CN115807130A - Method for rapidly detecting monkeypox virus - Google Patents
Method for rapidly detecting monkeypox virus Download PDFInfo
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- CN115807130A CN115807130A CN202211436074.0A CN202211436074A CN115807130A CN 115807130 A CN115807130 A CN 115807130A CN 202211436074 A CN202211436074 A CN 202211436074A CN 115807130 A CN115807130 A CN 115807130A
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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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
Technical Field
The invention belongs to the field of microbial detection application, relates to a monkeypox virus rapid detection method, and particularly relates to a monkeypox virus rapid detection method based on CRISPR/Cas12a and RPA.
Background
Monkeypox virus (Monkeypox virus) is an enveloped double-stranded DNA virus belonging to the orthopoxvirus genus of the poxviridae family, which causes the infectious disease Monkeypox. The main coding genes comprise an envelope protein gene (B6R) and a specific target sequence (F3L). Because the monkey pox virus has strong infectivity, the symptoms of the patient at the initial stage after infection include fever, headache, muscle ache, lymphadenectasis, chill, fatigue and the like, which are similar to the symptoms of the variola virus and can be transmitted by people, animals, blood, body fluid, spray and the like. The clinical consequences are serious, the monkey pox virus is detected at the initial stage of infection, the patient is treated in time, effective isolation measures are taken, and the control of epidemic spread is very important. The detection requirements of the method are fast, accurate and simple, namely, the method can realize fast detection and accurate analysis, and the operation flow is simple.
At present, the detection method recommended and widely applied by the monkeypox virus is nucleic acid detection by a real-time fluorescent quantitative PCR technology, has the advantages of rapidness and sensitivity, meets the requirement of 'quasi', but has longer time consumption, expensive instrument and equipment, is difficult to popularize in basic inspection institutions, and still needs to be promoted in the aspects of 'quickness' and 'simplicity'. Recombinase Polymerase Amplification (RPA) is a nucleic acid detection technique that can be used in place of PCR. Strand exchange occurs in double-stranded DNA by searching for homologous sequences in the double-stranded DNA at 37-42 ℃ for a protein-DNA complex formed by binding a recombinase capable of binding single-stranded nucleic acids (oligonucleotide primers) to the primers, DNA synthesis is initiated by the action of a strand-displacing DNA polymerase, and the displaced DNA strand is then bound to a single-stranded DNA binding protein (SSB) to prevent further displacement. The RPA system can realize the amplification product with a detectable level within ten minutes, has the characteristics of high sensitivity and quick reaction, and meets the 'quick' requirement in the detection of the monkeypox virus.
CRISPR/Cas12a (clustered regulated amplified short palindromic repeats amplified polymorphic gene) is an endonuclease capable of realizing a DNA (deoxyribonucleic acid) cutting function under the guidance of specific RNA (ribonucleic acid), can be used for genome editing, has nonspecific side branch cutting activity, can realize the cutting of a fluorescent probe to generate fluorescence, can be used as a tool for in-vitro detection of biomolecules at normal temperature, and can meet the 'quasi' requirement in monkey pox virus detection. The guide RNA needs to be combined with a target sequence, and the 5' end of the target sequence is required to have a pro-spacer adjacent motif (PAM sequence) so as to stimulate the enzymatic activity to the maximum extent and realize the detection of specific nucleic acid.
The monkey pox virus is subjected to specific rapid detection by RPA normal-temperature amplification combined with CRISPR/Cas12a, and rapid screening detection of the monkey pox virus in places such as basic health institutions, airports, railway stations and the like can be realized by means of a simple and convenient ultraviolet imaging mode.
Disclosure of Invention
In order to solve the technical problems in the background art, the invention provides a method for rapidly detecting the monkeypox virus, which has strong specificity and high sensitivity.
In order to achieve the purpose, the invention adopts the following technical scheme:
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.
Preferably, the sequence of crRNA8 of the monkeypox virus specific target sequence F3L adopted by the invention is:
UAAUUUCUACUAAGUGUAGAUGCGGGAUACAUCAUCUAUUAUAGC。
preferably, the monkeypox virus detection primers adopted by the invention also comprise a specific primer B6R-RPA-6 for amplifying a monkeypox virus envelope protein gene B6R, and 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.
Preferably, the sequence of crRNA1 of the monkeypox virus envelope protein gene B6R adopted by the invention is:
UAAUUUCUACUAAGUGUAGAUUUCAACAUGUACUGUACCCACUAUGA。
preferably, the monkey pox virus detection primers adopted by the invention also comprise a specific primer B6R-RPA-4 for amplifying a monkey pox virus envelope protein gene B6R, and a forward primer F of the specific primer B6R-RPA-4 is CCATCACCATGTATCGACGTAA; the reverse primer R of the specific primer B6R-RPA-4 is CACGGTAGCAATTATGGAACTTATATTGGTCA.
Preferably, the sequence of crRNA6 of the monkey pox virus envelope protein gene B6R adopted by the invention is as follows:
UAAUUUCUACUAAGUGUAGAUAUCCAGUGGAUGAUGGUCCCGACG。
the application of the monkey pox virus detection primer in detecting monkey pox virus, in particular in quickly detecting monkey pox virus.
The application of the primer for detecting the monkeypox virus as the monkeypox virus detection kit.
A monkey pox virus rapid detection method based on the primer for monkey pox virus detection is characterized in that: the method for rapidly detecting the monkeypox virus comprises the following steps:
1) Obtaining a sample to be tested, wherein the sample to be tested is vesicle, pustule liquid or dry scab of skin lesion of a patient;
2) Configuring 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.
Preferably, the detection system adopted by the invention also comprises buffer solution 1, lbaCas12a, crRNA, F-Q fluorescence quenching probe, sterile enzyme-free deionized water,
Rehydration、
Lyophilized powder and
Magnesium Acetate(MgOAC)；
the buffer solution 1 comprises the following components in percentage by weight: 500mM NaCl, 100mM Tris-HCl, 100mM MgCl 2 And 100. Mu.g/ml Recombinant Albumin, the pH of buffer 1 is 7.9;
the F-Q fluorescent 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 configuring the detection system in the step 2) is as follows:
2.1 Obtaining an amplification product: take 29.5ul
Rehydration buffer、
Lyophilized powder, 2.4u10mM RPA upstream primer and 2.4u10mM RPA downstream primer; the RPA upstream primer and the RPA downstream primer are both the above-mentioned monkey pox virus detection primers; in that
Hydration buffer addition
Freeze-drying powder and dissolving
Freezing the powder, adding the above primer for detecting monkeypox virus, mixing, and centrifuging at 3000g for 10s; adding 10ul of sterile enzyme-free deionized water, adding 3.5ul of a sample to be detected, and fully and uniformly mixing; 2.5ul of water was added
Heating MgOAC solution in 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 crRNA is respectively matched with a monkey pox virus detection primer;
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 for 60 minutes at 37 ℃, and then placing under 485nm exciting light to observe the luminescence effect, wherein if fluorescence appears, the sample to be detected is positive; if no fluorescence appears, the sample to be detected is negative.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a monkeypox virus rapid detection method, which is a monkey pox molecule detection kit and a detection method generated by combining an RPA amplification technology and a CRISPR/Cas12a reaction system. According to the invention, based on the CRISPR/Cas12a lateral branch cutting function, crRNA sequences and RPA primers are designed aiming at F3L genes and B6R genes of the monkeypox virus respectively to realize quick, simple, convenient, sensitive and specific detection, so that the invention is beneficial to portable detection and popularization and application of the monkeypox virus in customs, airports, railway stations or remote areas.
Drawings
FIG. 1 time/fluorescence change curves of each F3L/crRNA in CRISPR/Cas12a assay;
FIG. 2 is a graph showing the effect of visualization of the results of the detection of each F3L/crRNA in FIG. 1 under 485nm excitation light at 60 minutes;
fig. 3 is a time/fluorescence change curve of each B6R/crRNA in CRISPR/Cas12a detection;
FIG. 4 is a graph showing the effect of visualization of the results of the detection of each B6R/crRNA in FIG. 1 under 485nm excitation light at 60 minutes;
FIG. 5 is a time/fluorescence change curve of the amplification product of each monkeypox virus F3LRPA primer used in the F3L/crRNA7-CRISPR/Cas12a assay;
FIG. 6 is a time/fluorescence change curve of the amplification product of each monkeypox virus F3LRPA primer used in the F3L/crRNA8-CRISPR/Cas12a assay;
FIG. 7 is a time/fluorescence change curve of the amplification product of each monkeypox virus B6RRPA primer used in the B6R/crRNA1-CRISPR/Cas12a assay;
FIG. 8 is a time/fluorescence change curve of the amplification product of each monkeypox virus B6RRPA primer used in the B6R/crRNA6-CRISPR/Cas12a assay;
FIG. 9 shows the monkeypox virus B6R recombinant plasmids at different concentrations (1 x 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-RPA6 for a time/fluorescence change curve in the detection of B6R/crRNA1-CRISPR/Cas12 a;
FIG. 10 is a graph showing the effect of visualization of the detection results of FIG. 9 at various concentrations under 485nm excitation light at 60 minutes;
FIG. 11 shows the monkeypox virus B6R recombinant plasmids at different concentrations (1 x 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 for time/fluorescence change curve in B6R/crRNA6-CRISPR/Cas12a detection;
FIG. 12 is a graph showing the effect of visualization of the detection results of FIG. 11 at various concentrations under 485nm excitation light at 60 minutes;
FIG. 13 shows the monkeypox virus F3L recombinant plasmid at different concentrations (1 x 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 F3L-RPAncb3 for time/fluorescence change curve in F3L/crRNA7-CRISPR/Cas12a detection;
FIG. 14 is a graph showing the effect of visualization of the detection results at 485nm excitation light at 60 minutes for each concentration in FIG. 13;
FIG. 15 shows the monkeypox virus F3L recombinant plasmid at different concentrations (1 x 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 the F3L-RPAncb3 amplification product was used for time/fluorescence change curve in the F3L/crRNA8-CRISPR/Cas12a assay;
FIG. 16 is a graph showing the effect of the detection results at 485nm excitation light on the detection results at respective concentrations in FIG. 15 at 60 minutes;
FIG. 17 is a time/fluorescence change curve of amplification products of monkeypox B6R pseudovirus B6R-RPA6 in CRISPR/Cas12a detection of B6R/crRNA6 with amplification products of B6R/crRNA1, B6R-RPA 4;
FIG. 18 is a graph showing the effect of visualization of the detection results at 485nm excitation light at 60 minutes for each concentration in FIG. 17;
FIG. 19 is a time/fluorescence change curve of the amplification product of monkeypox F3L pseudovirus F3L-RPAncb3 in CRISPR/Cas12a detection of F3L/crRNA7, F3L/crRNA 8;
FIG. 20 is a graph showing the effect of visualization of the detection results of FIG. 19 at various concentrations under 485nm excitation light at 60 minutes;
FIG. 21 is a time/fluorescence change curve of amplification products 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 assay for B6R/crRNA 1;
FIG. 22 is a graph showing the effect of visualization of the detection results at 485nm excitation light at 60 minutes for each concentration in FIG. 21;
FIG. 23 is a time/fluorescence change curve of amplification products 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 assay for B6R/crRNA 6;
FIG. 24 is a graph showing the effect of the detection result at 485nm excitation light on the detection result at each concentration in FIG. 23 at 60 minutes;
FIG. 25 is a time/fluorescence change curve of amplification products of F3L-RPAncb3 of Monkey Pox (MP) F3L pseudovirus, HSV1 (herpes simplex virus type 1), and HSV2 (herpes simplex virus type 2) in the CRISPR/Cas12a assay for F3L/crRNA 7;
FIG. 26 is a graph showing the effect of visualization of the detection results at 485nm excitation light at 60 minutes for each concentration in FIG. 25;
FIG. 27 is a time/fluorescence change curve of amplification products of F3L-RPAncb3 of Monkey Pox (MP) F3L pseudovirus, HSV1 (herpes simplex virus type 1), and HSV2 (herpes simplex virus type 2) in the CRISPR/Cas12a assay for F3L/crRNA 8;
FIG. 28 is a graph showing the effect of visualization of the measurement results at 485nm excitation light at 60 minutes in FIG. 27.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified. The crRNA, DNA primers and recombinant plasmids used in the experiment are all synthesized by the Kinry organism company.
Example 1: specific RPA primer design
Monkeypox virus F3L, B6R genomic sequences were downloaded from NCBI (http:// www. NCBI. Nlm. Nih. Gov/genome /). GenBank > NC — 003310.1:164845-165798 design B6R specific primers, geneID: MT250197.1 (46192.. 466539) was designed with F3L specific primers. RPA primers were designed according to the principle of RPA primer design and confirmed for specificity and identification availability in BLAST search.
The sequence of the experimentally designed RPA primer is as follows:
F3L gene
F3L-RPA-1F：ATGGAGAAGCGAGAAGTTAATAAAGC
F3L-RPA-1R：ACTAAGAAGTTTATCTACAGCCAATT
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：TCCAACGATACTCCTCCTCGT
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: cas12acrRNA for screening and verifying F3L and B6R optimality
According to the CRISPR/Cas12a primer design principle, 4 crRNAs aiming at the B6R gene are designed in a TTTNPAM mode:
crRNA1：UAAUUUCUACUAAGUGUAGAUUUCAACAUGUACUGUACCCACUAUGA
crRNA3：UAAUUUCUACUAAGUGUAGAUCACGUGUCAAAGACUAAUACAGAG
crRNA6：UAAUUUCUACUAAGUGUAGAUAUCCAGUGGAUGAUGGUCCCGACG
crRNA7：UAAUUUCUACUAAGUGUAGAUUGAUUAUGUCUCUGAACUAUAUGA
6 crRNAs against the F3L gene were designed:
crRNA3：UAAUUUCUACUAAGUGUAGAUGUUACUCUCUCCUAGUAUUCAGAA
crRNA5：UAAUUUCUACUAAGUGUAGAUUUGAUCCUCUCUAACCAGAAAAGC
crRNA7：UAAUUUCUACUAAGUGUAGAUACCGGAAUAACAUCAUCAAAAGAC
crRNA8：UAAUUUCUACUAAGUGUAGAUGCGGGAUACAUCAUCUAUUAUAGC
crRNA9：UAAUACGACUCACUAUAGGGUAAUUUCUACUAAGUGUAGAUCAUCUGCCUUAUCGAAUACUCUUC
crRNA10：UAAUACGACUCACUAUAGGGUAAUUUCUACUAAGUGUAGAUGUUGUGAAGAAAAAAAUGGAAAUA
the following reaction system is prepared: CRISPR/Cas12a reaction system: final concentration was 33nMcrRNA, final concentration was 33nM lbaca12a, 3ul reaction buffer 1, nuclease-free water, final concentration was 50nMF-Q fluorescence quenching probe: 5'-FAM-TTATT-Quencher-3'. The specific reaction process is as follows: 3ul buffer 1, lbaCas12a, crRNA, F-Q fluorescence quenching probe and 20ul sterile enzyme-free deionized water were mixed well and pre-incubated at room temperature for 10 minutes. Adding 3.5ul of recombinant B6R and F3L plasmids, reacting in a transparent tube at 37 ℃ for 60 minutes, and then placing under 485nm exciting light to observe the luminous effect.
As shown in fig. 1-4, the crRNA screening results of the CRISPR/Cas12a detection system show that in the F3L detection system, the crRNA7 and the crRNA8 generate visible fluorescence, and the crRNA8 can generate brighter fluorescence under the excitation wavelength of 485nm of the transmission light source, so that the F3L-crRNA8 is used for detection in subsequent experiments. In the B6R detection system, the crRNA1, the crRNA6 and the crRNA7 generate visible fluorescence, wherein the crRNA1 and the crRNA6 have higher fluorescence intensity and are used for subsequent B6R gene detection. In the invention, the fluorescence intensity increases with the increase of the detection time, and the fluorescence which can be distinguished can be reached in about 20 minutes at the fastest speed.
Example 3: RPA primer most suitable for screening and verifying crRNA and sensitivity detection
3.1F3L/B6RcrRNA the most suitable RPA primer screening
The RPA primer in example 1 was used for CRISPR/Cas12a detection. The following amplification reaction system is configured: 29.5ul
Rehydration buffer、
Lyophilized powder, 2.4ul10mM RPA upstream primer and 2.4ul10mM RPA downstream primer. Adding the hydration buffer into the freeze-dried powder to dissolve the powder, adding the upstream and downstream primers, mixing, centrifuging for 3000g and 10s, adding 10ul of sterile enzyme-free deionized water, adding 3.2ul of a substrate to be amplified, namely recombinant plasmid,mixing well, adding 2.5ul
Heating MgOAC solution in metal bath at 37 deg.c to start amplification for 10 min; CRISPR/Cas12a reaction system: final concentration 33nM crRNA, final concentration 33nM lbaca12a, 3ul reaction buffer 1, nuclease-free water, final concentration 50nM f-Q fluorescence quenching probe: 5'-FAM-TTATT-Quencher-3'. Respectively configuring F3L-crRNA8, B6R-crRNA1 and B6R-crRNA6 detection systems: 3ul buffer 1, lbaCas12a, crRNA (F3L-crRNA 8, B6R-crRNA1, B6R-crRNA6, respectively), F-Q fluorescence quenching probe and 20ul sterile enzyme-free deionized water were mixed well and pre-incubated at room temperature for 10 minutes. Taking 3.5ul of RPA products, wherein five RPA products including RPA1, RPA2, ncb1, ncb2 and ncb3 are respectively given to a detection system of F3L crRNA 8; wherein the B6R crRNA1 detection system is respectively administered with five RPA products of RPA1, RPA5, RPA6, ncb1 and ncb 2; wherein the B6R crRNA6 detection system respectively gives three RPA products of RPA4, ncb3 and ncb 4. The fluorescence intensity was monitored by heating at 37 ℃ for 60 minutes.
As shown in FIG. 5-FIG. 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 primers can realize negative and positive discrimination within 20 minutes, and are used for subsequent sensitivity detection and pseudovirus sample detection.
3.2 assay System sensitivity verification
F3L and B6R plasmids (1X 10) were prepared at different concentrations 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), F3L-RPA-ncb3, B6R-RPA6, B6R-RPA4 amplification reactions were carried out according to the system of example 1, respectively.
The F3L-RPA-ncb3, B6R-RPA6 and B6R-RPA4 products were used in Cas12a detection reactions of F3L-crRNA, B6R-crRNA1 and B6R-crRNA6, respectively, in the reaction system according to example 2. After reacting for 60 minutes in a transparent tube, the reaction tube is placed under 485nm exciting light to observe the luminous effect.
As shown in figure 9-figure 16, the sensitivity detection result of the F3L-RPA-CRISPR/Cas12a identification systemIt was shown that the sensitivity of the B6RcrRNA1 assay system of the present invention was 1X 10 7 The sensitivity of the identification system of Copies/uL, B6RcrRNA6 is 1 × 10 8 Copies/uL plasmid, and has good distinguishing effect within 20 minutes. The sensitivity of the F3L-crRNA8 identification system is 1 x 10 3 Copies/uL, with good discrimination in 40 minutes.
3.3 verification of detection Effect of Simplepox pseudovirus sample
Samples of F3L and B6R monkeypox pseudovirus (next san 12099ES03, 12100ES 03) were used for 5 minutes at 95 ℃ and heat-inactivated to liberate nucleic acids. F3L-RPA-ncb3, B6R-RPA6, and B6R-RPA4 nucleic acid amplification reactions were carried out according to the system of example 3. The F3L-RPA-ncb3, B6R-RPA6 and B6R-RPA4 products were used in Cas12a detection reactions of F3L-crRNA8, B6R-crRNA1 and B6R-crRNA6, respectively, in the reaction system according to example 2.
As shown in FIGS. 17-20, the monkey pox RPA-CRISPR/Cas12a identification system is verified in pseudoviruses in the invention. The detection method can be used for detecting the virus sample, and the detection is convenient and rapid, has higher sensitivity, and can observe the detection result visually and visually.
3.4 specificity verification of RPA-CRISPR/Cas12a identification system
Samples of F3L and B6R monkeypox pseudovirus (next san 12099ES03, 12100ES 03) were used for 5 minutes at 95 ℃ and heat-inactivated to liberate nucleic acids. The herpes simplex virus type 1 and herpes simplex virus type 2 samples are subjected to nucleic acid extraction according to a Tiangen virus DNA extraction kit (the cargo number is YDP 438), and the extraction mode is detailed in the specification. Using sample nucleic acids of herpes simplex virus type 1 and herpes simplex virus type 2, and monkeypox pseudovirus, F3L-RPA-ncb3, B6R-RPA6, and B6R-RPA4 nucleic acid amplification reactions were carried out, respectively, according to the system of example 3. The RPA products were used in Cas12a detection reactions of F3L-crRNA8, B6R-crRNA1, and B6R-crRNA6, respectively, in the reaction system according to example 3. As shown in fig. 21-fig. 28, the monkeypox RPA-CRISPR/Cas12a identification system of the invention showed fluorescence in monkeypox pseudovirus only, and no fluorescence was generated in other virus samples and negative controls, indicating that the detection method is suitable for monkeypox virus sample detection, and the detection has specificity.
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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