CN118547093B - Primer group and kit for detecting betelnut yellow phytoplasma based on CRISPR/Cas12a method and use method of primer group and kit - Google Patents
Primer group and kit for detecting betelnut yellow phytoplasma based on CRISPR/Cas12a method and use method of primer group and kit Download PDFInfo
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Abstract
The invention discloses a primer group and a kit for detecting phytoplasma areca catechu yellow based on a CRISPR/Cas12a method and a use method thereof, wherein the primer comprises an upstream primer sequence shown as SEQ ID NO.1 and a downstream primer sequence shown as SEQ ID NO. 2. The kit comprises a primer for amplifying the DNA to be detected, a nucleic acid amplification reagent, crRNA, ssDNA fluorescent reporter probes and Cas12a enzyme. The detection method of the kit comprises the following steps: s1, extracting DNA of a sample to be detected; s2, amplifying the DNA obtained in the step S1; s3, mixing the crRNA, the ssDNA fluorescent reporter probe and the Cas12a enzyme with the nucleic acid amplification product obtained in the step S2 for reaction, and detecting the reaction product; s4, determining a detection result through fluorescence detection. The primer set designed by the invention has strong specificity, high detection sensitivity which is 100 times that of common PCR amplification, the detection limit can reach 2.29 multiplied by 10 2 copies/mu L, the response speed is high, the result judgment is simple, and the operation is simple and convenient.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a betel nut yellow phytoplasma visualization detection primer group based on a CRISPR/Cas12a system, a kit and a use method thereof.
Background
Betel (Areca catechu l.) is a typical tropical cash and ornamental crop belonging to the family palmaceae (ARECACEAE) and genus betel (Areca) as perennial evergreen arbor.
Arecae semen yellow disease (yellow LEAF DISEASE of ARECA PALM, YLD) is an infectious disease that is lethal to Arecae semen. The disease was first found in Muvattupuzha region of india in 1914. Found in Tunchang county of Hainan province in 1981. To date, the etiolation of betel nuts spread to almost all betel nut planting areas in india and hainan provinces, which currently pose a threat to hainan betel nut production, landscaping, ecological environment and travel.
Betel nut yellowing disease caused by phytoplasma (Phytoplasma) is mainly transmitted by leafhoppers and plant hoppers. The phytoplasma is proved to be the etiology of the betel nut yellowing disease by the methods of electron microscope observation, dinar staining, semen cuscutae transmission, medium insect sugarcane spot sleeve wax cicada (Proutista moesta) transmission, serology, nest PCR detection and the like. The plant pathogen has low content and uneven distribution in the betel nut plant, and the plant pathogen content changes with seasons, so that accurate identification of betel nut yellowing plant pathogen becomes a difficulty in practical application.
The current detection methods for the betel nut yellow phytoplasma include nested PCR (nested-PCR) technology, real-time fluorescent quantitative PCR (Real-time PCR) technology, loop-mediated isothermal amplification (loop mediated isothermal amplification, LAMP) and microdroplet digital PCR technology, for example, the following published patent documents:
Chinese patent CN116356052A discloses a nest type primer group, a kit and a detection method for specifically detecting the phytoplasma areca, and provides a novel nest type molecular detection primer pair for detecting the phytoplasma areca and a nest type molecular detection method for detecting the areca yellowing disease.
Chinese patent CN116676401a discloses a primer probe combination, kit and method for detecting betel nut yellow phytoplasma based on real-time fluorescence quantitative PCR technique. The invention provides a Probe AM-Probe and a specific primer AM-f/AM-r based on the gene sequence design of the areca yellow phytoplasma 16SrDNA, and an established kit and a detection method.
Chinese patent CN111850151A discloses a micro-drop digital PCR detection kit, a method and application of the micro-drop digital PCR detection kit for the areca-nut yellow phytoplasma, the invention designs a specific primer Atf/Atr and a probe AtProbe based on a phytoplasma tuf gene sequence, establishes a ddPCR detection technology for the areca-nut yellow phytoplasma, and adopts the established ddPCR technology to detect and analyze samples of the areca-nut yellow diseases and different parts of diseased tissues in different regions of Hainan province of China.
In the practical detection by applying the detection method provided by the prior art, the specificity, sensitivity, detection efficiency or convenience of the prior detection technology are not high enough. For example, improved nested PCR methods remain less sensitive and are time-consuming and laborious; the Real-time PCR and ddPCR methods require expensive equipment. Therefore, the existing molecular detection technology can not meet the requirements of accurately and rapidly identifying and detecting the areca catechu yellow phytoplasma in practical application, and the RPA-CRISPR/Cas12a technology provides a new thought for solving the problem.
Disclosure of Invention
The invention aims to solve the problems that the existing phytoplasma detection technology depends on large instruments and equipment, has low detection accuracy and sensitivity and the like, and provides a primer group for detecting the phytoplasma areca catechu yellowing, a detection kit and a detection using method thereof, which are used for rapidly and accurately detecting the phytoplasma areca catechu yellowing.
The invention firstly provides a primer group for detecting the betel nut yellow phytoplasma, wherein the primer comprises an upstream primer sequence shown as SEQ ID NO.1 and a downstream primer sequence shown as SEQ ID NO.2.
Secondly, the invention also provides a kit for detecting the betel nut yellow phytoplasma, which comprises a primer, a nucleic acid amplification reagent, crRNA, a ssDNA fluorescent reporter probe and Cas12a enzyme; the primer comprises an upstream primer sequence shown as SEQ ID NO. 1 and a downstream primer sequence shown as SEQ ID NO. 2.
In some embodiments of the invention, the crRNA is the sequence shown as SEQ ID NO. 3.
In some embodiments of the invention, the nucleic acid amplification reagent is an RPA or RAA amplification reagent.
In the present invention, both the recombinase polymerase amplification technique (recombinase polymerase amplification, RPA) and the recombinase-mediated amplification technique (recombinase-aid amplification, RAA) are common isothermal amplification techniques, in which a nucleic acid is rapidly amplified at a constant temperature of 39℃using a recombinase, a single-stranded binding protein and a DNA polymerase during the amplification process. The difference is that the sources of the recombinases are different, the recombinases of the RPA system are derived from T4 phage, and the recombinases of the RAA system are derived from bacteria or fungi. In some embodiments of the invention, nucleic acid amplification reagents for commercial RPA or RAA systems are commercially available for reaction.
In some embodiments of the invention, the nucleotide sequence of the ssDNA fluorescent reporter probe is: 6-FAM-TTATT-BHQ1.
In some preferred embodiments of the present invention, the ssDNA fluorescent reporter probe is linked at each end to a fluorophore selected from at least one of 6-FAM, TAMRA, TET, HEX, cy, cy5, and ROX.
In some more preferred embodiments of the present invention, the ssDNA fluorescent reporter probe has a nucleotide sequence of 6-FAM-TTATT-BHQ1.
The invention also provides a use method for detecting the betel nut yellow phytoplasma by using the kit, which comprises the following steps:
S1, extracting DNA of a sample to be detected;
S2, performing a nucleic acid amplification reaction on the DNA obtained in the step S1 by using the primer and the nucleic acid amplification reagent to obtain a nucleic acid amplification product;
S3, mixing the crRNA, the ssDNA fluorescent reporter probe and the Cas12a enzyme with the nucleic acid amplification product obtained in the step S2, and performing CRISPR/Cas12a reaction on the nucleic acid amplification product;
s4, determining a detection result through fluorescence detection.
In some embodiments of the invention, the nucleic acid amplification reaction in step S2 is a recombinase-mediated isothermal nucleic acid amplification reaction, including an RPA amplification reaction or a RAA amplification reaction. In some embodiments of the invention, the optimal temperature for the CRISPR/Cas12a reaction in step S3 is 39 ℃, and the time for the CRISPR/Cas12a reaction is 15-30 min.
In the invention, CRISPR/Cas12a is used as an emerging biotechnology and can be used for rapid isothermal detection of pathogenic bacteria. Casl2a can specifically recognize and cleave ssDNA rich in T nucleotide PAM sequence under the guidance of crRNA, and cleave target strand at specific site to achieve detection purpose. Coupling the RPA amplification technology with Casl a, performing target DNA amplification by using the RPA/RAA amplification technology, adding a CRISPR system, and activating trans-cleavage activity after the Cas12a-crRNA complex binds to the target DNA, and cleaving a labeled fluorescent group in the system to generate a fluorescent signal. The invention establishes a more accurate and rapid detection method aiming at the Hainan betel nut yellow phytoplasma by combining CRISPR/Cas12a and RPA/RAA. The specificity, sensitivity and repeatability analysis show that the betel nut yellow phytoplasma detection method established by the invention has high accuracy and sensitivity, can finish result detection in a short time, and is suitable for real-time on-site detection.
The invention has the beneficial effects that:
1) The specificity is strong: the designed primer has obvious specificity, and can accurately distinguish the target strain from other strains.
2) The sensitivity is high: the sensitivity of RPA (RAA) -CRISPR/Cas12a detection is 100 times that of common PCR amplification, and the detection limit can reach 2.29 multiplied by 10 2 copies/. Mu.L.
3) The response speed is high: the RPA/RAA technology can complete high-efficiency gene amplification within 30 min.
4) The result judgment is simple: the reaction result can be observed by naked eyes under a blue light lamp, and the color is colorless and negative, and the green is positive.
5) The operation is simple and convenient: only simple constant temperature instrument equipment is needed.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 shows the result of specific detection of RPA primer in example 3 of the present invention, in which M is DL2000 and templates in lanes 1-7 are ddH 2 O, burkholderia andropogonis, peanut witches' -broom, respectively
(16SrIIA_KC593280.1)、Lethal Yellowing Disease of Coconut (16SrIV-A_KU925788.1)、JWB-J549、PaWB-FJFZ、DMP-17-Plasmid.
FIG. 2 shows the sensitivity test results of the RPA amplification method of example 4 of the present invention, in which M is DL2000, lane 1 is a negative control, and the concentrations of plasmids in lanes 2-14 are 2.29X10 -2 copies/. Mu.L, 2.29X10 -1 copies/. Mu.L, 2.29X10 0 copies/. Mu.L, 2.29X 10 1 copies/. Mu.L, 2.29X 10 2 copies/. Mu.L, 2.29X 10 3 copies/. Mu.L, 2.29X 10 4 copies/. Mu.L, 2.29X 10 5 copies/. Mu.L, 2.29X 10 6 copies/. Mu.L, 2.29X 10 7 copies/. Mu.L, 2.29X 10 8 copies/. Mu.L, 2.29X 10 9 copies/. Mu.L, and 2.29X 10 10 copies/. Mu.L, respectively.
FIG. 3 shows the result of detection of cleavage activity of crRNA binding to Cas12a in example 5 of the present invention, wherein M is DL2000 and lanes 1-4 are, respectively, only DNA templates are added; b. only adding DNA template and crRNA; c. only DNA templates and Cas12a were added; d. complete DNA template, crRNA, and Cas12a were added.
FIG. 4 shows the results of a specific assay of CRISPR/Cas12a method according to example 7 of the present invention, wherein test tube 1 is the bacterial leaf spot of Areca catechu, test tube 2 is the synthetic gene plasmid of Phytoplasma grifolii rp, test tube 3 is the synthetic gene plasmid of Leucocalyxa Cocois lethal xanthophyll rp, test tube 4 is the DNA sample of Zaoma, test tube 5 is the DNA sample of Paulownia, test tube 6 is the DNA sample of Areca catechu xanthophyll, and test tube 6 is the self-constructed gene plasmid of Leucocalyxa catechu xanthophyll rp.
FIG. 5 shows the results of CRISPR/Cas12a sensitivity assay in example 8 of the present invention, wherein tubes 1-11 correspond to the betel nut yellow phytoplasma positive plasmids with concentrations of 2.29×10 -2 copies/. Mu.L, 2.29×10 -1 copies/. Mu.L, 2.29×10 0 copies/. Mu.L, 2.29×10 1 copies/. Mu.L, 2.29×10 2 copies/. Mu.L, 2.29×10 3 copies/. Mu.L, 2.29×10 4 copies/. Mu.L, 2.29×10 5 copies/. Mu.L, 2.29×10 6 copies/. Mu.L, 2.29×10 7 copies/. Mu.L, 2.29×10 8 copies/. Mu.L, NC blank.
FIG. 6 shows the results of the practical application of the test method in example 9 of the present invention, wherein test tubes 1-12 correspond to DMP-17, S97, C018Y-2, C021-1, C026-1, C046-1, B027-2, B029-2, B030-1, B042-2, and mountain ephedra leaf-2, respectively, and NC test tubes are blank controls.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 RPA (RAA) primer design and Synthesis of crRNA and ssDNA
The rpgene of the betel nut yellow phytoplasma (uploaded GenBank, serial number OR 286210) is amplified by a nested PCR method and compared with the rpgene sequences of different groups and subgroups of phytoplasmas, and RPA (RAA) amplification primers and corresponding crRNA (the sequences are shown as SEQ ID NO. 1-3) aiming at the phytoplasmas are designed. The sequences are shown in Table 1.
TABLE 1 primers and sequences for amplification and detection of Phytoplasma areca yellow
Example 2 RPA amplification
1) Extraction of target DNA: the DNA extraction of diseased betel nut leaves was performed using a plant genome DNA extraction kit from Tiangen company.
2) The general primer of rp gene is used to amplify betel leaf genome DNA, and the amplified fragment is recovered by gel and then connected with T carrier.
3) The primers in example 1 were used for RPA amplification using plasmid DNA of the Phytoplasma areca and genomic DNA of betel leaves as templates, and a blank control was set (TwistAmp Basic TwistDX kit).
The RPA amplification system is as follows: to the RPA reaction tube containing the lyophilized enzyme powder, PRIMER FREE Rehydration buffer 29.5.5. Mu.L of each of the 10. Mu.M concentration of the upstream and downstream primers was added 2.4. Mu.L, 1. Mu.L of the template, and finally 2.5. Mu.L of the magnesium acetate solution was added, and ddH 2 O12.2. Mu.L was added, whereby the total reaction system was 50. Mu.L.
Reaction conditions of RPA: the RPA reaction system is fully and evenly mixed, and amplified for 30min under the condition of 39 ℃. The RPA amplification was observed by electrophoresis on an agarose gel having a concentration of 1.5%.
Example 3 specificity detection of RPA primers
The sample information used in this example is shown in table 2.
TABLE 2 samples for RPA primer specific detection
The specific detection of RPA primers was performed using the primers of example 1, with the following specific procedures:
1) RPA amplification: RPA amplification was performed using two sets of primers designed in example 1, with the templates being the samples listed in table 2. Each of the lyophilized powders was added with 2.4. Mu.L (10. Mu.M) of the downstream primer, PRIMER FREE Rehydration buffer 29.5.5. Mu.L of the template, 1. Mu.L of the magnesium acetate, 2.5. Mu.L of ddH 2 O, and 50. Mu.L of the reaction system was supplemented. The reaction conditions were 39℃30 min.
2) Purification of amplification products: the amplified product was added to chloroform isoamyl alcohol (1:1), shaken and centrifuged at 12000 rpm min.
3) The aqueous phase component in step 2) was aspirated and the amplification results were analyzed by 1.5% agarose gel electrophoresis, as shown in FIG. 1, in which M is DL2000 and the templates of lanes 1-7 are ddH2O、Burkholderia andropogonis、Peanut witches'-broom (16SrIIA_KC593280.1)、Lethal Yellowing Disease of Coconut (16SrIV-A_KU925788.1)、JWB-J549、PaWB-FJFZ、DMP-17-Plasmid., respectively, showing that: and (3) performing RPA amplification detection by adopting primers YLD-F33-2 and YLD-R33, wherein amplified products are obtained from betel nut yellowing and paulownia bush samples, and other samples have no amplified products. The paulownia bush sample is a 16SrI-D subgroup phytoplasma, and belongs to the 16SrI group phytoplasma together with the used betel nut yellowing sample, so that the primer combination can detect the phytoplasma from betel nuts, and can distinguish the 16SrI group from other phytoplasma, but cannot distinguish betel nut yellowing phytoplasma from paulownia bush which is the 16SrI group.
Example 4 sensitivity detection of RPA amplification method
The primer in example 1 was used for sensitivity detection of the RPA amplification method, and the specific procedure was as follows:
1) Preparation of templates with different concentration gradients: for the gene positive plasmid of the phytoplasma areca yellow constructed in the laboratory, the plasmid concentration was detected by Nanodrop 2000, and the plasmids were diluted to 2.29×10 10 copies/. Mu.L, 2.29×10 9 copies/. Mu.L, 2.29×10 8 copies/. Mu.L, 2.29×10 7 copies/. Mu.L, 2.29×10 6 copies/. Mu.L, 2.29×10 5 copies/. Mu.L, 2.29×10 4 copies/. Mu.L, 2.29×10 3 copies/. Mu.L, 2.29×10 2 copies/. Mu.L, 2.29×10 1 copies/. Mu.L, 2.29×10 0 copies/. Mu.L, 2.29×10 -1 copies/. Mu.L and 2.29×10 -2 copies/. Mu.L, respectively.
2) RPA amplification: RPA amplification was performed using the two primers designed in example 1, and the plasmids diluted at the above-described different concentrations as templates. Each of the lyophilized powders was added with 2.4. Mu.L (10. Mu.M) of the downstream primer, PRIMER FREE Rehydration buffer 29.5.5. Mu.L of the template, 1. Mu.L of the magnesium acetate, 2.5. Mu.L of ddH 2 O, and 50. Mu.L of the reaction system was supplemented. The reaction conditions were 39℃for 30min.
3) Purification of amplification products: the amplified product was added to chloroform isoamyl alcohol (1:1) and shaken 12000 rpm and centrifuged for 5min.
4) The aqueous phase component in step 3) was aspirated and analyzed for amplification results using 1.5% agarose gel electrophoresis. As a result, FIG. 2 shows that M is DL2000, lane 1 is a negative control, and the plasmid concentrations in lanes 2-14 are 2.29×10 -2 copies/. Mu.L, 2.29×10 -1 copies/. Mu.L, 2.29×10 0 copies/. Mu.L, 2.29×10 1 copies/. Mu.L, 2.29×10 2 copies/. Mu.L, 2.29×10 3 copies/. Mu.L, 2.29×10 4 copies/. Mu.L, 2.29×10 5 copies/. Mu.L, 2.29×10 6 copies/. Mu.L, 2.29×10 7 copies/. Mu.L, 2.29×10 8 copies/. Mu.L, 2.29×10 9 copies/. Mu.L, and 2.29×10 10 copies/. Mu.L, respectively. 2-14 are YLD-F33-2 and YLD-R33 amplification products. The results in the figure show that: the lowest copy number of the positive plasmid was detected to be 2.29×10 2 copies/. Mu.L by agarose gel electrophoresis after RPA amplification with the primer pairs YLD-F33-2 and YLD-R33.
The results show that the primer pair YLD-F33-2 and YLD-R33 has good sensitivity and can be used for detecting the betel nut yellow phytoplasma.
Example 5 crRNA cleavage Activity assay
Using the crrnas in table 1, the cleavage activity of Cas12a protein was detected by the following procedure:
1) The general primer of rp gene is used to amplify betel nut leaf genome DNA, and then the amplified fragment is cut and recovered. Four groups of experiments are carried out by taking the template as the template: a. adding only a DNA template; b. only adding DNA template and crRNA; c. only DNA templates and Cas12a were added; d. complete DNA template, crRNA, and Cas12a were added. The system details are shown in Table 3.
TABLE 3 cleavage Activity detection System
2) The prepared system was subjected to instantaneous centrifugation in a centrifuge, and then reacted in a constant temperature incubator at 37℃for 1 h. After removal, 1. Mu.L of Protein K was added to each tube, and the mixture was left at room temperature for 2 min hours; electrophoresis was then performed using a 1% agarose gel.
As shown in fig. 3, only crRNA1 and Cas12a have cleavage activity after binding, M in the figure is DL2000, lanes 1-4 are systems to which only DNA template and crRNA are added, systems to which only DNA template and Cas12a are added, and systems to which only whole DNA template, crRNA and Cas12a are added, respectively. Only the system added with the DNA template, the Cas12a enzyme and the crRNA1 simultaneously has three bands through electrophoresis detection, and the sizes of the three bands are consistent with the expected band sizes, so that the Cas12a protein is cut into rp gene fragments only after the crRNA1 is combined.
Example 6 RPA establishment of CRISPR/Cas12a method
The RPA product of the betel nut yellow phytoplasma in example 2 above was used as a DNA template for CRISPR/Cas 12 a. To the PCR tube, 9.9. Mu.L of DEPC water, 2. Mu.L of 10 XBuffer, 2. Mu.L of crRNA, 2. Mu.L of the obtained RPA product of example 2, 1.6. Mu.L of ssDNA fluorescence reporter probe at a concentration of 10. Mu.M, 0.5. Mu. L RNase Inhibitor and 2. Mu.L of Cas12a enzyme were sequentially added, the total reaction system was 20. Mu.L, and the initial concentrations of the systems are shown in Table 4. The ssDNA fluorescent reporter probe nucleotide sequences used in this example were: 6-FAM-TTATT-BHQ1, wherein 6-FAM and BHQ1 are fluorophores. In other embodiments, the fluorophore may also be selected from TET, HEX, cy, cy5, and ROX. The prepared system is immediately placed in a constant temperature incubator at 37 ℃ for 30 minutes, and the reaction result is observed under a blue light.
TABLE 4 CRISPR/Cas12 reaction System
Example 7 RPA-specificity analysis of CRISPR/Cas12a method
The samples listed in table 2 were subjected to RPA amplification as in example 2, with the product as CRISPR/Cas12a reaction template. CRISPR/Cas12a cleavage reactions were performed following the procedure of example 6.
CRISPR/Cas12a cleavage system and conditions: 2. Mu.L of 10 XCas 12 buffer, 2. Mu.L of crRNA (1. Mu.M), 1.6. Mu.L of ssDNA fluorescent reporter probe (10. Mu.M), 2. Mu.L of Cas12a enzyme (1. Mu.M), 2. Mu.L of target DNA of different samples, 0.5. Mu. L RNase Inhibitor DEPC water were added to a reaction total system of 20. Mu.L, and after incubation at 37℃for 30 minutes in a 37℃incubator, the reaction results were observed under a blue light.
As shown in FIG. 4, the reaction tubes of the No. 5, 6 and 7 samples, i.e. the paulownia bush disease sample, the betel nut yellow phytoplasma sample and the betel nut yellow phytoplasma plasmid, are green, and the detection results of other common samples are as same as the negative control, and do not show fluorescence, and are negative. The result shows that the RPA-CRISPR/Cas12a method established by the invention has good specificity.
Example 8 RPA sensitivity test of CRISPR/Cas12a method
The sensitivity test is carried out on the established RPA-CRISPR/Cas12a method of the betel nut yellow phytoplasma:
after gradient dilution of the plasmid with the gene rp of the phytoplasma areca yellow, the RPA amplification product is used as a CRISPR/Cas12a reaction template, 2 mu L of 10 XCas 12 buffer,2 mu L of crRNA (1 mu M), 1.6 mu L of ssDNA fluorescent reporter probe (10 mu M), 2 mu LCas a enzyme (1 mu M) and 2 mu L of plasmid DNA with different concentrations of the gene rp of the phytoplasma areca yellow are added, DEPC water is added into 0.5 mu L RNase Inhibitor to the reaction total system of 20 mu L, and after incubation is carried out for 30 minutes at 37 ℃ in a 37 ℃ constant temperature incubator, the reaction result is observed under a blue light.
The experimental result is shown in figure 5, and the detection limit of the RPA-CRISPR/Cas12a method can reach 2.29 multiplied by 10 2 copies/. Mu.L, which proves that the method has higher sensitivity.
Example 9 practical application of the detection method
The established RPA-CRISPR/Cas12a method of the betel nut yellow phytoplasma is practically applied:
The samples used are shown in table 5,
TABLE 5 sample collection sites, numbers and groups to which they belong
The samples listed in table 5 were subjected to RPA amplification as in example 2, with the product as CRISPR/Cas12a reaction template. The CRISPR/Cas12a reaction was performed following the procedure of example 6.
CRISPR/Cas12a reaction system and conditions: 2. Mu.L of 10 XCas 12 buffer, 2. Mu.L of crRNA (1. Mu.M), 1.6. Mu.L of ssDNA fluorescent reporter probe (10. Mu.M), 2. Mu.L of Cas12a enzyme (1. Mu.M), 2. Mu.L of target DNA of different samples, 0.5. Mu. L RNase Inhibitor DEPC water were added to a reaction total system of 20. Mu.L, and after incubation at 37℃for 30 minutes in a 37℃incubator, the reaction results were observed under a blue light.
As shown in FIG. 6, the green fluorescence was detected in the samples of the diseased betel palm leaves containing 16SrI group phytoplasma, but not in the samples of the mountain ephedra leaves containing 16SrXXXII phytoplasma.
The embodiment shows that the detection method has the advantages of rapidness, simplicity in operation, strong specificity, high sensitivity and the like, and is suitable for real-time on-site detection.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
The crRNA and sequence used in the invention are shown in Table 6:
table 6 crRNA and sequence
It should be understood that the above description is not intended to limit the invention to the particular embodiments disclosed, but to limit the invention to the particular embodiments disclosed, and that various changes, modifications, additions and substitutions can be made by those skilled in the art without departing from the spirit and scope of the invention.
Claims (3)
1. A kit for detecting betelnut yellow phytoplasma based on a CRISPR/Cas12a method, which is characterized by comprising a primer for amplifying DNA to be detected, a nucleic acid amplification reagent, crRNA, ssDNA fluorescent reporter probes and Cas12a enzyme; the primer comprises an upstream primer of a sequence shown as SEQ ID NO. 1 and a downstream primer of a sequence shown as SEQ ID NO. 2;
The nucleic acid amplification reagent is RPA or RAA amplification reagent;
The crRNA sequence is a sequence shown as SEQ ID NO. 3; the nucleotide sequence of the ssDNA fluorescent reporter probe is as follows: 6-FAM-TTATT-BHQ1.
2. The method for detecting the phytoplasma areca yellow by using the kit as claimed in claim 1, which is characterized by comprising the following steps:
S1, extracting DNA of a sample to be detected;
S2, performing a nucleic acid amplification reaction on the DNA obtained in the step S1 by using the primer and the nucleic acid amplification reagent to obtain a nucleic acid amplification product;
S3, mixing the crRNA, the ssDNA fluorescent reporter probe and the Cas12a enzyme with the nucleic acid amplification product obtained in the step S2 for reaction, and detecting the reaction product;
s4, determining a detection result through fluorescence detection.
3. The method of claim 2, wherein the nucleic acid amplification reaction in step S2 is a recombinase-mediated isothermal nucleic acid amplification reaction, including an RPA amplification reaction or a RAA amplification reaction.
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