CN115807130A - Method for rapidly detecting monkeypox virus - Google Patents

Abstract

The invention belongs to the field of microbial detection application, and relates to a method for quickly detecting a monkeypox virus, which comprises the following steps: 1) Obtaining a sample to be detected, wherein the sample to be detected is vesicle, pustule liquid or dry scab of skin lesion of a patient; 2) Preparing an amplification detection system comprising a monkey pox virus detection primer; 3) Placing the sample to be detected obtained in the step 1) in a detection system for rapid detection to obtain whether the sample to be detected has the monkeypox virus. The invention provides a method for quickly detecting monkeypox virus, which has strong specificity and high sensitivity.

Description

Rapid detection method for monkeypox virus

technical field

The invention belongs to the field of microbial detection applications, and relates to a rapid detection method for monkeypox virus, in particular to a rapid detection method for monkeypox virus based on CRISPR/Cas12a and RPA.

Background technique

Monkeypox virus (Monkeypox virus) is an enveloped double-stranded DNA virus belonging to the Orthopoxvirus genus of the Poxviridae family that causes the infectious disease monkeypox. Its main coding genes include envelope protein gene (B6R) and specific target sequence (F3L). Because monkeypox virus is highly contagious, the initial symptoms of patients after infection include fever, headache, muscle aches, swollen lymph nodes, chills, and fatigue. Foam spreads. Its clinical consequences are serious. It is extremely important to treat the patients in time if the monkeypox virus is detected in the early stage of infection, and to take effective isolation measures to control the spread of the epidemic. Its detection requirements are "fast, accurate, and simple", that is, rapid detection, accurate analysis, and simple operation process.

At present, the recommended and widely used detection method for monkeypox virus is real-time fluorescence quantitative PCR technology for nucleic acid detection, which has the advantages of fast and sensitive, and meets the requirements of "accuracy", but it takes a long time and the equipment is expensive. It is difficult to popularize in grassroots inspection institutions, and it still needs to be improved in terms of "fast" and "simple". Recombinase Polymerase Amplification (RPA) is known as a nucleic acid detection technology that can replace PCR. Under the condition of 37-42°C, the protein-DNA complex formed by the combination of the recombinase capable of binding single-stranded nucleic acid (oligonucleotide primer) and the primer searches for homologous sequences in the double-stranded DNA to perform strand exchange in strand replacement DNA synthesis is initiated by DNA polymerase, and then the replaced DNA strand binds to single-strand DNA binding protein (SSB) to prevent further replacement. The RPA system can achieve a detectable level of amplification products within ten minutes, and has the characteristics of high sensitivity and fast response, which meets the "fast" requirements in the detection of monkeypox virus.

CRISPR/Cas12a (clustered regularly interspaced short palindromicrepeats crispr associated gene) is an endonuclease that can achieve DNA cutting function under the guidance of specific RNA, which can be used as genome editing. The cleavage produces fluorescence, which can be used as a tool for in vitro detection of biomolecules at room temperature, and can meet the "quasi" requirements in the detection of monkeypox virus. Its guide RNA needs to be combined with the target target sequence, and the 5' end of the target target sequence is required to have a protospacer adjacent motif (PAM sequence) so as to stimulate the enzyme activity to the greatest extent and realize the detection of specific nucleic acids.

Using RPA normal temperature amplification combined with CRISPR/Cas12a for specific and rapid detection of monkeypox virus, with the help of a simple ultraviolet imaging method, rapid screening and detection of monkeypox virus can be realized in primary health institutions, airports, railway stations and other places.

Contents of the invention

In order to solve the above-mentioned technical problems in the background technology, the present invention provides a rapid detection method for monkeypox virus with strong specificity and high sensitivity.

In order to achieve the above object, the present invention adopts the following technical solutions:

A primer for detection of monkeypox virus, characterized in that: the primer for detection of monkeypox virus includes a specific primer F3L-RPA for amplifying the specific target sequence F3L of monkeypox virus, and the specific primer F3L-RPA The forward primer F of the specific primer F3L-RPA is TCCAACGATACTCCTCTCTCGT; the reverse primer R of the specific primer F3L-RPA is AGGCCCCACTGATTCAATACG.

Preferably, the crRNA8 sequence of the monkeypox virus-specific target sequence F3L used in the present invention is:

UAAUUUCUACUAAGUGUAGAUGCGGGAUACAUCAUCUAUUAUAUAGC.

Preferably, the monkeypox virus detection primers used in the present invention also include a specific primer B6R-RPA-6 for amplifying the monkeypox virus envelope protein gene B6R, and the positive expression of the specific primer B6R-RPA-6 is The direction primer F is CGTTGTTATGCGTACTACCTGC; the reverse primer R of the specific primer B6R-RPA-6 is GCAACTTAGTGTCATGGTGGAA.

As preferably, the crRNA1 sequence of the monkeypox virus envelope protein gene B6R used in the present invention is:

UAAUUUCUACUAAGUGUAGAUUUCAACAUGUACUGUACCCACUAUGA.

Preferably, the monkeypox virus detection primers used in the present invention also include a specific primer B6R-RPA-4 for amplifying the monkeypox virus envelope protein gene B6R, and the positive expression of the specific primer B6R-RPA-4 is The forward primer F is CCATCATCCACATGTATCGACGGTAA; the reverse primer R of the specific primer B6R-RPA-4 is CACGGTAGCAATTTATGGAACTTATATTGGTCA.

As preferably, the crRNA6 sequence of the monkeypox virus envelope protein gene B6R used in the present invention is:

UAAUUUCUACUAAGUGUAGAUAUCCAGUGGAUGAUGGUCCCGACG.

The application of the aforementioned primers for detection of monkeypox virus in the detection of monkeypox virus, especially the application in the rapid detection of monkeypox virus.

Application of the monkeypox virus detection primers as described above as a monkeypox virus detection kit.

A monkeypox virus rapid detection method based on the aforementioned monkeypox virus detection primers, characterized in that: the monkeypox virus rapid detection method comprises the following steps:

1) Obtain the sample to be tested, which is the vesicle of the patient's skin lesions, pustular fluid or dry scab;

2) configuring an amplification detection system comprising primers for detection of monkeypox virus;

3) Put the sample to be tested obtained in step 1) into the detection system for rapid detection to obtain whether the sample to be tested has monkeypox virus.

Rehydration、

冻干粉以及

Magnesium Acetate(MgOAC)；Preferably, the detection system used in the present invention also includes buffer 1, LbaCas12a, crRNA, FQ fluorescence quenching probe, sterile enzyme-free deionized water,

Rehydration,

lyophilized powder and

Magnesium Acetate (MgOAC);

The contents of each component in the buffer 1 are: 500mM NaCl, 100mM Tris-HCl, 100mM MgCl ₂ and 100μg/ml Recombinant Albumin, and the pH of the buffer 1 is 7.9;

The F-Q fluorescence quenching probe is: 5'-FAM-TTATT-Quencher-3';

The crRNA is crRNA8 of monkeypox virus specific target sequence F3L, crRNA1 of monkeypox virus envelope protein gene B6R or crRNA6 of monkeypox virus envelope protein gene B6R;

The specific implementation of configuring the detection system in the step 2) is:

Rehydration buffer、

冻干粉，2.4ul10mM RPA上游引物、2.4ul10mM RPA下游引物；所述RPA上游引物以及RPA下游引物均是如前所记载的猴痘病毒检测用引物；在

Rehydration buffer中加入

冻干粉并溶解

冻粉末，加入如前所记载的猴痘病毒检测用引物，混合后在3000g的条件下离心10s；加入无菌无酶去离子水10ul，加入待测样本3.5ul，充分混匀；加入2.5ul

MgOAC溶液37℃金属浴加热启动扩增10分钟，获取扩增产物；2.1) Obtain the amplification product: take 29.5ul

Rehydration buffer,

Freeze-dried powder, 2.4ul10mM RPA upstream primer, 2.4ul10mM RPA downstream primer; the RPA upstream primer and RPA downstream primer are all monkeypox virus detection primers as previously described;

Added to Rehydration buffer

Freeze-dried powder and dissolved

Frozen powder, add the monkeypox virus detection primers as described above, mix and centrifuge at 3000g for 10s; add 10ul of sterile enzyme-free deionized water, add 3.5ul of the sample to be tested, and mix well; add 2.5ul

MgOAC solution was heated in a metal bath at 37°C to start the amplification for 10 minutes to obtain the amplification product;

2.2) Prepare reagents for detection: Take 1uM crRNA1ul, 1uM LbaCas12a1ul, 3ul buffer 1, 20ul sterile enzyme-free deionized water and 1.5ul of 10uMF-Q fluorescence quenching probe and mix thoroughly, and put the mixture at 25±0.5℃ pre-incubating for 10 minutes to obtain detection reagents; the crRNAs are respectively matched with monkeypox virus detection primers;

The concrete implementation of described step 3) is:

Add 3.5ul of the amplification product obtained in step 2.1) to the detection reagent obtained in step 2.2), heat at 37°C for 60 minutes, and observe the luminescence effect under 485nm excitation light. If there is fluorescence, The sample to be tested is positive; if no fluorescence appears, the sample to be tested is negative.

Compared with the prior art, the present invention has the following beneficial effects:

The invention provides a rapid detection method for monkeypox virus, which is a monkeypox molecular detection kit and detection method by combining RPA amplification technology and CRISPR/Cas12a reaction system. Through the side branch cutting function based on CRISPR/Cas12a, the present invention designs crRNA sequences and RPA primers respectively for the F3L gene and B6R gene of monkeypox virus to realize fast, simple, sensitive and specific detection. Portable detection and promotion application in airports, railway stations or remote areas.

Description of drawings

Fig. 1 Time/fluorescence curves of each F3L/crRNA in CRISPR/Cas12a detection;

Figure 2 is a visualization effect diagram of each F3L/crRNA detection result in Figure 1 under 485nm excitation light at 60 minutes;

Figure 3 is the time/fluorescence curve of each B6R/crRNA in CRISPR/Cas12a detection;

Fig. 4 is a visualization effect diagram of each B6R/crRNA detection result in Fig. 1 under 485nm excitation light at 60 minutes;

Figure 5 is the time/fluorescence curve of the amplification products of each monkeypox virus F3LRPA primer used in the detection of F3L/crRNA7-CRISPR/Cas12a;

Figure 6 is the time/fluorescence curve of the amplification products of each monkeypox virus F3LRPA primer used in the detection of F3L/crRNA8-CRISPR/Cas12a;

Figure 7 is the time/fluorescence curve of the amplification products of each monkeypox virus B6RRPA primer used in the detection of B6R/crRNA1-CRISPR/Cas12a;

Figure 8 is the time/fluorescence curve of the amplification products of each monkeypox virus B6RRPA primer used in the detection of B6R/crRNA6-CRISPR/Cas12a;

Figure 9 shows the monkeypox virus B6R recombinant plasmid at different concentrations (1*10 ¹¹ copies/ul, 1*10 ¹⁰ copies/ul, 1*10 ⁹ copies/ul, 1*10 ⁸ copies/ul, 1*10 ⁷ copies /ul, 1*10 ⁶ copies/ul, 1*10 ⁵ copies/ul, 1*10 ⁴ copies/ul, 1*10 ³ copies/ul) the amplification product of B6R-RPA6 is used for B6R/crRNA1-CRISPR /Time/fluorescence curve in Cas12a detection;

Figure 10 is a visualization effect diagram of the detection results of each concentration in Figure 9 under 485nm excitation light at 60 minutes;

Figure 11 shows the monkeypox virus B6R recombinant plasmid at different concentrations (1*10 ¹¹ copies/ul, 1*10 ¹⁰ copies/ul, 1*10 ⁹ copies/ul, 1*10 ⁸ copies/ul, 1*10 ⁷ copies /ul, 1*10 ⁶ copies/ul, 1*10 ⁵ copies/ul, 1*10 ⁴ copies/ul, 1*10 ³ copies/ul) of B6R-RPA4 amplification products for B6R/crRNA6-CRISPR /Time/fluorescence curve in Cas12a detection;

Figure 12 is a visualization effect diagram of the detection results of each concentration in Figure 11 under 485nm excitation light at 60 minutes;

Figure 13 shows the monkeypox virus F3L recombinant plasmid at different concentrations (1*10 ¹¹ copies/ul, 1*10 ¹⁰ copies/ul, 1*10 ⁹ copies/ul, 1*10 ⁸ copies/ul, 1*10 ⁷ copies /ul, 1*10 ⁶ copies/ul, 1*10 ⁵ copies/ul, 1*10 ⁴ copies/ul, 1*10 ³ copies/ul) the amplification product of F3L-RPAncb3 is used for F3L/crRNA7-CRISPR /Time/fluorescence curve in Cas12a detection;

Figure 14 is a visualization effect diagram of the detection results of each concentration in Figure 13 under 485nm excitation light at 60 minutes;

Figure 15 shows the monkeypox virus F3L recombinant plasmid at different concentrations (1*10 ¹¹ copies/ul, 1*10 ¹⁰ copies/ul, 1*10 ⁹ copies/ul, 1*10 ⁸ copies/ul, 1*10 ⁷ copies /ul, 1*10 ⁶ copies/ul, 1*10 ⁵ copies/ul, 1*10 ⁴ copies/ul, 1*10 ³ copies/ul) the amplification product of F3L-RPAncb3 is used for F3L/crRNA8-CRISPR /Time/fluorescence curve in Cas12a detection;

Figure 16 is a visualization effect diagram of the detection results of each concentration in Figure 15 under 485nm excitation light at 60 minutes;

Figure 17 is the time/fluorescence curve of the amplification product of monkeypox B6R pseudovirus B6R-RPA6 in the CRISPR/Cas12a detection of B6R/crRNA1 and B6R-RPA4;

Figure 18 is a visualization effect diagram of the detection results of each concentration in Figure 17 under 485nm excitation light at 60 minutes;

Figure 19 is the time/fluorescence curve of the amplification product of monkeypox F3L pseudovirus F3L-RPAncb3 in the CRISPR/Cas12a detection of F3L/crRNA7 and F3L/crRNA8;

Figure 20 is a visualization effect diagram of the detection results of each concentration in Figure 19 under 485nm excitation light at 60 minutes;

Figure 21 is the time/time of the amplification product of B6R-RPA6 of monkeypox (MP) B6R pseudovirus, HSV1 (herpes simplex virus type 1) and HSV2 (herpes simplex virus type 2) in the CRISPR/Cas12a detection of B6R/crRNA1 Fluorescence change curve;

Figure 22 is a visualization effect diagram of the detection results of each concentration in Figure 21 under 485nm excitation light at 60 minutes;

Figure 23 is the time/time of the amplification product of B6R-RPA4 of monkeypox (MP) B6R pseudovirus, HSV1 (herpes simplex virus type 1) and HSV2 (herpes simplex virus type 2) in the CRISPR/Cas12a detection of B6R/crRNA6 Fluorescence change curve;

Figure 24 is a visualization effect diagram of the detection results of each concentration in Figure 23 under 485nm excitation light at 60 minutes;

Figure 25 is the time/time of the amplification product of F3L-RPAncb3 of monkeypox (MP) F3L pseudovirus, HSV1 (herpes simplex virus type 1) and HSV2 (herpes simplex virus type 2) in the CRISPR/Cas12a detection of F3L/crRNA7 Fluorescence change curve;

Figure 26 is a visualization effect diagram of the detection results of each concentration in Figure 25 under 485nm excitation light at 60 minutes;

Figure 27 is the time/time of the amplification product of F3L-RPAncb3 of monkeypox (MP) F3L pseudovirus, HSV1 (herpes simplex virus type 1) and HSV2 (herpes simplex virus type 2) in the CRISPR/Cas12a detection of F3L/crRNA8 Fluorescence change curve;

Fig. 28 is a visualization effect diagram of the detection results of each concentration in Fig. 27 under 485nm excitation light at 60 minutes.

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from commercial sources unless otherwise specified. The crRNA, DNA primers, and recombinant plasmids used in this experiment were all synthesized by GenScript Biotech.

Example 1: Design of specific RPA primers

Monkeypox virus F3L, B6R genome sequences were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/genome/). GenBank>NC_003310.1:164845-165798 designed B6R-specific primers, GeneID: MT250197.1 (46192..466539) designed F3L-specific primers. RPA primers were designed according to the principles of RPA primer design, and their specificity and identification availability were confirmed by BLAST search.

The sequence of the RPA primers designed for the experiment is as follows:

F3L gene

F3L-RPA-1F: ATGGAGAAGCGAGAAGTTAATAAAGC

F3L-RPA-1R: ACTAAGAAGTTTTATCTACAGCCAATT

F3L-RPA-2F: AGCTCTGTATGATCTTCAACGTAGTGCTATG

F3L-RPA-2R: GATGACATAACTAAGAAGTTTATCTACAGC

F3L-RPA-ncb1F: TGCTGATACACGGCCTACAG

F3L-RPA-ncb1R: GGAGAGTTACTAGGCCCCAC

F3L-RPA-ncb2F: GGATGCTGATACACGGCCTA

F3L-RPA-ncb2R: AGGCCCCACTGATTCAATACG

F3L-RPA-ncb3F: TCCAACGATACTCCTCTCTCGT

F3L-RPA-ncb3R: AGGCCCCACTGATTCAATACG

B6R gene

B6R-RPA-1F: CGATTTCCGTTGTTACGTTGTTATGCGTAC

B6R-RPA-1R: CAGACAGCATTTGGATCCAAAGAATGATAT

B6R-RPA-4F: CCATCATCCACATGTATCGACGGTAA

B6R-RPA-4R: CACGGTAGCAATTTATGGAACTTATATTGGTCA

B6R-RPA-5F: TGCGTACTACCTGCTGTTGTTT

B6R-RPA-5R: CGTTGCAACTTAGTGTCATGGT

B6R-RPA-6F: CGTTGTTATGCGTACTACCTGC

B6R-RPA-6R: GCAACTTAGTGTCATGGTGGAA

B6R-RPA-ncb1F: TGCGTACTACCTGCTGTTGT

B6R-RPA-ncb1R: CGCATTAGGACACGTGACAG

B6R-RPA-ncb2F: TGCGTACTACCTGCTGTTGTT

B6R-RPA-ncb2R: TGAAGAGGTTGACATTCCGCA

B6R-RPA-ncb3F: CACTAACGGGGTCTCCATCA

B6R-RPA-ncb3R: ACGTCTTTCGAGAGTTTGCTCA

B6R-RPA-ncb4F: CACTAACGGGGTCTCCATCA

B6R-RPA-ncb4R: ACCCATAATTGTCAACGCCAT

Example 2: Screening and verification of the best Cas12acrRNA for F3L and B6R

According to the principles of CRISPR/Cas12a primer design, four crRNAs targeting the B6R gene were designed in TTTNPAM mode:

crRNA1: UAAUUUCUACUAAGUGUAGAUUUCAACAUGUACUGUACCCACUAUGA

crRNA3: UAAUUUCUACUAAGUGUAGAUCACGUGUCAAAGACUAAUACAGAG

crRNA6: UAAUUUCUACUAAGUGUAGAUAUCCAGUGGAUGAUGGUCCCGACG

crRNA7: UAAUUUCUACUAAGUGUAGAUUGAUUAUGUCUCUGAACUAUAUGA

Six crRNAs targeting the F3L gene were designed:

crRNA3: UAAUUUCUACUAAGUGUAGAUGUUACUCUCUCCUAGUAUUCAGAA

crRNA5: UAAUUUCUACUAAGUGUAGAUUUGAUCCUCUCUAACCAGAAAAGC

crRNA7: UAAUUUCUACUAAGUGUAGAUACCGGAAUAACAUCAUCAAAAGAC

crRNA8: UAAUUUCUACUAAGUGUAGAUGCGGGAUACAUCAUCAUAUUAUAGC

crRNA9: UAAUACGACUCACUAUAGGGUAAUUUCUACUAAGUGUAGAUCAUCUGCCUUAUCGAAUACUCUUC

crRNA10: UAAUACGACUCACUAUAGGGUAAUUUCUACUAAGUGUAGAUGUUGUGAAGAAAAAAAUGGAAAUA

Configure the following reaction system: CRISPR/Cas12a reaction system: final concentration is 33nMcrRNA, final concentration is 33nMLbaCas12a, 3ul reaction buffer 1, nuclease-free water, final concentration is 50nMF-Q fluorescence quenching probe: 5'-FAM-TTATT -Quencher-3'. The specific reaction process is: fully mix 3ul of buffer solution 1, LbaCas12a, crRNA, F-Q fluorescence quenching probe and 20ul of sterile enzyme-free deionized water, and pre-incubate at room temperature for 10 minutes. Add 3.5ul of recombinant B6R and F3L plasmids, react in a transparent tube at 37°C for 60 minutes, and then place it under 485nm excitation light to observe the luminescent effect.

As shown in Figure 1-Figure 4, the crRNA screening results of the CRISPR/Cas12a detection system show that in the F3L detection system, crRNA7 and crRNA8 produce visible fluorescence, and crRNA8 can produce brighter fluorescence under the transmission light source with an excitation wavelength of 485nm , so F3L-crRNA8 was used for the detection of subsequent experiments. In the B6R detection system, crRNA1, crRNA6 and crRNA7 produce visible fluorescence, among which crRNA1 and crRNA6 have higher fluorescence intensity and are used for subsequent B6R gene detection. In the present invention, the fluorescence intensity increases as the detection time increases, and reaches distinguishable fluorescence in about 20 minutes at the fastest.

Example 3: Screening and verification of the most suitable RPA primers and sensitivity detection for crRNA

3.1 Screening of the most suitable RPA primers for F3L/B6RcrRNA

Rehydration buffer、

冻干粉，2.4ul10mM RPA上游引物、2.4ul10mM RPA下游引物。将Rehydration buffer加入冻干粉溶解粉末，加入上下游引物，混合后3000g10s离心，加入无菌无酶去离子水10ul，加入待扩增底物即重组质粒3.2ul，充分混匀，加入2.5ul

MgOAC溶液37℃金属浴加热启动扩增10分钟；CRISPR/Cas12a反应体系：终浓度是33nM crRNA、终浓度是33nM LbaCas12a、3ul反应缓冲液1、无核酸酶水、终浓度是50nMF-Q荧光淬灭探针：5’-FAM-TTATT-Quencher-3’。分别配置F3L-crRNA8、B6R-crRNA1、B6R-crRNA6检测体系：将3ul缓冲液1、LbaCas12a、crRNA(分别是F3L-crRNA8、B6R-crRNA1、B6R-crRNA6)、F-Q荧光淬灭探针以及20ul无菌无酶去离子水后充分混合，室温预孵育10分钟。取RPA产物3.5ul，其中F3L crRNA8的检测体系分别给予RPA1、RPA2、ncb1、ncb2以及ncb3五种RPA产物；其中B6R crRNA1检测体系分别给予RPA1、RPA5、RPA6、ncb1以及ncb2五种RPA产物；其中B6R crRNA6检测体系分别给予RPA4、ncb3以及ncb4三种RPA产物。37℃，60分钟加热，监测荧光强度。The RPA primers in Example 1 were used for CRISPR/Cas12a detection. Configure the amplification reaction system as follows: 29.5ul

Rehydration buffer,

Freeze-dried powder, 2.4ul10mM RPA upstream primer, 2.4ul10mM RPA downstream primer. Add Rehydration buffer to freeze-dried powder to dissolve the powder, add upstream and downstream primers, mix and centrifuge at 3000g for 10s, add 10ul of sterile enzyme-free deionized water, add 3.2ul of the recombinant plasmid to be amplified, mix well, and add 2.5ul

MgOAC solution was heated in a metal bath at 37°C to start amplification for 10 minutes; CRISPR/Cas12a reaction system: final concentration of 33nM crRNA, final concentration of 33nM LbaCas12a, 3ul reaction buffer 1, nuclease-free water, final concentration of 50nMF-Q fluorescence quenching Killing probe: 5'-FAM-TTATT-Quencher-3'. Configure F3L-crRNA8, B6R-crRNA1, B6R-crRNA6 detection system respectively: 3ul buffer 1, LbaCas12a, crRNA (respectively F3L-crRNA8, B6R-crRNA1, B6R-crRNA6), FQ fluorescence quenching probe and 20ul no Mix well after deionized water without enzymes, and pre-incubate at room temperature for 10 minutes. Take 3.5ul of RPA products, among which five RPA products of RPA1, RPA2, ncb1, ncb2 and ncb3 were given to the detection system of F3L crRNA8; five kinds of RPA products of RPA1, RPA5, RPA6, ncb1 and ncb2 were given respectively to the detection system of B6R crRNA1; The B6R crRNA6 detection system gave RPA4, ncb3 and ncb4 three RPA products respectively. Heat at 37°C for 60 minutes and monitor the fluorescence intensity.

As shown in Figure 5-8, the optimal RPA primer for F3L crRNA8 is RPA-ncb3, the optimal RPA primer for B6R crRNA1 is RPA6, and the optimal RPA primer for B6R crRNA6 is RPA4. The above primers can distinguish between negative and positive within 20 minutes, and are used for subsequent sensitivity detection and pseudovirus sample detection.

3.2 Sensitivity verification of detection system

Configure different concentrations of F3L and B6R plasmids (1×10 ³ , 1×10 ⁴ , 1×10 ⁵ , 1×10 ⁶ , 1×10 ⁷ , 1×10 ⁸ , 1×10 ⁹ , 1×10 ¹⁰ , 1×10 ¹¹ copies/ul), referring to the system in Example 1, the amplification reactions of F3L-RPA-ncb3, B6R-RPA6, and B6R-RPA4 were respectively carried out.

The F3L-RPA-ncb3, B6R-RPA6, and B6R-RPA4 products were used in the Cas12a detection reactions of F3L-crRNA, B6R-crRNA1, and B6R-crRNA6, respectively, and the reaction system was referred to in Example 2. After reacting in a transparent tube for 60 minutes, place it under 485nm excitation light to observe the luminescent effect.

As shown in Figure 9-Figure 16, the sensitivity detection results of the F3L-RPA-CRISPR/Cas12a identification system show that the sensitivity of the B6RcrRNA1 identification system in the present invention is 1×10 ⁷ Copies/uL, and the sensitivity of the B6RcrRNA6 identification system is 1×10 ⁸ Copies/uL plasmid with good discrimination within 20 minutes. The sensitivity of the F3L-crRNA8 identification system is 1×10 ³ Copies/uL, and it has a good discrimination effect within 40 minutes.

3.3 Validation of the detection effect of monkeypox pseudovirus samples

Use F3L and B6R monkeypox pseudovirus samples (Yisheng 12099ES03, 12100ES03) to inactivate by heating at 95°C for 5 minutes to release the nucleic acid. Referring to the system of Example 3, the nucleic acid amplification reactions of F3L-RPA-ncb3, B6R-RPA6 and B6R-RPA4 were respectively carried out. The F3L-RPA-ncb3, B6R-RPA6, and B6R-RPA4 products were used in the Cas12a detection reactions of F3L-crRNA8, B6R-crRNA1, and B6R-crRNA6, respectively, and the reaction system was referred to in Example 2.

As shown in Figures 17-20, in the present invention, the monkeypox RPA-CRISPR/Cas12a identification system has been verified in pseudoviruses. It shows that the detection method can be used for detection of virus samples, and the detection is convenient, fast, and has high sensitivity, and the detection results can be observed intuitively and visible to the naked eye.

3.4 RPA-CRISPR/Cas12a identification system specificity verification

Use F3L and B6R monkeypox pseudovirus samples (Yisheng 12099ES03, 12100ES03) to inactivate by heating at 95°C for 5 minutes to release the nucleic acid. For herpes simplex virus type 1 and herpes simplex virus type 2 samples, the nucleic acid was extracted according to Tiangen virus DNA extraction kit (article number: YDP438). See the instructions for the extraction method for details. Using sample nucleic acids of herpes simplex virus type 1, herpes simplex virus type 2, and monkeypox pseudovirus, the F3L-RPA-ncb3, B6R-RPA6, and B6R-RPA4 nucleic acid amplification reactions were respectively performed with reference to the system of Example 3. The RPA products were used in the Cas12a detection reactions of F3L-crRNA8, B6R-crRNA1 and B6R-crRNA6 respectively, and the reaction system was referred to Example 3. As shown in Figure 21-Figure 28, in the present invention, the monkeypox RPA-CRISPR/Cas12a identification system shows fluorescence only in monkeypox pseudoviruses, and no fluorescence occurs in other virus samples and negative controls, indicating that the detection method is applicable to Monkeypox virus sample detection, and the detection is specific.

Claims (10)

1. A monkey pox virus detection primer is characterized in that: the primer for detecting the monkeypox virus comprises a specific primer F3L-RPA for amplifying a specificity target sequence F3L of the monkeypox virus, wherein a forward primer F of the specific primer F3L-RPA is TCCAACGATACTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTG; the reverse primer R of the specific primer F3L-RPA is AGGCCCCACTGATTCAATACG.

2. The monkey pox virus detection primer according to claim 1, characterized in that: the crRNA8 sequence of the monkey pox virus specific target sequence F3L is:

UAAUUUCUACUAAGUGUAGAUGCGGGAUACAUCAUCUAUUAUAGC。

3. the monkey pox virus detection primer according to claim 1 or 2, characterized in that: the monkey pox virus detection primer also comprises a specific primer B6R-RPA-6 for amplifying a monkey pox virus envelope protein gene B6R, wherein a forward primer F of the specific primer B6R-RPA-6 is CGTTGTTATGCGTACTACCTGC; the reverse primer R of the specific primer B6R-RPA-6 is GCAACTTAGTGTCATGGAA.

4. The monkey pox virus detection primer according to claim 3, characterized in that: the sequence of the crRNA1 of the monkey pox virus envelope protein gene B6R is as follows:

UAAUUUCUACUAAGUGUAGAUUUCAACAUGUACUGUACCCACUAUGA。

5. the monkeypox virus detection primer according to claim 4, characterized in that: the primer for detecting the monkeypox virus also comprises a specific primer B6R-RPA-4 for amplifying a monkeypox virus envelope protein gene B6R, wherein a forward primer F of the specific primer B6R-RPA-4 is CCATCACCAATGTATCGACGGAGCGTAA; the reverse primer R of the specific primer B6R-RPA-4 is CACGGTAGCAATTATGGAACTTATATTGGTCA.

6. The monkeypox virus detection primer according to claim 5, characterized in that: the sequence of the crRNA6 of the monkey pox virus envelope protein gene B6R is as follows:

UAAUUUCUACUAAGUGUAGAUAUCCAGUGGAUGAUGGUCCCGACG。

7. the use of the primer for monkey pox virus detection according to any one of claims 1 to 6 for the detection of monkey pox virus, in particular for the rapid detection of monkey pox virus.

8. Use of the monkey pox virus detection primers according to any of claims 1 to 6 as a monkey pox virus detection kit.

9. A method for rapidly detecting a monkeypox virus based on the primer for detecting a monkeypox virus according to any one of claims 1 to 6, characterized in that: the method for rapidly detecting the monkeypox virus comprises the following steps:

1) Obtaining a sample to be detected, wherein the sample to be detected is vesicle, pustule liquid or dry scab of skin lesion of a patient;

2) Configuring an amplification detection system comprising the simian pox virus detection primer according to any one of claims 1 to 6;

3) Placing the sample to be detected obtained in the step 1) in a detection system for rapid detection to obtain whether the sample to be detected has the monkeypox virus.

10. The monkeypox virus rapid detection method according to claim 9, characterized in that: the detection system also comprises buffer solution 1, lbaCas12a, crRNA, F-Q fluorescent quenching probes, sterile enzyme-free deionized water, twistAmp Rehydration, twistAmp freeze-dried powder and TwistAmp Magnesium Acetate;

the buffer solution 1 comprises the following components in percentage by weight: 500mM NaCl, 100mM Tris-HCl, 100mM MgCl ₂ And 100 μ g/ml Recombinant Albumin, the pH of buffer 1 is 7.9;

the F-Q fluorescence quenching probe is as follows: 5'-FAM-TTATT-Quencher-3';

the crRNA is crRNA8 of a monkey pox virus specific target sequence F3L, crRNA1 of a monkey pox virus envelope protein gene B6R or crRNA6 of a monkey pox virus envelope protein gene B6R;

the specific implementation manner of the detection system configured in the step 2) is as follows:

2.1 Obtaining an amplification product: 29.5ul twist Amp Rehydration buffer, twist Amp freeze-dried powder, 2.4ul10mM RPA upstream primer and 2.4ul10mM RPA downstream primer; the RPA upstream primer and the RPA downstream primer are both the monkey pox virus detection primers of any one of claims 1 to 6; according to the technical scheme, twistAmp freeze-dried powder is added into TwistAmp Rehydation buffer, twistAmp freeze-dried powder is dissolved, the primers for detecting the monkey pox virus of any one of claims 1 to 6 are added, and after mixing, the mixture is centrifuged for 10s under the condition of 3000 g; adding 10ul of sterile enzyme-free deionized water, adding 3.5ul of a sample to be detected, and fully and uniformly mixing; adding 2.5ul twist Amp MgOAC solution into the mixture, heating the mixture in a metal bath at 37 ℃ to start amplification for 10 minutes to obtain an amplification product;

2.2 Preparation of detection reagent: fully mixing 1uM crRNA1ul, 1uM LbaCas12a1ul, 3ul buffer solution 1, 20ul sterile enzyme-free deionized water and 1.5ul 10uMF-Q fluorescent quenching probe, and pre-incubating the mixture for 10 minutes at the temperature of 25 +/-0.5 ℃ to obtain a reagent for detection; the crRNAs are matched with the monkeypox virus detection primers of any one of claims 1 to 6 respectively;

the specific implementation manner of the step 3) is as follows:

adding 3.5ul of the amplification product obtained in the step 2.1) into the detection reagent obtained in the step 2.2), heating at 37 ℃ for 60 minutes, and then placing under 485nm exciting light to observe the luminous effect, wherein if fluorescence appears, the sample to be detected is positive; if no fluorescence appears, the sample to be detected is negative.

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