CN116606961A - CrRNA for EB virus nucleic acid detection and application thereof - Google Patents
CrRNA for EB virus nucleic acid detection and application thereof Download PDFInfo
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Abstract
The application relates to the field of biotechnology, and particularly discloses crRNA for EB virus nucleic acid detection and application thereof. Compared with the single ERA isothermal amplification detection, the ERA combined CRISPR/Cas12a detection is more sensitive, the operation of the test strip is simpler, and electrophoresis is not needed.
Description
Technical Field
The application relates to the field of biotechnology, in particular to crRNA for EB virus nucleic acid detection and application thereof.
Background
Nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) is one of the common malignant tumors, and detection of EB virus (Epsteinbarr virus, EBV) is an important index for early screening of nasopharyngeal carcinoma, disease progression, therapeutic efficacy and prognosis monitoring. At present, two methods for detecting EB virus are mainly used clinically, namely, a serological method is used for detecting EB virus antibodies; the other is a fluorescent quantitative PCR method for detecting the EBV DNA virus nucleic acid. Compared with the virus antibody, the detection result of the virus nucleic acid can reflect the infection, replication and content conditions of the EB virus more stably, so the detection of the EBV DNA nucleic acid becomes an important means for screening and diagnosing the nasopharyngeal carcinoma. The fluorescent quantitative PCR detection is a 'gold standard' for clinical EB virus nucleic acid detection, and the detection sensitivity of the current commercial kit is 100-500copies/ml.
Although fluorescence quantitative PCR is a gold standard for EB virus nucleic acid detection, the sensitivity is high, 100-500copies can be detected, but for early-stage infected patients, the detection sensitivity of the method is insufficient, and false negative results are easy to appear to cause missed detection; secondly, fluorescent quantitative PCR is implemented by relying on professional instruments and special training, is suitable for laboratory detection, and is not suitable for the requirements of rapid diagnosis of field detection and community screening. Therefore, the EB virus nucleic acid detection method with convenient research and development operation and higher sensitivity is an urgent need for diagnosing EB virus and similar pathogens, and has important clinical application value.
The recombinase polymerase amplification (Recombinase polymerase amplification, RPA) is a novel nucleic acid isothermal amplification technology, and can effectively amplify nucleic acid within 10-20min at 37-42 ℃, and the amplification efficiency can reach billions times. Compared with PCR, the Enzyme Recombination Amplification (ERA) technology is an upgrade version of RPA, has lower ERA and RPA temperature requirements, does not need special instruments, has shorter reaction time, is simple, convenient, efficient and quick, has high specificity and sensitivity, and is a nucleic acid detection technology capable of replacing PCR. There are many detection methods for detecting ERA amplification results, including agarose gel electrophoresis, real-time fluorescent quantitative detection, lateral flow test strips, and other detection means. Agarose gel electrophoresis is complex, the amplified product is required to be purified and then subjected to electrophoresis detection, and aerosol is easy to generate, so that the environment is polluted. The fluorescence method or the test strip method is used for detecting the added specific probes, and the ERA probes are complex in design, high in price and not suitable for popularization.
CRISPR/Cas gene editing techniques are derived from immune defense systems within certain bacteria and archaea, and DNA or RNA can be edited by artificial engineering. Mainly consists of a Cas protein with nuclease function and a specific guide nucleic acid sequence, and CRISPR/Cas systems such as Cas9, cas12, cas13 and Cas14 and the like are mainly developed at present, wherein Cas9, cas12 and Cas14 are RNA-guided DNA nucleases, and Cas13 is RNA editing enzyme. In addition to accurate targeted cleavage of the target nucleic acid sequence, cas12, cas13, and Cas14 proteins also have a "side cleavage effect". When Cas12, cas13 and Cas14 proteins form double chains with target DNA (Cas 12 targeted double-stranded dsDNA; cas14 targeted single-stranded ssDNA) or RNA sequence (Cas 13 targeted RNA) under the guidance of guide RNA through base complementary pairing specificity, the enzyme activity of Cas protein can be activated, and Cas enzyme can cleave single-stranded target DNA or RNA molecule in a non-specific manner, namely 'accessory cleavage', and by utilizing the characteristic, after the target DNA or RNA molecule is transformed into a signal molecule, cas12, cas13 and Cas14 proteins can be applied to quantitative and qualitative detection of nucleic acid, so that the detection sensitivity is greatly improved.
There is no current study disclosing the use of Enzymatic Recombinant Amplification (ERA) in combination with CRISPR/Cas12a to detect epstein barr virus nucleic acids.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provide an EB virus nucleic acid detection kit and a detection method based on enzyme recombination amplification combined with CRISPR/Cas12a, and the application utilizes an enzymatic recombination isothermal amplification technology (ERA) combined with a CRISPR/Cas12a gene editing technology (Clustered Regularly Interspaced Short Palindromic Repeats/Cas12 a) to enable EB virus nucleic acid detection to be simple in operation, higher in sensitivity and more suitable for rapid diagnosis of field detection and community screening.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a crRNA for EB virus nucleic acid detection, the crRNA comprising at least one of LMP1-crRNA1, LMP1-crRNA2, LMP1-crRNA3, LMP2-crRNA1, LMP2-crRNA2 and LMP2-crRNA3, the nucleotide sequence of the LMP1-crRNA1 being SEQ ID NO:4, a step of; the nucleotide sequence of the LMP1-crRNA2 is SEQ ID NO:5, a step of; the nucleotide sequence of the LMP1-crRNA3 is SEQ ID NO:6, preparing a base material; the nucleotide sequence of the LMP2-crRNA1 is SEQ ID NO:7, preparing a base material; the nucleotide sequence of the LMP2-crRNA2 is SEQ ID NO:8, 8; the nucleotide sequence of the LMP2-crRNA3 is SEQ ID NO:9.
the application designs the six crRNAs with high efficiency for cutting the target sequence, can specifically identify the EBV, and can improve the sensitivity and accuracy of detection.
The second object of the application is to provide a primer group for detecting the crRNA, wherein the nucleotide sequence of the primer group is shown in SEQ ID NO:10 to 21.
The primer group for detecting the crRNA is screened out, so that the sensitivity for detecting the crRNA can be improved, and the sensitivity for detecting viruses is higher.
The third object of the present application is to provide the application of the crRNA in preparing EB virus nucleic acid detection products.
Preferably, the test product comprises a test strip or a test kit.
The fourth object of the application is to provide an EB virus nucleic acid detection product based on enzyme recombination and amplification combined with CRISPR/Cas12a, wherein the detection test strip comprises the primer group and a CRISPR/Cas12a detection system;
the CRISPR/Cas12a detection system includes a Cas12a protein, the crRNA described above, and a ssDNA reporting system.
Preferably, the Cas12a protein is LbCas12a. The concentration of LbCas12a is 1 μm; the crRNA concentration was 5. Mu.M.
As a preferred embodiment of the EB virus nucleic acid detection product based on the enzyme recombinant amplification combined with CRISPR/Cas12a, the ssDNA reporting system comprises a ssDNA probe with the concentration of 0.1-10 mu M. Preferably the ssDNA probe is at a concentration of 5 μm.
The application optimizes the concentration of LbCAs12a, crRNA, ssDNA probe, so that the detection of EB virus nucleic acid is more accurate, the detection sensitivity is high, and the specificity is strong.
In a fifth aspect, the present application provides a method for detecting EB virus nucleic acid for the purpose of non-disease diagnosis and treatment, comprising the steps of:
(1) Taking a sample to be detected, and extracting an EB virus genome;
(2) ERA enzymatic recombination isothermal amplification: amplifying the EB virus genome obtained in the step (1) by using the primer set;
(3) CRISPR/Cas12a detection: taking the amplified product in the step (2), adding a ssDNA probe, cas12a protein and crRNA, performing CRISPR/Cas12a detection, and reading a detection signal (a test strip method/a fluorescence method). The test strip method is suitable for on-site detection and community screening rapid diagnosis, and the fluorescence method is suitable for laboratory high-throughput inspection.
As a preferred embodiment of the method for detecting EB virus nucleic acid of the present application, in the step (2), the concentration of the EB virus genome is 10 by amplifying the nucleic acid in the range of 10min to 20min with a gradient of 5min 6 copies/μL。
As a preferred embodiment of the method for detecting EB virus nucleic acid according to the present application, in the step (3), the concentration of the amplified product is diluted to 10 2 ～10 5 copies/μL。
The sixth object is to provide the application of the EB virus nucleic acid detection product in the detection of EB virus nucleic acid.
Compared with the prior art, the application has the following beneficial effects:
the application provides crRNA for EB virus nucleic acid detection and application thereof, and the detection method is based on enzyme recombination amplification combined with CRISPR/Cas12a to detect EB virus nucleic acid, does not need professional and special instruments, is convenient to operate, has short reaction time (less than 1 hour), and is more suitable for rapid diagnosis of field detection and community screening. Compared with the single ERA isothermal amplification detection, the ERA combined CRISPR/Cas12a detection is more sensitive, the operation of the test strip is simpler, and electrophoresis is not needed.
Drawings
FIG. 1 is a graph showing the results of amplification efficiencies of different primer combinations for LMP1 target sequences;
FIG. 2 is a graph showing the results of amplification efficiencies of different primer combinations for LMP2 target sequences;
FIG. 3 is a graph of test results for optimizing primer usage for ERA amplification;
FIG. 4 is a graph of test results for optimizing activator dosage;
FIG. 5 is a graph of experimental results of the amount of optimized templates (LMP 1 target sequence, LMP2 target sequence);
FIG. 6 is a graph of the minimum limit of detection for fluorescent ERA amplification detection;
FIG. 7 is a graph I of crRNA screening results of the optimized CRISPR/Cas12a detection of example 4;
FIG. 8 is a graph II of crRNA screening results of the optimized CRISPR/Cas12a detection of example 4;
FIG. 9 is a graph showing the results of the different ERA amplification times of example 4;
fig. 10 is a graph of the results for different Cas12a amounts of example 4;
FIG. 11 is a graph showing the results of different types of buffers in example 4;
FIG. 12 is a graph showing the results of different volumes of F-Q probes of example 4;
fig. 13 is a graph of the results of different Cas12a reaction template dosages of example 4;
FIG. 14 is a graph showing the results of F-B probes of example 4 at different concentrations;
fig. 15 is a graph showing the results of Cas12a test strip method of example 4 at different reaction times;
FIG. 16 is a graph showing the results of the minimum limit of detection (LOD) for ERA/CRISPR-Casl2a strip system detection;
FIG. 17 is a graph of the specificity results of the ERA/CRISPR-Cas12a strip system for detecting EBV;
FIG. 18 is a graph of the results of clinical application of the ERA/CRISPR-Cas12a strip system;
FIG. 19 is a graph of the results of clinical application of ERA/CRISPR-Cas12a fluorescence system;
FIG. 20 is a detection flow diagram of the ERA/CRISPR-Cas12a reaction.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
The "cloning" referred to in the examples below is a prior art approach.
Example 1 design of crRNA
1. The EBV-LMP-2A gene sequence (SEQ ID NO: 1) is queried through NCBI, two sections of proper target DNA sequences (LMP 1 target sequence, LMP2 target sequence, conserved sequence, ERA primer design suitable and PAM site of Cas 12A) are screened and determined, and the target DNA sequence (SEQ ID NO: 2-3) is cloned into pUC57 plasmid vector (Kirschner) and used as a follow-up experimental template.
2. For the EBV-LMP-2A target sequence, 6 crRNAs were designed using CRISPR-DT software, and the best crRNA was selected experimentally. The application designs and synthesizes 6 crRNAs with better cutting efficiency through PCR and in vitro transcription (shown in table 1) by different crRNA cutting efficiencies of different target DNA sequence designs.
TABLE 1
Example 2 design of ERA primers
The primer is screened by using a fluorescence ERA method (such as a reaction system in Table 2), so that pollution caused by gel running of electrophoresis after uncapping can be avoided. The procedure was followed according to the instructions of ERA kit (from Jiangsu Mida Gene technology Co., ltd.) at 1.10 4 The copies/. Mu.l plasmid is used as a primer pair with no amplification or poor amplification efficiency for template elimination,then with lower concentration of plasmid (2 x 10 3 copies/. Mu.l) was used as template to screen out primer pairs with better amplification efficiency.
TABLE 2ERA fluorescence reaction System
Transferring the premixed solution of the reaction system into a reaction tube; 1 μl of activator was added to the reaction tube cap, the cap was closed, and the mixture was centrifuged briefly for a few seconds, and the activator was added to the premix. Sufficiently oscillating for a short time, uniformly mixing and rapidly centrifuging again; the reaction tube was incubated in a QPCR apparatus (37 ℃) for 30 minutes, and fluorescence values were collected every 1 minute.
The primer sequences were as follows:
1. designing a primer and a fluorescent probe aiming at an LMP1 target sequence:
probe PB1: GTGACGAAGCAGCAGACGGCGGATATGGGAA (FAM-dT) (THF) (BHQ 1-dT) CAGAATGAGGTGG (C3-SPACER);
LMP1-F1:F3295 AAATGGAAAGGCAGTGCGGCAATCAGAAG(SEQ ID NO：10)；
LMP1-F2:F3315 AATCAGAAGGGGGAGTGCGTAGTGTTGTG(SEQ ID NO：11)；
LMP1-F3:F3332 CGTAGTGTTGTGGGAAGCGGCAGTGTAAT(SEQ ID NO：12)；
LMP1-F4:F3439 GGTGAGGCAAGGCTGTGGGGTAACCGTAG(SEQ ID NO：13)；
LMP1-R1:R3584 ACAGCCCACACCCTTTTCGCCTGAATCCG(SEQ ID NO：14)；
LMP1-R2:R3595 ATGACACTCGCACAGCCCACACCCTTTTC(SEQ ID NO：15)；
LMP1-R3:R3634 ATGCCCCAGCAAGCCGCAGCGACTTTCCG(SEQ ID NO：16)；
2. designing a primer and a fluorescent probe aiming at an LMP2 target sequence:
probe PB2: CAGTAAGGACAAGGAAAGAAGGCCAGAGGAA (FAM-dT) (THF) (BHQ 1-dT) GGAAAGATGAGCG (C3-SPACER);
LMP2-F1:F840 GATGAGGAGCAGGCATAAAAGTCCAAACAG(SEQ ID NO：17)；
LMP2-F2:F862 CCAAACAGGACACAGAGTACCACCAGGAGT(SEQ ID NO：18)；
LMP2-F3:F871 ACACAGAGTACCACCAGGAGTAGTCTTAGT(SEQ ID NO：19)；
LMP2-R1:R1172 TAAGCACTTTAATCCCTCTCTCACACCCAG(SEQ ID NO：20)；
LMP2-R2:R1247 TGGATTCAGCCCAAGCCACACCTAACTCAT(SEQ ID NO：21)。
referring to fluorescent FIGS. 1-2, primer combinations with higher efficiency, such as LMP1 target sequences selected from LMP1-F3 and LMP1-R3, LMP2 target sequences selected from LMP2-F3+LMP2-R1, LMP2-F3+LMP2-R2, are selected.
Example 3 optimization of the reaction conditions for ERA enzymatic recombinant isothermal amplification
1. Primer amount, activator amount, template (LMP 1 target sequence, LMP2 target sequence) amount for ERA amplification are optimized, and the lowest detection limit LOD is detected.
1) In the reaction system of Table 2, the amount of the upstream and downstream primer pairs (5. Mu.M each) was optimized (1.5. Mu.l, 2.0. Mu.l, 2.5. Mu.l) under the same conditions, and for example, LMP1-F3+LMP1-R3 or LMP2-F3+LMP2-R1 primers were selected for fluorescence ERA amplification, see FIG. 3.
2) In the reaction system of Table 2, the amount of the activator used (1.0. Mu.l, 1.25. Mu.l) was optimized under the same conditions, and fluorescent ERA amplification was performed, as shown in FIG. 4.
3) In the reaction system of Table 2, the amount of template (2.0. Mu.l, 4.0. Mu.l) was optimized under the same conditions, and fluorescent ERA amplification was performed, as shown in FIG. 5.
4) The detection of the lowest detection limit was performed on the templates (200, 20, 2 copies/. Mu.l) diluted 10-fold in accordance with the above-described optimized reaction conditions, and the determination of positive fluorescence signal-to-noise ratio (S/N) value of 2 or more was made, referring to FIG. 6.
TABLE 3 optimized ERA reaction System
Example 4 optimization of reaction conditions for CRISPR/Cas12a detection
The Cas12a protein recognizes and cleaves target DNA while activating its attendant cleavage activity, cleaving non-target DNA. Therefore, for Cas12a protein with high affinity to AT-rich sequences, an F-Q probe for fluorescence detection (sequence 5 '-FAM-ttatatt-BHQ-3') and a test strip F-B probe (sequence 5 '-FAM-ttatatt-Biotin-3') were designed, and Cas12a cleavage efficiency was observed using two methods. ssDNA probes were synthesized by kusnezoff biosystems.
The fluorescence detection method is relatively suitable for the optimization of a laboratory method, so that the condition optimization of the Cas12a reaction is performed by using the fluorescence method.
Using CRISPR-DT software, 3 crRNAs were designed for each target sequence and synthesized by the overlap primer method (reference example 1).
1. crRNA screening
ERA products (the concentration of the template in amplification is 200 cp/. Mu.l) are taken as templates, the Cas12a system is added for reaction, and after full mixing, the ERA products are placed in a QPCR instrument (37 ℃) for incubation for 30 minutes, and fluorescent values are collected every 1 minute. To exclude the effect of ERA system on Cas12a reaction system, a blank control (DEPC water for template target DNA of Cas12a reaction) and a negative control (water for template at ERA amplification) were set, crrnas with high fluorescence value in experimental group and low fluorescence value in negative control group were selected. The red line is the negative control of Cas12a experiment, the green line is the negative control of ERA experiment, the gray line is ERA experiment group (template is 200 cp/. Mu.l), the template is selected to be amplified, crRNA with low background of the control group is selected, LMP1 fragment is comprehensively compared to select LMP1-crRNA1, LMP2 fragment is selected to select LMP2-crRNA1, refer to FIGS. 7-8.
After crrnas with better cleavage efficiency are obtained by screening, ERA reaction time involved in Cas12a reaction, cas12a usage, buffer type, template usage, and F-Q and F-B probe usage are optimized.
TABLE 3Cas12a reaction System
Note that: the F-Q or F-B probe was synthesized in the Kirschner BioCo., ltd. And the sequence was 5'-FAM-TTATTATT-Biotin-3'.
2. In the reaction system of table 3, the ERA amplification time (15, 20, 25 min) was optimized, the fluorescence value of Cas12a reaction of ERA product was selected, the maximum fluorescence value (S/N) was selected, and the ERA amplification time was selected for 20min, under other conditions.
The fluorescence value generated at 25min is lower than 20min because the target detected by the CRISPR-Cas12a system is ERA product, and the detection is affected by ERA amplification. dNTPs are continuously consumed and interact with Mg in ERA7 system when ERA amplification time is prolonged 2+ The reaction forms an insoluble magnesium pyrophosphate solution, which becomes a solution. The amplified turbidity product then migrates to the Cas12a system, which becomes correspondingly turbid, resulting in a decrease in fluorescence value. Refer to fig. 9.
3. In the reaction system of table 3, the fluorescence values of the Cas12a dose (1, 2, 3 μl) reactions were optimized, the maximum fluorescence value (S/N) was selected, and the Cas12a dose was 1 μl, under the other conditions, referring to fig. 10.
4. In the reaction system of Table 3, the fluorescence values of different types of buffers (NEB 2.1Buffer,NEB 3.1Buffer,NEB 4Buffer) were compared under the same conditions, and the maximum fluorescence value (S/N) was selected, and NEB 2.1Buffer was selected for the buffers, see FIG. 11.
5. In the reaction system of Table 3, the fluorescence values of the reactions of the F-Q probes (1, 1.5, 2, 2.5. Mu.l) of different volumes were compared under the same conditions, and the maximum fluorescence value (S/N) was selected, and the F-Q probe was selected to have a volume of 2. Mu.l, see FIG. 12. The more F-Q probes, the more fluorescence, but the corresponding noise, the highest signal to noise ratio (S/N) is selected.
6. In the reaction system of Table 3, the fluorescence values of reactions with the addition of different volumes of template amounts (2, 4, 6, 8. Mu.l) were compared under the other conditions, the maximum fluorescence value (S/N) was selected, and the template amount was selected to be 8. Mu.l, see FIG. 13.
7. Optimization of F-B probes:
the molecular markers FAM and biotin are arranged at two ends of the probe, the two markers are equivalent to the antigen, and the "hook effect" is caused by the improper concentration of the antigen, so that the concentration of the probe needs to be optimized, and probes with different volumes are diluted by the test strip 2 diluent. F-B probe concentrations were diluted to different concentration gradients (10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.625. Mu.M, 0.1. Mu.M), and then the empty strip was placed in the diluted probe dilution and left for several seconds, the strip was observed, and the lowest probe concentration where T line disappeared was the optimal concentration. Referring to FIG. 14 (concentration of F-B probe selected to be 2.5. Mu.M).
8. Cas12a test strip method reaction time optimization:
under the condition that other conditions of the reaction system are the same, incubating in a water bath at 37 ℃ for different times (10, 20, 30 and 40 min), after the reaction is finished, sucking 5 μl of the product into a test strip sample absorption pad, inserting a buffer solution, comparing the strip color of the T line, and setting the template as 2×10 4 The copies/. Mu.l ERA product is shown in FIG. 15 (Cas 12a strip reaction time 30 min).
9. ERA-Cas12a minimum limit of detection (LOD):
according to the optimized reaction conditions, a 10-fold dilution of the template (10 5 、10 4 、10 3 、10 2 Lowest limit of detection was performed by copies/. Mu.l, see FIG. 16 (option 10) 3 copies/μl)。
Example 5 method for detecting EB Virus nucleic acid for the purposes of non-disease diagnosis and treatment
The embodiment provides a method for detecting EB virus nucleic acid for non-disease diagnosis and treatment purposes, which comprises the following steps:
(1) MP1 target sequence and LMP2 target sequence were obtained as described in example 1;
(2) Using the primer group obtained by screening in the example 2, performing a reaction by using the ERA enzymatic recombination isothermal amplification conditions optimized in the example 3, and amplifying the EBV-LMP-2A target sequence obtained in the step (1) to obtain an ERA amplification product;
(3) CRISPR/Cas12a detection: taking the ERA amplification product in the step (2), and concentrating the ERA amplification product to a concentration of 10 5 The copies/. Mu.L was diluted down to 10 with a gradient of 10 5 、10 4 、10 3 、10 2 copies/. Mu.L, using ERA/CRISPR-Casl2a dipstick system (using optimized CRISPR/Cas12a detectionReaction conditions) were tested, with no enzyme water as a negative control, three replicates per sample, and the strip change was observed to determine the lower limit of detection (LOD) for the CRISPR/Cas12a-ERA system concentration, test flow is referenced in fig. 20. The minimum limit of detection (LOD) of ERA/CRISPR-Casl2a test strip system detection is 10 3 copies/μl。
Comparative example 1
In comparison with example 5, the detection of EB virus nucleic acid by the method does not involve the use of CRISPR/Cas12a, but only ERA detection alone (step (2) of reference example 5), and the lowest detection limit (10 4 cobies/ul) (clinical samples cannot be detected by ERA alone).
Test examples
The detection method of example 5 was used to detect clinical samples, and the specificity and sensitivity of ERA-CRISPR/Cas12a detection results were evaluated using clinical qPCR results as a standard.
1. And (3) specificity verification:
nucleic acid samples of the a-stream virus, the b-hepatitis virus and the EBV virus collected from clinic are detected respectively by using the optimized detection method (ERA/CRISPR-Cas 12a reaction system), and the strip change condition is observed to determine whether the ERA/CRISPR-Cas12a test strip system detects the EBV specifically or not, and refer to fig. 17-18.
To evaluate the clinical application of ERA/CRISPR-Cas12a fluorescence system, this study clinically collected more nucleic acid samples of epstein barr virus (14 cases, refer to fig. 19), alphavirus, hepatitis a virus and hepatitis b virus for detection.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present application.
Claims (10)
1. A crRNA for epstein barr virus nucleic acid detection, characterized in that the crRNA comprises at least one of LMP1-crRNA1, LMP1-crRNA2, LMP1-crRNA3, LMP2-crRNA1, LMP2-crRNA2 and LMP2-crRNA3, the nucleotide sequence of LMP1-crRNA1 is SEQ ID NO:4, a step of; the nucleotide sequence of the LMP1-crRNA2 is SEQ ID NO:5, a step of; the nucleotide sequence of the LMP1-crRNA3 is SEQ ID NO:6, preparing a base material; the nucleotide sequence of the LMP2-crRNA1 is SEQ ID NO:7, preparing a base material; the nucleotide sequence of the LMP2-crRNA2 is SEQ ID NO:8, 8; the nucleotide sequence of the LMP2-crRNA3 is SEQ ID NO:9.
2. the primer set for detecting crRNA of claim 1, wherein the primer set has a nucleotide sequence as set forth in SEQ ID NO:10 to 21.
3. The use of crRNA according to claim 1 for the preparation of epstein barr virus nucleic acid detection products.
4. The use of claim 3, wherein the test product comprises a test strip or a test kit.
5. An epstein barr virus nucleic acid detection product based on enzyme recombinant amplification in combination with CRISPR/Cas12a, characterized in that the detection product comprises the primer set of claim 2 and a CRISPR/Cas12a detection system;
the CRISPR/Cas12a detection system comprises a Cas12a protein, the crRNA of claim 1, and a ssDNA reporting system.
6. The epstein-barr virus nucleic acid detection product of claim 5, characterized in that said ssDNA reporter system comprises ssDNA probes with a concentration of 0.1 to 10 μm.
7. A method for detecting epstein barr virus nucleic acid for non-disease diagnosis and treatment purposes, comprising the steps of:
(1) Taking a sample to be detected, and extracting an EB virus genome;
(2) ERA enzymatic recombination isothermal amplification: amplifying the EB virus genome obtained in step (1) with the primer set according to claim 2;
(3) CRISPR/Cas12a detection: taking the amplified product in step (2), adding ssDNA probe, cas12a protein and crRNA according to claim 1, performing CRISPR/Cas12a detection, and reading the detection signal.
8. The method according to claim 7, wherein in the step (2), the EB virus genome is amplified with a gradient of 5min in the range of 10min to 20min, and the concentration of the EB virus genome is 10 6 copies/μL。
9. The method according to claim 7, wherein in the step (3), the concentration of the amplified product is diluted to 10 2 ～10 5 copies/μL。
10. The use of the epstein barr virus nucleic acid detection product according to claim 5 or 6 for detecting epstein barr virus nucleic acid.
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