CN112266971A

CN112266971A - Probe, kit and method for detecting phomopsis brassicae by RAA fluorescence method

Info

Publication number: CN112266971A
Application number: CN202011026665.1A
Authority: CN
Inventors: 陈吴健; 林晓佳; 任琰; 张明哲; 吴志毅; 程帆; 吴颖; 应清界; 郭利川
Original assignee: Jiangsu Qitian Gene Biotechnology Co ltd; Zhejiang Academy Of Science & Technology For Inspection & Quarantine
Current assignee: Jiangsu Qitian Gene Biotechnology Co ltd; Zhejiang Academy Of Science & Technology For Inspection & Quarantine
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2021-01-26

Abstract

The invention discloses a probe, a kit and a method for detecting sclerotinia rot of rape by an RAA fluorescence method. The invention can rapidly detect the rape stem-based canker pathogen by utilizing the RAA technology, has simple and convenient operation, greatly shortens the used time, does not need large-scale instruments and equipment, and is suitable for large-scale screening. The detection method provided by the invention has the advantages of high sensitivity, good specificity, simple and rapid operation and low requirement on the quality of detection materials. The invention solves the problems of time and labor waste, long time period, low efficiency, requirement of professional and expensive instruments, high false positive rate and the like in the detection of the rape stem-base canker pathogen by the detection method in the prior art. The method can analyze the result only by reacting for 5-20 minutes at 39 ℃. Has the characteristics of rapidness, sensitivity and the like, has very important significance for detecting the spread of the rape stem-based canker pathogen in the future, and has great application prospect.

Description

Probe, kit and method for detecting phomopsis brassicae by RAA fluorescence method

Technical Field

The invention belongs to the technical field of molecular biology detection, and particularly relates to a probe, a kit and a method for detecting sclerotinia rot of colza by an RAA fluorescence method.

Background

Rape stem canker is one of the most serious fungal diseases on rape, mainly comprising 2 species: the 2 pathogenic bacteria of Leptosphaeria biglobosa and L.maculons have similar morphological characteristics, but have different capacities of causing rape stem infection and basal ulcer. The former is weak in pathogenicity, generally causes damage to the middle and upper parts of the stems, causes little loss, and is distributed in some rape producing countries including China. The latter is strong in pathogenicity, causes the base of the stem to be attacked, is a main factor causing serious prevalence of rape black shank and rape yield loss, is distributed in major rape producing countries such as Australia, Canada, America, Europe and the like, and is not reported in China.

The rape stem canker pathogen (Leptosphaeria maculans) is a pathogen which is widely distributed in the world and seriously harms the rape industry. Because the stem-based canker pathogen of rape has strong pathogenicity, the pathogenic bacteria cause serious reduction of the yield of European, American and Australian rapeseeds, and the yield loss caused by L.maculans is about 10 to 20 percent in general years, and can reach 30 to 50 percent or even higher when the yield loss is serious. It is estimated that the economic loss of rapeseed due to l.maculans is more than 3 billion euros each year worldwide. In recent years, edible rapeseeds in China are mainly imported from foreign countries, the climate of rape production areas in China is similar to that of Europe, America and Australia, and the current rape main cultivars in China are highly susceptible to diseases. Therefore, the strengthening of quarantine and supervision is to control the introduction of the germ into China, and is an urgent task of various port plant quarantine departments in China at present. Therefore, an efficient and rapid detection technology is urgently needed to be established for preventing the rape stem base canker from spreading to China.

The pathogenic bacteria of stem-based canker of Brassica napus are of the genus Leptosphaeria maculans of the genus Micrococcus (Fungi), Ascomycota (Ascomycota), Ascomycota (Dothidymycoceae), Geospora (Pleosporales), Geosporaceae (Pleosporaceae), and Leptosphaera (Leptosphaeria). It not only infects rape, but also harms nearly 30 cruciferae plants such as cabbage, cabbage and mustard, causing stem base ulcers, plant lodging and death. The planting area of cruciferous crops in China is huge, wherein the planting area of rape is kept 667 kilohm since 2002²The yield reaches 1000-1300 ten thousand, and once the rape stem-based canker pathogen is not found in China, the rape stem-based canker pathogen causes huge loss to the economy of China, so that L.maculans is listed in the entry plant quarantine pest entry of the people's republic of China in 2007.

The existing laboratory detection technology for the phomopsis brassicae mainly comprises a separation culture method, a molecular biological method and the like.

1. Isolation culture method

The method comprises the following steps: soaking suspected susceptible seeds in sterile water for 12h, standing overnight in a refrigerator at-20 ℃, disinfecting the surface with 1% sodium hypochlorite for 5min, sterilizing and washing for 3 times, sucking dry by using filter paper, placing on a PDA culture medium, culturing for 48h, and observing, wherein if a bacterial colony is circular, the surface is white, flat, the edge is irregular, hyphae are white and radial, and nodular hyphae are visible in the hyphae, so that the bacterial colony can be judged to be the bacterial of the stem base of rape canker.

2. Molecular biological method

2.1 Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) utilizes genomic DNA or cDNA to change from double strand to single strand at high temperature of 95 ℃, primers and single strand are combined according to the principle of base complementary pairing at about 60 ℃, and DNA Polymerase synthesizes a complementary strand along the direction from phosphoric acid to pentose (5'-3') at about 72 ℃. PCR detection methods are widely used due to their rapid, accurate and sensitive characteristics. The method is used by Chen Qing et al to detect the stem base canker germ of rape.

2.2 double priming oligonucleotide primer method (DPO method)

A double-priming oligonucleotide primer (DPO) is a novel PCR primer design method, wherein the primer comprises two independent regions, oligomeric hypoxanthine is used for connection, the hydrogen bond bonding force of the hypoxanthine is weak, and in the amplification process, if more than 3 basic groups at the 5 'end or the 3' end of the DPO primer are mismatched, the primer can be separated from a template and cannot normally react, so that the specificity of the primer is greatly enhanced, meanwhile, a secondary structure is difficult to form between the DPO primer and the primer, the double-priming oligonucleotide primer is insensitive to annealing temperature, and a person such as Longyang establishes a technology for quickly detecting the stem ulcer germs of rape.

2.3 real-time fluorescent quantitative PCR method

Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR) is a method for measuring the total amount of products after each Polymerase Chain Reaction (PCR) cycle by using fluorescent chemical substances in DNA amplification reaction, and is a method for quantitatively analyzing a specific DNA sequence in a sample to be detected by internal reference. SYBR Green real-time fluorescent PCR technology has been used for quantitative detection of rape stem-based canker pathogen.

2.4 Loop-mediated isothermal amplification (LAMP)

Loop-mediated isothermal amplification (LAMP) is a rapid nucleic acid amplification method, wherein 4 specific LAMP primers are designed for 6 different regions of a target gene sequence, Bst DNA polymerase with strand displacement activity is used for incubation for 30-60 min under an isothermal condition (about 65 ℃) to complete a nucleic acid amplification reaction, and people in the world and the like detect the phoma gracilis through the technology.

In conclusion, the traditional methods such as the separation culture method have long period and are tedious to operate, so that the current requirements for quick clearance are difficult to meet; the PCR technology has the advantages of high speed, strong specificity and high sensitivity, is one of the important means for screening the rape stem-based canker germs at the port of China at present, but needs expensive instruments, and is equipped with excellent laboratories and professional operators. The LAMP method does not need an expensive PCR instrument, is isothermal and sensitive, has strong specificity, is simple to operate and is easy to observe results, but the LAMP technology requires multiple pairs of primers and has higher requirements on target genes, so the LAMP method is difficult to popularize and apply in basic laboratories.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an RAA fluorescence detection method, a primer probe and a kit for sclerotinia sclerotiorum.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a probe for detecting the phomopsis brassicae by an RAA fluorescence method,

the probe is 5 'terminal sequence/fluorescent reporter group// A THF// quenching group/3' terminal sequence; wherein:

the nucleotide sequence of the 5' end is as follows: CTTAGAGGAGCTAGAGTCCTTATCTTCT, respectively;

the nucleotide sequence of the 3' end is as follows: TAAGGAGCTCTAGGC, respectively;

the THF is at 1 base position;

the fluorescent reporter group is selected from any one of FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy 5;

the quenching group is selected from any one of TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.

As an improvement of the probe for detecting the phomopsis brassicae by the RAA fluorescence method, the nucleotide sequence of the probe is as follows: CTTAGAGGAGCTAGAGTCCTTATCTTCTAATAAGGAGCTCTAGGC are provided.

The invention also provides a kit for detecting the stem-based canker of rape by RAA fluorescence method, which comprises the probe, the upstream primer and the downstream primer;

the upstream primer is as follows:

5’-CGCCTAGGTTGCCCTAACCTAGAATCTATAAG-3’；

the downstream primer is:

5’-CTRCTACTGCTGCTAAGCTTACGCGCAAGGC-3’。

as an improvement of the RAA fluorescence detection kit for the stem-base canker of rape, the storage concentration of the probe and the primer in the kit is not limited, and for the accuracy of the test, the final concentration of the probe in a reaction system is 0.02-0.05 mM when the probe is used, and the final concentration of the upstream primer and the downstream primer in the reaction system is 0.05-0.1 mM when the upstream primer and the downstream primer are used.

The kit for detecting the stem-based canker pathogenic bacteria of the rape RAA by the fluorescence method is further improved as follows: the kit also comprises a RAA basic fluorescence universal reaction reagent and a reaction buffer solution.

Wherein: the RAA basic fluorescence universal reaction reagent is freeze-dried powder which is subjected to low-temperature freeze drying and is provided by Jiangsu Qitian gene biotechnology limited; is a basic reaction unit with a commodity number of F08017. The skilled person can also select other products capable of serving as RAA-based fluorescence universal reagents as alternatives according to the principle of the RAA fluorescence detection method.

As a further improvement of the RAA fluorescence detection kit for the phomopsis brassicae of the invention, the reaction buffer solution consists of the following reagents: the storage concentration in the kit is not limited, and for the accuracy of the test, Tris-HCl Buffer (pH7.6) with the concentration of 450mM, MgAc with the concentration of 260mM and PEG 10000 with the concentration of 10% W/V (namely, 10g/100ml) are generally used as each component in the reaction Buffer; the balance being water as solvent.

As a further improvement of the rape stem-based ulcer germ RAA fluorescence detection kit, the kit also comprises the following reagents: a negative quality control material, a positive quality control material and/or a critical positive quality control material;

further, the positive quality control substance is a genomic DNA fragment containing the Brassica napobrassica var, the storage concentration of the positive quality control substance in the kit is not limited, and the use concentration of the positive quality control substance is 1.0 × 10 in general for the accuracy of the test⁴copies/uL; any concentration higher than the concentration can be selected as a positive quality control product;

the critical positive quality control substance is a genome DNA fragment containing the rape stem-based canker pathogen, the storage concentration of the critical positive quality control substance in the kit is not limited, and for the accuracy of the test, the use concentration of the critical positive quality control substance is 1.0 multiplied by 10 under the general condition³copies/uL；

The negative quality control product is a reagent (namely, a DNA plasmid without the genome fragment of the phomopsis brassicae) which does not contain the genome fragment of the phomopsis brassicae.

Specifically, the invention discloses a probe for detecting sclerotinia rot of rape by an RAA fluorescence method, wherein the nucleotide sequence of the probe is as follows: CTTAGAGGAGCTAGAGTCCTTATCTTCTAATAAGGAGCTCTAGGC, the probe is modified in the following manner:

5 'terminal sequence/fluorescent reporter group// A THF// quencher group/3' terminal sequence.

The probes specifically used in the present invention were synthesized by modification as follows:

5’-CTTAGAGGAGCTAGAGTCCTTATCTTC/i6FAMdT//A THF//iBHQdT/AAGGAGCTCTAGGC-3’。

the design key points are as follows: the fluorescent probe is modified in the middle of the probe, a fluorescent reporter group is modified at the position 28bp away from the 5 'end base number, a quencher group is modified at the position 15bp away from the 3' end base number, and the fluorescent reporter group and the quencher group are separated by 2 base positions, wherein 1 base is used for modifying tetrahydrofuran residues.

The method for detecting the phomopsis brassicae by using the kit comprises the following steps:

s1, extracting DNA of a sample to be detected; extracting the DNA of the sclerotinia rot germs by a CTAB method, or extracting the DNA of a sample to be detected by a commercialized DNA extraction kit;

s2, preparation of reaction Buffer: taking 47 mu L of reaction buffer solution, adding 2 mu L of mixture of the probe and the primer, and fully and uniformly mixing; adding the prepared 49 mu L of reagent into the split-packaged and freeze-dried RAA basic fluorescence universal reaction reagent freeze-dried powder, and fully dissolving and uniformly mixing the reagent; then adding 1 mul of extracted DNA of a sample to be detected as a template, wherein the total volume is 50 mul, and fully and uniformly mixing;

s3, putting the mixture into a fluorescence detection instrument for detecting FAM to perform real-time RAA fluorescence reaction; the real-time RAA fluorescence method reaction conditions are set as follows: the reaction temperature is 39 ℃, and the reaction time is 5-20 min;

s4, result analysis:

within the reaction time (for example, 20min), the signal detected by the FAM fluorescence detector is obviously increased, the increase is 30% of the initial amount of fluorescence, and the amplification is judged to be positive; when the signal of the FAM fluorescence instrument is not increased during the reaction time (for example, 20min), the FAM fluorescence instrument is judged to be negative.

Compared with the prior art, the invention has the beneficial effects that: the invention solves the problems of time and labor waste, long time period, low efficiency, requirement of professional and expensive instruments, high false positive rate and the like in the detection of the rape stem-base canker pathogen by the detection method in the prior art. The invention adopts RAA technology, and has the characteristics of short detection time, high sensitivity, strong specificity, simple operation and the like. The result can be analyzed only by reacting for 5-20 minutes at 39 ℃. Has the characteristics of rapidness, sensitivity, simple and convenient operation and the like, has very important significance for detecting the spread of the rape stem-based canker pathogen in the future, and has great application prospect.

In conclusion, the method can be used for rapidly detecting the stem-based canker of the rape by utilizing the RAA technology, is simple and convenient to operate, greatly shortens the used time, does not need large-scale instruments and equipment, and is suitable for large-scale screening. The detection method provided by the invention has the advantages of high sensitivity, good specificity, simple and rapid operation and low requirement on the quality of detection materials.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a graph of the detection amplification with sensitivity in example 1 of the present invention;

FIG. 2 is an amplification chart of the detection of an actual sample of the phoma brassicae of the present invention;

FIG. 3 is the detection and amplification chart of the specificity of the example of the invention for the phoma brassicae samples.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The inventive content is not limited to this.

In the following examples, both the primer probe and the DNA plasmid were synthesized by Biotechnology engineering (Shanghai) Ltd;

example 1 sensitivity of RAA fluorescence detection method for Phosphaera rapae:

selecting a specific conserved gene of the phomopsis brassicae as a target detection gene, obtaining a gene sequence of the phomopsis brassicae through the National Center for Biotechnology Information (NCBI) (http:// www.ncbi.nlm.nih.gov), performing multi-sequence comparison, and selecting a segment of conserved sequence from the gene sequence, wherein the sequence is as follows: TACGCGTAAGAAGCGTGCCTTAGAGTCTATAGGGAGCCGCCTAGGTTGCCCTAACCTAGAATCTATAAGGGGAACCTTAGAGGAGCTAGAGTCCTTATCTTCTAATAAGGAGCTCTAGGCGCCCTTAGCTATAGTATTAGCCTTGCGCGTAAGCTTAGCAGCAGTAGTAGTACCTTTTACTAGCTCCTACTATTTAGCCTTGCTCTCCTTAAGGAGCACTAAGACCCTCTGCTTCTTATCCTTAAGAAGGTCTTAGCTCTTACCTGCCTACTTCCTTGCTAGGAAGGATAGCGTAGAATATTAGGCAAGCTTAGGGCTATTAGTTTGTTAGTATAGATCTTACTAAGCGTTA, respectively; DNA plasmids were synthesized according to the above sequence of Competition Biotechnology engineering (Shanghai) Ltd, and the size of the plasmid was 352 bp.

Designing according to the RAA technical primer and probe design principle, and finally determining through screening and evaluation:

an upstream primer sequence 5'-CGCCTAGGTTGCCCTAACCTAGAATCTATAAG-3';

the downstream primer sequence 5 '-CTRCTACTGCTGCTAAGCTTACGCGCAAGGC-3';

the probe sequence and the modification method are as follows:

5’-CTTAGAGGAGCTAGAGTCCTTATCTTC/i6FAMdT//A THF//iBHQdT/AAGGAGCTCTAGGC-3’。

wherein, FAM is a fluorescence reporter group, BHQ is a fluorescence quenching group, THF is a tetrahydrofuran residue, and the modification method is FAM: 6-Carboxyfluoroscein; THF, tetrahydrofuran; BHQ is black hole queue; 3' phosphate to block interaction.

TABLE 1, table of information relating to primers and probes

TABLE 2 kit composition of the invention

In order to facilitate the sensitivity determination of the kit, the DNA plasmid of the stem-based ulcer germ synthesized by the company of Biotechnology engineering (Shanghai) is transformed into Escherichia coli, expanded and cultured at 37 ℃ for 8 hours, the DNA plasmid of the stem-based ulcer germ is extracted by a commercial plasmid extraction kit, and the concentration of the DNA plasmid is diluted to 10 after the concentration determination¹⁰Copies/ul for standby;

mu.l of 10¹⁰Copies/ul soybean stem ulcersPreparing the pathogen DNA plasmid into working standard products with different gradients, which are respectively as follows:

working standard 1, containing 1.0 × 10⁴DNA fragment of gene of Copies/ul rape stem base canker germ.

Working standard 2, containing 1.0 × 10³DNA fragment of gene of Copies/ul rape stem base canker germ.

Working standard 3, containing 1.0 × 10²DNA fragment of gene of Copies/ul rape stem base canker germ.

Working standard 4, containing 1.0 × 10¹DNA fragment of gene of Copies/ul rape stem base canker germ.

Preparing a reaction Buffer: adding 16 mu L of the mixture of the probe and the primer into 376 mu L of reaction buffer solution by suction, and fully and uniformly mixing; sucking and mixing uniformly, and then adding 49 mu L of reagent into 7 RAA basic fluorescence universal reaction reagent tubes (50 mu L of system freeze-dried powder) respectively to fully dissolve and mix uniformly the freeze-dried powder;

respectively adding 1 mu L of negative quality control material, 4 standard works, 3 standard works, 2 standard works and 1 standard works as templates into 5 prepared tubes, fully mixing each reaction tube uniformly, wherein the total volume of each reaction tube is 50 mu L;

the detection instrument adopts an RAA-F1620 fluorescence detector provided by Jiangsu Qitian gene biotechnology limited;

the instrument is set as follows: the reaction temperature was 39 ℃ and the reaction time was 20 minutes.

The mixed reaction tube was placed in a RAA-F1620 fluorescence detector and reacted at 39 ℃ for 20 minutes.

The results of the detection are shown in FIG. 1. The results show that the fastest 2 minutes amplification was evident, with all standard works amplified within 10 minutes.

Description of the drawings: when the fluorescence increase amount was 30% of the initial fluorescence amount, the sample was judged to be positive.

Example 2

The primer probes were the same as in example 1.

The kit was the same as in Table 2 of example 1.

Sample source and DNA extraction:

the sample is provided by the scientific and technical research institute of inspection and quarantine in Zhejiang province and is Canadian rapeseeds 1 and 2 and Australian rapeseeds 1 and 2; extracting DNA from seeds of local green vegetables and broccoli, and storing the extracted DNA at-80 ℃ for later use.

Preparing a reaction Buffer: taking 8 reaction tubes, respectively sucking 47 mu L of reaction buffer solution, adding 2 mu L of mixture of the probe and the primer, and fully and uniformly mixing; adding 49 mu L of the uniformly mixed reagent into an RAA basic fluorescence universal reaction reagent tube (50 mu L of system freeze-dried powder) to fully dissolve and uniformly mix the freeze-dried powder;

respectively adding 1 mu L of negative quality control substances and 1 mu L of extracted sample DNA (the concentration is 20 ng/mu L) into 8 prepared tubes, taking 1 mu L of positive quality control substances as a template, fully mixing each reaction tube uniformly, and ensuring that the total volume of each reaction tube is 50 mu L;

The results of the detection are shown in FIG. 2. The results showed that amplification was evident at 3 minutes. The positive signals of the rapeseeds in Canada and Australia appear, and the positive signals of the local green vegetables and broccoli seeds do not appear.

Example 3

The primer probe and the positive quality control substance sequence are the same as those in example 1.

The kit was the same as in Table 2 of example 1.

Same as watch 2

Sample source and DNA extraction:

the DNA of the stem-base canker pathogenic bacteria Leptosphaeria maculans, Leptosphaeria biglobosa, Alternaria brassicae, Alternaria alternata, Stachybotrys fulva, Stachybotrys fulvescens, Cercospora brassica, Phoma alphagladiata and Sclerotia sclerotiorum is provided by Zhejiang provincial institute, and the extracted DNA is stored at-80 ℃ for later use.

Extracting DNA of a sample to be detected; the extraction of the DNA of the sclerotinia rot pathogen of rape stem is carried out by a CTAB method, and the DNA of a sample to be detected can also be extracted by a commercial DNA extraction kit.

Preparing a reaction Buffer: taking a 1.5ml PE tube, sucking 423 mu L of reaction buffer solution and adding into the PE tube, sucking 18 mu L of mixture of the probe and the primer and adding into 423 mu L of reaction buffer solution, and fully and uniformly mixing;

respectively adding 49 mu L of the uniformly mixed prepared reaction liquid into 9 RAA basic fluorescence universal reaction reagent tubes (50 mu L of system freeze-dried powder) to fully dissolve and uniformly mix the freeze-dried powder; adding 49 mu L of the uniformly mixed buffer solution into an RAA basic fluorescence universal reaction reagent tube, and fully dissolving and uniformly mixing the freeze-dried powder;

respectively adding 1 mu L of negative quality control substances, Leptosphaeria maculans, Leptosphaeria brassicae biglobosa, Alternaria brassicae brassica, Alternaria alternata, Staphylotrichu fuliginea, Staphylotrichum fuliginea, Cercospora brassicae Cercospora brassiccus, Phoma grapevicola alpha globata and Sclerotinia sclerotiorum DNA into 8 prepared tubes, wherein 1 mu L of each DNA is used as a template, each reaction tube is fully mixed, and the total volume of each reaction tube is 50 mu L;

The results of the detection are shown in FIG. 3. The result shows that only the DNA of the samples of the sclerotinia rot pathogen of rape stem is obviously amplified, and other samples and negative quality control products are not amplified and are negative.

Example 3 shows that the kit of the invention has good specificity, and can distinguish the similar species of the phomopsis brassicae and the common diseases on rape.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

<110> scientific and technical research institute for inspection and quarantine in Zhejiang province

JIANGSU QITIAN GENE BIOTECHNOLOGY Co.,Ltd.

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gcccttagct atagtattag ccttgcgcgt aagcttagca gcagtagtag taccttttac 180

tagctcctac tatttagcct tgctctcctt aaggagcact aagaccctct gcttcttatc 240

cttaagaagg tcttagctct tacctgccta cttccttgct aggaaggata gcgtagaata 300

ttaggcaagc ttagggctat tagtttgtta gtatagatct tactaagcgt ta 352

Claims

A probe for detecting rape stem-based canker pathogen by an RAA fluorescence method is characterized in that:

the probe is 5 'terminal sequence/fluorescent reporter group// A THF// quenching group/3' terminal sequence; wherein:

the nucleotide sequence of the 5' end is as follows: CTTAGAGGAGCTAGAGTCCTTATCTTCT, respectively;

the nucleotide sequence of the 3' end is as follows: TAAGGAGCTCTAGGC, respectively;

the THF is at 1 base position.
A probe for detecting the phomopsis brassicae by an RAA fluorescence method is characterized in that: the fluorescent reporter group is selected from any one of FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy 5.
The probe for detecting the phomopsis brassicae by the RAA fluorescence method is characterized in that: the quenching group is selected from any one of TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
4. The probe for detecting phoma brassicae by the RAA fluorescence method according to claim 1, wherein the probe comprises: the nucleotide sequence of the probe is as follows:

CTTAGAGGAGCTAGAGTCCTTATCTTCTAATAAGGAGCTCTAGGC。
5. the kit for detecting the stem-based canker pathogenic bacteria of the rape by the RAA fluorescence method is characterized in that: the kit comprises the probe as claimed in any one of claims 1 to 4 and an upstream primer and a downstream primer;

the nucleotide sequence of the upstream primer is as follows: CGCCTAGGTTGCCCTAACCTAGAATCTATAAG

The nucleotide sequence of the downstream primer is as follows: CTRCTACTGCTGCTAAGCTTACGCGCAAGGC are provided.
6. The RAA fluorescence detection kit for phomopsis brassicae according to claim 5, wherein the RAA fluorescence detection kit comprises: the final concentration of the probe is 0.02-0.05 mM, and the final concentrations of the upstream primer and the downstream primer are both 0.05-0.1 mM.
7. The RAA fluorescence detection kit for phomopsis brassicae according to claim 5 or 6, wherein the RAA fluorescence detection kit comprises: the kit also comprises a RAA basic fluorescence universal reaction reagent and a reaction buffer solution.
8. The RAA fluorescence detection kit for phomopsis brassicae according to claim 7, wherein the RAA fluorescence detection kit comprises: the reaction buffer comprises: Tris-HCl Buffer (pH7.6) at a concentration of 450 mM; 260mM of MgAc; 10% W/V PEG 10000.
9. The RAA fluorescence detection kit for phomopsis brassicae according to claim 8, wherein the RAA fluorescence detection kit comprises: the kit also comprises the following reagents: a negative quality control material, a positive quality control material and/or a critical positive quality control material;

the positive quality control product is a genome DNA fragment containing the rape stem base canker pathogen, and the concentration is 1.0 multiplied by 10⁴Copies/μl；

The critical positive quality control product is a genome DNA fragment containing the rape stem base canker pathogen, and the concentration is 1.0 multiplied by 10³Copies/μl；

The negative quality control product is a reagent which does not contain genome fragments of the sclerotinia rot pathogen of rape.
10. A method for detecting phoma brassicae using the kit of any one of claims 5 to 9, comprising the steps of:

s1, extracting DNA of a sample to be detected;

s2, preparation of reaction Buffer: taking 47 mu L of reaction buffer solution, adding 2 mu L of mixture of the probe and the primer, and fully and uniformly mixing; adding 49 mu L of the prepared reagent into a tube filled with the freeze-dried RAA basic fluorescence universal reaction reagent freeze-dried powder, and fully dissolving and uniformly mixing the reagent; then adding 1 mul of extracted DNA of a sample to be detected as a template, wherein the total volume is 50 mul, and fully and uniformly mixing;

s3, putting the mixture into a fluorescence detection instrument for detecting FAM to perform real-time RAA fluorescence reaction; the real-time RAA fluorescence method reaction conditions are set as follows: the reaction temperature is 39 ℃, and the reaction time is 5-20 min;

s4, result analysis:

the signal detected by the FAM fluorescence detector is obviously increased within the reaction time, the increase is 30 percent of the initial amount of fluorescence, and the amplification is judged to be positive; within the reaction time, the signal of the FAM fluorescence instrument is not increased, and the FAM fluorescence instrument is judged to be negative.