Prepare CRISPR / Cas12a detection reagent freeze-dried pellets: Using a precise liquid dispensing system, add 8 μl of the prepared CRISPR / Cas12a detection system freeze-dried mixed solution into liquid nitrogen to condense into round pellets. Wait about 10 seconds for the pellets to sink to the bottom of the liquid nitrogen. Pick up the pellets and transfer them to a vial that has been pre-cooled with dry ice.

[0121] After the reagent pellets are collected, they are transferred to the freeze dryer. The freeze drying procedure is shown in Table 4:

[0122] Table 4

[0123]

[0124]

[0125] 2. Detection Method

[0126] After placing the RPA freeze-dried reagent ball in the reaction tube, add 21μL of enzyme-free water to reconstitute it, and then add 2μL of Buffer B. Subsequently, add 2μL of the target to be tested. Then, add 8μL of enzyme-free water to reconstitute the CRISPR / Cas12a detection reagent freeze-dried ball, add it to the reaction tube lid, and gently cover it. Incubate at 37°C for 40 minutes for amplification. After the amplification is completed, centrifuge instantly to allow the reconstituted CRISPR / Cas12a detection system on the lid to be centrifuged 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 40 minutes.

[0127] Example 2 MPXV virus one-pot test strip detection kit and detection method

[0128] 1. Kit composition and preparation method

[0129] The kit of this embodiment mainly includes RPA / CRISPR system detection reagent freeze-dried pellets for MPXV virus detection. The composition and preparation method of the freeze-dried pellets are basically the same as those in Example 1, except that the composition of the mixed freeze-dried protective agent is different.

[0130] The composition of the mixed lyophilized protective agent for the MPXV virus test strip detection in this embodiment is an aqueous solution containing the following components: 16% w / v polyethylene glycol, 20% w / v trehalose, and 20% w / v mannitol, wherein the percentages are weight-volume percentages, g / ml.

[0131] 2. Detection Method

[0132] Method 1:

[0133] After placing the RPA freeze-dried reagent ball into the reaction tube, add 21μL of enzyme-free water to reconstitute it, and then add 2μL of Buffer B. Subsequently, add 2μL of the sample to be tested. Then, add 8μL of enzyme-free water to reconstitute the CRISPR / Cas12a detection reagent freeze-dried ball, add it to the reaction tube cap, and gently cover it. Incubate at 37°C for 40 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, and incubate at 37°C for 40 minutes. Finally, take 1μL of the reaction product, add 39μL of 5% PEG chromatography buffer, mix well, insert it into the lateral flow immunochromatography test strip (from Milenia GenLine HybriDetect kit, purchased from Mileniabiotec), and observe the results with the naked eye within 15 to 20 minutes.

[0134] Method 2:

[0135] Design a microfluidic chip for MPXV RPA / CRISPR detection, such as Figure 1 As shown, the microfluidic chip includes an RPA area, a CRISPR area, a mixing channel area, a CRISPR shearing area, and a test strip reading area.

[0136] The detection steps are as follows:

[0137] 1) Coat the RPA area with the RPA lyophilized reagent pellet and reconstitute it with 21 μL of enzyme-free water. Add 2 μL of Buffer B and 2 μL of the sample to be tested to the RPA area and incubate at 37°C for 40 minutes to allow for amplification.

[0138] 2) The CRISPR lyophilized reagent pellet is coated in the CRISPR zone and reconstituted with 8 μL of enzyme-free water. The RPA-amplified sample product and the reconstituted CRISPR reagent are then introduced into the mixing channel, where the two liquids are thoroughly mixed.

[0139] 3) The mixed solution enters the CRISPR cutting zone and incubates at 37°C for 40 minutes.

[0140] 4) The reaction products of the CRISPR cutting area flow into the lateral flow immunochromatographic test strip in the reading area of ​​the test strip, and the results are observed with the naked eye within 15 to 20 minutes.

[0141] The technical solution of the present invention is further illustrated by experiments below.

[0142] Experimental Example 1 Optimal Conditions for One-Pot Fluorescence Detection of MPXV Virus

[0143] 1. Design of primers, crRNA, and ssDNA reporter molecules

[0144] The present invention searches for the full gene sequence of monkeypox virus (MPXV) (Genome ID: NC_003310.1) through NCBI (www.ncbi.nlm.nih.gov), and selects the conserved region of the A27L gene (GenBank: ON563414) as the target region. According to the characteristics of Cas12a recognition of T-rich PAM sequences, a sequence with a length of 20bp is selected as the detection target.

[0145] The gRNA for Cas12a was designed based on the cleavage characteristics of the Cas12a protein. The fluorescence signal was detected using a constant temperature fluorescence detector, and FAM-BHQ-labeled ssDNA was used as a reporter. The sequence of ssDNA-FQ is shown in Example 1.

[0146] 2. Optimization of detection conditions

[0147] 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. The detection condition with the fastest and highest increase in fluorescence intensity within the same time period was selected as the preferred condition. The preferred results are shown in Table 5 below:

[0148] Table 5

[0149]

[0150]

[0151] The composition of the optimized RPA / CRISPR detection system is shown in Example 1.

[0152] Experimental Example 2: Screening of mixed lyophilized protective agents for the MPXV virus one-pot fluorescence detection kit

[0153] In the following experiments, reagents or conditions not specifically described were carried out according to the description in Example 1.

[0154] 1. Screening of single protective agent

[0155] The effects of sorbitol, PEG, trehalose, sucrose, ammonium acetate, xylitol, mannitol, galactose, and inositol as freeze-drying protectants for RPA detection system and CRISPR detection system on the fluorescence signal were studied.

[0156] 5.0 μl and 1.6 μl of 9 lyophilized protectants with a concentration of 15% (mass volume percentage, g / ml) were added to the RPA reagent and CRISPR reagent, respectively, to obtain the RPA detection system (Table 6) and CRISPR detection system (Table 7) containing a single protectant as follows.

[0157] Table 6

[0158]

[0159] Table 7

[0160]

[0161] The fluorescence intensity of the RPA and CRISPR detection systems containing different types of lyoprotectants was tested. Compared with the fluorescence intensity of the RPA and CRISPR detection systems without lyoprotectants, if the fluorescence signal intensity decreased by no more than 10% within the same time period, the protectant was considered to have no inhibitory effect on the detection system. The results are shown in Table 8:

[0162] Table 8

[0163]

[0164] The above results show that PEG, trehalose, sucrose, mannitol and inositol have no effect on the RPA / CRISPR detection system.

[0165] 2. Suitable concentration range of protective agent

[0166] The above lyophilized protective agents whose fluorescence signal intensity decreases by no more than 10% are selected, and the concentrations are configured at 1%-64%. The fluorescence intensity of the RPA detection system and the CRISPR detection system is tested at different concentrations of lyophilized protective agents. 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% 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 9:

[0167] Table 9

[0168]

[0169]

[0170] The above results show that the suitable concentration range of PEG is 4%-32%, the suitable concentration range of trehalose is 4%-40%, the suitable concentration range of sucrose is 2%-32%, the suitable concentration range of mannitol is 4%-40%, and the suitable concentration range of inositol is 4%-32%.

[0171] 3. Screening of freeze-dried protective agents for RPA constant temperature amplification reagents

[0172] 1) The contents of PEG, trehalose, sucrose, mannitol, and inositol were optimized by an orthogonal experimental design with 5 factors and 4 levels, as shown in Tables 10 and 11.

[0173] Table 10. Factor level table

[0174] factor Level 1 Level 2 Level 3 Level 4 PEG 0％ 10％ 20％ 32％ Trehalose 0％ 15％ 30％ 40％ sucrose 0％ 10％ 20％ 32％ Mannitol 0％ 15％ 30％ 40％ Inositol 0％ 10％ 20％ 32％

[0175] Table 11. Scheme

[0176]

[0177]

[0178] 2) Select all 16 of the above schemes and lyophilize the RPA isothermal amplification reagent. The lyophilization system is shown in Table 12:

[0179] Table 12

[0180]

[0181] 3) Morphological examination of freeze-dried pellets of RPA constant temperature amplification reagent:

[0182] The preparation method of the RPA constant temperature amplification reagent freeze-dried pellet was prepared according to Example 1. After the preparation was completed, the appearance of the RPA constant temperature amplification reagent freeze-dried pellet was observed to see if it was qualified. The test results are shown in Table 13:

[0183] Table 13

[0184]

[0185]

[0186] The above results show that RPA isothermal amplification reagent lyophilized protectant screening schemes 7, 8, 10, and 15 can be used for the next step of screening.

[0187] 4) Moisture content of RPA constant temperature amplification reagent freeze-dried pellets

[0188] The moisture content of the RPA constant temperature amplification reagent freeze-dried pellets that passed the above morphological inspection was measured using a calorimeter. The results are shown in Table 14:

[0189] Table 14

[0190] plan Moisture content in conclusion Option 7 5.98％ Unqualified Option 8 2.91％ qualified Plan 10 2.23％ qualified Plan 15 3.03％ qualified

[0191] The above results show that RPA isothermal amplification reagent lyophilized protectant screening schemes 8, 10, and 15 can be used for the next step of screening.

[0192] 5) Select the RPA freeze-dried reagent balls with qualified moisture content for performance inspection

[0193] The detection method of Example 1 was used. 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 15:

[0194] Table 15

[0195] plan Fluorescence intensity increase Option 8 100％ Plan 10 96％ Plan 15 92％

[0196] The above results show that RPA isothermal amplification reagent lyophilized protectant screening schemes 8, 10, and 15 can be used for the next step of screening.

[0197] 6) Select the qualified RPA constant temperature amplification reagent lyophilization protectant solution above and lyophilize the CRISPR / Cas12a detection reagent. The lyophilization system is shown in Table 16:

[0198] Table 16

[0199]

[0200] 7) Morphological examination of CRISPR / Cas12a detection reagent freeze-dried pellets:

[0201] The preparation method of the CRISPR / Cas12a detection reagent freeze-dried pellet was prepared according to Example 1. After the preparation was completed, the appearance of the CRISPR / Cas12a detection reagent freeze-dried pellet was observed to see if it was qualified. The test results are shown in Table 17:

[0202] Table 17

[0203] plan Formability Surface smoothness Completeness Resolubility in conclusion Option 8 3 3 3 3 qualified Plan 10 3 3 3 3 qualified Plan 15 2 2 2 1 Unqualified

[0204] The above results show that the CRISPR / Cas12a detection reagent lyophilized protectant screening schemes 8 and 10 are qualified and can be used for the next screening step.

[0205] 8) Moisture content of CRISPR / Cas12a detection reagent freeze-dried pellets

[0206] The moisture content of the CRISPR / Cas12a detection reagent freeze-dried pellets that passed the above morphological inspection was measured using a calorimeter. The results are shown in Table 18:

[0207] Table 18

[0208] plan Moisture content in conclusion Option 8 2.08％ qualified Plan 10 2.33％ qualified

[0209] Conclusion: Both Schemes 8 and 10 for screening lyophilized protectants for CRISPR / Cas12a detection reagents are qualified and can be used for the next step of screening.

[0210] 9) Select the CRISPR / Cas12a detection reagent freeze-dried pellets with qualified moisture content for performance inspection

[0211] Performance testing was performed according to the detection method in Example 1. The fluorescence results of the above scheme were compared with the fluorescence intensity of the unlyophilized RPA detection system and the CRISPR detection system. The results are shown in Table 19:

[0212] Table 19

[0213] plan Fluorescence intensity Option 8 88％ Plan 10 95％

[0214] The above results show that for the MPXV virus fluorescence detection kit and detection method of the present invention, Scheme 10 is the best mixed lyophilized protectant formula.

[0215] Experimental Example 3 Performance Verification of the One-Pot Fluorescence Detection Method for Freeze-dried MPXV Virus

[0216] 1. Sensitivity test

[0217] The performance test was carried out according to the test method in Example 1, wherein 10 -1 -10 5 aM of 2μL of the target to be tested, and the results are as follows Figure 2 Shown: Monkeypox virus concentration is 10 0 The fluorescence intensity can be clearly detected at aM, indicating that the fluorescence detection method of the kit of Example 1 has high sensitivity.

[0218] 2. Specificity test

[0219] The performance test was performed according to the detection method in Example 1, wherein the plasmid standards of monkeypox, smallpox, cowpox, and vaccinia viruses were used as test samples, such as Figure 3 As shown, the results show that the fluorescence detection method of the kit of Example 1 is specific to the plasmid standard of monkeypox virus.

[0220] 3. Repeatability test

[0221] The performance test was performed according to the detection method in Example 1, wherein 2 μL of the target to be tested was added to each tube at low, medium and high concentrations, and each concentration was repeated 20 times before the subsequent operation. Figure 4 As shown, the results show that the fluorescence intensity detection of different concentrations of the target to be tested using the kit of Example 1 has good repeatability.

[0222] 4. Clinical sample testing

[0223] Five monkeypox-positive clinical patients were collected and named P1, P2, P3, P4, and P5. Six types of specimens, including anal swabs, blister fluid, saliva, throat swabs, blood, and urine, were collected from each patient. Negative and positive controls were also set up. The freeze-dried RPA / CRISPR one-pot fluorescence detection method was used to detect the above specimens. The results are as follows: Figure 5 As shown: Anal swabs, blister fluid, saliva, and throat swabs can all test positive, and the most suitable specimen types are anal swabs, blister fluid, and saliva.

[0224] The above experimental results show that the MPXV virus fluorescence detection kit and detection method provided in Example 1 have excellent detection performance and can meet the requirements of actual detection.

[0225] Experimental Example 4: Optimal Conditions for One-Pot Test Strip Detection of MPXV Virus

[0226] This experimental example adjusted the various conditions of the test strip detection based on Example 2, and compared the fluorescence intensity of the target gene at the same concentration under different conditions. The detection conditions with the fastest color development speed and the most obvious color development within the same time period were selected as the preferred conditions. The preferred results are shown in Table 20 below.

[0227] Table 20

[0228] Optimal RPA primer concentration 1~3μM Optimal Buffer B concentration 1.6~2.0μL Optimal RPA reaction volume 20-25 μL Optimal RPA reaction temperature 37℃ Optimal RPA response time 40-60 minutes Optimal CRISPR reaction volume 8-10 μL Optimal CRISPR reaction temperature 35～40℃ Optimal CRISPR reaction time 40-60 minutes Optimal crRNA and Cas12a concentration and ratio 1:1 Optimal CRISPR reaction buffer Buffer2.1 Optimal reporter concentration 30~60μM Optimal reaction product volume 1-2 μL Optimal test strip buffer concentration 4～8％ Optimal test strip buffer volume 35-45 μL Optimal test strip reaction time 10-20 minutes

[0229] The composition of the optimized RPA / CRISPR detection system is shown in Example 2.

[0230] Experimental Example 5: Screening of mixed lyophilized protective agents for the MPXV virus one-pot test strip detection kit

[0231] In the following experiments, reagents or conditions not specifically described were performed according to the description in Example 2.

[0232] 1. Screening of single protective agent

[0233] The effects of sorbitol, PEG, trehalose, sucrose, ammonium acetate, xylitol, mannitol, galactose, and inositol as freeze-drying protectants for RPA detection system and CRISPR detection system on the colorimetric signal were studied.

[0234] 5 μL and 1.6 μL of 9 lyophilized protective agents with a concentration of 15% (mass volume percentage, g / mL) were added to the RPA reagent and CRISPR reagent, respectively, to obtain the RPA detection system (Table 21) and CRISPR detection system (Table 22) containing a single protective agent as follows:

[0235] Table 21

[0236]

[0237] Table 22

[0238]

[0239] The color intensity of the RPA detection system and CRISPR detection system containing different types of lyoprotectants was tested and compared with the color intensity of the RPA detection system and CRISPR detection system without lyoprotectants. The results are shown in Table 23:

[0240] Table 23

[0241]

[0242] The above results show that sorbitol, PEG, trehalose, and mannitol have no effect on the color development of the test strips of the RPA / CRISPR detection system.

[0243] 2. Suitable concentration range of protective agent

[0244] The above lyophilized protective agents that have no effect on the RPA / CRISPR detection system were selected and prepared at concentrations of 1%-48%. The effects of different concentrations of lyophilized protective agents on the color development of 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 24:

[0245] Table 24

[0246]

[0247] The above results show that the suitable concentration range of sorbitol is 4%-40%, the suitable concentration range of PEG is 4%-32%, the suitable concentration range of trehalose is 2%-40%, and the suitable concentration range of mannitol is 2%-40%.

[0248] 3. Screening of freeze-dried protective agents for RPA constant temperature amplification reagents

[0249] 1) The contents of sorbitol, PEG, trehalose, and mannitol were optimized by an orthogonal experimental design with 4 factors and 3 levels, as shown in Tables 25 and 26.

[0250] Table 25. Factor level table

[0251] factor Level 1 Level 2 Level 3 sorbitol 0％ 20％ 40％ PEG 0％ 16％ 32％ Trehalose 0％ 20％ 40％ Mannitol 0％ 20％ 40％

[0252] Table 26. Scheme

[0253]

[0254]

[0255] 2) Select all nine of the above schemes and lyophilize the RPA isothermal amplification reagent. The lyophilization system is shown in Table 27:

[0256] Table 27

[0257]

[0258] 3) Morphological examination of freeze-dried pellets of RPA constant temperature amplification reagent:

[0259] The preparation method of the RPA constant temperature amplification reagent freeze-dried pellet was prepared according to Example 1. After the preparation was completed, the appearance of the RPA constant temperature amplification reagent freeze-dried pellet was observed to determine whether it was qualified. The test results are shown in Table 28:

[0260] Table 28

[0261]

[0262]

[0263] The above results indicate that RPA isothermal amplification reagent freeze-dried protective agent screening schemes 2, 5, and 9 can be used for further screening.

[0264] 4) Moisture content of RPA constant temperature amplification reagent freeze-dried pellets

[0265] The moisture content of the RPA constant temperature amplification reagent freeze-dried pellets that passed the above morphological inspection was measured using a calorimeter. The results are shown in Table 29:

[0266] Table 29

[0267] plan Moisture content in conclusion Option 2 1.66％ qualified Option 5 2.84％ qualified Option 9 2.03％ qualified

[0268] The above results indicate that RPA isothermal amplification reagent freeze-dried protective agent screening schemes 2, 5, and 9 can be used for further screening.

[0269] 5) Select the RPA freeze-dried reagent balls with qualified moisture content for performance inspection

[0270] The detection method of Example 2 was used. 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. The results are shown in Table 30:

[0271] Table 30

[0272] plan Color intensity Option 2 powerful Option 5 powerful Option 9 weak

[0273] The above results indicate that RPA isothermal amplification reagent freeze-dried protective agent screening schemes 2 and 5 can be used for further screening.

[0274] 6) Screening of freeze-dried protective agents for CRISPR / Cas12a detection reagents

[0275] Select the above qualified RPA constant temperature amplification reagent lyophilization protectant solution and lyophilize the CRISPR / Cas12a detection reagent. The lyophilization system is shown in Table 31:

[0276] Table 31

[0277]

[0278]

[0279] 7) Morphological examination of CRISPR / Cas12a detection reagent freeze-dried pellets:

[0280] The preparation method of the CRISPR / Cas12a detection reagent freeze-dried pellet was prepared according to Example 1. After the preparation was completed, the appearance of the CRISPR / Cas12a detection reagent freeze-dried pellet was observed to see if it was qualified. The test results are shown in Table 32:

[0281] Table 32

[0282] plan Formability Surface smoothness Completeness Resolubility in conclusion Option 2 3 3 3 3 qualified Option 5 3 3 3 3 qualified

[0283] The above results show that the CRISPR / Cas12a detection reagent lyophilized protectant screening schemes 2 and 5 have qualified appearance and can be used for further screening.

[0284] 8) Moisture content of CRISPR / Cas12a detection reagent freeze-dried pellets

[0285] The moisture content of the CRISPR / Cas12a detection reagent freeze-dried pellets that passed the above morphological inspection was measured using a calorimeter. The results are shown in Table 33:

[0286] Table 33

[0287] plan Moisture content in conclusion Option 2 2.56％ qualified Option 5 5.28％ Unqualified

[0288] The above results show that the CRISPR / Cas12a detection reagent lyophilized protectant screening scheme 2 is qualified.

[0289] 9) Select the CRISPR / Cas12a detection reagent freeze-dried pellets with qualified moisture content for performance inspection

[0290] The performance test was performed according to the detection method 1 in Example 2. 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. The results are shown in Table 34:

[0291] Table 34

[0292]

[0293]

[0294] The above results show that for the MPXV virus test strip detection kit and detection method of the present invention, Scheme 2 is the optimal mixed lyophilized protective agent formula.

[0295] Experimental Example 6 Performance Verification of the One-Pot Test Strip Detection Method for Freeze-Dried MPXV Virus

[0296] The detection method of this experimental example was performed according to Method 1 in Example 2.

[0297] 1. Sensitivity test

[0298] The performance test was carried out according to the test method 1 in Example 2, wherein 10 -1 -10 5 aM of 2μL of the target to be tested, the results are as follows Figure 6 (Sensitivity) shows that the lowest virus concentration that can be detected by the test strip detection method of the kit of Example 2 is 10 1 aM, high sensitivity.

[0299] 2. Specificity test

[0300] The performance test was performed according to the detection method 1 in Example 2, wherein the plasmid standards of monkeypox, smallpox, cowpox, and vaccinia viruses were used as test samples. The results are as follows Figure 6 As shown in (Specificity), the test strip detection method of the kit of Example 2 can only detect the plasmid standard of monkeypox virus, indicating that the detection method of the present invention has high specificity.

[0301] 3. Repeatability test

[0302] Performance testing was performed according to the first detection method in Example 2, with 2 μL of the target to be tested added to each tube at low, medium, and high concentrations, respectively. Each concentration was repeated 20 times before the subsequent steps were performed. The results are shown in Table 35. The results demonstrate that the kit of Example 2 has good reproducibility when used for strip testing of monkeypox virus at different concentrations.

[0303] Table 35

[0304]

[0305] Experimental Example 7 Performance Verification of Freeze-dried MPXV Virus One-Pot Test Strip Detection Method (Using Microfluidic Chip)

[0306] The detection method of this experimental example was performed according to Method 2 in Example 2.

[0307] 1. Sensitivity test

[0308] The designed concentration gradient is 10 -1 -10 5 The samples to be tested were processed according to the experimental steps in Example 2. Figure 7As shown in the results, the lowest virus concentration that can be detected by the test strip detection method (using microfluidic chip) in Example 2 is 10 1 aM, high sensitivity.

[0309] 2. Specificity test

[0310] The concentration was selected as 10 5 aM monkeypox, smallpox, cowpox, and vaccinia viruses were used as test samples. The experimental procedures in Example 2 were followed. Figure 7 As shown, the results indicate that the test strip detection method (using a microfluidic chip) in Example 2 is specific for detecting the plasmid standard of monkeypox virus.

[0311] 3. Repeatability test

[0312] Low, medium, and high concentrations of MPXV were selected as test samples, and the experimental procedures in Example 2 were followed, with 20 replicates for each concentration. As shown in Table 36, the results demonstrate good reproducibility when using the test strip method in Example 2 (using a microfluidic chip) for MPXV at different concentrations.

[0313] Table 36

[0314]

[0315] 4. Clinical sample testing

[0316] Five monkeypox positive clinical patients were collected and named P1, P2, P3, P4, and P5. Six types of specimens, including anal swab, blister fluid, saliva, throat swab, blood, and urine, were collected from each patient. Negative and positive controls were also set up. The experimental procedures in Example 2 were followed and the results were as follows. Figure 8 As shown: both blister fluid and saliva can test positive, and the most suitable specimen types are blister fluid and saliva.

[0317] The above experimental results show that the monkeypox virus test strip detection kit and detection method provided in Example 2 have excellent detection performance and can meet the requirements of actual detection.

[0318] In summary, the present invention solves the problems of room-temperature storage and transportation of RPA / CRISPR detection reagents for monkeypox virus, supports POCT (point-of-care testing) and home self-testing; and ensures high sensitivity and specificity of detection, realizing early, rapid, portable and simple operation of MPXV detection, overcoming the traditional method's dependence on expensive equipment and professionals, and providing an efficient and accurate solution for large-scale screening.

Claims

1. A one-pot fluorescence detection reagent for monkeypox virus, 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 constant temperature amplification system, and the RPA constant temperature amplification system includes the following components: RPA constant temperature amplification reagent 1 20-25μL, Buffer A 48-60% v / v, Forward primer 0.04-0.05 μM, Rear primer 0.04-0.05μM, Mixed lyophilized protective agent 20-25% 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 0.2-0.33μM, gRNA 0.2-0.33 μM, ssDNA reporter molecule 1.2-3μM, Buffer 2.1 8.0-13.3% v / v, Mixed lyophilized protective agent 16.0-26.7% v / v; Wherein, the mixed lyophilization protective agent consists of the following components: Polyethylene glycol 20% w / v, Trehalose 15% w / v, Sucrose 32% w / v, Mannitol 30% w / v; The Buffer A is from the DNA isothermal rapid amplification kit of Anpu Future Biotechnology Co., Ltd., with the catalog number WLB8201KIT; the Buffer 2.1 is from New England Biolabs and is a component of EnGen Lba Cas12a NEB#M0653.

2. The one-pot fluorescence detection reagent for monkeypox virus according to claim 1, characterized in that: The RPA constant temperature amplification system includes the following components: RPA constant temperature amplification reagent 1 25μL, Buffer A 60% v / v, Forward primer 0.04 μM, The rear primer 0.04 μM, Mixed lyophilized protective agent 20.0% v / v; The CRISPR / Cas12a detection system includes the following components: Cas12a 0.25μM, gRNA 0.25 μM, ssDNA reporter molecule 1.5μM, Buffer 2.1 10% v / v, Mix lyophilized protective agent 20% v / v.

3. A one-pot test strip for monkeypox virus, characterized by: It includes RPA lyophilized reagents and CRISPR / Cas12a detection system lyophilized reagents; The RPA lyophilized reagent is a lyophilized reagent made from an RPA constant temperature amplification system, and the RPA constant temperature amplification system includes the following components: RPA constant temperature amplification reagent 1 20-25μL, Buffer A 48-60% v / v, Forward primer 0.04-0.05 μM, Rear primer 0.04-0.05μM, Mixed lyophilized protective agent 20-25% 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 0.2-0.33μM, gRNA 0.2-0.33 μM, ssDNA reporter molecule 1.2-3μM, Buffer 2.1 8.0-13.3% v / v, Mixed lyophilized protective agent 16.0-26.7% v / v; Wherein, the mixed lyophilization protective agent consists of the following components: Polyethylene glycol 16% w / v, Trehalose 20% w / v, Mannitol 20% w / v; The Buffer A is from the DNA isothermal rapid amplification kit of Anpu Future Biotechnology Co., Ltd., with the catalog number WLB8201KIT; the Buffer 2.1 is from New England Biolabs and is a component of EnGen Lba Cas12a NEB#M0653.

4. The one-pot test strip detection reagent for monkeypox virus according to claim 3, characterized in that: The RPA constant temperature amplification system includes the following components: RPA constant temperature amplification reagent 1 25μL, Buffer A 60% v / v, Forward primer 0.04 μM, The rear primer 0.04 μM, Mixed lyophilized protective agent 20% v / v; The CRISPR / Cas12a detection system includes the following components: Cas12a 0.25μM, gRNA 0.25 μM, ssDNA reporter molecule 1.5μM, Buffer 2.1 10% v / v, Mix lyophilized protective agent 20% v / v.

5. The detection reagent according to any one of claims 1 to 4, characterized in that: The front primer includes at least one of the primers having nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; The rear primer includes at least one of the primers having nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4; The nucleotide sequence of the gRNA is shown in SEQ ID NO.

5.

6. The detection 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 detection reagent according to any one of claims 1 to 6 in the preparation of a monkeypox virus detection kit.

8. A monkeypox virus detection kit, characterized in that include: Buffer B and the detection reagent according to any one of claims 1-6; the Buffer B comes from the DNA Constant Temperature Rapid Amplification Kit of Anpu Future Biotechnology Co., Ltd., with the product number WLB8201KIT.

9. A method for detecting monkeypox virus for non-diagnostic purposes using the monkeypox virus detection kit according to claim 8, characterized in that: The steps include: Step 1: Redissolve the RPA lyophilized reagent and the CRISPR / Cas12a detection system lyophilized reagent separately; 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: Add the solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent to the reaction system and incubate the reaction; Step 4: Detection by fluorescence detection or color development on a test strip.

10. A method for detecting monkeypox virus for non-diagnostic purposes using the monkeypox virus detection kit 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 instant centrifugation.

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