Nucleic acid combination, kit and method for detecting monkey pox virus based on CRISPR (clustered regularly interspaced short palindromic repeats) technology

Abstract

The invention belongs to the technical field of in-vitro detection, and particularly relates to a nucleic acid combination, a kit and a method for detecting monkey pox virus based on a CRISPR (clustered regularly interspaced short palindromic repeats) technology. The detection reagent provided by the invention comprises an RPA freeze-drying reagent and a CRISPR / Cas12a detection system freeze-drying reagent, the RPA freeze-drying reagent comprises an RPA isothermal amplification reagent, Buffer A, a front primer, a rear primer and a mixed freeze-drying protective agent; the CRISPR / Cas12a detection system freeze-drying reagent comprises Cas12a, gRNA (guide ribonucleic acid), ssDNA (single-stranded deoxyribonucleic acid) reporter molecules, Buffer 2.1 and a mixed freeze-drying protective agent. The composition of the mixed freeze-drying protective agent is optimized according to the composition of a detection system and a specific detection method. The kit solves the problems of normal-temperature storage and transportation of RPA / CRISPR detection reagents of the monkey pox virus, has the characteristics of simplicity and convenience in operation, rapidness in detection, high sensitivity, suitability for on-site and large-scale screening and the like, provides an efficient and accurate solution for large-scale screening, and has a very good application prospect.

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 that belongs to the genus Orthopoxvirus in the Poxviridae family, along with smallpox and vaccinia viruses. It can be transmitted through contact with body fluids, skin or mucosal wounds, respiratory droplets, and contaminated objects. Eating undercooked meat products from infected animals also increases the risk of infection. Although MPXV patients often develop 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 identify monkeypox infection. Existing immunological, molecular biological, microbiological and other methods are still 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 the field of point-of-care testing (POCT). Although RPA has been used to detect MPXV with good sensitivity, the problem of non-specific products cannot be ignored. Utilizing the characteristics of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), guide RNA (gRNA) tightly binds to Cas protein, thereby activating the precise cleavage of target DNA or RNA sequences by Cas protein. Therefore, RPA is combined with CRISPR, while ensuring the rapid, efficient and accurate detection process.

[0004] However, existing methods require opening the lid and transferring part of the amplification product, which poses a risk of aerosol contamination. At the same time, nucleic acid detection reagents not only need to be stored at -20°C, but repeated freezing and thawing during transportation or use will reduce their detection performance. Therefore, it is crucial to develop a detection reagent and method that is stable at room temperature and does not require opening the lid. Summary of the invention

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

[0006] A one-pot fluorescence detection reagent for monkeypox virus, comprising a RPA lyophilized reagent and / or a CRISPR / Cas12a detection system lyophilized 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 0.04-0.05μM,

[0011] Post 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 0.2-0.33μM,

[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 comprises 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 comprises 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 is 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 0.25μM,

[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 comprises 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 detection reagent for monkeypox virus, comprising a RPA freeze-dried reagent and / or a CRISPR / Cas12a detection system freeze-dried 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 0.04-0.05μM,

[0046] The rear primer is 0.04-0.05 μM,

[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 0.2-0.33μM,

[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 comprises 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 comprises 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 is 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 0.25μM,

[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 comprises 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 quenching group.

[0078] The present invention also provides use of the above 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 above-mentioned monkeypox virus detection kit, comprising the following steps:

[0081] Step 1, respectively re-dissolve the RPA lyophilized reagent and the CRISPR / Cas12a detection system lyophilized reagent;

[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, adding a solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent to the reaction system and incubating 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 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 capable of one-pot RPA / CRISPR detection of monkeypox virus and a kit made thereof. 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 and can meet practical application requirements.

[0088] 2. The present invention realizes for the first time the one-pot RPA / CRISPR detection reagent of monkeypox virus to be made into a lyophilized 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 the CRISPR / Cas12a detection in two reaction tubes. There is no operation of transferring the reaction solution, which can effectively avoid the contamination problem of nucleic acid aerosol.

[0090] 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.

[0091] 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

[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 reproducibility of the one-pot fluorescence detection method for freeze-dried MPXV virus.

[0096] Figure 5This is a graph showing 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 graph of clinical sample test results for the one-pot test strip detection method for freeze-dried MPXV virus (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 products.

[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 are from the DNA constant temperature rapid amplification kit (Cat. No.: WLB8201KIT) of Anpu Future Biotechnology Co., Ltd.; Buffer 2.1 is from New England Biolabs and is a component of 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, and the freeze-dried balls include RPA freeze-dried reagent balls made by freeze-drying according to the RPA constant temperature amplification system in Table 1 and CRISPR detection reagent freeze-dried balls made by freeze-drying according to 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 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; the preferred front primer in this embodiment is RPA-A27-F2, and the preferred rear primer is RPA-A27-R2.

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

[0114]

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

[0116] 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.

[0117] The composition of the mixed lyophilized protective agent 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 mass volume percentages, g / ml.

[0118] The specific method of making the above RPA / CRISPR detection system into freeze-dried balls is as follows:

[0119] 1. Prepare RPA freeze-dried reagent balls: Use a precise liquid dispensing system to drop 25 μL of the prepared freeze-dried mixed solution of the RPA constant temperature amplification system into liquid nitrogen to condense into round balls. Wait for about 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.

[0120] 2. Prepare freeze-dried pellets of CRISPR / Cas12a detection reagent: Use a precise liquid dispensing system to drop 8 μl of the prepared freeze-dried mixed solution of the CRISPR / Cas12a detection 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.

[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 the RPA freeze-dried reagent ball is placed in the reaction tube, 21μL of enzyme-free water is added to reconstitute it, and then 2μL of Buffer B is added. Subsequently, 2μL of the target to be tested is added. Then, the CRISPR / Cas12a detection reagent freeze-dried ball is added to 8μL of enzyme-free water to reconstitute it, added to the reaction tube lid, and the lid is gently closed. 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 lid to the bottom of the tube. Finally, place the reaction tube in a constant temperature fluorescence detector, collect fluorescence data every 1 minute, and continue collecting 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 balls for MPXV virus detection. The composition and preparation method of the freeze-dried balls 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 mass 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 lid, and gently cover the lid. 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 lid 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 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) The RPA lyophilized reagent ball was coated on the RPA area, and 21 μL of enzyme-free water was added to re-dissolve it. Then 2 μL of Buffer B and 2 μL of the sample to be tested were added to the RPA area and incubated at 37°C for 40 minutes for amplification.

[0138] 2) The CRISPR lyophilized reagent ball 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 reagent enter the mixing channel area together, and the two liquids in the mixing channel are fully mixed.

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

[0140] 4) The reaction products of the CRISPR shearing area flow into the lateral flow immunochromatography test strip in the strip reading area, 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. Primer and crRNA, ssDNA reporter molecule design

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

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

[0146] 2. Optimization of testing conditions

[0147] This experimental example adjusted the various detection conditions on the basis of 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 Table 5 below:

[0148] Table 5

[0149]

[0150]

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

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

[0153] In the following experiments, reagents or conditions not specifically described were performed according to the description of 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 lyoprotectants for RPA detection system and CRISPR detection system on fluorescence signals were studied.

[0156] 5.0 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 the CRISPR reagent, respectively, to obtain the RPA detection system (Table 6) and CRISPR detection system (Table 7) containing a single protective agent as follows.

[0157] Table 6

[0158]

[0159] Table 7

[0160]

[0161] The fluorescence intensity of the RPA detection system and the CRISPR detection system containing different types of lyophilized protective agents was tested. Compared with the fluorescence intensity of the RPA detection system and the CRISPR detection system without lyophilized protective agents, if the fluorescence signal intensity decreased by no more than 10% during the same period of time, it was considered that the protective agent had no inhibitory effect on the detection system. The results are shown in Table 8:

[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 freeze-dried protective agent whose fluorescence signal intensity decreases by no more than 10% is 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 agent. Compared with the fluorescence intensity of the RPA detection system and the CRISPR detection system without freeze-dried protective agent, if the fluorescence signal intensity decreases by no more than 10% 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 isothermal amplification reagents

[0172] 1) The contents of PEG, trehalose, sucrose, mannitol and inositol were optimized by 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) All 16 schemes above were selected, and the RPA isothermal amplification reagent was freeze-dried. The freeze-drying system was shown in Table 12:

[0179] Table 12

[0180]

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

[0182] The preparation method of the RPA constant temperature amplification reagent freeze-dried ball is prepared according to Example 1. After the preparation is completed, the appearance of the RPA constant temperature amplification reagent freeze-dried ball is observed to see whether it is 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 pellet

[0188] 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 14:

[0189] Table 14

[0190] plan Moisture content in conclusion Solution 7 5.98％ Failure Solution 8 2.91％ qualified Solution 10 2.23％ qualified Solution 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 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 15:

[0194] Table 15

[0195] plan Fluorescence intensity increase Solution 8 100％ Solution 10 96％ Solution 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 above qualified RPA constant temperature amplification reagent lyophilization protectant solution, and lyophilize the CRISPR / Cas12a detection reagent. The lyophilization system is shown in Table 16:

[0198] Table 16

[0199]

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

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

[0202] Table 17

[0203] plan Formability Surface smoothness Completeness Resolubility in conclusion Solution 8 3 3 3 3 qualified Solution 10 3 3 3 3 qualified Solution 15 2 2 2 1 Failure

[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 step of screening.

[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 cardiometer, and the results are shown in Table 18:

[0207] Table 18

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

[0209] Conclusion: Both Schemes 8 and 10 for screening lyophilized protectants of CRISPR / Cas12a detection reagents were 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] The performance test 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 unfreeze-dried RPA detection system and the CRISPR detection system. The results are shown in Table 19:

[0212] Table 19

[0213] plan Fluorescence intensity Solution 8 88％ Solution 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 protective agent 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 detection 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 samples to be tested. 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, respectively, 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 detected 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 swab, blister fluid, saliva, throat swab, blood, and urine, were extracted 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 on the basis of Example 2, 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 color development speed and the most obvious color development 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℃ Best RPA Response Time 40~60min Optimal CRISPR reaction volume 8-10 μL Optimal CRISPR reaction temperature 35～40℃ Optimal CRISPR reaction time 40~60min Optimal crRNA and Cas12a concentration and ratio 1:1 Optimal CRISPR reaction buffer Buffer2.1 Optimal reporter concentration 30~60μM Optimal reaction product volume 1~2μL Optimal test strip buffer concentration 4～8％ Optimal test strip buffer volume 35~45μL Optimal test strip reaction time 10~20min

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

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

[0231] In the following experiments, reagents or conditions not specifically described were performed according to the description of 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 lyoprotectants 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 the 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 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 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 the concentration was configured to be 1%-48%. 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 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 isothermal amplification reagents

[0249] 1) The contents of sorbitol, PEG, trehalose and mannitol were optimized by 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) All the above 9 schemes are selected, and the RPA isothermal amplification reagent is freeze-dried. The freeze-drying system is shown in Table 27:

[0256] Table 27

[0257]

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

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

[0260] Table 28

[0261]

[0262]

[0263] The above results show that RPA isothermal amplification reagent lyophilized protectant screening schemes 2, 5, and 9 can be used for further screening.

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

[0265] 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 29:

[0266] Table 29

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

[0268] The above results show that RPA isothermal amplification reagent lyophilized protectant 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 for the operation. 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 30:

[0271] Table 30

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

[0273] The above results show that RPA isothermal amplification reagent lyophilized protectant 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 freeze-dried pellets of CRISPR / Cas12a detection reagent:

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

[0281] Table 32

[0282] plan Formability Surface smoothness Completeness Resolubility in conclusion Solution 2 3 3 3 3 qualified Solution 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 cardiometer, and the results are shown in Table 33:

[0286] Table 33

[0287] plan Moisture content in conclusion Solution 2 2.56％ qualified Solution 5 5.28％ Failure

[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 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 is performed according to method 1 in Example 2.

[0297] 1. Sensitivity test

[0298] The performance test was carried out according to the detection 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 samples to be tested. 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 strong specificity.

[0301] 3. Repeatability test

[0302] The performance test was performed according to the detection method 1 in Example 2, wherein 2 μL of the target to be tested at low, medium and high concentrations were added to each tube, and each concentration was repeated 20 times before the subsequent operation. The results are shown in Table 35, which show that the test strips of monkeypox virus of different concentrations were tested using the kit of Example 2 with good repeatability.

[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 is performed according to method 2 in Example 2.

[0307] 1. Sensitivity test

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

[0309] 2. Specificity test

[0310] Select a concentration of 10 5 aM monkeypox, smallpox, cowpox, and vaccinia viruses were used as test samples. The experimental steps 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 samples to be tested, and the experimental steps in Example 2 were followed, with each concentration repeated 20 times. As shown in Table 36, the results showed that the test strip detection method (using a microfluidic chip) in Example 2 was used to detect monkeypox viruses of different concentrations with good repeatability.

[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 extracted from each patient. Negative and positive controls were also set. The experimental procedures in Example 2 were followed. The results are shown in Figure 2. 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 problem 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, realizes early, rapid, portable and simple operation of MPXV 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 fluorescence detection reagent for monkeypox virus, characterized in that: It includes RPA lyophilized reagent and / or CRISPR / Cas12a detection system lyophilized reagent; The RPA lyophilized reagent is a lyophilized reagent made from an RPA constant temperature amplification system, and the RPA constant temperature amplification system includes the following components: RPA constant temperature amplification reagent 1 / 20-25μL, Buffer A48-60% v / v, Forward primer 0.04-0.05μM, Post 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 0.2-0.33μM, ssDNA reporter molecule 1.2-3μM, Buffer 2.18.0-13.3% v / v, Mixed lyophilized protective agent 16.0-26.7% v / v; Wherein, the mixed lyophilization protective agent comprises the following components: Polyethylene glycol 10-20% w / v, Trehalose 4-15% w / v, Sucrose 20-32% w / v, Mannitol 15-30% w / v.

2. The one-pot fluorescence detection reagent for monkeypox virus according to claim 1, characterized in that: The RPA isothermal 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 is 0.04 μM, Mixed lyophilized protective agent 20.0% v / v; The CRISPR / Cas12a detection system includes the following components: Cas12a 0.25μM, gRNA 0.25μM, ssDNA reporter molecule 1.5μM, Buffer 2.110% v / v, Mixed lyophilized protective agent 20% v / v; Wherein, the mixed lyophilization protective agent comprises the following components: Polyethylene glycol 20% w / v, Trehalose 15% w / v, Sucrose 32% w / v, Mannitol 30% w / v.

3. A one-pot test strip for monkeypox virus, characterized in that: It includes RPA lyophilized reagent and / or CRISPR / Cas12a detection system lyophilized reagent; The RPA lyophilized reagent is a lyophilized reagent made from an RPA constant temperature amplification system, and the RPA constant temperature amplification system includes the following components: RPA constant temperature amplification reagent 1 / 20-25μL, Buffer A48-60% v / v, Forward primer 0.04-0.05μM, Post 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 0.2-0.33μM, ssDNA reporter molecule 1.2-3μM, Buffer 2.18.0-13.3% v / v, Mixed lyophilized protective agent 16.0-26.7% v / v; Wherein, the mixed lyophilization protective agent comprises the following components: Polyethylene glycol 4-16% w / v, Trehalose 2-20% w / v, Mannitol 2-20% w / v.

4. The one-pot test strip detection reagent for monkeypox virus according to claim 3, characterized in that: The RPA isothermal 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 is 0.04 μM, Mixed lyophilized protective agent 20% v / v; The CRISPR / Cas12a detection system includes the following components: Cas12a 0.25μM, gRNA 0.25μM, ssDNA reporter molecule 1.5μM, Buffer 2.110% v / v, Mixed lyophilized protective agent 20% v / v; Wherein, the mixed lyophilization protective agent comprises the following components: Polyethylene glycol 16% w / v, Trehalose 20% w / v, Mannitol 20% w / 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 with 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 to 6.

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, respectively re-dissolve the RPA lyophilized reagent and the CRISPR / Cas12a detection system lyophilized reagent; Step 2, adding the Buffer B and the test sample to the solution of the reconstituted RPA freeze-dried reagent, and incubating the reaction; Step 3, adding a solution of the reconstituted CRISPR / Cas12a detection system lyophilized reagent to the reaction system and incubating the reaction; Step 4, detection by fluorescence detection or color development on a test strip.

10. 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 instantaneous centrifugation.

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