CN117363792A - Method for dual detection of verticillium dahliae based on RPA-CRISPR and application - Google Patents
Method for dual detection of verticillium dahliae based on RPA-CRISPR and application Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to a method for dual detection of verticillium dahliae based on RPA-CRISPR and application thereof. According to the invention, through research and analysis of fallen leaf type and non-fallen leaf type specific genes of the verticillium dahliae, a dual detection method of verticillium dahliae pathotype based on an RPA-CRISPR system is provided for the first time, an RPA primer group and crRNA sequence meeting detection requirements are designed and screened, and simple, rapid, accurate and sensitive detection of verticillium dahliae pathotype is realized through a dual detection means.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for dual detection of verticillium dahliae based on RPA-CRISPR and application thereof.
Background
Cotinus coggygriaCotinus coggygria) Is of the family Rhus vernicifluaAnacardiaceae) Cotinus genusCotinus) The leaves of the plants, deciduous shrubs or small trees turn red in autumn, and have great ornamental value and economic value. Cotinus coggygria wilt diseaseThe pathogenic bacteria are Verticillium dahliaeVerticillium dahliae) Verticillium dahliae can be classified into deciduous and nondeciduous types according to their pathotypes. Verticillium dahliae is a soil-borne pathogenic fungus that infects plant roots by forming attached shoots, causing serious verticillium wilt of a variety of plants worldwide, including woody trees, commercial crops (sunflower and cotton), vegetables, fruits and flowers, causing tremendous economic loss. Because of the broad host range and long-lasting soil viability of verticillium dahliae, many control measures are difficult to achieve the desired effect, accurate, sensitive and rapid detection of verticillium dahliae is critical to limiting the entry of pathogens into new regional environments and early management of verticillium wilt.
Currently, a number of methods have been developed based on the Polymerase Chain Reaction (PCR) that can be used to detect verticillium dahliae, such as conventional PCR, nested PCR and qPCR. PCR detection techniques are limited in field application because of the appropriate training, techniques and expensive and complex thermal cycling equipment required for conventional detection methods. Isothermal DNA amplification methods, such as loop-mediated isothermal amplification (LAMP) and Recombinant Polymerase Amplification (RPA), may be more suitable alternatives to in-situ PCR detection. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have been developed as sensitive nucleic acid detection tools, with Cas12 and Cas13 families capable of collective cleavage (referred to as trans-cleavage); when the Cas protein crRNA binary complex detects and binds to a substrate, it can activate targeted cis-cleavage and nonspecific "trans-cleavage" activity of single-stranded/double-stranded DNA or single-stranded RNA. Several CRISPR/Cas-based detection methods, including DETECTER, HOLMES, SHERLOCK and CDetection, have been used as tools for molecular detection using this trans-cleavage activity as a signal amplifier. In combination with LAMP and RPA isothermal amplification methods, CRISPR/Cas provides a robust method for accurate, rapid and specific detection and has been established for rapid and sensitive detection of plant pathogens. However, there is still little dual detection of verticillium dahliae.
Disclosure of Invention
The invention provides a method for dual detection of verticillium dahliae based on RPA-CRISPR and application thereof. According to the invention, the specific target genes based on two pathotypes are combined by Cas12 and Cas13, and different fluorescent report groups are adopted for reading, so that a method for double detection of the verticillium dahliae is established, and the pathotypes of the verticillium dahliae can be distinguished, thereby providing more accurate basis and foundation for controlling the smoke tree wilt caused by the verticillium dahliae.
The invention provides a primer combination for detecting verticillium dahliae, which comprises an RPA primer pair and/or crRNA;
the RPA primer pair comprises any one or more of the following:
(1) A primer comprising a sequence shown as SEQ ID NO. 1-2;
(2) A primer comprising a sequence as shown in SEQ ID NO. 3-4;
the crRNA comprises a sequence shown in any one of SEQ ID NO.5-7 and SEQ ID NO. 10-13.
Preferably, the RPA primer pair and target gene of crRNA include a Dahurian round colony leaf type specific gene (GenBank: AJ 302674.2) and a Dahurian round non-fallen leaf type specific gene (GenBank: AJ 302675.1).
According to the primer combination for detecting verticillium dahliae, the primer combination comprises a primer with a sequence shown as SEQ ID NO.1-2 and crRNA with a sequence shown as any one of SEQ ID NO. 5-7.
And/or the primer combination comprises a primer with a sequence shown as SEQ ID NO.3-4 and crRNA with a sequence shown as any one of SEQ ID NO. 10-13.
Preferably, the RPA primer pair of the Verticillium dahliae comprises an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2.
Preferably, the RPA primer pair of the non-verticillium dahliae comprises an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4.
Preferably, the crRNA sequence of the Verticillium dahliae comprises one of SEQ ID NO.5 SEQ ID NO.6 SEQ ID NO. 7.
Preferably, SEQ ID NO.6 is a crRNA for detecting Verticillium dahliae.
Preferably, the crRNA sequence of the non-fallen verticillium dahliae comprises one of SEQ ID NO.10 SEQ ID NO.11 SEQ ID NO.12 SEQ ID NO. 13.
Preferably, SEQ ID NO.12 is a sample of crRNA from Verticillium dahlia.
Preferably, the RPA-CRISPR/Cas13 is used to detect verticillium dahliae and the RPA-CRISPR/Cas12 is used to detect non-verticillium dahliae.
The invention also provides a kit for detecting the verticillium dahliae, which comprises the primer combination for detecting the verticillium dahliae.
According to the kit for detecting verticillium dahliae, the kit further comprises a Cas13 protein and a ssRNA fluorescent probe comprising a sequence shown as SEQ ID NO. 8.
And/or, cas12 protein and ssDNA fluorescent probe comprising the sequence shown as SEQ ID No. 14.
The excitation light of the 5' -end fluorescent group of the ssRNA fluorescent probe and the ssDNA fluorescent probe are different.
According to the kit for detecting verticillium dahliae, the ssRNA fluorescent probe comprises a sequence shown as SEQ ID NO. 8.
And/or, the ssDNA fluorescent probe comprises a sequence shown as SEQ ID NO. 14.
The invention also provides a method for detecting the pathogenic type of the verticillium dahliae based on the RPA-CRISPR system, which utilizes the primer combination for detecting the verticillium dahliae or the kit for detecting the verticillium dahliae.
The method for detecting the pathotype of the verticillium dahliae based on the RPA-CRISPR system comprises the following steps of:
(1) Taking the DNA of the sample to be detected as a template to perform double RPA reaction;
(2) Carrying out enzyme digestion reaction on the dual RPA product;
wherein the Cas13 protein is used to cleave verticillium dahliae and/or the Cas12 protein is used to cleave verticillium dahliae;
(3) Fluorescence detection is performed after cleavage reaction.
Preferably, the 15 mu L RPA double reaction system comprises 9 mu L1X reaction buffer, RPA primers (10 mu M) of 1 mu L, 0.8 mu L magnesium acetate (MgAc, 14 mM), 1 mu L DNA template and the balance of purified water.
According to the method for detecting the pathotype of the verticillium dahliae based on the RPA-CRISPR system, the verticillium dahliae is detected based on the RPA-CRISPR/Cas13, and 20 mu l of the reaction system comprises 100 nM LwaCas13a, 1.75U/mu l T RNA polymerase, 1U/mu l RNase inhibitor and 2 mu l template DNA.
Preferably, 100 nM LwaCas13a (10. Mu.M) (Tolobio, china), 100 nM crRNA, 1 Xreaction buffer (Tolobio), 1 mM rNTP Mix (New England Biolabs), 1.75U/. Mu. l T7 RNA polymerase (New England Biolabs), 1U/. Mu.l RNase inhibitor (Sangon), 125nM ssRNA fluorescent probe and 2. Mu.l template DNA are included.
And/or, detecting the non-verticillium dahliae based on the RPA-CRISPR/Cas12, wherein 20 μl reaction system comprises: 100 nM Cas12a, 200nM crRNA, 200nMssDNA fluorescent probe and 2. Mu.l template DNA.
Preferably, 100 nM of Cas12a (New England Biolabs), 200nM of crRNA, 1 XNEB 2.1 buffer (New England Biolabs), 200nM of ssDNA fluorescent probe and 2. Mu.l of template DNA are included.
Preferably, the method comprises the steps of:
s1, extracting genome DNA of a sample to be detected, and carrying out double RPA amplification by taking the genome DNA of the sample to be detected as a template to obtain a double RPA product;
s2, constructing a Verticillium dahliae CRISPR/Cas13 detection system by taking the RPA product as a template. The 40. Mu.l reaction system consisted of 100 nM LwaCas13a (10. Mu.M) (Tolobio, china), 100 nM crRNA, 1 Xreaction buffer (Tolobio), 1 mM rNTP Mix (New England Biolabs), 1.75U/. Mu. l T7 RNA polymerase (New England Biolabs), 1U/. Mu.l RNase inhibitor (Sangon), 125nM ssRNA fluorescent probe labeled with FAM fluorescent group, 100 nM Cas12a (New England Biolabs), 200nM crRNA, 1 XNEB 2.1 buffer (New England Biolabs), 200nM ssDNA fluorescent probe labeled with HEX fluorescent group, and 4. Mu.l RPA reaction product were reacted at 37 ℃.
The invention also provides application of the primer combination for detecting the verticillium dahliae or the kit for detecting the verticillium dahliae in detecting the verticillium dahliae.
Depending on the application, FAM fluorophores label the 5' end of ssRNA fluorescent probes.
And/or, the HEX fluorophore marks the 5' end of the ssDNA fluorescent probe.
The beneficial effects are that:
the invention provides a primer combination, a method and application for detecting verticillium dahliae. Wherein, the RPA primer pair and crRNA can detect pathotype of verticillium dahliae. The primer combination and the method provided by the invention combine the RPA amplification technology and the CRISPR detection technology, realize the rapid detection of the verticillium dahliae and can distinguish the pathotype of the verticillium dahliae. The primer combination and the method are applied to the detection of the verticillium dahliae, and have important significance for the prevention and treatment of the cotinus coggygria wilt caused by verticillium dahliae infection.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or of the prior art, the following description will make a brief introduction to the drawings used as required in the description of the embodiments or of the prior art.
Fig. 1 is a schematic diagram of detection of deciduous and non-deciduous verticillium using lwaca 13a and LbCas12a provided by the invention. The LbCas12 a-specific ssDNA reporter was labeled with HEX fluorescein and the LwaCas13 a-specific ssRNA reporter was labeled with FAM fluorescein.
FIG. 2 is a dual RPA test of verticillium dahliae of deciduous (D) and non-deciduous (ND) species provided in example 1 of the present invention. ND+D represents the simultaneous addition of gDNA from both deciduous and non-deciduous Verticillium. D and ND represent the addition of DNA from deciduous and non-deciduous Verticillium respectively. N is the negative control of the RPA reaction (DEPC water), P is the positive control of the RPA reaction, and M is Marker.
Fig. 3 is a crRNA screen for detecting verticillium dahliae based on RPA-CRISPR/Cas13 provided in example 2 of the present invention. crRNA1-N, crRNA2-N and crRNA3-N represent crRNA negatives, i.e., no RPA reaction product was added and replaced with DEPC water.
Fig. 4 is a crRNA screen of non-verticillium dahliae based on RPA-CRISPR/Cas12 detection provided in example 3 of the present invention. crRNA1-N, crRNA2-N, crRNA3-N and crRNA4-N indicate crRNA negativity, i.e., no RPA reaction product was added and replaced with DEPC water.
Fig. 5 is a multiplex assay fluorescence reading for verticillium dahliae by LbCas12a and LwaCas13a provided in example 4 of the present invention. In the presence of a dahlia colony leaf type target gene, a FAM signal can be detected; HEX signal can be detected in the presence of a non-deciduous target gene of Verticillium dahliae; n is a negative control (DEPC water).
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a dual detection method of verticillium dahliae pathotype based on an RPA-CRISPR system, which is shown in figure 1 and comprises the following main processes:
(1) Dual RPA reactions.
(2) And (3) carrying out enzyme digestion reaction on the dual RPA product, wherein Cas13 is used for cutting the verticillium dahliae, and Cas12 is used for cutting the non-verticillium dahliae.
(3) After the cleavage reaction, a real-time fluorometer is used for reading.
The following may illustrate the details of the present invention.
Example 1 establishment of Dual RPA detection System
1. Experimental method
(1) RPA primer design:
the method comprises the steps of performing sequence comparison and analysis on published dahlia colony leaf type and non-fallen leaf type specific genes, respectively designing four pairs of RRA primers by combining with an RPA primer design principle, screening, respectively screening 1 pair of high-specificity and high-amplification-efficiency RPA primers, and finally screening to obtain fallen leaf type RPA primers with the sequence:
an upstream primer: RPA-D-F2 5'-TACGGGTCGAAGTTGTTGCCCGCCTGCCGC-3' (SEQ ID NO. 1);
a downstream primer: RPA-D-F3 5'-CGAAGTTGTTGCCCGCCTGCCGCAACTGGG-3' (SEQ ID NO. 2);
the final screening result shows that the sequence of the non-fallen leaf type RPA primer is:
an upstream primer: RPA-ND-F2 5'-CATCATCCCTCTCGCAGGTGGCAGTCATCC-3' (SEQ ID NO. 3);
a downstream primer: RPA-ND-R4 5'-CCCAGCATGTGTCATGGTCCCCGGCTGGAC-3' (SEQ ID NO. 4).
(2) Dual RPA reaction:
the kit used in the RPA double reaction process is a RAA kit (B00000) of Jiangsu Qihan gene biotechnology limited company, and each RPA reaction comprises 9 mu L of 1 x reaction buffer, 1 mu L of RPA primer (10 mu M) respectively, 0.8 mu L of magnesium acetate (MgAc, 14 mM) and 1 mu L of purified water for supplementing the DNA template to 15 mu L. And adding other reaction liquids except MgAc into the freeze-dried powder tube, fully redissolving the freeze-dried powder uniformly, adding a magnesium acetate solution into the tube cover of each reaction tube, covering the tube cover, flicking with fingers to fully mix the reagents in the tubes, centrifuging at a low speed for 3-5s, and reacting at 37 ℃ for 15min. When the reaction products are detected by agarose gel electrophoresis, the dual RPA reaction products are evenly mixed with chloroform in a ratio of 1:1, and the mixture is centrifuged for 2 min at 12000 and g. The supernatant was aspirated and analyzed by electrophoresis, and the result of electrophoresis is shown in FIG. 2.
2. Test results
As shown in fig. 2, N is a negative control of RPA reaction, P is a positive control of RPA reaction, lane nd+d is dual RPA product of larvicia and non-larvicia, ND is non-larvicia RPA product, and D is larvicia product.
Example 2 establishment of an RPA-CRISPR/Cas 13-based detection System for Verticillium dahlia
1. Experimental method
(1) Specific crrnas and probe sequences of verticillium dahliae were designed as shown in the following table:
(2) Preparing crRNA:
for the convenience of preservation and the requirement of amplification in the subsequent experiments, the crRNA is synthesized into DNA, and is transcribed into crRNA in vitro when in use, and a T7 promoter sequence GAAATTAATACGACTCACTATAGGG (SEQ ID NO. 9) is required to be added at the 5' end. The DNA sequence was synthesized by Shanghai Biotechnology Co. The DNA sequences for transcription into crRNA are specifically as follows:
the DNA template of crRNA was annealed to double-stranded DNA using taq10 x PCR buffer (TaKaRa) with the annealing system: crRNAs IVT tempaltes (10. Mu.M) 1. Mu.L, T7-3G IVT (10. Mu.M) 1ul,10 XPCR buffer 1. Mu.L, DEPCH 2 O was made up to a total system of 10ul.
And (3) carrying out an annealing reaction, wherein the annealing process comprises the following steps: 95 ℃ for 5min;94℃to 25℃C (0.1℃C/s).
10ul rNTP buffer mix,2ul T7 RNA polymerase mix and 8. Mu.l DEPC H were added to the annealed product obtained in the previous step according to the description of Hi Scribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) 2 O. Double stranded DNA was transcribed into crRNA by incubation overnight at 37 ℃; then 20. Mu.l DEPC H was added 2 O, 2. Mu.l DNase I, 37℃for 15min. Then according to the manufacturer's instructions, RNA Clean was used&The crRNA was purified using the Concentrator Kit (APEx BIO) using DEPC H 2 The concentration of O was adjusted to 10 ng/ul (split into 50ul per tube, stored at-80 ℃).
(3) RPA-CRISPR/Cas13 reaction system
CRISPR-Cas13a detection system was 100 nM LwaCas13a (10. Mu.M) (Tolobio, china), 100 nM crRNA, 1 Xreaction buffer (Tolobio), 1 mM rNTP Mix (New England Biolabs), 1.75U/. Mu. l T7 RNA polymerase (New England Biolabs), 1U/. Mu.l RNase inhibitor (Sangon), 125nM ssRNA fluorescent probe labeled with FAM fluorophore and 2. Mu.l DNA (1 ng/. Mu.l) template were reacted in a 20. Mu.l reaction at 37℃with a real-time fluorometer (Bio-rad) for 60 cycles per minute reading data.
2. Test results
(1) Screening of crRNA: the foregoing experiment provided 3 different crrnas. 3 different crRNAs are applied to detection of the verticillium dahliae; the extracted verticillium dahliae genome DNA is added into an RPA reaction unit for pre-amplification, and then an amplification product is placed into a CRISPR-Cas13a detection system, and the result is observed through fluorescence reaction. The reaction temperature was 37℃and the fluorescence detection time was 60min, and the detection results are shown in FIG. 3.
As can be seen from fig. 3: all reactions with template addition had fluorescent signal generation, and negative control was established without false positive. From FIG. 3, it can be derived that the optimal crRNA is crRNA2. The subsequent experiments used crRNA2 as the common crRNA sequence. The results also demonstrate that the established RPA-CRISPR-Cas13a detection method can detect dahlia lare.
(2) Optimization of CRISPR/Cas13 reaction system
In optimizing the above reaction system, the final concentrations of LwaCas13a, T7 RNA polymerase, RNase inhibitor and RPA amplification products are optimized as follows: the concentrations of lwaca 13a were 25, 50, 75, 100 and 125nM, respectively; the concentrations of T7 RNA polymerase were 1, 1.25, 1.5, 1.75 and 2U/. Mu.l, respectively; RPA amplification products were 0.5, 1, 1.5, 2 and 2.5. Mu.l, respectively; the concentration of RNase inhibitor was 0.8, 1, 1.2, 1.4 and 1.6U/. Mu.l, respectively. The reaction concentrations as described in the experimental methods above were finally selected.
Example 3 establishment of a non-Larix dahlia detection System based on RPA-CRISPR/Cas12
1. Experimental method
(1) Design of specific crRNA and probe sequences of non-Larix dahliae as shown in the following Table
(2) RPA-CRISPR/Cas12 reaction system
The reaction was performed by reacting 100 nM Cas12a (New England Biolabs), 200nM crRNA, 1 XNEB 2.1 buffer (New England Biolabs), 200nM ssDNA fluorescent probe labeled with HEX fluorescent groups, and 2. Mu.l template DNA in a 20. Mu.l reaction system at 37℃and detecting with a real-time fluorometer (Bio-rad), reading the data once per minute for 60 cycles.
2. Test results
(1) Screening of crRNA: the foregoing experiment provided 4 different crrnas. Applying 4 different crrnas to detection of non-verticillium dahliae; the extracted genome DNA of the non-fallen verticillium dahliae is added into an RPA reaction unit for pre-amplification, and then an amplification product is placed into a CRISPR-Cas12 detection system, and the result is observed through fluorescence reaction. The reaction temperature was 37℃and the fluorescence detection time was 60min, and the detection results are shown in FIG. 4.
As can be seen from fig. 4: all reactions with template addition had fluorescent signal generation, and negative control was established without false positive. From fig. 4, it can be derived that the optimal crRNA is crRNA3. The subsequent experiments used crRNA3 as the common crRNA sequence. The results also demonstrate that the established RPA-CRISPR-Cas12a detection method can detect non-deciduous verticillium dahliae.
(2) Optimization of CRISPR/Cas12 reaction system
In optimizing the above reaction system, the final concentrations of the LbaCas12a, crRNA, HEX fluorescent probe and RPA amplification product are optimized as follows: the concentration of LbaCas12a is (25, 50, 75, 100, and 125 nM), respectively; the concentration of crRNA is crRNA (50, 100, 150, 200, and 250 nM), respectively; the concentration of the HEX fluorescent probe was HEX reporter (50, 100, 150, 200, and 250 nM), respectively; RPA amplification products were 0.5, 1, 1.5, 2 and 2.5. Mu.l, respectively; the reaction concentrations as described in the experimental methods above were finally selected.
Example 4 establishment of a dual detection System for detecting pathotype of Verticillium dahliae based on RPA-CRISPR/Cas
1. The experimental method comprises the following steps: dual RPA-CRISPR/Cas detection
mu.L of RPA amplification product was added to 36. Mu.L of ssRNA fluorescent probe containing 100 nM LwaCas13a (10. Mu.M) (Tolobio, china), 100 nM crRNA, 1 Xreaction buffer (Tolobio), 1 mM rNTP Mix (New England Biolabs), 1.75U/. Mu. L T7 RNA polymerase (New England Biolabs), 1U/. Mu.L RNase inhibitor (Sangon), 125nM labeled with FAM fluorophore; 100 nM Cas12a (New England Biolabs), 200nM crRNA, 1 XNEB 2.1 buffer (New England Biolabs), 200nM ssDNA fluorescent probe labeled with HEX fluorescent groups, the composition of the reaction solution was a mixture of Cas12 and Cas13 reaction solutions described above, and the RPA reaction product was used as a template for the reaction using a real-time PCR instrument (Bio-Rad).
2. Test results
As shown in fig. 5, the ssDNA probe of LbCas12a was labeled with a HEX fluorophore, while the ssRNA probe of LwaCas13a was labeled with a FAM fluorophore. The most suitable crRNA3 was selected in CRISPR/Cas12 for detection of DNA from non-verticillium dahliae, whereas crRNA2 was selected in CRISPR/Cas13 for detection of DNA from verticillium dahliae. The fluorescent signal intensities were read by a real-time PCR instrument and displayed simultaneously by two types of fluorescent signals corresponding to fallen leaves and non-fallen leaf pathotypes. In the double reaction, only the FAM groups emitted fluorescent signals from the deciduous strains, and only the HEX fluorescent channels emitted signals from the non-deciduous strains.
In addition, mixing of the lards and non-lards with healthy soil samples revealed that only FAM fluorescence signals could be detected in the sample (D) with lards and only HEX fluorescence signals could be detected in the sample (ND) with non-lards, as shown in the following table. Taken together, these results indicate that the established double detection can distinguish between pathotypes of verticillium dahliae.
In the table: d = verticillium dahliae; ND = non-deciduous verticillium dahliae; NA = negative control (healthy soil); negative control of n=rpa-CRISPR/Cas 13.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A primer combination for detecting verticillium dahliae, characterized in that the primer combination comprises an RPA primer pair and/or crRNA;
the RPA primer pair comprises any one or more of the following:
(1) A primer comprising a sequence shown as SEQ ID NO. 1-2;
(2) A primer comprising a sequence as shown in SEQ ID NO. 3-4;
the crRNA comprises a sequence shown in any one of SEQ ID NO.5-7 and SEQ ID NO. 10-13.
2. The primer combination for detecting verticillium dahliae according to claim 1, wherein the primer combination comprises a primer having a sequence as shown in SEQ ID No.1-2 and crRNA having a sequence as shown in any one of SEQ ID No. 5-7;
and/or the primer combination comprises a primer with a sequence shown as SEQ ID NO.3-4 and crRNA with a sequence shown as any one of SEQ ID NO. 10-13.
3. A kit for detecting verticillium dahliae, comprising the primer combination for detecting verticillium dahliae of claim 1 or 2.
4. A kit for detecting verticillium dahliae according to claim 3, further comprising Cas13 protein and ssRNA fluorescent probe comprising the sequence as set forth in SEQ ID No. 8;
and/or, cas12 protein and ssDNA fluorescent probe comprising the sequence shown as SEQ ID No. 14;
the excitation light of the 5' -end fluorescent group of the ssRNA fluorescent probe and the ssDNA fluorescent probe are different.
5. The kit for detecting verticillium dahliae according to claim 4, wherein the ssRNA fluorescent probe comprises a sequence as shown in SEQ ID No. 8;
and/or, the ssDNA fluorescent probe comprises a sequence shown as SEQ ID NO. 14.
6. A method for detecting the pathotype of verticillium dahliae based on an RPA-CRISPR system, which is characterized by using the primer combination for detecting verticillium dahliae according to any one of claims 1 to 2 or the kit for detecting verticillium dahliae according to any one of claims 3 to 4.
7. The method for detecting the pathotype of verticillium dahliae based on the RPA-CRISPR system according to claim 6, comprising the following steps:
(1) Taking the DNA of the sample to be detected as a template to perform double RPA reaction;
(2) Carrying out enzyme digestion reaction on the dual RPA product;
wherein the Cas13 protein is used to cleave verticillium dahliae and/or the Cas12 protein is used to cleave verticillium dahliae;
(3) Fluorescence detection is performed after cleavage reaction.
8. The method for detecting the pathotype of the verticillium dahliae based on the RPA-CRISPR system according to claim 7, wherein the 20. Mu.l reaction system for detecting the verticillium dahliae based on the RPA-CRISPR/Cas13 comprises 100 nM LwaCas13a, 1.75U/. Mu. l T7 RNA polymerase, 1U/. Mu.l RNase inhibitor and 2. Mu.l template DNA;
and/or, detecting the non-verticillium dahliae based on the RPA-CRISPR/Cas12, wherein 20 μl reaction system comprises: 100 nM Cas12a, 200nM crRNA, 200nMssDNA fluorescent probe and 2. Mu.l template DNA.
9. Use of the primer combination for detecting verticillium dahliae according to any one of claims 1 to 2 or the kit for detecting verticillium dahliae according to any one of claims 3 to 4 for detecting verticillium dahliae.
10. The use according to claim 9, wherein FAM fluorophores label the 5' end of the ssRNA fluorescent probe;
and/or, the HEX fluorophore marks the 5' end of the ssDNA fluorescent probe.
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