CN115976254A - RPA-LFD primers and detection method for plant pathogen Fusarium oxysporum - Google Patents

Abstract

The invention belongs to the fields of biotechnology and plant disease epidemiology. The invention discloses a composition for detecting plant pathogenic Fusarium oxysporum based on the RPA-LFD method, including a primer pair and a probe for detecting the pathogen. The present invention also simultaneously discloses an RPA-LFD kit for rapidly detecting plant Fusarium oxysporum comprising the above-mentioned primer pair and probe, and discloses a method for rapidly detecting plant pathogens such as strawberries using the above-mentioned kit. The present invention establishes a rapid, specific, and highly sensitive quantitative monitoring technology system for strawberry stem rot, and the establishment of the method is very useful for early diagnosis, early warning, forecasting, and prevention and control of diseases caused by FOSC such as stem rot. Significance.

Description

RPA-LFD primers and detection method for plant pathogen Fusarium oxysporum

technical field

The invention belongs to the field of biotechnology and plant disease epidemiology, and relates to the detection of recombinase polymerase amplification (recombinase ploymerase amplification, RPA) combined with lateral flow dipstick (LFD) for monitoring plants, soil and other places. The recombinase polymerase amplification primer set of pathogenic Fusarium oxysporum and its application method belong to the technical field of plant disease epidemiology, dynamic monitoring and early warning.

Background technique

Fusarium sp. is an important fungus in soil, and Fusarium oxysporum Species Complex (FOSC) has a wide range of hosts and strong pathogenicity, and is listed as one of the top ten plant pathogenic fungi in the world. , Serious threat to the production of plants such as eggplant, capsicum and other economic plants.

The inventor's research group found that strawberry stalk rot caused by FOSC is the most serious disease to strawberry production at present in the research of the early stage of the invention. The vascular bundles at the base of the stem of the infected plant turn brown, and the aboveground part of the plant wilts because it cannot absorb water, causing considerable economic losses. The pathogen will be transmitted from the mother plant to the seedlings through the stolons, resulting in a large number of dead seedlings after transplanting. Disinfecting the soil before transplanting strawberries and root irrigation during transplanting are the main means to prevent and control strawberry stem rot. It is very important and can have an intuitive guiding significance for scientific drug use. The inventor's research group also found that the saffron bulb rot caused by FOSC is also the most serious disease in saffron production in the research of the early stage of the invention, and one of its main sources of infection is the bulb. It is of great significance to quickly test the bulbs, soil, etc., and carry out precise control based on the test results.

With the development of molecular technology, molecular detection technologies such as polymerase chain reaction (PCR) and quantitative PCR have been successfully used to detect plant pathogens. However, PCR detection requires expensive materials and instruments, takes a long time and has low detection sensitivity. Therefore, developing a rapid and portable method for pathogen detection is of great significance to the sustainable development of agricultural production. Loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) is a new, convenient, fast, highly sensitive and cheap nucleic acid amplification method. LAMP does not require thermal denaturation and can complete the reaction under constant temperature conditions. . However, LAMP detection generally takes about 1 hour at a temperature of 40-60°C to complete the detection. At present, in the field of detection of plant pathogenic fungi, LAMP detection of botrytis cinerea, rice blast, gibberella and other diseases has been reported. RPA is a newly invented new technology for in vitro nucleic acid isothermal amplification detection. Compared with LAMP, its most significant advantage is that the reaction can be completed within 5-20 minutes at 25°C-43°C similar to room temperature. The test result can be observed within 5 minutes of the lateral flow test strip. Moreover, the operation is simple and the equipment cost is low. There are relatively few reports on the field of RPA detection of plant pathogenic fungi. Therefore, it has better application potential in portable nucleic acid detection.

Although rapid detection techniques such as LAMP detection of various plant pathogenic fungi have been reported, there are problems such as false positives in actual field detection of actual samples, resulting in little practical application. One of the main reasons leading to this result is that most rapid detection technologies use highly conserved factors such as ITS, tublin, and EF as detection primers for design. In fact, these single factors with high homology among different fungi are not enough to distinguish individual species. And in the same species, there are many SNPs in such factors, which makes this kind of technology feasible under the strictly controlled environmental conditions in the laboratory and small groups of pathogens, but in the actual field environment, the false positive rate of detection results and High error rate.

Therefore, one of the key points and difficulties of plant pathogenic fungus RPA technology is to find out the specificity factors unique to different species to be detected, and the specific primers designed according to the specificity factors can be truly specific in field detection.

Contents of the invention

The technical problem to be solved by the present invention is to provide an RPA-LFD primer and detection method for the plant pathogen Fusarium oxysporum.

In order to solve the above-mentioned technical problems, the present invention provides a composition for rapid detection of FOSC, said composition comprising primer pairs and probes for detection;

The primer pair:

Upstream primer: 5'-TCAACTGGCATCGTCAACATCACCGAAGTAA-3'

Downstream primer: 5'-BIOTIN-CCAGGCATGACGAAGTTGATAGGTTGAAAGC-3'

The probe sequence:

5'-6-FAM-GAGGATGTTGATTGCTATTCTGATGGGTGGG/idSp/CAGCACAACACTGCTGCTA-C3Spacer-3'.

The present invention also simultaneously provides a kind of RPA-LFD kit that is used for fast detection plant Fusarium oxysporum, and described RPA-LFD kit comprises above-mentioned composition, also comprises reaction A buffer (reconstitution buffer), B buffer (magnesium acetate), HybriDetect colloidal gold test strips, ddH ₂ O.

That is, the RPA-LFD kit according to the present invention includes the concentration of the recombinase polymerase amplification primer mixture and the LFD primer mixture: 10 μM forward primer, 10 μM reverse primer, 10 μM probe, A buffer , B buffer, ddH ₂ O. The detection solution was 48 μL in total, and 2 μL of the DNA template to be tested (concentration: 10 ng/μL) was added to form a 50 μL detection reaction system. The 50 μL reaction system contained 29.4 μL of A buffer, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 0.6 μL of 10 μM probe, 2.5 μL of B buffer, 2 μL of DNA template, and double distilled water to make up to 50 μL.

The present invention also provides a method for quickly detecting plant pathogens such as strawberries by using the above kit, comprising the following steps;

(1) Extract sample nucleic acid;

(2) Use the composition to perform RPA amplification reaction on the sample nucleic acid extracted in step (1). The 50 μL system contains 29.4 μL of Abuffer, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, and 0.6 μL of 10 μM probe, 2.5 μL of B buffer, 2 μL of DNA template (for both positive control and negative control, the concentration is 10 ng/μL), make up to 50 μL with double distilled water; the RPA-LFD amplification reaction program is: 39 ° C, 8 min;

(3) Take 10 μL of the reaction product obtained in step (2) and add it to 190 μL ddH ₂ O. After mixing, take 50 μL of the diluted amplification product and drop it into the sample hole of the HybriDetect colloidal gold test strip. Record the control line and Detection line, determine the test result (interpretation result):

If the control line is visible but the detection line is invisible, this is a negative result, that is, it is determined that the test object corresponding to the DNA template does not contain the plant pathogen Fusarium oxysporum;

If both the detection line and the control line are visible, this is a positive result; that is, it is determined that the test object corresponding to the DNA template contains the plant pathogen Fusarium oxysporum;

If neither the detection line nor the control line is visible, it means that the experiment operation is wrong or the kit is damaged, and the experiment needs to be repeated.

The recombinase polymerase amplification (RPA-LFD) method, primer probe composition and kit for monitoring FOSC in plants, soil, etc. of the present invention are expected to complete the RPA reaction and LFD detection within 10min, thereby obtaining more efficient It is fast, easy to operate, sensitive, and does not need to rely too much on professional equipment and professional technicians. It is a visual detection method suitable for rapid field screening and provides technical support for early diagnosis of diseases. It belongs to the latest point-of-care ) rapid detection technology has broad application prospects.

Although RPA is an existing technology, the technical solution of the present invention cannot be easily obtained. The present invention designs, screens and invents primer pairs and probes capable of clinical detection (point-of-care) rapid diagnosis of FOSC based on the specificity factors of plant pathogenic FOSC, and its supporting detection technical conditions. The method of the invention can monitor whether plants, soil, seeds and seedlings contain FOSC germs, overcomes the shortcomings of previous PCR and LAMP detection methods, and has the advantages of high sensitivity, high specificity, fast timeliness and the like.

The invention process of the present invention comprises:

(1) Design specific primers and probes according to the difference between the FOSC genome sequence and other bacterial species;

(2) Screening of primers;

(3) Specific detection of primers and probes;

(4) Optimization of RPA-LFD reaction conditions;

(5) Detection sensitivity of RPA-LFD.

details as follows:

1. Design of primers and probes:

Select common phytopathogens in soil and plants, especially other Fusarium: F. tricinctum, F. fujikuroi, F. graminearum, Fusarium solani (F.solani), F. proliferatum, F. equiseti, Stagonosporopsis sp., Phoma sp., other common pathogenic fungi cryptic anthracnose (CoIletotrichum.aenigma ), C. fructicola, C. gloeosporioides, C. siamense, Botrytis cinerea to design FOSC-specific primers, and find primers by screening The suitable conditions are 31-32bp in length. Too short primers will affect the amplification speed and detection sensitivity; too long amplified fragments will form secondary structures and affect the amplification. The present invention has discovered several phytopathogenic FOSC-specific factors through the comparative genomics research between FOSC and other various strains, and considering specificity, accuracy and practical feasibility, etc., selects one of them in the phytopathogenic The unique and specific biological functions of FO are not clear. Factors that do not exist in other common pathogenic fungi, especially in the detection environment, are designed on the basis of genetic analysis of FOSC populations (N>100) and according to sequence differences. Primers, including 3 pairs of primers and 1 probe as shown in Table 1:

Table 1 Primer sequence information

2. Screening of primers

PCR uses a 25 μl reaction system, as follows:

Table 2 PCR reaction system

The PCR amplification program was: 3min pre-denaturation at 95°C, denaturation at 95°C for 15s, annealing at 60°C for 15s, extension at 72°C for 15s, 35 cycles, and PCR products were verified on 2.0% agarose gel at 254nm (UV).

After multiple rounds of screening, the results showed that since the result corresponding to primer 1 was positive and stable, while the results corresponding to primer pair 2 and primer pair 3 were negative or unstable, therefore, primer pair 1 in Table 1 is suitable for use in RPA detection of FOSC, while primer pair 2 and 3 are not suitable.

The screened primer pairs were tagged with Biotin, and purified using HPLC like the corresponding probes, as shown in Table 3

Table 3 RPA-LFD detection primer probe label

3. Specific detection of primers and probes

In order to verify the specificity of primers and probes, use primer pair 1 and probes in Table 3 to perform RPA-LFD detection on each strain. The reaction system is 50 μL: A buffer 29.4 μL, FOSC template DNA and ddH ₂ O total 13.5 μL , 2 μL of each primer, 0.6 μL of probe, 2.5 μL of B buffer, when B buffer is added, the reaction starts immediately, the mixed solution is inverted 6-8 times to mix well, and reacted at a constant temperature of 38°C for 10 minutes. After amplification, use LFD test paper strips are tested. For the tested FOSC samples, the product can be amplified, and the detection line and control line of the HybriDetect colloidal gold test strip can be seen; for other species of bacteria to be detected, the product cannot be amplified, and the HybriDetect colloidal gold test strip detects that the product cannot be amplified. The lines are invisible and only the control lines are visible. The reaction result is shown in Figure 2, which further demonstrates that the primer pair 1 and the probe combination of the present invention have specificity to FOSC.

Reference strains include F. tricinctum, F. fujikuroi, F. graminearum, F. solani, F. proliferatum), F.equiseti, C.aenigma, C.fructicola, C.gloeosporioides, C.siamense ), Botrytis cinerea, Stagonosporopsis sp., Phoma sp.

4. Optimization of RPA-LFD reaction conditions

For the purpose of establishing the optimal reaction time and optimal reaction temperature of RPA-LFD for rapid detection of FOSC using the primer pair 1 and probe combination of the present invention, the above primer pair 1 and probe were used to add a concentration of 10 ng/ For 2 μL of template, the reaction temperatures were set to 33°C, 36°C, 39°C, 42°C, 45°C, 48°C and 51°C, respectively, and the reaction time was 10 min. The detection results of the amplification products are shown in Figure 3a. Between 39°C and 45°C, there are clear bands in the detection area of the HybriDetect colloidal gold test strip. According to the stability of the reaction, we choose 39°C as the optimal reaction temperature. Set the reaction time to 4, 6, 8, 10, 12, and 14 minutes respectively, and the reaction temperature to 39°C. The detection results of the amplified products are shown in Figure 3b. There are clear Strip, combined with reality, choose 8min as the best time. Subsequently, 39°C for 8 min was used as the optimal reaction condition for FOSC.

5. Sensitivity verification of the detection system

Using the optimized FOSC RPA-LFD detection system for strawberry stem rot fungus to detect the DNA solution of the gradient dilution of the pure mycelia of the strawberry stem rot fungus, the results showed that at the DNA concentration of 10ng/μL, 1ng/μL, 100pg/μL, 10pg /μL and 1pg/μL, both can be effectively amplified, and there is still a band in the detection area of the HybriDetect colloidal gold test strip. The results show that the theoretical detection line of the RPA-LFD detection system is 1pg. /μL DNA.

The present invention establishes a rapid, specific, and highly sensitive quantitative monitoring technology system for strawberry stem rot, and the establishment of the method is very useful for early diagnosis, early warning, forecasting, and prevention and control of diseases caused by FOSC such as stem rot. Significance.

Compared with the prior art, the present invention has the following technical advantages:

(1) Strong specificity and rapid identification: The results of the detection method show that common fungi are all negative results, and only FOSC such as Strawberry Stem Root Rot have positive results. The entire detection process only takes less than 10 minutes, which overcomes the shortcomings of requiring a lot of professional knowledge to make judgments in the traditional tissue separation process, and the results are difficult to judge, and overcomes the limitation of long time required by ordinary PCR and LAMP.

(2) High sensitivity: Sensitivity detection is carried out through hyphae genome DNA. RPA-LFD technology can detect the lowest concentration of pathogens at 1pg/μL, and the sensitivity is significantly higher than ordinary PCR and LAMP detection. It overcomes the shortcomings of high detection limit of ordinary PCR and LAMP.

(3) Good practicability and wide application range: it can monitor germs in plants, soil, etc. It is suitable for dynamic monitoring of germs and provides a basis for its prediction and forecast.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Fig. 1 is the determination of primer specificity;

In Figure 1:

a is the assay result of the primer pair numbered 1; b is the assay result of the primer pair numbered 2; c is the assay result of the primer pair numbered 3;

Lane Maker: DNA Maker; Lane 1-10: Fusarium oxysporum (F.oxysporum); Lane 11: Fusarium tricinctus (F.tricinctum); Lane 12: Fusarium tengura (F.fujikuroi); Lane 13: Grass F. graminearum; Lane 14: F. solani; Lane 15: F. proliferatum; Lane 16: F. equiseti; Lane 17 Lane 18: C. fructicola; Lane 19: C. gloeosporioides; Lane 20: C. siamense; 21: Botrytis cinerea; lane 22: Stagonosporopsis sp.; lane 23: Phoma sp.; lane 24: blank control.

Fig. 2 is the determination of primer probe combination specificity;

1-10: F. oxysporum; 11: F. tricinctum; 12: F. fujikuroi; Lane 13: F. graminearum; 14: Fusarium solani (F.solani); 15: Fusarium solani (F.proliferatum); 16: Fusarium equiseti (F.equiseti); 17: Anthracnose occultum (C.aenigma); 18: Fruit C. fructicola; 19: C. gloeosporioides; 20: C. siamense; 21: Botrytis cinerea; 22: Stagonosporopsis sp.; 23: Phoma sp.; 24: blank control.

Fig. 3 is the impact diagram of reaction temperature and time on the RPA-LFD detection system of FOSC;

a: Effect of different time on RPA-LFD detection; 1-7: Reaction temperatures are 33°C, 36°C, 39°C, 42°C, 45°C, 48°C and 51°C;

b: Effects of different temperatures on RPA-LFD detection; 1-6: The reaction times are 4, 6, 8, 10, 12, and 14 min, respectively.

Figure 4 is the sensitivity diagram of the RPA-LFD detection system; 1-6: the concentrations are 10ng/μL, 1ng/μL, 100pg/μL, 10pg/μL, 1pg/μL, 100fg/μL, respectively.

Figure 5 is the RPA-LFD detection results of different field samples;

1: positive control, 2-5: strawberry samples from which FOSC was isolated; 6-9: strawberry samples from which FOSC was not isolated.

Detailed ways

The present invention will be further described below through the embodiments and in conjunction with the accompanying drawings. The following description does not limit the protection scope of the present invention in any sense, but is only for illustration.

The main reagents and instruments used in the following examples are: A buffer (Anpu Future Biotechnology Co., Ltd.), B buffer (Anpu Future Biotechnology Co., Ltd.), HybriDetect colloidal gold test strip (Anpu Future Biotechnology Co., Ltd. ), ddH ₂ O water, molecular mass standard DNA Maker (TaKaRa Bioengineering Company Genome), PEG200 (Sangon Bioengineering Co., Ltd.), PCR instrument.

Embodiment 1, the screening of specific primer

First, based on the FOSC genome sequence, by comparing the common genera in soil and plants: three-line Fusarium (F.tricinctum), Fujikura Fusarium (F.fujikuroi), Fusarium graminearum (F.graminearum,), solanaceous F. solani, F. proliferatum, F. equiseti, Stagonosporopsis sp., Phoma sp., other pathogenic fungi commonly found on strawberries (CoIletotrichum.aenigma), C. fructicola, C. gloeosporioides, C. siamense, Botrytis cinerea sequence difference sites were designed 3 Specific primers and 1 probe for Fusarium oxysporum, as described in Table 1.

The specificity of the primers described in Table 1 was verified using conventional PCR, and the reaction system was described in Table 2.

The PCR amplification program was: 3min pre-denaturation at 95°C, denaturation at 95°C for 15s, annealing at 60°C for 15s, extension at 72°C for 15s, 35 cycles, and PCR products were verified on 2.0% agarose gel at 254nm (UV). The amplification results are shown in Figure 1. Primer pair 1 can only amplify a homozygous target fragment for the stem rot pathogen, while other genera cannot amplify a band or have mixed bands. The size of the amplified fragment of primer pair 1 was 167bp, and the coincidence degree was 100% verified by sequencing.

The screened primer pairs were tagged with Biotin and purified using HPLC as the corresponding probes, as described in Table 3.

Right now,

The primer pair:

Upstream primer: 5'-TCAACTGGCATCGTCAACATCACCGAAGTAA-3'

Downstream primer: 5'-Biotin-CCAGGCATGACGAAGTTGATAGGTTGAAAGC-3'

The probe sequence:

5'-6-FAM-GAGGATGTTGATTGCTATTCTGATGGGTGGG/idSp/CAGCACAACACTGCTGCTA-C3Spacer-3'.

Embodiment 2, the specificity analysis of detection system

The specificity analysis of the detection system was carried out by using the labeled primers and probes obtained after screening. F. oxysporum, which caused diseases such as strawberry stem rot and saffron bulb rot, was used as a positive control, and three-line Fusarium (F. tricinctum), Fusarium tengura (F.fujikuroi), Fusarium graminearum (F.graminearum), Fusarium solani (F.solani), Fusarium solani (F.proliferatum), Fusarium equisetum (F. equiseti), C. aenigma, C. fructicola, C. gloeosporioides, C. siamense, Botrytis cinerea, Stagonosporopsis sp., Phoma sp., 13 kinds of fungi were used as negative controls, and ddH ₂ O was used as blank control for testing.

The detection solution was 48 μL in total, and 2 μL of the DNA template to be tested (concentration: 10 ng/μL) was added to form a 50 μL detection reaction system. The 50 μL reaction system contained 29.4 μL of A buffer, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 0.6 μL of 10 μM probe, 2.5 μL of B buffer, 2 μL of DNA template (for positive control and negative control, the concentration Both were 10ng/μL), ddH ₂ O was added to 50μL. The RPA-LFD amplification reaction program is: 39°C, 8min,

The amplified product was detected with HybriDetect colloidal gold test strip. After the amplification was completed, 10 μL of the amplified product was added to 190 μL ddH ₂ O. After mixing, 50 μL of the diluted amplified product was dropped into the HybriDetect colloidal gold test strip. Sample hole, record control line and detection line interpretation result in 5min, reaction result is as shown in Figure 2, with the sample detection line of stem base rot fungus DNA as template visible, detection result is positive; With other 13 kinds of fungi DNA as template and The detection line of the ddH ₂ O control sample was not visible, and the test results were all negative. The detection results showed that the screened RPA-LFD primer-probe set could specifically detect the stem rot pathogen.

Embodiment 3, the optimization of RPA-LFD detection condition

Use the screened RPA-LFD primer probe set to optimize the detection conditions. The optimization results are shown in Figure 3. When the reaction time is 10 minutes, the detection area of the HybriDetect colloidal gold test strip has a clear According to the stability of the reaction, 39°C was selected as the optimal reaction temperature, and there were clear bands in the detection area of the 8-10min HybriDetect colloidal gold test strip. The results showed that the established RPA-LFD detection system was the best The reaction conditions were 39°C, 8min.

The optimized reaction system and amplification conditions are:

The 50 μL reaction system contains 29.4 μL of A buffer, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 0.6 μL of 10 μM probe, 2.5 μL of B buffer, and 2 μL of DNA template (for positive control and negative control, the concentrations are 10ng/μL), ddH ₂ O made up to 50μL. The RPA-LFD amplification reaction program is: 39°C, 8min.

Forward primer: 5'-TCAACTGGCATCGTCAACATCACCGAAGTAA-3'

Reverse primer: 5'-Biotin-CCAGGCATGACGAAGTTGATAGGTTGAAAGC-3'

Probe sequence:

5'-6-FAM-GAGGATGTTGATTGCTATTCTGATGGGTGGG/idSp/CAGCACAACACTGCTGCTA-C3Spacer-3'.

Embodiment 4, the sensitivity detection of primer

With the optimized FOSCRPA-LFD detection system and conditions (as described in Example 3), the detection results of the serially diluted bacterial DNA solution are shown in Figure 4, when the DNA concentration is 10ng/μL, 1ng/μL, 100pg/μL, At 10pg/μL and 1pg/μL, both can effectively amplify, and there is still a band in the detection area of the HybriDetect colloidal gold test strip, and the repeatability of the same sample is reliable. The results show that the detection limit of the established RPA-LFD detection system for FOSC 1pg/μL DNA.

Embodiment 5, the plant of simulation inoculation FOSC is detected

Use sterilized insects to stab the base of 5-week-old healthy strawberry tissue-cultured seedlings, spray 5mL of FOSC spore liquid with a concentration of 10 ⁶ cfu/mL at the base of each strawberry stem, and place it in a light culture at 28°C±5°C Daytime at 25°C for 12h/darkness at 25°C for 12h. After 5 days, use 20mM KOH-60% PEG 200 lysate to extract genomic DNA from diseased plants.

20mM KOH-60% PEG 200 lysate configuration method: the system is 40mL, 24mL PEG 200, 0.93mL 2MKOH, ddH ₂ O make up to 40mL, pH is 13.3-13.5, the extraction process is as follows: 10mg plant sample tissue (strawberry Stem base tissue) was added to 100 μL of 20 mM KOH-60% PEG 200 extract solution and soaked at room temperature for 15 minutes.

Take 1 μL of the lysate directly as a DNA template, perform amplification and detection according to the optimized reaction system and amplification conditions obtained in Example 3 above, and verify the detection results with the tissue separation results.

At the same time, strawberry tissue culture seedlings were inoculated with sterile water and anthrax bacteria as experimental controls (except for the change in the type of inoculated bacteria, the rest were the same as inoculated with FOSC spore liquid). The results obtained are as follows in Table 4:

Table 4

Test results Tissue separation results Subject 1 (vaccinated with FOSC) Positive Positive Experimental object 2 (vaccinated with sterile water) Negative Negative Subject 3 (vaccinated with anthrax bacteria) Negative Negative Subject 4 (vaccinated with F. graminearum) Negative Negative

The results in Table 4 show that RPA-LFD detection can accurately detect whether strawberry plants are infected with FOSC.

Embodiment 6, RPA-LFD detection field sample

In order to verify the field practicability of the established RPA-LFD method, eight strawberry stem bases were randomly collected from the field for RPA-LFD detection.

Strawberry samples from which FOSC was isolated were taken as samples 2 to 5, and strawberry samples from which FOSC was not isolated were taken as samples 6 to 9; the DNA sample of FOSC was used as a positive control (sample 1);

Use 20mM KOH-60%PEG 200 to quickly extract tissue genomic DNA (as described in Example 5), after the sample reacts at 39°C for 8min, use LFD to detect, the detection results are shown in Figure 5, the positive control and 4 strawberry samples wherein (Samples 2-5) had bands in both the control area and the detection area, and there were no bands in the detection area of 4 strawberry samples (samples 6-9). The test results were verified with the results of tissue separation. The results showed that RPA-LFD detection It can accurately detect whether grass plants are infected with FOSC.

Comparative example 1. Change the primers to the following:

Upstream primer: 5'-AACTGGCATCGTCAACATCACCGAAGT-3'

Downstream primer: 5'-BIOTIN-CATGACGAAGTTGATAGGTTGAAA-3'

The rest were detected in the same manner as in Example 4, and the results showed that when the DNA concentration was as low as 10pg/μL, there were still bands in the detection area of the HybriDetect colloidal gold test strip, so the detection limit of FOSC was 10pg/μL DNA.

Comparative example 2, change the primer to the following

Upstream primer: 5'-CTCAACTGGCATCGTCAACATCACCGAAGTAA-3'

Downstream primer: 5'-BIOTIN-CCAGGCATGACGAAGTTGATAGGTTGAAAGCTA-3'

The rest were detected in the same manner as in Example 2, and the results showed that only samples 1 and 3 were positive, and the rest were negative. Therefore, the detection result of this comparative example 2 is incorrect.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (6)

1. The composition for detecting the plant pathogenic fusarium oxysporum based on the RPA-LFD method is characterized by comprising a primer pair and a probe for detecting the pathogenic fusarium oxysporum;

the primer pair comprises:

an upstream primer: 5'-TCAACTGGCATCGTCAACATCACCGAAGTAA-3'

A downstream primer: 5'-Biotin-CCAGGCATGACGAAGTTGATAGGTTGAAAGC-3'

The probe sequence is as follows:

5’-6-FAM-GAGGATGTTGATTGCTATTCTGATGGGTGGG/idSp/CAGCACAACACTGCTGCTA-C3 Spacer-3’。

2. the composition of claim 1, wherein the downstream primer is labeled with Biotin at the 5' end, the fluorophore at the 5' end of the probe is FAM, and the fluorophore at the 3' end is C3 Spacer.

3. An RPA-LFD kit for rapid detection of fusarium oxysporum of a plant comprising the composition of claim 1 or 2.

4. The RPA-LFD kit according to claim 3, wherein: the RPA-LFD kit also comprises a reaction A buffer, a reaction B buffer, a HybriDetect colloidal gold test strip and ddH ₂ O。

5. A method for rapidly detecting Phytophthora infestans such as strawberry using the kit according to claim 3 or 4, comprising the steps of;

(1) Extracting sample nucleic acid;

(2) Carrying out RPA amplification reaction on the sample nucleic acid extracted in the step (1) by adopting a composition, wherein a 50 mu L system contains 29.4 mu L of A buffer, 2 mu L of 10 mu M forward primer, 2 mu L of 10 mu M reverse primer, 0.6 mu L of 10 mu M probe, 2.5 mu L of B buffer, 2 mu L of DNA template and double distilled water to make up to 50 mu L; the RPA-LFD amplification reaction program is as follows: at 39 ℃ for 8min;

(3) Taking 10 mu L of the reaction product obtained in the step (2), adding the reaction product into 190 mu L of ddH ₂ And O, dripping 50 mu L of diluted amplification product into a sample adding hole of a HybriDetect colloidal gold test strip after uniformly mixing, recording a control line and a detection line within 5min, and judging the test result.

6. The method for rapidly detecting Phytophthora infestans such as strawberry according to claim 5, wherein the method for determining the test result is as follows:

if the control line is visible, the detection line is invisible, and the negative result is obtained, namely, the object to be detected corresponding to the DNA template is judged to contain no plant pathogenic fusarium oxysporum;

if the detection line and the control line are both visible, the result is a positive result; namely, judging that the object to be detected corresponding to the DNA template contains plant pathogenic fusarium oxysporum;

if the detection line and the control line are invisible, the detection needs to be carried out again.

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