CN115976254A - RPA-LFD primer and detection method for plant pathogenic fusarium oxysporum - Google Patents
RPA-LFD primer and detection method for plant pathogenic fusarium oxysporum Download PDFInfo
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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 an RPA-LFD method, which comprises a primer pair and a probe for detecting the pathogenic fusarium oxysporum. The invention also discloses an RPA-LFD kit containing the primer pair and the probe for rapidly detecting the plant fusarium oxysporum, and a method for rapidly detecting the plant pathogenic bacteria such as strawberry by using the kit. The invention establishes a quantitative monitoring technical system with high speed, specificity and sensitivity for the strawberry stem rot, and the establishment of the method has very important significance for early diagnosis, early warning, prediction, prevention and control of diseases caused by FOSC such as stem rot and the like.
Description
Technical Field
The invention belongs to the field of biotechnology and plant disease epidemiology, and relates to a recombinase polymerase amplification primer set for detecting plant pathogenic fusarium oxysporum in places such as plants and soil by combining Recombinase Polymerase Amplification (RPA) technology with lateral flow test strip (LFD) detection and a using method thereof, belonging to the technical field of plant disease epidemiology, dynamic monitoring and early warning.
Background
Fusarium sp is an important fungus in soil, wherein Fusarium oxysporum Complex (FOSC) has wide hosts and strong pathogenicity, is listed as one of ten kinds of plant pathogenic fungi in the world, and seriously threatens the production of various vegetables such as eggplants, hot peppers and other plants in economy.
The research of the inventor in the prior period of invention finds that the strawberry stem rot caused by the FOSC is the disease which has the most serious harm to the strawberry production at present. The vascular bundles at the base of the infected plant stem become brown, the overground part of the plant wilts due to the fact that the plant cannot absorb water, considerable economic loss is caused, pathogenic bacteria can be transmitted to the sub-seedlings from the mother plant through stolons, and a large amount of seedling death phenomena occur after transplanting. The method is a main means for preventing and treating the strawberry stem rot by disinfecting soil before transplanting strawberries and irrigating roots during transplanting, is vital to detect whether fusarium oxysporum which can cause the strawberry stem rot exists in the soil and strawberry seedlings before using medicines, and can generate visual guiding significance for scientific medicine application. The research of the subject group of the inventor in the earlier stage of the invention also finds that the rot disease of the corm of the saffron caused by the FOSC is also the most serious disease in the production of the saffron, one of the main infection sources of the rot disease is seed balls, and if the detection can be rapidly carried out on the seed balls, soil and the like before planting and the accurate control can be carried out according to the detection result, the rot disease has important significance.
With the development of molecular techniques, molecular detection techniques such as Polymerase Chain Reaction (PCR) and quantitative PCR have been successfully used for detecting phytopathogens, however, PCR detection requires expensive materials and instruments, takes a long time, and has low detection sensitivity. Therefore, the development of a rapid and portable pathogenic bacterium detection method has important significance on the sustainable development of agricultural production. Loop-mediated isothermal amplification (LAMP) is a novel, convenient and rapid nucleic acid amplification method with extremely high sensitivity and low price, and the LAMP can complete reaction under the constant temperature condition without thermal denaturation. However, LAMP detection generally requires about 1 hour at a temperature of 40-60 ℃ to complete detection. At present, the LAMP detection of various diseases such as botrytis cinerea, rice blast, gibberellic disease and the like in the field of detection of plant pathogenic fungi is reported. RPA is a novel in vitro nucleic acid isothermal amplification detection technology which is newly invented, and compared with LAMP, the kit has the most remarkable advantages that the reaction can be completed within 5-20min under the condition that the temperature is 25-43 ℃ and is similar to room temperature, and the detection result can be observed by using a lateral flow test strip for 5 min. And the operation is simple, and the equipment cost is low. The detection field of the RPA to detect the plant pathogenic fungi is relatively few reports. Therefore, the method has better application potential in portable nucleic acid detection.
Although rapid detection technologies such as LAMP detection of various plant pathogenic fungi have been reported, the problems of false positive and the like exist when the method is actually applied to field detection of actual samples, so that the practical application is rare. One of the main reasons for this is that most rapid detection techniques use highly conserved factors such as ITS, tublin, EF, etc. as detection primers. Indeed, these single factors of high homology between different fungi alone are not sufficient to distinguish the individual species. In the same species, the factors have a plurality of SNPs, so that the technology is feasible under the environmental conditions strictly controlled by a laboratory and the population with small germs, and the false positive rate and the error rate of the detection result are high in the actual field environment.
Therefore, one of the key points and difficulties of the plant pathogenic fungus RPA technology is that specific factors unique to different species to be detected need to be found, and a specific primer designed according to the specific factors can really have specificity when being detected in the field.
Disclosure of Invention
The invention aims to solve the technical problem of providing an RPA-LFD primer and a detection method for plant pathogen fusarium oxysporum.
In order to solve the technical problems, the invention provides a composition for rapidly detecting FOSC, which comprises a primer pair and a probe for detection;
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’。
the invention also provides an RPA-LFD kit for rapidly detecting the fusarium oxysporum, which comprises the composition, a reaction A buffer (redissolving buffer), a reaction B buffer (magnesium acetate), a HybriDetect colloidal gold test strip and ddH 2 O。
That is, the RPA-LFD kit according to the present invention comprises recombinase polymerase amplification primer mixture and LFD primer mixture at concentrations of: 10 μ M forward primer, 10 μ M reverse primer, 10 μ M probe, A buffer, B buffer, ddH 2 And O. The total volume of the detection solution is 48 mu L, and 2 mu L of DNA template to be detected (the concentration is 10 ng/. Mu.L) is added to form a 50 mu L detection reaction system. The 50. Mu.L reaction 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 invention also provides a method for rapidly detecting plant germs such as strawberries and the like by using the kit, which comprises the following steps;
(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 and 2 mu L of DNA template (the concentration is 10 ng/mu L in the case of positive control and negative control), and double distilled water is added 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) and adding the reaction product into the reactor190μL 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 (interpretation result):
if the control line is visible and the detection line is invisible, the result is a negative result, 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 error of the experiment operation or the damage of the kit is indicated, and the experiment needs to be carried out again.
The recombinase polymerase amplification (RPA-LFD) method, the primer probe composition and the kit for monitoring the FOSC at plants, soil and the like complete RPA reaction and LFD detection within 10min, so that the visual detection method which is more efficient, rapid, simple and sensitive and does not depend on professional instruments and equipment and professional technicians is obtained, the visual detection method can be applied to field rapid screening, technical support is provided for early disease diagnosis, the method belongs to the latest Point-of-care rapid detection technology, and the method has wide application prospect.
Although RPA is prior art, the solution of the present invention is not readily available. The invention designs, screens and invents a primer pair and a probe which can rapidly diagnose the FOSC in clinical detection (point-of-care) according to the specific factor of the plant pathogenic FOSC, and a matched detection technical condition thereof. The method can monitor whether the plant, soil, seed and seedling contain FOSC germ, overcomes the defects of the traditional PCR and LAMP detection method, and has the advantages of high sensitivity, high specificity, quick time effect and the like.
The invention process comprises the following steps:
(1) Designing specific primers and probes according to the difference sites between the FOSC genome sequence and other strains;
(2) Screening a primer;
(3) Detecting the specificity of the primer and the probe;
(4) Optimizing the RPA-LFD reaction condition;
(5) Detection sensitivity of RPA-LFD.
The method comprises the following specific steps:
1. design of primers and probes:
the selection of the common phytopathogens in soil and plants, in particular other fusarium species: fusarium trilobum (f.tricinctum), fusarium graminearum (f.fujikuroi), fusarium graminearum (f.graminearum), fusarium solani (f.solani), fusarium solani (f.proliferum), fusarium equiseti (f.equiseti), stagonosporopsis sp, phoma sp., common cryptomorpha anthracis (coietiochloa) which is another pathogenic fungus, fusarium oleaginosum (c.fructicola), colletotrichum gloeosporioides (c.gloeosporioides), fusarium samaraense (c.siemenses), botrytis cinerea (Botrytis cinerea), FOSC specific primers were designed, and it was found by screening that the primer lengths were 31 to 32bp, respectively, that too short primers affected the amplification rate and detection sensitivity; an excessively long amplified fragment may form a secondary structure that affects amplification. The invention discovers several specific factors of the plant pathogenic FOSC through comparative genomics research between the FOSC and other various strains, comprehensively considers the reasons of specificity, accuracy rate, practical feasibility and the like, selects one of the specific factors with unclear specific biological function in the plant pathogenic FO, and designs a plurality of pairs of primers according to sequence difference on the basis of carrying out genetic analysis on the FOSC population (N > 100) by using factors which do not exist in other common pathogenic fungi, particularly in detection environments, wherein 3 pairs of primers and 1 probe are shown in a table 1:
TABLE 1 primer sequence information
2. Screening of primers
The PCR was carried out using a 25. Mu.l reaction system as follows:
TABLE 2 PCR reaction System
The PCR amplification procedure was: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 15s,35 cycles, PCR products were verified on a 254nm (UV) 2.0% agarose gel.
After multiple rounds of screening, the results showed that primer pair 1 in table 1 was suitable for the RPA detection of FOSC, while primer pairs 2 and 3 were not suitable, since primer pair 1 corresponded to positive and stable results, while the results corresponded to primer pair 2 and primer pair 3 were negative or unstable.
The selected primer pairs were labeled with Biotin and purified by HPLC as the corresponding probes, as shown in Table 3
TABLE 3 RPA-LFD detection primer Probe labeling
3. Specific detection of primers and probes
To verify primer and probe specificity, RPA-LFD detection was performed on each strain using primer pair 1 and probe in table 3, with a reaction system of 50 μ L: a buffer 29.4. Mu.L, template DNA of FOSC and ddH 2 13.5 mu L of O, 2 mu L of each primer, 0.6 mu L of probe and 2.5 mu L of B buffer, when the B buffer is added, the reaction starts immediately, the mixed solution is inverted for 6-8 times and mixed evenly, the constant temperature reaction is carried out for 10min at 38 ℃, and after the amplification is finished, the LFD test strip is used for detection. Aiming at the FOSC sample to be tested, products can be amplified, and a detection line and a control line of the HybriDetect colloidal gold test strip are visible; aiming at other species of germs to be detected, products cannot be amplified, the detection line of the HybriDetect colloidal gold test strip is invisible, only the control line is visible, the reaction result is shown in figure 2, and the combination of the primer pair 1 and the probe has the specificity on the FOSC.
Reference strains include fusarium trilobata (f.tricinctum), fusarium graminearum (f.fujikuroi), fusarium graminearum (f.graminearum), fusarium solani (f.solani), fusarium solani (f.proliferatum), fusarium equiseti (f.equiseti), fusarium graminearum (c.aesnigrum), anthracnose fructicola (c.fructicola), colletotrichum gloeosporioides (c.gloeosporioides), fusarium siamensis (c.siemense), botrytis cinerea (Botrytis cinerea), stagonospodopsis sp.
4. Optimization of RPA-LFD reaction conditions
Aiming at establishing the optimal reaction time and the optimal reaction temperature for rapidly detecting the RPA-LFD of the FOSC by using the combination of the primer pair 1 and the probe, 2 mu L of template with the concentration of 10 ng/mu L is added into a reaction system by using the primer pair 1 and the probe, the reaction temperature is respectively set to be 33 ℃, 36 ℃, 39 ℃, 42 ℃, 45 ℃, 48 ℃ and 51 ℃, and the reaction time is 10min. The detection result of the amplification product is shown in figure 3a, a clear band is formed in the detection area of the HybriDetect colloidal gold test strip at the temperature of 39-45 ℃, and the optimal reaction temperature is 39 ℃ according to the stability of the reaction. The reaction time was set to 4, 6, 8, 10, 12, and 14min, respectively, the reaction temperature was 39 ℃, the detection result of the amplification product was as shown in fig. 3b, a clear band was present in the 8-10min HybriDetect colloidal gold test strip detection zone, and 8min was selected as the optimum time in combination with the actual conditions. Subsequently, the best reaction condition of the FOSC is that the temperature is 39 ℃ for 8min.
5. Sensitivity verification of detection systems
The detection result of the optimized FOSC strawberry stem rot pathogen RPA-LFD detection system on the DNA solution of the gradient diluted pure hyphae of the stem rot pathogen shows that the DNA concentration can be effectively amplified when the DNA concentration is 10 ng/mu L,1 ng/mu L, 100 pg/mu L,10 pg/mu L and 1 pg/mu L, the detection area of the HybriDetect colloidal gold test strip still has a strip, and the result shows that the detection line of the stem rot pathogen DNA of the established RPA-LFD detection system is 1 pg/mu L of DNA theoretically.
The invention establishes a quantitative monitoring technical system with high speed, specificity and sensitivity for the strawberry stem rot, and the establishment of the method has very important significance for early diagnosis, early warning, prediction, prevention and control of diseases caused by FOSC such as stem rot and the like.
Compared with the prior art, the invention has the following technical advantages:
(1) Strong specificity and rapid identification: the detection method results show that common fungi are negative results, and positive results only occur to FOSC such as strawberry stem rot. The whole detection process is only within 10 minutes, the defects that a large amount of professional knowledge is needed to make judgment and the result is difficult to judge in the traditional tissue separation process are overcome, and the limitation that the time needed by common PCR and LAMP is long is overcome.
(2) The sensitivity is high: sensitivity detection is carried out through hypha genome DNA, the lowest germ concentration can be detected to be 1 pg/mu L by the RPA-LFD technology, and the sensitivity is obviously higher than that of common PCR and LAMP detection. Overcomes the defect of high detection limit of common PCR and LAMP.
(3) Good practicability and wide application range: the method can monitor germs on plants, soil and the like, is suitable for dynamic monitoring of germs, and provides basis for prediction and forecast of the germs.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a primer specificity assay;
in fig. 1:
a is the measurement result of the primer set of number 1; b is the result of measurement of the primer set of SEQ ID No. 2; c is the measurement result of the primer set of No. 3;
lane Maker: DNA marker; lanes 1-10: fusarium oxysporum (f. Oxysporum); lane 11: fusarium trilobate (f.tricinctum); lane 12: teng Cang fusarium (f. Fujikuroi); lane 13: fusarium graminearum (f.graminearum); lane 14: fusarium solani (f.solani); lane 15: fusarium (f. Proliferum); lane 16: fusarium equiseti (f.equiseti); lane 17: cryptoporus anthracis (c.aenigma); lane 18: fruit anthrax (c.fructicola); lane 19: colletotrichum gloeosporioides (c.); lane 20: siamenose anthrax (c.siamense); lane 21: botrytis cinerea; lane 22: stagonosporangis sp.; lane 23: phoma stem (Phoma sp.); lane 24: blank control.
FIG. 2 is a primer probe combination specificity assay;
1-10: fusarium oxysporum (f.oxysporum); 11: fusarium trilobate (f.tricinctum); 12: teng Cang fusarium (f. Fujikuroi); lane 13: fusarium graminearum (f.graminearum); 14: fusarium solani (f.solani); 15: fusarium (f. Proliferum); 16: fusarium equiseti (f.equiseti); 17: cryptococcus anthracis (c.aenigma); 18, fruit anthrax (c. Fructicola); 19: colletotrichum gloeosporioides (c.); 20: siamense (C.siemens); 21: botrytis cinerea; 22: stagonosporangis sp.;23: phoma stem (Phoma sp.); 24: blank control.
FIG. 3 is a graph of the effect of reaction temperature and time on the RPA-LFD detection system of FOSC;
a, influence of different time on RPA-LFD detection; 1-7: the reaction temperatures are 33 ℃, 36 ℃, 39 ℃, 42 ℃, 45 ℃, 48 ℃ and 51 ℃ respectively;
b, influence of different temperatures on RPA-LFD detection; 1-6: the reaction times were 4, 6, 8, 10, 12, and 14min, respectively.
FIG. 4 is a graph of the sensitivity of the RPA-LFD detection system; 1-6: the concentrations were 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, and 100 fg/. Mu.L, respectively.
FIG. 5 shows the results of the RPA-LFD detection of different field samples;
1: positive control, 2-5: isolating a strawberry sample of FOSCs; 6-9: strawberry samples with no FOSC isolated.
Detailed Description
The invention is further described below by way of examples and with reference to the accompanying drawings, which do not in any way limit the scope of the invention, but are merely illustrative.
The main reagents and instruments used in the following examples were: 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 marker (TaKaRa bioengineering company genome), PEG200 (biological engineering for biological engineering)Gmbh), PCR instrument.
Example 1 screening of specific primers
First, based on the FOSC genomic sequence, by aligning the genera common in soil and plants: fusarium trilobata (f.tricinctum), fusarium graminearum (f.fujikuroi), fusarium graminearum (f.graminearum), fusarium solani (f.solani), fusarium exuberans (f.proliferum), fusarium equiseti (f.equiseti), fusarium equiseti (Stagonosporopsis sp., phoma sp., other common pathogenic fungi on strawberries cryptosporidium anthracis (coilotrichum aenigrma), anthrax fructicola (c.fructicola), colletotrichum gloeosporioides (c.gloeosporioides), fusarium siamenses (c.siense), botrytis cinerea (Botrytis cinerea) sequence difference sites 3 pairs of fusarium oxysporum specific primers and 1 probe were designed as described in table 1.
The primer specificities as described in table 1 were verified using conventional PCR, and the reaction system is described in table 2.
The PCR amplification procedure was: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 15s,35 cycles, PCR products were verified on a 254nm (UV) 2.0% agarose gel. The amplification result is shown in FIG. 1, and primer pair 1 can only amplify a homozygous target fragment for P.stipitis, while other genera can not amplify a band or have a hybrid band. The size of the amplified fragment of the primer pair 1 is 167bp, and the conformity is verified to be 100% by sequencing.
The selected primer pairs were labeled with Biotin and purified by HPLC as with the corresponding probes, as described in table 3.
That is to say that the first and second electrodes,
the primer pair is as follows:
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’。
example 2 specificity analysis of detection System
Using the markers obtained by screeningThe latter primers and probes were used for specific analysis of the detection system, using f.oxysporum causing diseases such as strawberry stem-base rot and west safflower corm rot as positive control, using fusarium trilobate (f.tricinctum), teng Cang (f.fujikuroi), fusarium graminearum (f.graminearum), fusarium solani (f.solani), fusarium solani (f.proliferum), fusarium equiseti (f.equiseti), fusarium graminearum (c.aesnigrum), fusarium fructicum (c.aesnigma), colletotrichum fructicum (c.fructicola), colletotrichum collodionum (c.gloeosporioides), fusarium sambucinum (c.siemanense), griseofur (Botrytis cinerea), gonospodopsis stasp.sp.sp., phoma negative control, and using h 13 fungi as negative control 2 O was tested as a blank control.
The total volume of the detection solution is 48 mu L, and 2 mu L of DNA template to be detected (the concentration is 10 ng/. Mu.L) is added to form a 50 mu L detection reaction system. The 50. Mu.L reaction system contained 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 (the concentration was 10 ng/. Mu.L in both positive and negative controls), ddH 2 O make up to 50. Mu.L. The RPA-LFD amplification reaction program is as follows: the temperature is controlled at 39 ℃ for 8min,
detecting the amplification product by using a HybriDetect colloidal gold test strip, and after the amplification is finished, adding 190 mu L ddH into 10 mu L of amplification product 2 In 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 the interpretation results of a control line and a detection line within 5min, wherein the reaction result is shown in figure 2, the sample detection line taking the DNA of the phomopsis longipes as a template is visible, and the detection result is positive; DNA of other 13 fungi as template and ddH 2 And the detection line of the sample of the O control is invisible, and the detection results are negative. The detection result shows that the RPA-LFD primer probe group obtained by screening can specifically detect the phomopsis longissima.
Example 3 optimization of the conditions for RPA-LFD detection
The screened RPA-LFD primer probe group is used for optimizing the detection condition, the optimization result is shown in figure 3, when the reaction time is 10min, a clear strip is formed in the detection zone of the HybriDetect colloidal gold test strip at 39-45 ℃, according to the stability of the reaction, 39 ℃ is selected as the optimal reaction temperature, and a clear strip is formed in the detection zone of the HybriDetect colloidal gold test strip at 8-10min, and the result shows that the optimal reaction condition of the established RPA-LFD detection system is 39 ℃ for 8min.
The optimized reaction system and the amplification conditions are as follows:
a50. Mu.L reaction system contained 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 (10 ng/. Mu.L for both positive and negative controls), and ddH 2 O make up to 50. Mu.L. The RPA-LFD amplification reaction program is as follows: at 39 ℃ for 8min.
A forward primer: 5'-TCAACTGGCATCGTCAACATCACCGAAGTAA-3'
Reverse primer: 5'-Biotin-CCAGGCATGACGAAGTTGATAGGTTGAAAGC-3'
The probe sequence is as follows:
5’-6-FAM-GAGGATGTTGATTGCTATTCTGATGGGTGGG/idSp/CAGCACAACACTGCTGCTA-C3 Spacer-3’。
example 4 detection of sensitivity of primers
The detection result of the pathogen DNA solution diluted in a gradient by using the optimized FOSCRPA-LFD detection system and conditions (as described in example 3) is shown in FIG. 4, when the DNA concentration is 10 ng/muL, 1 ng/muL, 100 pg/muL, 10 pg/muL and 1 pg/muL, the amplification can be effectively performed, the detection area of the HybriDetect colloidal gold test strip still has a strip, the repeatability of the same sample is reliable, and the result shows that the detection limit of the established RPA-LFD detection system on FOSC is 1 pg/muL DNA.
Example 5 detection of plants mock inoculated with FOSC
Pricking base of stem of 5-week-old healthy strawberry tissue culture seedling with sterilized insects, spraying 5mL of 10-concentration water to the base of stem of each strawberry 6 cfu/mL FOSC spore solution, 28 ℃. + -. 5 ℃ light incubator, day 25 ℃,12 h/dark 25 ℃,12h, 5 days later using 20mM KOH-60% PEG200 lysate extraction of diseased plant genomic DNA.
20mM KOH-60% PEG200 lysate preparation method: the system is 40mL,24mL PEG200, 0.93mL 2M KOH, ddH 2 Complement of O to 40mLThe pH value is 13.3-13.5, and the extraction process specifically comprises the following steps: 10mg of plant sample tissue (stem base part tissue of strawberry) was added to 100. Mu.L of 20mM KOH-60% PEG200 extract solution and soaked at room temperature for 15 minutes.
Taking 1 μ L lysate directly as DNA template, and amplifying and detecting according to the optimized reaction system and amplification conditions obtained in the above example 3; and (5) verifying the detection result and the tissue separation result.
Meanwhile, inoculating sterile water, inoculating anthrax bacteria and the like to the strawberry tissue culture seedlings are set as experiment controls (except for changing the species of the inoculated bacteria, the rest of the inoculation controls are the same as the mode of inoculating the FOSC spore solution). The results obtained are in particular as given in table 4 below:
TABLE 4
| The result of the detection | Tissue isolation results | |
| Subject 1 (inoculation FOSC) | Positive for | Positive for |
| Experimental object 2 (inoculation sterile water) | Negative of | Negative of |
| Subject 3 (inoculation anthrax) | Negative of | Negative of |
| Subject 4 (inoculation F.graminearum) | Negative of | Negative of |
The results in Table 4 show that the RPA-LFD assay can accurately detect whether strawberry plants are infected with FOSC.
Example 6 detection of RPA-LFD on field samples
In order to verify the field practicability of the established RPA-LFD method, 8 strawberry stem base parts are randomly collected from the field to carry out RPA-LFD detection.
The FOSC separated strawberry sample is taken as a sample 2 to 5, and the FOSC not separated strawberry sample is taken as a sample 6 to 9; the DNA sample of the FOSC was used as a positive control (sample 1);
the tissue genome DNA was rapidly extracted by PEG200 using 20mM KOH-60 (as described in example 5), and after the samples were reacted at 39 ℃ for 8min, the detection was carried out using LFD, and the detection results are shown in FIG. 5, wherein the positive control and the detection zone of 4 strawberry samples (samples 2 to 5) both had bands, and the detection zone of 4 strawberry samples (samples 6 to 9) had no band, and the results were verified against the tissue isolation results, showing that the RPA-LFD detection can accurately detect whether the grass plants were infected with FOSC.
Comparative example 1, the primers were changed as follows:
an upstream primer: 5'-AACTGGCATCGTCAACATCACCGAAGT-3'
A downstream primer: 5'-BIOTIN-CATGACGAAGTTGATAGGTTGAAA-3'
The test was carried out in the same manner as in example 4, and the results obtained showed that: when the DNA concentration is as low as 10 pg/mu L, a band still exists in a detection area of the HybriDetect colloidal gold test strip, so that the detection limit of the FOSC is 10 pg/mu L of DNA.
Comparative example 2, the primers were changed as follows
An upstream primer: 5'-CTCAACTGGCATCGTCAACATCACCGAAGTAA-3'
A downstream primer: 5'-BIOTIN-CCAGGCATGACGAAGTTGATAGGTTGAAAGCTA-3'
The test was carried out identically to example 2, and the results obtained show: only samples 1, 3 were positive, the remainder were negative. Therefore, the detection result of this comparative example 2 is incorrect.
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.
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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