Primer probe combination for detecting banana fusarium wilt TR4, RPA-LFD detection method and application

Abstract

The invention relates to the technical field of molecular biology, in particular to a primer probe combination for detecting banana fusarium wilt TR4, an RPA-LFD detection method and application. The primer probe combination comprises an upstream amplification primer, a downstream amplification primer and a probe, the nucleotide sequence in the upstream amplification primer is as shown in SEQ ID NO: 1, the nucleotide sequence in the downstream amplification primer is as shown in SEQ ID NO: 2, and the probe is characterized in that on the basis of the nucleotide sequence as shown in SEQ ID NO: 3, the 5'end is connected with a fluorophore, the 31st basic group A is replaced by dSpacer, and the 3 'end is connected with ddC. The primer probe combination disclosed by the invention is applied to RPA-LFD detection of banana fusarium wilt TR4, and has the advantages of good specificity and high sensitivity. The invention also provides an RPA-LFD detection method of banana fusarium wilt TR4, and the RPA-LFD detection method has the advantage of high detection speed in addition to good specificity and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a primer-probe combination for detecting banana wilt disease TR4, an RPA-LFD detection method and an application thereof. Background Art

[0002] Banana Fusarium wilt is a priority for international plant quarantine. The pathogen is currently classified into subspecies FOC1, FOC2, TR4, and STR4, with TR4 being the most widespread and damaging. Due to the lack of effective treatments and resistant varieties, the disease is extremely difficult to control in production. Therefore, rapid and accurate detection of the pathogen is crucial to enable timely destruction and isolation measures to prevent its spread.

[0003] Currently, accurate detection of this disease is primarily achieved through molecular methods, including standard PCR, isothermal amplification (RPA), and LAMP. Standard PCR is cumbersome, time-consuming, and prone to nonspecific amplification. However, there are no reports of using RPA for detection. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a primer-probe combination, RPA-LFD detection method and application for detecting banana wilt TR4, aiming to improve the detection efficiency, specificity and sensitivity of banana wilt TR4.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A first aspect of the present invention provides a primer-probe combination for detecting banana wilt TR4, wherein the primer-probe combination comprises an upstream amplification primer, a downstream amplification primer, and a probe; The sequence of the upstream amplification primer is: Biotin-5'-GACTTACGGTGACGTGCATATTACGTAGC-3'; The sequence of the downstream amplification primer is: 5'-CGCCAAATATTACGAAATAACAGCATTA-3'; The probe is based on the nucleotide sequence shown in SEQ ID No. 3, with a fluorescent group connected to the 5' end, the 31st base A replaced by dSpacer, and the 3' end connected to ddC; SEQ ID No.3: 5'-CTGATCGCTGGCTGCAGCTACTCATGCCACACTCCACCTATCGACAG-3'.

[0006] A second aspect of the present invention provides a kit for detecting banana wilt disease TR4, wherein the kit comprises the primer-probe combination described above.

[0007] The kit for detecting banana wilt TR4 further comprises a nucleic acid extraction composition; the nucleic acid extraction composition comprises a lysis solution, chloroform or a chloroform substitute, a binding solution, a washing solution and an eluent.

[0008] The kit for detecting banana wilt TR4, wherein the lysate comprises the following components: 1% to 2% by mass of CTAB, 0.5 to 1.0 M NaCl, 0.05 to 0.08 M Tris, 20 to 50 mM EDTA, and a pH of 7.0 to 7.5.

[0009] The kit for detecting banana wilt TR4, wherein the binding solution comprises the following components: 2-3M guanidine thiocyanate, 20mM Tris-HCl, 20mM EDTA, pH=8.5.

[0010] In the kit for detecting banana wilt TR4, the washing solution comprises the following components: 10 mM Tris-HCl and 50-60% ethanol by volume; and the eluent comprises the following components: 10 mM Tris-HCl, pH=7.0-7.5.

[0011] A third aspect of the present invention provides a method for detecting banana wilt TR4 by RPA-LFD, comprising the following steps: S01. Extract DNA from suspected diseased parts of banana pseudostems or fungal cultures; S02. Perform an RPA reaction using the primer-probe combination described above to obtain an amplified product; S03. Dilute the amplified product and add it dropwise to the test paper card for testing to obtain the test result.

[0012] The RPA-LFD method for detecting banana wilt TR4, wherein the extraction of DNA from the fungal culture comprises the following steps: S11. Place the white mycelium from the suspected diseased area or the surface of the fungal culture into a centrifuge tube, add lysis buffer and zirconium oxide beads, shake, and centrifuge. S12. Transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform or chloroform substitute, invert to mix, and centrifuge. The solution will separate from top to bottom into a milky white liquid layer, an intermediate impurity layer, and a lower solvent layer. S13. Transfer the milky white liquid layer to a new centrifuge tube, add an equal volume of binding buffer, and mix thoroughly to obtain a mixed solution; S14. Use a purification column to extract the nucleic acid in the mixed solution to obtain a nucleic acid extract.

[0013] Beneficial effects: The present invention provides a primer-probe combination for detecting banana Fusarium wilt TR4. The combination is applied to detecting banana Fusarium wilt TR4 and has the characteristics of good specificity and high sensitivity, and can detect banana Fusarium wilt in the early stage of infection.

[0014] The present invention also provides an application of a primer-probe combination in detecting banana wilt TR4 by RPA-LFD. Compared with conventional PCR, detection by RPA-LFD takes less time and has better specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the test result of the test paper card in Example 1.

[0016] Figure 2 This is the test result of the test paper card part of Comparative Example 1.

[0017] Figure 3 This is the test result of the test paper card part of Comparative Example 2.

[0018] Figure 4 This is the test result of the test paper card of Example 2.

[0019] Figure 5 This is the test result of the test paper card of Example 3. DETAILED DESCRIPTION

[0020] The present invention provides a primer-probe combination for detecting banana Fusarium wilt TR4, an RPA-LFD detection method, and its application. To further clarify the objectives, technical solutions, and effects of the present invention, the present invention is further described below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0021] The present invention provides a primer-probe combination for detecting banana wilt TR4, wherein the primer-probe combination comprises an upstream amplification primer, a downstream amplification primer and a probe; The sequence of the upstream amplification primer is: Biotin-5′-GACTTACGGTGACGTGCATATTACGTAGC-3′, the nucleotide sequence of which is SEQ ID NO: 1; The sequence of the downstream amplification primer is: 5'-CGCCAAATATTACGAAATAACAGCATTA-3', the nucleotide sequence of which is SEQ ID NO: 2; The probe is based on the nucleotide sequence shown in SEQ ID No. 3, with a fluorescent group connected to the 5' end, the 31st base A replaced by dSpacer, and the 3' end connected to ddC; SEQ ID No.3: 5'-CTGATCGCTGGCTGCAGCTACTCATGCCACACTCCACCTATCGACAG-3'.

[0022] Specifically, the fluorescent group can be FAM or FITC.

[0023] The present invention also provides a kit for detecting banana wilt TR4, the kit comprising the primer-probe combination described above; Specifically, the kit may further include: amplification lyophilized powder, a reagent, nuclease-free water, and an activator. The kit of this embodiment can be used to complete an RPA reaction to obtain a corresponding amplification product.

[0024] Furthermore, the kit may also include: a nucleic acid extraction composition and a test paper card. The kit of this embodiment can not only be used to complete the RPA reaction, but also can be used for sample nucleic acid extraction and complete the entire RPA-LFD detection.

[0025] Preferably, the nucleic acid extraction composition comprises: a lysis solution, chloroform or a chloroform substitute, a binding solution, a washing solution and an elution solution.

[0026] Preferably, the lysis solution consists of the following components: 1% to 2% by mass of CTAB, 0.5 to 1.0 M NaCl, 0.05 to 0.08 M Tris, 20 to 50 mM EDTA, pH = 7.0 to 7.5.

[0027] Preferably, the binding solution consists of the following components: 2-3 M guanidine thiocyanate, 20 mM Tris-HCl, 20 mM EDTA, pH=8.5.

[0028] Preferably, the washing solution consists of the following components: 10 mM Tris-HCl and 50-60% ethanol by volume.

[0029] Preferably, the eluent consists of the following components: 10 mM Tris-HCl, pH = 7.0-7.5.

[0030] The present invention also provides a method for detecting banana wilt TR4 using RPA-LFD, which may specifically include the following steps: A01. Obtain a suspected diseased area or fungal culture from the banana pseudostem as a sample. Place a 0.5- to 1-soybean-sized sample into a 1.5 mL centrifuge tube. The method for obtaining suspected diseased areas from the banana pseudostem is as follows: Take the pseudostem of the banana to be tested, which is close to the ground, and find 2-3 suspected diseased areas. Use a knife or scissors to take a 1-3g piece from each area. Then cut all the samples into small pieces as small as possible, so that the length and width of the sample pieces do not exceed 3mm and the height does not exceed 2mm. All the samples are mixed evenly. The method for obtaining fungal culture is as follows: frozen purified fungi are activated and inoculated into PDA culture medium under sterile operation. The culture medium is placed in an environment of 28-30°C for 3-7 days. When the culture medium plate is covered with white mycelium, the mycelium is scraped off. A02. Add 400–600 μL of lysate and 0.7–0.9 g of zirconium oxide beads (0.6–0.8 mm diameter) to a centrifuge tube. Place the tube in a mini shaker and shake at maximum speed (3,000–10,000 rpm) for 1–3 minutes. Centrifuge briefly for 3–5 seconds. Take 150-300 μL of supernatant and place it in another new 1.5 mL centrifuge tube. Add an equal volume of chloroform or Meiji Bio's chloroform substitute BDP, invert and mix 10 times, and centrifuge for 5-10 seconds. The solution can be seen to separate from the top to the bottom into a milky white liquid layer, an impurity layer in the middle, and a solvent layer at the bottom. Carefully aspirate 200 μL of the upper milky white liquid and add it to another new 1.5 mL centrifuge tube. Then add an equal volume of binding solution and pipette up and down 3 to 4 times to mix thoroughly to obtain a mixed solution. A03. Place the purification column on the collection tube, add the entire mixture to the column, securely cap, and centrifuge at 10,000 rpm for 60 seconds at room temperature. Discard the filtrate in the collection tube, reinstall the purification column, pipette 300-500 μL of washing solution into the purification column, close the lid tightly, and centrifuge at 8000-10000 rpm for 60 seconds at room temperature. Discard the filtrate in the collection tube, reinstall the purification column, close the lid tightly, and centrifuge the empty tube at 10,000 rpm for 60 seconds at room temperature to remove the residual washing solution in the purification column; Transfer the purification column to a new collection tube, draw 40-60 μL of eluate and add it to the center of the membrane of the purification column. Let it stand at room temperature for 1 minute, and centrifuge at 8000-10000 rpm for 60 seconds. The collection tube contains the nucleic acid extract. A04. 10 minutes in advance, allow the reaction buffer and activator buffer to fully dissolve at room temperature. Once dissolved, gently vortex for 3-5 seconds to mix thoroughly, and centrifuge for 2-3 seconds. Remove the reaction tube containing the amplification freeze-dried powder, open the lid, and add the reagents in the order shown in the table below. For multiple samples, prepare a premix excluding the nucleic acid template and then add it to the amplification freeze-dried powder.

[0031] After adding the above ingredients to the lyophilized powder, add 2 μL of activator buffer to each tube cap, carefully close the cap, and centrifuge for 2-3 seconds to allow the activator to enter the reaction mixture. Immediately invert the tube 8-10 times or vortex for 3-5 seconds to mix, and then immediately centrifuge for 2-3 seconds to allow all the reagents to sink to the bottom of the tube. Immediately place the reaction tube in a metal bath (38-39°C) and incubate for 10-15 minutes. The metal bath can be opened in advance and heated to 38-39°C for later use. At the same time, set up a negative control (nuclease-free water instead of template DNA) to detect false positives. A05. After the RPA incubation is complete, carefully open the lid and pipette 3-5 μL of the amplified product. Dilute it 40-60 times with diluent. Mix thoroughly. Add 60-80 μL of the diluted RPA amplified product to the test card. Wait 2-3 minutes and observe the results. The results displayed within 1-5 minutes are the final result. Results displayed after 5 minutes cannot be used as the basis for determining positive or negative results. Result judgment: When there are lines at both C and T in the test paper card (fuzzy lines are also counted), it is judged as positive; if there is a line only at C, it is judged as negative; if there is no line at C (regardless of whether there is a line at T), the test paper card is invalid and needs to be replaced for retesting.

[0032] Example 1 Specificity Detection A primer-probe combination comprises an upstream amplification primer, a downstream amplification primer and a probe; The sequences of the upstream amplification primers are: Biotin-5'-GACTTACGGTGACGTGCATATTACGTAGC-3'; The sequences of the downstream amplification primers are: 5'-CGCCAAATATTACGAAATAACAGCATTA-3'; The sequence of the probe is: (6-FAM)-5'-CTGATCGCTGGCTGCAGCTACTCATGCCAC-dSPacer- CTCCACCTATCGACAG-3′-ddC.

[0033] The primer-probe combination provided by the present invention was used to detect nucleic acids extracted from pure cultured mycelia of 37 different banana wilt fungi. Among them, numbers 1-29 belong to different VCGs of banana wilt (VCG types do not overlap), belonging to four subspecies, namely FOC1, FOC2, TR4, and STR4. In addition, 7 wilts of other species were detected, including cotton wilt, cucumber wilt, tomato wilt, tobacco wilt, cabbage wilt, and melon wilt. The sample information is shown in Table 1 below: Table 1

[0034] The specific steps of the detection method refer to the above-mentioned RPA-LFD method for detecting banana wilt TR4. The results are as follows Figure 1 shown.

[0035] See Table 1 and Figure 1 Of the 37 DNA samples, 30 were related to banana Fusarium wilt, and 7 were related to other species. Four of the 30 banana Fusarium wilt samples were TR4, numbered 2, 13, 16, and 28. The first three gave strong positive results, while the last gave a weak positive. This indicates that the method can detect all TR4 samples, with no false negatives. The remaining 33 non-TR4 DNA templates, with the exception of sample 37, which showed a weak positive, were all negative, demonstrating good specificity. The negative control water was negative, with no false positives, confirming the reliability of the test results.

[0036] Comparative Example 1 a second primer-probe combination, comprising an upstream amplification primer, a downstream amplification primer, and a probe; Nucleotide sequence of upstream amplification primer (SEQ ID No. 4): 5'-ATTGGAGCGACTTATAGTGATAAGCCAT-3'; Nucleotide sequence of downstream amplification primer (SEQ ID No.5): Biotin-5'-CGCCAAATATTACGAAATAGGACGAAAT-3'; Nucleotide sequence of the probe: (6-FAM)-5'-(SEQ ID No.6)-dSPacer-(SEQ ID No.7)-3'-ddC (6-FAM)-5'-TTCTGTGGTTTATCCGGAGTCAGAGTTCACT-dSPacer- TGGACGAGGGTCTTATC-3′-ddC; Primer-probe combination 2 was used to detect the samples in Table 1. The specific steps of the detection method refer to the above-mentioned RPA-LFD method for detecting banana wilt TR4.

[0037] The final test results showed that only TR4 target samples 2 and 28 were detected, while samples 13 and 16 were not detected, and samples 5, 9, 18, and 22 that did not belong to TR4 were detected, indicating that the detection effectiveness and specificity of primer-probe combination 2 were very poor. Figure 2 shown.

[0038] Comparative Example 2 a primer-probe combination three, comprising an upstream amplification primer, a downstream amplification primer, and a probe; Nucleotide sequence of upstream amplification primer (SEQ ID No.8): 5'-CTCAATGTGCGGTGAGAGAATGCGAACTAT-3'; Nucleotide sequence of downstream amplification primer (SEQ ID No.9): Biotin-5'-TCTCATGTACGCCTAGTACGCGCTTCCTGAT-3'; Nucleotide sequence of the probe: (6-FAM)-5'-(SEQ ID No.10)-dSPacer-(SEQ ID No.11)-3'-ddC (6-FAM)-5'-TAGTTGCGACATCGGTTACGATCGTTCCTA-dSPacer- GAGGTTGAAATCTGAAC-3′-ddC; Primer-probe combination three was used to detect the samples in Table 1. The specific steps of the detection method refer to the above-mentioned RPA-LFD method for detecting banana wilt TR4.

[0039] The final test results showed that only TR4 target samples 13 and 16 were detected, while samples 2 and 28 were not detected, and samples 7, 9, 15, 18, and 22 that did not belong to TR4 were detected, indicating that the detection effectiveness and specificity of primer-probe combination 3 were very poor. Figure 3 shown.

[0040] Example 2 Sensitivity Test In this embodiment, primer-probe combination 1 is used as primer and probe; From a banana plant infected with banana Fusarium wilt TR4, an appropriate amount of sample was removed from the pseudostem close to the ground for DNA extraction. This was then used as the stock solution and a two-fold dilution method was used to obtain a total of 12 groups of DNA templates with different concentrations of 2, 4, 8, 16, 32, 64, 128, 356, 712, and 1424 times. These samples, along with a negative control, were used for RPA-LFD detection to test the sensitivity of the method. For the specific steps of the detection method, refer to the RPA-LFD method for banana Fusarium wilt TR4 detection described above.

[0041] Figure 4 For the test results, Figure 4 It can be seen that after the original liquid template was diluted 512 times, fuzzy bands were still observed, and the target bands disappeared after the template was diluted 1024 times. This shows that if the amount of infected bacteria in a sample infected with banana wilt is reduced by about 500 times, that is, in the early stage of infection, it can still be detected in the absence of symptoms, indicating that this method has extremely high sensitivity.

[0042] Example 3 Obtain samples infected with banana wilt disease, including one plant with obvious symptoms, one with moderate symptoms, one without symptoms (but confirmed to be infected by QPCR method), and one uninfected sample. Then set up positive control and negative control respectively, and use the primer-probe combination 1 of the present invention and refer to the above-mentioned RPA-LFD method for detecting banana wilt TR4. The test results are as follows: Figure 5 shown.

[0043] from Figure 5 The results show that all three positive samples were detected. Although the asymptomatic sample had a lighter band (T line), it was clearly positive. The negative sample had no band, and both the positive and negative controls were normal, indicating that the test results were reliable. These results demonstrate that this method can detect positive samples even in the absence of symptoms, demonstrating its effectiveness.

[0044] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A primer-probe combination for detecting banana wilt TR4, characterized in that: The primer-probe combination includes an upstream amplification primer, a downstream amplification primer and a probe; The sequence of the upstream amplification primer is: Biotin-5'-GACTTACGGTGACGTGCATATTACGTAGC-3'; The sequence of the downstream amplification primer is: 5'-CGCCAAATATTACGAAATAACAGCATTA-3'; The probe is based on the nucleotide sequence shown in SEQ ID No. 3, with a fluorescent group connected to the 5' end, the 31st base A replaced by dSpacer, and the 3' end connected to ddC; SEQ ID No.3: 5'-CTGATCGCTGGCTGCAGCTACTCATGCCACACTCCACCTATCGACAG-3'.

2. A kit for detecting banana wilt TR4, characterized in that: The kit comprises the primer-probe combination according to claim 1.

3. The test kit for detecting banana wilt TR4 according to claim 2, wherein Also includes: Amplification lyophilized powder, reagent, nuclease-free water, activator.

4. The kit for detecting banana wilt TR4 according to claim 3, wherein Also includes test strip cards.

5. The kit for detecting banana wilt TR4 according to claim 3, wherein Also includes: a nucleic acid extraction composition; The nucleic acid extraction composition comprises: a lysis solution, chloroform or a chloroform substitute, a binding solution, a washing solution and an elution solution.

6. The kit for detecting banana wilt TR4 according to claim 5, characterized in that The lysis solution includes the following components: 1% to 2% CTAB by mass, 0.5 to 1.0 M NaCl, 0.05 to 0.08 M Tris, 20 to 50 mM EDTA, and pH = 7.0 to 7.

5.

7. The kit for detecting banana wilt TR4 according to claim 5, characterized in that The binding solution includes the following components: 2-3 M guanidine thiocyanate, 20 mM Tris-HCl, 20 mM EDTA, pH=8.

5.

8. The kit for detecting banana wilt TR4 according to claim 5, characterized in that: The washing solution includes the following components: 10mM Tris-HCl and 50-60% ethanol by volume; the eluent includes the following components: 10mM Tris-HCl, pH=7.0-7.

5.

9. A method for detecting banana wilt TR4 using RPA-LFD, characterized in that: The steps include: S01. Extract DNA from suspected diseased parts of banana pseudostems or fungal cultures; S02. Performing an RPA reaction using the primer-probe combination according to claim 1 to obtain an amplified product; S03. Dilute the amplified product and add it dropwise to the test paper card for testing to obtain the test result.

10. The method for detecting banana wilt TR4 using RPA-LFD according to claim 9, wherein: The DNA extraction of fungal culture comprises the following steps: S11. Place the white mycelium from the suspected diseased area or the surface of the fungal culture into a centrifuge tube, add lysis buffer and zirconium oxide beads, shake, and centrifuge. S12. Transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform or chloroform substitute, invert to mix, and centrifuge. The solution will separate from top to bottom into a milky white liquid layer, an intermediate impurity layer, and a lower solvent layer. S13. Transfer the milky white liquid layer to a new centrifuge tube, add an equal volume of binding buffer, and mix thoroughly to obtain a mixed solution; S14. Use a purification column to extract the nucleic acid in the mixed solution to obtain a nucleic acid extract.

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