CN114836570A - Method for visually detecting chaetomium chrysogenum leaf blight by using CRISPR/Cas12a system - Google Patents
Method for visually detecting chaetomium chrysogenum leaf blight by using CRISPR/Cas12a system Download PDFInfo
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Abstract
The invention provides a method for visually detecting Chaetomium chrysosporium C.globosum by using a CRISPR/Cas12a system, which comprises the following steps: amplifying C.globosum specific gene segments of the Chrysanthemum morifolium Ramat by means of a PCR technology; under the mediation of crRNA, Cas12a specifically recognizes the amplification product and activates the nuclease activity, thereby cutting ssDNA (reporter) with any base sequence modified with FAM and BHQ1 at two ends to form a FAM-ssDNA fragment and a BHQ1-ssDNA fragment; FAM-ssDNA and BHQ1-ssDNA can show fluorescence under the irradiation of a blue light transilluminator, thereby realizing the detection of Chaetomium chrysolepis C. The method is simple and portable to operate, does not depend on large-scale instruments and equipment, is low in cost, high in sensitivity and strong in specificity, and has a certain application prospect.
Description
Technical Field
The invention belongs to the field of biology and chemistry, and relates to a method for visually detecting Chaetomium globosum (C.globosum, Cg) of Chaetomium globosum by using a CRISPR/Cas12a system.
Background
Regularly Clustered interspaced short palindromic repeats (CRISPR) are an immune mechanism against invasion in many bacteria and archaea. CRISPR and CRISPR associated proteins (Cas) are referred to as CRISPR/Cas systems. Cas12a is a class 2V-type Cas protein that has no cleavage activity when inactivated, and upon binding to a specific crRNA, the Cas12a conformation will change, binding to the crRNA to form a Cas12a-crRNA binary complex. The compound can specifically recognize target DNA and activate the endonuclease activity of the target DNA. When Cas12a cleaves DNA double strands in cis sequentially, the active site will be exposed continuously, exhibiting trans cleavage activity, and non-specifically cleaving surrounding ssDNA. Scientists have designed a variety of highly sensitive, highly specific nucleic acid detection platforms for the properties of Cas12 a.
Chrysanthemum morifolium (Chrysanthemum morifolium Ramat) is a Chrysanthemum of Compositae, and is one of the traditional medicinal cultivated plants in China and eight Chinese medicinal materials of Zhejiang province, namely Zhe eight flavors. At present, the perennial planting area of the chrysanthemum morifolium ramat in Zhejiang Hangzhou is 7 ten thousand mu, the annual total value is more than 1 hundred million yuan, and the total amount accounts for about 50 percent of the total chrysanthemum sales in China. Modern pharmacological research shows that the traditional Chinese medicine composition has multiple effects of diminishing inflammation, resisting oxidation and cancers, relaxing blood vessels, reducing blood fat, improving intestinal microenvironment and the like. The leaf blight is the main leaf disease of Hangzhou white chrysanthemum, the yellow part of the diseased leaves firstly turns yellow, and the yellow part gradually turns brown and necroses. The disease spreads from local to whole veins, presents brown to reddish brown marginal lesion spots with wavy edges and darker color. The border of disease is obvious, and its outer margin sometimes has a yellow light band with different width, then the disease spot gradually extends toward the base of the leaf until the whole leaf becomes brown to grey brown. The economic benefit of agricultural products is seriously influenced by the generation of leaf blight. However, the traditional separation and identification method has a long detection period, complicated operation steps, time and labor consumption and is difficult to meet the requirement of convenient and fast detection in the field, so that the limitation of the traditional separation and identification method is overcome, the frontier technology is introduced, and the accurate, fast, convenient, sensitive and specific detection technology for the c.
The detection technology of chaetomium globosum C.globosum of Hangzhou white chrysanthemum leaf blight mainly depends on a molecular biological or immunological detection method. Molecular biology relies on the amplification of the ITS sequence of a gene of interest, followed by species information determination by evolutionary tree analysis. The whole process needs 7-10 days, and rapid detection cannot be carried out. Or the expression level of the specific gene is determined by a fluorescent quantitative PCR method, but the detection method has higher technical requirements on instruments and detection personnel, needs expensive experimental equipment and is difficult to realize on-site instant detection; the immunology uses two specific probes to respectively carry out real-time fluorescence GHI on a leaf M1N extracting solution and a seed soaking solution of naturally infected bacterial blight and bacterial streak, and the detection method has good specificity, but has low sensitivity, long time consumption and high cost, so the method is not suitable for large-scale detection of a basic layer.
Disclosure of Invention
The invention aims to overcome the technical defects of the traditional Chaetomium globosum C.globosum detection, and provides a simple, quick, sensitive, specific and portable detection method by taking a specific gene fragment as a detection target.
Therefore, the technical scheme adopted by the invention is as follows:
a method for visually detecting Chaetomium chrysosporium C.globosum by using a CRISPR/Cas12a system comprises the following steps:
the method comprises the following steps: performing PCR amplification on a specific target gene fragment in a C.globosum genome of Chrysanthemum morifolium leaf blight Chaetomium globosum;
the primer sequences used for PCR amplification include:
C.g-PCR-F, the nucleotide sequence of which is shown in SEQ ID No. 1;
C.g-PCR-R, the nucleotide sequence of which is shown in SEQ ID No. 2;
step two: adding the amplified PCR product into a reaction tube containing a CRISPR/Cas system, and incubating, wherein the CRISPR/Cas system comprises specific crRNA aiming at a target gene, CRISPR/Cas12a protein and a reporter sequence for fluorescence detection of an enzyme-labeling instrument, and the crRNA sequence is shown as SEQ ID No. 3; under the mediation of crRNA, CRISPR/Cas12a protein specifically recognizes an amplified fragment of a specific gene fragment and activates the activity of nuclease, so that a reporter sequence modified with FAM and BHQ1 at two ends is cut to form FAM-ssDNA and BHQ 1-ssDNA;
step three: fluorescence appears between FAM-ssDNA and BHQ1-ssDNA under a blue light projector, and visual detection of Chaetomium chrysosporium C.globosum is realized.
In a preferred embodiment of the present invention, in the first step, the PCR step comprises: pre-denaturation at 95 ℃ for 3 min; 30s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃ for 30 times in a total cycle; extension for 10min at 72 ℃.
In a preferred embodiment of the present invention, in the second step, the incubation temperature is 37 ℃ and the incubation time is 30 min.
As a preferred embodiment of the invention, the reporter sequence is ssDNA FQ reporter used for fluorescence detection of a microplate reader, and the sequence is as follows: 5 '-FAM-NNNNN-BHQ 1-3', NNN refers to any base sequence.
As a preferred embodiment of the present invention, in step two, the concentration of Cas12a is 100nM, the concentration ratio of Cas12a to crRNA is 1:1-1:5, and the concentration of Reporter sequence is 250-2000 nM.
As a preferable scheme of the invention, in the second step, the reaction temperature of the CRISPR/Cas12a system is 35-40 ℃, and the reaction time is 10-90 min;
as a preferable scheme of the invention, in the third step, the reaction tube directly shows fluorescence under the irradiation of a blue light transmission instrument, and the visual detection of Chaetomium chrysosporium C.globosum is realized.
From the above description of the invention, the invention provides a novel method for visually detecting Chaetomium chrysosporium C.globosum of Hangzhou rhizoctonia solani by using a CRISPR/Cas12a system. Compared with the existing detection method, the method has the advantages of high sensitivity, strong specificity, rapidness, high efficiency, convenience in operation, user friendliness, low price and the like, and is a novel instant detection platform for Chaetomium chrysosporium C.
Drawings
Fig. 1 is a working principle diagram of the present invention.
FIG. 2 is a graph showing the detection of a positive sample in the present invention.
FIG. 3 shows the influence of different Hangzhou Chrysanthemum Chaetomium globosum C.globosum content samples on the detection;
FIG. 4 shows the relationship between the content of Chaetomium chrysosporium C.globosum and fluorescence intensity after gel electrophoresis analysis.
FIG. 5 is a graph showing the results of the test feasibility test of the present invention.
FIG. 6 is a diagram showing the results of the test for detecting specificity of Chaetomium chrysosporium C.
Detailed Description
The technical solution of the present invention is further illustrated and described by the following detailed description in conjunction with the accompanying drawings:
CRISPR/Cas12a system for visually detecting Chaetomium chrysolepis C
Pretreating an actual sample by a thermal cracking method, under an aseptic condition, taking 1mL of sample uniform solution into a sterilized centrifuge tube, centrifuging at 8000 Xg for 2min, and discarding the supernatant; resuspending with 200 μ L sterilized deionized water, centrifuging at 8000 Xg for 2min, and discarding the supernatant; adding 100 μ L of sterilized deionized water, and boiling at 100 deg.C for 10 min; the thermal lysate is centrifuged at 8000 Xg for 2min, and the supernatant is taken as the substrate (i.e., Target DNA) for the subsequent amplification reaction, as shown in FIG. 1 for the specific operation steps. The PCR amplification technology is utilized to amplify the target fragment, and the primers used by the specific gene fragment system are SEQ ID No.1 and SEQ ID No. 2.
Specifically, SEQ ID No.1 sequence:
5’-CGTTACCTATACCGTTGC-3’；
SEQ ID No.2 sequence:
5’-AGAGCGAGATGTATGCTACT-3’；
constructing CRISPR/Cas12a reaction system, adding 250nM Cas12a, 100nM crRNA (SEQ ID No. 3: 5'-UAAUUUCUACUAAGUGUAGAUUGGCCUCUCUGAGUCUUCUGUACU-3';), 1000nM FAM-Reporter-BHQ1(5 '-FAM-NNNNN-BHQ 1-3'; NNN stands for arbitrary base sequence), 2 uL amplification product, 2 uL buffer C (2 uL buffer C; (R, N, and N, 2, N50mM NaCl，10mM Tris-HCl，10mM MgCl 2 1mM DTT, pH 7.9) and 20. mu.L of RNase-free water was made up. And (3) incubating at 37 ℃ for 20min, immediately putting the reaction solution under a blue light irradiator for analyzing the detection result, wherein the figure 2 is a detection chart of the positive sample in the invention.
Secondly, the method detects the sensitivity characterization of the Chaetomium globosum C
In order to examine the detection sensitivity of the invention to Chaetomium chryseum C.globosum in a Hangzhou white chrysanthemum sample, samples with different contents of 6.5 ng/mu l, 650 pg/mu l, 65 pg/mu l, 6.5 pg/mu l, 650 fg/mu l, 65 fg/mu l, 6.5 fg/mu l, 650 ag/mu l and 65 ag/mu l Chaetomium chryseum C.globosum are subjected to PCR amplification by using the primers used by the specific gene fragment system, and then the specific gene fragment system is analyzed by using the CRISPR/Cas12a system visual detection method. FIG. 3 shows the influence of different Hangzhou white chrysanthemum Chaetomium globosum C.globosum content samples on the detection of fluorescence intensity, and the low 65 ag/mu l Hangzhou white chrysanthemum Chaetomium globosum C.g can be directly identified by naked eye observation. FIG. 4 shows the corresponding amplicon run analysis showing that the detection limit of the method of the invention is 65 ag/. mu.l.
Thirdly, detecting Chaetomium globosum C.globosum in Hangzhou white chrysanthemum sample by using the method
The CRISPR/Cas12a system visual detection method is used for detecting different samples of 65 ag/mu l Chaetomium chryseum C.globosum with the lowest detection limit respectively. As shown in FIG. 5, the fluorescence detection condition of the specific gene fragment system of (i) Cas12a + crRNA + substrate (ii) Cas12a + crRNA (iii) Cas12a + substrate (iii) crRNA + substrate (iii) shows that only Cas12a containing substrate and specific crRNA can generate visible fluorescence. The CRISPR/Cas12a system visual detection method has good detection capability.
Fourth, detection specificity characterization of the methods of the invention
In order to characterize the specificity of the CRISPR/Cas12a system visual detection method for detecting C.globosum of Chrysanthemum chaenomeles Hangzhou, the detection of the method is carried out by using a C.globosum isolate of Chrysanthemum chaenomeles Hangzhou and Fusarium oxysporum F.in and Alt isolate of Fusarium nivale as detection samples. Through observation by a blue light irradiator, fluorescence detection conditions of chaetomium globosum and non-chaetomium globosum C.globosum are shown as shown in fig. 6; the method comprises the steps of utilizing Hangzhou white chrysanthemum Alternaria alternata (Alt), rhizoctonia solani Fusarium incarnatum (Fin) and Chaetomium oxysporum C.globosum (C.g) clinical isolates as detection samples, carrying out sample pretreatment, carrying out PCR amplification on primers SEQ ID No.1 and SEQ ID No.2 used in a C.globosum specific gene fragment system, utilizing C.globosum crRNA (SEQ ID No. 3) to enter a CRISPR/Cas12a system visual detection system for analysis, and displaying through naked eye observation and heat map, the Hangzhou white chrysanthemum chaetomium C.g clinical isolate is detected and shown to be positive under a blue light transmission instrument; the fluorescence detection of the specificity system of the fusarium graminearum F.in and the alternaria alternata Alt shows negative. The CRISPR/Cas12a system visualization detection method has good accuracy and specificity. The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, i.e. the scope of the invention should be determined by the following claims and their description.
Sequence listing
<110> China metering university
<120> method for visually detecting chaetomium chrysogenum leaf blight by using CRISPR/Cas12a system
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgttacctat accgttgc 18
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agagcgagat gtatgctact 20
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<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
uaauuucuac uaaguguaga uuggccucuc ugagucuucu guacu 45
Claims (7)
1. The method for visually detecting chaetomium chrysogenum C.globosum by using the CRISPR/Cas12a system is characterized by comprising the following steps:
the method comprises the following steps: performing PCR amplification on a specific target gene fragment in a C.globosum genome of Chrysanthemum morifolium leaf blight Chaetomium globosum;
the primer sequences used for PCR amplification include:
C.g-PCR-F, the nucleotide sequence of which is shown in SEQ ID No. 1;
C.g-PCR-R, the nucleotide sequence of which is shown in SEQ ID No. 2;
step two: adding the amplified PCR product into a reaction tube containing a CRISPR/Cas system, and incubating, wherein the CRISPR/Cas system comprises specific crRNA aiming at a target gene, CRISPR/Cas12a protein and a reporter sequence for fluorescence detection of an enzyme-labeling instrument, and the crRNA sequence is shown as SEQ ID No. 3; under the mediation of crRNA, CRISPR/Cas12a protein specifically recognizes an amplified fragment of a specific gene fragment and activates the activity of nuclease, so that a reporter sequence modified with FAM and BHQ1 at two ends is cut to form FAM-ssDNA and BHQ 1-ssDNA;
step three: fluorescence appears between FAM-ssDNA and BHQ1-ssDNA under a blue light projector, and visual detection of Chaetomium chrysosporium C.globosum is realized.
2. The method for visually detecting chaetomium globosum C.globosum by using CRISPR/Cas12a system according to claim 1, wherein the CRISPR/Cas12a system comprises the following steps: in the first step, the PCR step is: pre-denaturation at 95 ℃ for 3 min; 30s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃ for 30 times in a total cycle; and then extended for 10min at 72 ℃.
3. The method for visually detecting chaetomium globosum C.globosum by using CRISPR/Cas12a system according to claim 1, wherein the CRISPR/Cas12a system comprises the following steps: in the second step, the incubation temperature is 37 ℃ and the incubation time is 30 min.
4. The method for visually detecting chaetomium chrysogenum C.globosum by using CRISPR/Cas12a system according to claim 1, wherein the method comprises the following steps: the reporter sequence is ssDNA FQ reporter used for fluorescence detection of a microplate reader, and the sequence is as follows: 5 '-FAM-NNNNN-BHQ 1-3', NNN refers to any base sequence.
5. The method for visually detecting chaetomium globosum C.globosum by using CRISPR/Cas12a system according to claim 1, wherein the CRISPR/Cas12a system comprises the following steps: in step two, the concentration of Cas12a is 100nM, the concentration ratio of Cas12a to crRNA is 1:1-1:5, and the concentration of Reporter sequence is 250-2000 nM.
6. The method for visually detecting chaetomium globosum C.globosum by using CRISPR/Cas12a system according to claim 1, wherein the CRISPR/Cas12a system comprises the following steps: in the second step, the reaction temperature of the CRISPR/Cas12a system is 35-40 ℃, and the reaction time is 10-90 min.
7. The method for visually detecting Chaetomium globosum C.globosum according to CRISPR/Cas12a system of claim 1, characterized in that in the third step, the reaction tube directly shows fluorescence under the irradiation of a blue light transmission instrument, so that the visual detection of Chaetomium globosum C.globosum is realized.
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| CN116656850A (en) * | 2023-07-27 | 2023-08-29 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116656850A (en) * | 2023-07-27 | 2023-08-29 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof |
| CN116656850B (en) * | 2023-07-27 | 2023-10-31 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof |
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