CN117143871A - Rapid detection method for rice fusarium oxysporum CRISPR-Cas12a, combined product and kit thereof - Google Patents
Rapid detection method for rice fusarium oxysporum CRISPR-Cas12a, combined product and kit thereof Download PDFInfo
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Abstract
The application relates to a rapid detection method of rice bacterial rhizoctonia cerealis CRISPR-Cas12a, a combined product and a kit thereof. The CRISPR-Cas system comprising Cas12a and crRNA can be used with the system. The combined product and the kit provided by the application are used for detecting the ITS gene of the rice fusarium wilt, and the provided method has the advantages of higher detection sensitivity and specificity, convenience in detection, short time consumption and low cost.
Description
Technical Field
The application relates to the technical field of molecular biological detection, in particular to a rapid detection method of rice bacterial Rhizoctonia cerealis CRISPR-Cas12a, a combination product and a kit thereof.
Background
The glume burkholderia (Burkholderia glumae) is also called rice bacterial rhizoctonia cerealis, is an important pathogenic bacterium capable of causing rice bacterial diseases, and is listed in the entry plant quarantine pest directory of China. Glamoae can harm a variety of grasses, not only do it attack the grain, but it also causes the rice seedlings to decay, resulting in yield loss. In addition, the pathogen is also a conditional pathogen for chronic granulomatous diseases of human and livestock. Studies have shown that with global warming, the area of gummy up will gradually increase, with more severe occurrences, resulting in increased yield loss. And B, the glumae can overwintere in weeds and soil in diseased fields, can also be spread along with the rice seeds with bacteria for a long distance, and has high quarantine risk. The rice seed with bacteria is a main way of long-distance transmission of diseases and is also an important primary infection source, so that the rice seed health detection is developed aiming at a rapid and accurate quarantine detection technology for pathogen development, and the method is one of important ways of preventing disease transmission and diffusion, improving the control level of rice diseases and ensuring the sustainable and healthy development of the rice industry in China. The detection method of rice bacterial rhizoctonia cerealis which is commonly used at present mainly comprises the traditional separation culture method, the serological method and the PCR-based molecular biological detection method, including RPA, fluorescent PCR and the like. However, the conventional detection method is generally complex in steps and long in time consumption; the whole operation process of the conventional molecular biology detection method depends on laboratory instruments and equipment and professional technicians, so that the requirement of on-site rapid detection cannot be met in most cases.
As an emerging genome editing and regulating tool, the advent and development of CRISPR technology has greatly changed the direction of advance in the biological arts. In recent years, the working principle of the CRISPR system and related proteins thereof is gradually revealed, and the technology plays a vital role in various fields due to the excellent sensitivity and specificity. With its novel nuclease activity, the door to develop new methods for nucleic acid detection has been opened. CRISPR/Cas systems have also been developed as a rapid, portable, low cost, high sensitivity molecular detection tool.
Cas12 is a class ii v CRISPR effector protein that binds to target double stranded DNA and cleaves genomic DNA under the guidance of crRNA. After Cas12 specifically binds and cleaves target dsDNA, it has the activity of nonspecifically cleaving single-stranded DNA (ssDNA), and a nucleic acid detection system named DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) has been developed using this activity of Cas 12. The detection combines the RPA isothermal amplification technique with Cas12, the amplification product activates the accessory cleavage activity of Cas12, the cleavage substrate generates a fluorescent signal, and single molecule level sensitivity is achieved. The system has wide application potential in the aspects of single nucleotide polymorphism analysis, cancer screening, bacterial and virus infection detection, drug resistance screening and the like. Because the target and the substrate of the Cas12 are DNA, the stability is strong, so the system has low requirements on experimental operation environment and can be applied to on-site rapid detection. Meanwhile, after the Cas12 is specifically activated by the target, the single-chain reporter molecule can be cut in a non-specific way, and the step has a signal amplification effect, so that the technology has higher sensitivity compared with the traditional nucleic acid detection technology such as a probe method.
However, since Cas12 cleaves the target DNA molecule of RPA amplification and activated Cas12 cleaves single stranded DNA (ssDNA) molecules including primers, thereby impeding the progress of the amplification reaction, RPA nucleic acid amplification and CRISPR/Cas12 signal detection must be performed separately in this system. The step-by-step operation increases the complexity of operation and the risk of nucleic acid pollution, and the whole detection time is longer, so that the rapid detection application on the basic level site is not facilitated.
Disclosure of Invention
In view of the defects of the prior art and the development of molecular biological detection technology, the application establishes a visual detection technology capable of accurately detecting rice bacterial rhizoctonia cerealis based on a CRISPR/Cas report system, and realizes the rapid field detection of rice bacterial rhizoctonia cerealis on rice seeds and plants.
To solve the technical problems described above, the present application aims to provide a combination product comprising a CRISPR-Cas system comprising Cas12a and crrnas, wherein the sequence of the crrnas is Bg-crRNA designed for ITS gene sequence of bordetella glumae.
In one embodiment of the application, there is provided a combination product comprising a CRISPR-Cas system, wherein the Bg-CrRNA has the sequence:
5’-UAAUUUCUACUAAGUGUAGAUGCCGUAUUCCAAUUGA GCC-3’。
preferably, the combination product comprising a CRISPR-Cas system according to the present application, wherein the Cas12a is selected from one of FnCas12a, asCas12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a or Lb4Cas12 a.
In another embodiment, the combination product comprising a CRISPR-Cas system of the present application further comprises a buffer system and a single stranded DNA reporter.
Preferably, the Buffer system is a 3.1NE Buffer, wherein DEPC-H is used 2 O。
Preferably, the combination product comprising the CRISPR-Cas system, wherein the single-stranded DNA reporter is the test strip reporter FB2-DNA, preferably FAM-TTTTTTTATTTTTTT-Biotin.
In another aspect, the application provides a kit comprising a combination product comprising a CRISPR-Cas system as described above, and instructions for use.
In one embodiment, the kit of the application further comprises one or more of a nucleic acid extraction reagent, a positive control, and a negative control.
In another aspect, the application provides a method for rapidly detecting rice bacterial Rhizoctonia cerealis, comprising the steps of:
i) Extracting genome DNA of a sample to be detected;
ii) the genomic DNA obtained in step i) is tested using a kit as described above according to the instructions for use.
In one embodiment, in the method for rapidly detecting the bacterial Rhizoctonia cerealis of rice, the content of the components of the kit in a detection system is as follows: cas12a 2pM, FB2-DNA200nM, xoo-CrRNA 4nM.
In some embodiments, the kit further comprises an RNAse inhibitor and DTT in the method of the application for rapid detection of rice bacterial Rhizoctonia cerealis.
In one embodiment, in the method for rapidly detecting rice fusarium oxysporum according to the present application, it is preferable that the metal bath is heated at 37 ℃ in step ii) to react for 30min.
In addition, the application also provides a combined product containing the CRISPR-Cas system or application of the kit in detecting rice bacterial rhizoctonia cerealis.
Compared with the existing PCR nucleic acid detection technology, the application has the advantages that:
1. and (3) quick: the whole detection process from the sample to the detection result only needs 15-40min according to the template to be detected, the amplification efficiency and the crRNA cutting efficiency, and the visual result interpretation of the nucleic acid detection test paper strip is provided.
2. The accuracy is that: the application has extremely high sensitivity, can detect single nucleic acid molecules, and the detection limit of the application can reach 5.2 multiplied by 10 -5 The detection limit of ng/mu L is improved by one order of magnitude compared with that of a fluorescence PCR and fluorescence RPA detection method; meanwhile, since crrnas are sensitive to single base mismatches, the specificity is excellent.
3. Convenient: the detection rate of the application is higher than that of the traditional separation culture method, and because the application is carried out at the constant temperature of 37 ℃, the requirement on equipment is extremely low, and the reaction can be carried out by simple constant temperature equipment even by using the body temperature, thereby being beneficial to popularization in basic layer inspection mechanisms, being successfully applied to actual seed sample detection, having shorter detection time and simpler and more convenient operation and being suitable for field detection.
4. Low cost: the materials and enzymes used in the application are common, the dosage is small, and the micro-quantitative test analysis can be carried out, so the cost is relatively lower.
Drawings
FIG. 1 is a CRISPR-Cas12a strip specific test.
Fig. 2 is a CRISPR-Cas12a test strip sensitivity test, wherein 1:5.2 ng/. Mu.L; 2: 5.2X10 -1 ng/μL；3：5.2×10 -2 ng/μL；4：5.2×10 -3 ng/μL；5：5.2×10 -4 ng/μL；6：5.2×10 -5 ng/μL；7：5.2×10 -6 ng/μL；8：5.2×10 -7 ng/μL。
Fig. 3 is a CRISPR-Cas12a test strip natural seed sample test.
Detailed Description
The present application now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the application are shown. Indeed, the application may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The following definitions and methods will better define the present application and guide those of ordinary skill in the art in practicing the present application. Many modifications and other embodiments of the application will come to mind to one skilled in the art to which this application pertains having the benefit of the teachings presented in the descriptions and the associated drawings. Therefore, it is to be understood that the applications are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
Unless otherwise defined, all terms (including technical and scientific terms) used to describe the application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present application. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
The bacterial Rhizoctonia cerealis of the rice is Burkholderia glumae (Burkholderia glumae), can cause bacterial diseases of the rice, and is also called as the Rhizoctonia cerealis and the bacterial Rhizoctonia cerealis in the context.
In one aspect, the ITS gene sequence of Burkholderia glumae is the ITS gene sequence CP035901 of B.glumae in GeneBank, and Bg-CrRNA designed for the gene sequence includes but is not limited to the specific sequences of the application:
5’-UAAUUUCUACUAAGUGUAGAUGCCGUAUUCCAAUUGA GCC-3’。
other suitable sequences include crRNA sequences that have greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the specific sequences provided above. Sequences that can be prepared or modified according to standard techniques are also included.
The term "% identity" in the context of two or more nucleotide sequences or amino acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example,% identity is the entire length of the coding region relative to the sequences to be compared.
For sequence comparison, typically one sequence is used as a reference sequence, and the test sequence is compared to that sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. The percent identity can be determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al, 1990, J Mol Biol 215:3,403-410;Altschul et al, 1997,Nucleic Acids Res25:17,3389-402).
The CRISPR-Cas system disclosed by the application refers to a system comprising CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), namely a clustered regularly interspaced short palindromic repeat sequence and Cas (CRISPR associate), namely CRISPR effector protein. The presently known CRISPR/Cas systems are divided into 2 broad classes of 6 types, each type being divided into multiple subtypes, each type comprising multiple Cas proteins.
The Cas12a refers to Cas12a protein, also called Cpf1, is class II V CRISPR effector protein in a CRISPR/Cas system, can be combined with target double-stranded DNA under the guidance of crRNA and cut genome DNA, is RNA-guided DNA endonuclease, and can specifically cut double-stranded DNA under the guidance of crRNA.
In some embodiments, the single-stranded DNA reporter of the present application refers to a fluorophore-labeled single-stranded DNA (ssDNA) molecule, and when the target sequence is cleaved and the fluorophore-labeled single-stranded DNA reporter is incidentally cleaved, the corresponding result can be obtained by a fluorescence detection strategy.
The "kit" as described herein refers to any article of manufacture (e.g., package or container) comprising at least one device, which may further comprise instructions, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.
In some embodiments, the kit further comprises one or more of a nucleic acid extraction reagent, a positive control, and a negative control.
In some embodiments, the kit further comprises one or more of a buffer system, a pH adjuster, dntps, BSA and/or PEG of various molecular weight distributions, DTT, and water.
In some embodiments, the detection is performed at a constant temperature of 37℃to 42℃and a reaction time of 20min or more. Preferably at 37℃for 30min.
The sample to be detected suitable for the present application may be various substances suspected of containing rice fusarium, for example: the water source, soil and the like near the rice planting environment naturally also comprise rice plants and corresponding tissues. The variety of the rice is not limited, and can be japonica rice, java rice, indica rice, glutinous rice, mountain rice, palea rice or various penetrating lines, etc. The rice tissue may be cuttings, roots, stems, cells, protoplasts, leaves, pollen, embryos, cotyledons, hypocotyls, anthers, flowers and seeds of rice.
In some embodiments, the use further comprises the step of isolating DNA (particularly genomic DNA) from the suspected content of rice fusarium solani. The separation of the DNA fragments from the treated material may include the use of a separation solvent such as methanol, ethanol, water, acetone or combinations thereof. In some embodiments, kits of DNA isolation kits may be used, including, for example, DNA isolation protocols using the Dneasy Mericon food kit (Qiagen, germanten, MD, USA) or cetyl trimethylammonium bromide (CTAB). Other separation techniques include lysis, heating, alcohol precipitation, salt precipitation, organic separation, solid phase separation, silica crude membrane separation, CSCL gradient purification, or any combination thereof.
Embodiments of the present application will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present application, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
Example 1
1. Material reagent
1.1 test strains and seed Material
The test strains included 10 bacterial Rhizoctonia cerealis, 13 Burkholderia strains and 11 other pathogenic bacteria on rice, and a total of 34 strains, as shown in Table 1 below. Rice seeds are routinely collected and stored in the laboratory of the present inventors.
TABLE 1 test strains
* ATCC american type strain retention center; a BCCM belgium typical strain preservation center; CAIQ chinese inspection and quarantine science institute; a DSM german microorganism strain preservation center; ICMP International plant microorganism repository; NCPPB British plant pathogen repository
1.2 major instrument reagents
Lateral flow test strip detection kit (Beijing Baozhen Yinghui biotechnology Co., ltd.), RNase inhibitor (Tiangen Biochemical technology (Beijing) Co., ltd.), cas12a (United states NEB Co.), bacterial genomic DNA extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd.), plant genomic DNA extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd.), constant temperature metal bath (COYOTE).
2. Test method
2.1DNA extraction
Taking 1.5mL of strain culture broth of the test strain, centrifuging at 12000rpm for 2min, precipitating, and extracting genome DNA according to the operation steps of the specification by using a bacterial genome DNA extraction kit.
50g of seed sample was taken, and 100mL of sterile water was added thereto and immersed overnight at 4 ℃. 10mL of seed treatment solution is taken and centrifuged at 12000rpm for 5min, and the total DNA of the seed sample is extracted by using a plant genome DNA extraction kit according to the operation steps of the specification.
2.2 design of crRNA of bacterial Rhizoctonia cerealis
Bg-CrRNA was designed based on the ITS gene sequence of B.glumae in GenBank (CP 035901):
5’-UAAUUUCUACUAAGUGUAGAUGCCGUAUUCCAAUUGA GCC-3’。
2.3CRISPR-Cas12a detection System
The CRISPR-Cas12a test strip detection system comprises DEPC-H 2 O12.2. Mu.L, 3.1NE Buffer 2. Mu.L, lbaCas12a (Cpfl) (5. Mu. Mol/L) 0.4. Mu.L, RNAse Inhibitor (40U/. Mu.L) 0.5. Mu.L, DTT (0.1 mmol/L) 0.5. Mu.L, test strip reporter FB2-DNA (FAM-TTTTTTTATTTTTTT-Biotin) 2. Mu.L (final concentration 200 nM), bg-CrRNA (0.01 mmol/L) 0.4. Mu.L, the above solution was prepared as a mixture of 18. Mu.L, 2. Mu.L template DNA was added, and the mixture was heated in a metal bath at 37℃for 30min. Make up D after the reaction is finishedEPC-H 2 O to 50. Mu.L, and test strip is inserted for testing.
2.4 specificity and sensitivity test of RPA/CRISPR-Cas12a detection method
And (3) taking DNA of 10 rice bacterial rhizoctonia cerealis, 13 burkholderia strains and 11 rice other pathogenic bacteria as templates, performing specificity test, and judging the specificity of the established rice bacterial rhizoctonia cerealis detection method according to experimental results.
And (3) performing sensitivity test by using a 10-time gradient dilution of DNA extracted from rice bacterial rhizoctonia cerealis ATCC33617 as a template, and judging the sensitivity of the detection method according to a test result. And simultaneously comparing with a fluorescence PCR and fluorescence RPA detection method.
2.5 actual sample detection
And (3) taking the genome DNA extracted from 17 rice seed samples as a template, and adopting a CRISPR-Cas12a test strip detection system to test, so as to verify the application effect of the genome DNA on actual samples. And simultaneously comparing with a fluorescence PCR and fluorescence RPA detection method.
2.6RPA detection System
The RPA reaction used the Anpu future DNA isothermal amplification kit, and the reaction tube was charged with 29.4. Mu. L A buffer, 2. Mu.L upstream primer, 2. Mu.L downstream primer, 0.6. Mu.L probe, 11.5. Mu.L dd H2O and 2. Mu.L DNA, and finally 2.5. Mu.L buffer. The fluorescence apparatus was set at a constant temperature of 39℃and FAM channel fluorescence values were collected every 30sec for a reaction time of 20min.
2.7 real-time fluorescence PCR detection system
Real-time fluorescent PCR reaction system: the total system was 20. Mu.L, in which 2X Premix 10. Mu.L, each of the primers (10. Mu. Mol/L), the probe (10. Mu. Mol/L) was 0.5. Mu.L, the DNA template was 2. Mu.L, and the ultrapure water was 5.5. Mu.L.
Real-time fluorescent PCR reaction procedure: the first cycle was 95℃for 5min; 40 subsequent cycles of 95℃for 10s and 56℃for 1min.
3. Results
3.1 specificity test
The CRISPR-Cas12a test strip is adopted to test 34 tested strains, and the result shows that only 10 rice bacterial Rhizoctonia cerealis have positive detection strips, and other similar species and other pathogenic bacteria on rice have no detection strips, as shown in figure 1, so that the method can specifically detect the rice bacterial Rhizoctonia cerealis.
3.2 sensitivity test
10-fold gradient dilution of DNA extracted from Rhizoctonia cerealis ATCC33617 (concentration of 5.2 ng/. Mu.L-5.2X10) -7 ng/. Mu.L) as a template, and the results are shown in FIG. 2 and Table 2 below, the detection limit of rice bacterial Rhizoctonia cerealis CRISPR-Cas12a is 5.2X10 -5 The detection limit of ng/mu L is improved by one order of magnitude compared with that of a fluorescence PCR and fluorescence RPA detection method. And no instrument or equipment is needed, and the operation is simpler and more convenient.
Table 2 comparison of sensitivity test and detection method for detecting B.glumae DNA by CRISPR cas12a test strip
Note that: + indicates that the detection line has a stripe; -no bands indicating negative results or detection lines
3.3 sample detection
The DNA extracted from 17 natural rice seed samples is detected by using the established CRISPR-Cas12a test strip detection method, the test results are shown in the following table 3, the detection result of 7 seed rice bacterial Rhizoctonia cerealis is positive, the detection result is consistent with the fluorescent RPA method, and the detection rate is higher than that of the fluorescent PCR method and the traditional separation culture method. The method can be successfully applied to actual seed sample detection. The detection time is shorter, the operation is simpler and more convenient, and the method is suitable for on-site detection.
TABLE 3 Natural Rice seed sample detection results
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. The scope of the application is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.
Claims (11)
1. A combination product comprising a CRISPR-Cas system, wherein the CRISPR-Cas system comprises Cas12a and a crRNA, wherein the sequence of the crRNA is Bg-crRNA designed for an ITS gene sequence of bordetella glumae.
2. The combination product comprising a CRISPR-Cas system according to claim 1, wherein the Bg-CrRNA sequence is:
5’-UAAUUUCUACUAAGUGUAGAUGCCGUAUUCCAAUUGA GCC-3’。
3. the combination product comprising a CRISPR-Cas system according to claim 1, wherein the Cas12a is selected from one of FnCas12a, asCas12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a or Lb4Cas12 a.
4. The combination product comprising a CRISPR-Cas system according to claim 1, further comprising a buffer system and a single stranded DNA reporter.
5. The combination product comprising a CRISPR-Cas system according to claim 4, wherein said single stranded DNA reporter is a test strip reporter FB2-DNA, which is FAM-TTTTTTTATTTTTTT-Biotin.
6. A kit comprising a combination product comprising a CRISPR-Cas system according to any one of claims 1-5, and instructions for use.
7. The kit of claim 6, further comprising one or more of a nucleic acid extraction reagent, a positive control, and a negative control.
8. A method for rapidly detecting bacterial Rhizoctonia cerealis of rice is characterized by comprising the following steps:
i) Extracting genome DNA of a sample to be detected;
ii) the genomic DNA obtained in step i) is tested using the kit according to claim 6 according to the instructions for use.
9. The method for rapidly detecting bacterial blight of rice according to claim 8, wherein the components of the kit in the detection system comprise: cas12a 2pM, FB2-DNA200nM, bg-CrRNA 4nM.
10. The method for rapidly detecting bacterial blight of rice according to claim 9, wherein in step ii), the test strip is inserted after heating in a metal bath at 37 ℃ and reacting for 30min.
11. Use of a combination product comprising a CRISPR-Cas system according to any one of claims 1-5 or a kit according to any one of claims 6-7 for detecting bacterial rhizoctonia solani.
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