MIRA primer probe combination for detecting cucumber green mottle mosaic virus and application of MIRA primer probe combination

Abstract

The invention discloses an MIRA primer group for detecting a cucumber green mottle mosaic virus. The primer group comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown as any one of SEQ ID NO: 4 to SEQ ID NO: 6. The invention further provides an MIRA primer probe set for detecting the cucumber green mottle mosaic virus, the cucumber green mottle mosaic virus can be accurately detected within 10 min by means of the MIRA primer probe set, the detection sensitivity can reach 10 < 4 > copies / mu L, and excellent specificity is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a MIRA primer probe combination for detecting cucumber green mottle mosaic virus and application thereof. BACKGROUND

[0002] Cucumber green mottle mosaic virus (CGMMV) is a positive single-stranded RNA virus belonging to the genus Tobamovirus. Cucumber Green Mottle Mosaic Virus CGMMV is a plant pathogen transmitted by seeds, soil, agricultural operations and irrigation water, and is difficult to eradicate once it breaks out. Tobamovirus CGMMV is a plant pathogen transmitted by seeds, soil, agricultural operations and irrigation water, and is difficult to eradicate once it breaks out.

[0003] CGMMV is a plant pathogen transmitted by seeds, soil, agricultural operations and irrigation water, and is difficult to eradicate once it breaks out. CGMMV is a plant pathogen transmitted by seeds, soil, agricultural operations and irrigation water, and is difficult to eradicate once it breaks out.

[0004] Therefore, timely and accurate detection of CGMMV is the key to implementing effective prevention and control measures. SUMMARY

[0005] The present application relates to the technical field of biological medicine, in particular to a MIRA primer probe combination for detecting cucumber green mottle mosaic virus and application thereof.

[0006] The first object of the present application is to provide a MIRA primer set for detecting cucumber green mottle mosaic virus.

[0007] The second object of the present application is to provide the use of the MIRA primer set in the preparation of a product for detecting cucumber green mottle mosaic virus.

[0008] The third object of the present application is to provide a MIRA primer probe combination for detecting cucumber green mottle mosaic virus.

[0009] The fourth object of the present application is to provide a product for detecting cucumber green mottle mosaic virus.

[0010] The fifth objective of this invention is to provide a method for detecting cucumber green mottle mosaic virus for non-diagnostic purposes.

[0011] To achieve the above objectives, the present invention is implemented through the following solution: This invention claims protection for a MIRA primer set for detecting cucumber green mottle mosaic virus, the primer set containing an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in any one of SEQ ID NO: 1 to 3, and the nucleotide sequence of the downstream primer is shown in any one of SEQ ID NO: 4 to 6.

[0012] CGMMV-F1 (SEQ ID NO: 1): 5'-GAAGATGGCTTACAATCCGATCACACCTAG-3'; CGMMV-F2 (SEQ ID NO: 2): 5'-TTACAATCCGATCACACCTAGCAAACTTATTG-3'; CGMMV-F3 (SEQ ID NO: 3): 5'-AACTTATTGCGTTTAGTGCTTCTTATGTTCC-3'; CGMMV-R1 (SEQ ID NO: 4): 5'-CGTAGATATTAATTCTAGATTCCCAGATGCG-3'; CGMMV-R2 (SEQ ID NO: 5): 5'-TTCCTCAACGGTCCTGTGTTGAGGCCTATC-3'; CGMMV-R3 (SEQ ID NO: 6): 5'-TCCTGTGTTGAGGCCTATCTTCGTTTCGCTTC-3'.

[0013] Preferably, the primer set contains an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 4.

[0014] This invention also claims protection for the use of any of the MIRA primer sets described above in the preparation of products for detecting cucumber green mottle mosaic virus.

[0015] Preferably, the product is a detection reagent and / or a detection kit.

[0016] The present invention also claims protection for a MIRA primer-probe combination for detecting cucumber green mottle mosaic virus, comprising any of the MIRA primer sets described above and a probe with a nucleotide sequence as shown in SEQ ID NO: 7.

[0017] CGMMV-probe (SEQ ID NO: 7): 5'-TACCGCTTTCCAGACTCAAGCGGGAAGAGA[FAMdT][THF]C[BHQ1dT]TTCCGCGAGTCCCTGT-[3'C3spacer]; FAMdT is a fluorescent group, and BHQ1dT is a quenching group.

[0018] This invention also claims protection for a product for detecting cucumber green mottle mosaic virus, containing any of the MIRA primer sets described above.

[0019] Preferably, it also contains a probe with a nucleotide sequence as shown in SEQ ID NO: 7.

[0020] More preferably, it also contains MIRA amplification reaction reagents.

[0021] More preferably, the MIRA amplification reaction reagents include A Buffer, B Buffer, and lyophilized enzyme powder reaction tube reagents; wherein A Buffer, B Buffer, and lyophilized enzyme powder reaction tube reagents are all purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., product code WLRE8208KIT.

[0022] More preferably, the MIRA amplification reaction reagents include AD Buffer, B Buffer, and colloidal gold test strip RNA isothermal rapid amplification lyophilized enzyme powder reaction tube reagents; wherein AD Buffer, B Buffer, and colloidal gold test strip RNA isothermal rapid amplification lyophilized enzyme powder reaction tube reagents are all purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., product code WLRN8209KIT.

[0023] This invention also claims protection for a method for detecting cucumber green mottle mosaic virus for non-diagnostic purposes, comprising the following steps: S1. Extract RNA from the sample to be tested and reverse transcribe it into cDNA; S2. Using the cDNA obtained in step S1 as a template, perform MIRA amplification using any of the products described above to obtain MIRA amplification products; The MIRA amplification temperature is 37–42℃, and the amplification reaction time is 10–20 min; S3. Place the MIRA amplification product obtained in step S2 under blue light for observation. If the MIRA amplification product does not fluoresce under blue light, the sample to be tested does not contain cucumber green mottle mosaic virus; if the MIRA amplification product fluoresces under blue light, the sample to be tested contains cucumber green mottle mosaic virus.

[0024] Preferably, in step S2, when performing MIRA amplification, the amplification system comprises, based on a 50 μL amplification system: 1–13 μL of cDNA extracted in step S1, 2 μL of upstream primer from any of the above-mentioned products, 2 μL of downstream primer from any of the above-mentioned products, 0.1–1.2 μL of probe with nucleotide sequence as shown in SEQ ID NO: 7 from any of the above-mentioned products, 29.4 μL of A Buffer from any of the above-mentioned products, 2.5 μL of B Buffer from any of the above-mentioned products, 1 part of lyophilization reagent from any of the above-mentioned products, and water to a final volume of 50 μL; The concentrations of the upstream primer, downstream primer, and probe were 10 μM.

[0025] More preferably, the amplification system comprises 5 μL of RNA extracted in step S1, 2 μL of the upstream primer from any of the above-described products, 2 μL of the downstream primer from any of the above-described products, 0.6 μL of the probe with the nucleotide sequence shown in SEQ ID NO: 7 from any of the above-described products, 29.4 μL of Buffer A from any of the above-described products, 2.5 μL of Buffer B from any of the above-described products, one portion of the lyophilized reagent from any of the above-described products, and water to a final volume of 50 μL. The concentrations of the upstream primer, downstream primer, and probe were 10 μM.

[0026] This invention also claims protection for a method for detecting cucumber green mottle mosaic virus for non-diagnostic purposes, comprising the following steps: S11. Extract RNA from the sample to be tested and reverse transcribe it into cDNA; S12. Using the cDNA obtained in step S1 as a template, add a biotin-modified biotin to the 5' end of the downstream primer in the MIRA primer set of any of the above-mentioned products, and then perform MIRA amplification. After the amplification technology, the single nucleic acid test strip is used for detection. If red lines appear on both the control line and the test line of the single nucleic acid test strip, the sample to be tested contains cucumber green mottle mosaic virus; If a red line appears on the control line of a single nucleic acid test strip but not on the test line, the sample to be tested does not contain cucumber green mottle mosaic virus. If no red lines appear on either the control line or the test line of the single nucleic acid test strip, the test is invalid and needs to be repeated.

[0027] Preferably, the single nucleic acid test strip is purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., and the product code is WLFS8206.

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a MIRA primer set for detecting cucumber green mottle mosaic virus, comprising an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in any one of SEQ ID NO: 1-3, and the nucleotide sequence of the downstream primer is shown in any one of SEQ ID NO: 4-6. Furthermore, a MIRA primer-probe set for detecting cucumber green mottle mosaic virus is also provided, comprising the aforementioned MIRA primer set and a probe with the nucleotide sequence shown in SEQ ID NO: 7. Using the MIRA primer-probe set, rapid and accurate detection of cucumber green mottle mosaic virus can be achieved, with detection completed in as little as 10 minutes, and the detection sensitivity for cucumber green mottle mosaic virus reaching 10. 4 It has copies / μL and excellent specificity, and the detection process does not require the use of toxic reagents such as EB, making it safer for testing personnel and the environment. Attached Figure Description

[0029] Figure 1 The fluorescence curves of the amplification products after amplification with upstream and downstream primers in each combination in Example 1 are shown. Figure 2 The fluorescence curves of the MIRA amplification products corresponding to each template in Example 2 are shown below. Figure 3 The following are fluorescence curves of MIRA amplification products at different reaction temperatures in Example 3: A is the fluorescence curve of MIRA amplification products at a reaction temperature of 27℃; B is the fluorescence curve of MIRA amplification products at a reaction temperature of 32℃; C is the fluorescence curve of MIRA amplification products at a reaction temperature of 37℃; D is the fluorescence curve of MIRA amplification products at a reaction temperature of 42℃; E is the fluorescence curve of MIRA amplification products at a reaction temperature of 47℃; and F is the fluorescence curve of MIRA amplification products at a reaction temperature of 52℃. Figure 4 The image shows the fluorescence of the MIRA amplification products at different reaction times in Example 3. Figure 5 This is a fluorescence observation diagram of the MIRA products of different viruses in Example 4; Figure 6 This is a graph showing the detection results of the diluted MIRA amplification product in the test strip reading area in Example 5. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0031] The A Buffer, B Buffer, and lyophilized enzyme powder reaction tubes used in this invention embodiment were all purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., product code WLRE8208KIT; the single nucleic acid test strips were purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., product code WLFS8206; and the AD Buffer, B Buffer, and colloidal gold test strip-type RNA isothermal rapid amplification lyophilized enzyme powder reaction tubes were all purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., product code WLRN8209KIT.

[0032] Example 1: Design of MIRA Primer and Probe Set I. Experimental Methods 1. Primer and probe design Nucleotide sequences of cucumber green mottle mosaic virus and its related viruses (different isolates of CGMMV virus) were searched and downloaded from NCBI. These sequences were compared to identify specific primer candidate regions for cucumber green mottle mosaic virus. At the same time, the amplification fragments described in patents related to the detection of cucumber green mottle mosaic virus (patent publication numbers: CN116676418A, CN114507665A, CN102140536A, CN108456746A, and CN109468417A) were searched and compared, and no completely identical amplification fragments were found. Then, based on the design principles of MIRA primers and probes, MIRA-specific detection primers and probes for identifying and detecting cucumber green mottle mosaic virus were designed, as shown in Table 1.

[0033] Table 1 Primers and probes for MIRA-specific detection

[0034] Among them, "FAMdT" is a fluorescent group, "THF" is a tetrahydrofuran site, and "BHQ1dT" is a quenching group.

[0035] 2. Primer and probe set screening With 10 9 Using CGMMV standard plasmid copies / μL as templates, the primers and probes shown in Table 1 were screened as follows: 29.4 μL of A Buffer, 2 μL of CGMMV-F1 (SEQ ID NO: 1, 10 μM), 2 μL of CGMMV-R1 (SEQ ID NO: 4, 10 μM), 0.6 μL of CGMMV-probe (SEQ ID NO: 7, 10 μM), and 5 μL of template (10 μM) were added. 9Mix the following ingredients in a reaction tube: copies / μL of CGMMV standard plasmid, 2.5μL of B Buffer, one lyophilized enzyme powder reaction tube reagent, and 50μL of DEPC water. After thorough mixing, centrifuge the reaction solution quickly to the bottom of the reaction tube. Then, place the reaction tube in a fluorescence detection device (ABI 7500 Real-Time PCR instrument) to detect fluorescence and record the fluorescence curve. The fluorescence detection program of the fluorescence detection device is set as follows: constant temperature 42℃; fluorescence signal is collected every 30 seconds, and reaction time is 20 minutes. The CGMMV standard plasmid was constructed by inserting a 718bp CGMMV virus CP fragment (SEQ ID NO: 8) between the EcoRV sites of the pUC57 plasmid and the EcoRV site. A 718bp CGMMV virus CP fragment (SEQ ID NO: 8): A Buffer, B Buffer, and fluorescent RNA isothermal rapid amplification reagent powder were all purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., product code WLRE8208KIT.

[0036] Next, replace CGMMV-R1 (SEQ ID NO: 4, 10 μM) and CGMMV-probe (SEQ ID NO: 7, 10 μM) with the upstream and downstream primers in the MIRA primers and probes shown in Table 1, so that the upstream and downstream primers in Table 1 are combined in pairs, and record the fluorescence curves of the amplification products after amplification of each combination of upstream and downstream primers.

[0037] II. Experimental Results The fluorescence curves of the amplification products after amplification using upstream and downstream primers for each combination are shown in the figure. Figure 1 As shown in the results, the combination of upstream and downstream primers in Table 1 showed a certain amplification ability when amplifying CGMMV. However, the amplification effect was the best when CGMMV-F1 (nucleotide sequence shown in SEQ ID NO: 1) was used as the upstream primer and CGMMV-R1 (nucleotide sequence shown in SEQ ID NO: 4) was used as the downstream primer, and the amplification plateau phase was reached in the shortest time.

[0038] Therefore, CGMMV-F1, with a nucleotide sequence as shown in SEQ ID NO: 1, is used as the optimal upstream primer, and CGMMV-R1, with a nucleotide sequence as shown in SEQ ID NO: 4, is used as the optimal downstream primer.

[0039] Example 2: Detection limit test of MIRA primer and probe set for CGMMV detection I. Experimental Methods 10 10 The CGMMV standard plasmid with copies / μL was serially diluted 10-fold to obtain a copy number of 10. 1 copies / μL~10 10 CGMMV standard plasmid copies / μL.

[0040] The MIRA amplification system was set up as follows: 29.4 μL of A Buffer, 2 μL of CGMMV-F1 (SEQ ID NO: 1, 10 μM), 2 μL of CGMMV-R1 (SEQ ID NO: 4, 10 μM), 0.6 μL of CGMMV-probe (SEQ ID NO: 7, 10 μM), 5 μL of template, 2.5 μL of B Buffer, one lyophilized enzyme powder reaction tube reagent, and 50 μL of DEPC water were mixed thoroughly.

[0041] The MIRA amplification reaction program was: 42℃, reaction time 20 min.

[0042] With a copy number of 10 1 copies / μL~1010 CGMMV standard plasmid (copies / μL), CGMMV negative sample (cDNA sample obtained by reverse transcription of RNA extraction from healthy watermelon seed sample), and sterile double-distilled water were used as templates in the MIRA amplification system, and the fluorescence curves of the MIRA amplification products corresponding to each template were recorded.

[0043] II. Experimental Results The fluorescence curves of the MIRA amplification products corresponding to each template are shown in the figure. Figure 2 As shown, the results indicate that with a copy number of 10... 4 copies / μL~10 1 When using CGMMV standard plasmids with copies / μL as templates for the MIRA amplification system, no exponential growth phase amplification curve was observed when using CGMMV-F1 (nucleotide sequence shown in SEQ ID NO: 1) and CGMMV-R1 (nucleotide sequence shown in SEQ ID NO: 4) for amplification; however, when using plasmids with copy numbers ≥10... 5 When CGMMV standard plasmids of copies / μL are used as templates, a clear amplification curve can be obtained, indicating that the limit of detection for MIRA amplification of CGMMV using CGMMV-F1 with nucleotide sequences as shown in SEQ ID NO: 1 and CGMMV-R1 with nucleotide sequences as shown in SEQ ID NO: 4 is 10. 5 copies / μL.

[0044] Example 3 Optimization of MIRA amplification reaction I. Experimental Methods 1. Optimization of MIRA amplification temperature 10 respectively 5 Using CGMMV standard plasmid (copies / μL), CGMMV positive samples, negative samples, and sterile double-distilled water (blank control, CK) as templates, MIRA amplification was performed according to the following MIRA amplification system and MIRA amplification reaction degree, and the fluorescence curve of the MIRA amplification products was recorded. The CGMMV positive samples were: watermelon seed samples infected with the CGMMV virus, which were obtained by RNA extraction and reverse transcription to obtain CGMMV positive cDNA samples. CGMMV negative samples are shown in Example 2.

[0045] MIRA amplification system: Mix 29.4 μL of A Buffer, 2 μL of CGMMV-F1 (SEQ ID NO: 1, 10 μM), 2 μL of CGMMV-R1 (SEQ ID NO: 4, 10 μM), 0.6 μL of CGMMV-probe (SEQ ID NO: 7, 10 μM), 5 μL of template, 2.5 μL of B Buffer, one lyophilized enzyme powder reaction tube reagent, and bring the volume up to 50 μL of DEPC water.

[0046] MIRA amplification reaction procedure: 42℃, reaction time 20 min.

[0047] Next, the temperature in the MIRA amplification reaction program was adjusted to 27℃, 32℃, 37℃, 47℃ and 52℃ in sequence, and the fluorescence curves of the MIRA amplification products at different reaction temperatures were recorded.

[0048] 2. Optimization of MIRA amplification time 10 respectively 6 Using CGMMV standard plasmid (copies / μL), CGMMV positive samples, negative samples, and sterile double-distilled water (blank control, CK) as templates, amplification was performed according to the following MIRA amplification system and MIRA amplification reaction degree. The amplification products were collected and observed for fluorescence by irradiation with blue light. MIRA amplification system: Mix 29.4 μL of A Buffer, 2 μL of CGMMV-F1 (SEQ ID NO: 1, 10 μM), 2 μL of CGMMV-R1 (SEQ ID NO: 4, 10 μM), 0.6 μL of CGMMV-probe (SEQ ID NO: 7, 10 μM), 5 μL of template, 2.5 μL of B Buffer, one lyophilized enzyme powder reaction tube reagent, and bring the volume up to 50 μL of DEPC water.

[0049] MIRA amplification reaction procedure: 37℃, reaction time 20 min.

[0050] Next, the reaction time in the MIRA amplification reaction program was adjusted to 10 min and 15 min respectively, and the fluorescence of the MIRA amplification products at different reaction times was recorded and observed.

[0051] II. Experimental Results Fluorescence curves of MIRA amplification products at different reaction temperatures are shown in the figure. Figure 3 As shown, Figure 3 In the diagram, A represents the fluorescence curve of the MIRA amplification product at a reaction temperature of 27℃. Figure 3 B in the graph represents the fluorescence curve of the MIRA amplification product at a reaction temperature of 32℃. Figure 3In the figure, C represents the fluorescence curve of the MIRA amplification product at a reaction temperature of 37℃. Figure 3 In the diagram, D represents the fluorescence curve of the MIRA amplification product at a reaction temperature of 42℃. Figure 3 E in the figure represents the fluorescence curve of the MIRA amplification product at a reaction temperature of 47℃. Figure 3 F in the figure represents the fluorescence curve of the MIRA amplification product at a reaction temperature of 52℃.

[0052] The results showed that CGMMV had a certain amplification effect at all reaction temperatures. When the reaction temperatures were 37℃ and 42℃, there were obvious amplification curves that only showed the growth phase, indicating that the MIRA amplification effect was the best at these two reaction temperatures. Therefore, 37℃ was selected as the optimal MIRA amplification reaction temperature.

[0053] Figure 4 shows the fluorescence of MIRA amplification products at different reaction times. The results show that when the reaction time is 10 min, the fluorescence of the products increases significantly. 6 The MIRA amplification products corresponding to the CGMMV standard plasmid (copies / μL) and positive samples showed fluorescence under blue light irradiation. Moreover, the fluorescence intensity only increased slightly with the increase of reaction time. Therefore, 10 min was selected as the optimal reaction time for the MIRA amplification reaction.

[0054] Example 4: Specificity of the MIRA primer-probe set in detecting CGMMV I. Experimental Methods Nucleic acid samples were obtained from potato virus Y (PVY), zucchini yellow mosaic virus (ZYMV), maize chlorotic mottle virus (MCMV), lambsquarters mosaic virus (SoMV), and tomato chlorotic virus (ToCV); all viruses were collected from the field by the applicant's institution's laboratory over a long period of time.

[0055] Next, nucleic acid samples of potato virus Y, zucchini yellow mosaic virus, maize chlorotic mottle virus, lambsquarters mosaic virus, tomato chlorotic virus, positive samples (shown in Example 3), negative samples (shown in Example 2), and blank control (double-distilled water) were used as templates for MIRA amplification. Amplification was carried out according to the following MIRA amplification system and MIRA reaction procedure. The MIRA amplification products were collected and observed for fluorescence by irradiation with blue light.

[0056] The MIRA amplification system was set up as follows: 29.4 μL of A Buffer, 2 μL of CGMMV-F1 (SEQ ID NO: 1, 10 μM), 2 μL of CGMMV-R1 (SEQ ID NO: 4, 10 μM), 0.6 μL of CGMMV-probe (SEQ ID NO: 7, 10 μM), 5 μL of template, 2.5 μL of B Buffer, one lyophilized enzyme powder reaction tube, and 50 μL of DEPC water were mixed thoroughly.

[0057] The MIRA amplification reaction program was: 37℃, reaction time 10 min.

[0058] II. Experimental Results The fluorescence characteristics of MIRA products from different viruses are shown in the figure below. Figure 5 As shown, the results indicate that when using CGMMV-F1 with nucleotide sequences as shown in SEQ ID NO: 1 and CGMMV-R1 with nucleotide sequences as shown in SEQ ID NO: 4 for amplification, MIRA amplification products only show fluorescence when using CGMMV viral nucleic acid samples as templates; no fluorescence was observed when using other viruses and negative controls as templates, indicating excellent specificity.

[0059] Example 5: Detection of CGMMV using a MIRA primer-probe combination colloidal gold detection chromatography strip. I. Experimental Methods Add a biotin-modified biotin to the 5' end of CGMMV-R1, which has the nucleotide sequence shown in Table 1 of Example 1 as SEQ ID NO: 4, to obtain biotin-labeled CGMMV-R1 (5'-biotin-CGTAGATATTAATTCTAGATTCCCAGATGCG-3').

[0060] Next, the MIRA amplification system was set up as follows: 29.4 μL of AD Buffer, 2 μL of CGMMV-F1 (SEQ ID NO: 1, 10 μM), 2 μL of biotin-labeled CGMMV-R1 (10 μM), 0.6 μL of CGMMV-probe (SEQ ID NO: 7, 10 μM), 5 μL of template, 2.5 μL of B Buffer, 1 sample of colloidal gold test strip RNA isothermal rapid amplification lyophilized enzyme powder reaction tube reagent, and 50 μL of DEPC water were mixed thoroughly.

[0061] AD buffer, B buffer, and colloidal gold test strip RNA isothermal rapid amplification lyophilized enzyme powder reaction tube reagents were all purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., product code WLRN8209KIT; The B Buffer in the product with product code WLRN8209KIT is the same substance as the B Buffer in the product with product code WLRE8208KIT in Example 1; The MIRA amplification reaction program was: 42℃, reaction time 20 min.

[0062] Next, the reaction temperature in the MIRA amplification reaction program was adjusted to 37℃, and the test strip interpretation results of the MIRA amplification products at different reaction temperatures were recorded.

[0063] 10 respectively 3 copies / μL~10 8 CGMMV standard plasmid (copies / μL), CGMMV negative sample (shown in Example 2), and sterile double-distilled water (blank) were used as templates in the MIRA amplification system. MIRA amplification was performed and the MIRA amplification products were collected. Then, the MIRA amplification products were diluted 20 times, and 80 μL of the diluted MIRA amplification products were dropped onto the sample pad of the single nucleic acid test strip. The detection results of the test strip reading area were recorded within 5 minutes.

[0064] The single nucleic acid test strips were purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., with product code WLFS8206.

[0065] II. Experimental Results The detection results of the diluted MIRA amplification product in the test strip reading area are shown in the figure below. Figure 6 As shown, the results indicate that when combined with nucleic acid detection test strips, nucleotide sequences such as CGMMV-F1 contained in SEQ ID NO: 1, and biotin-labeled CGMMV-R1, the detection limit for CGMMV (cucumber green mottle mosaic virus) can still be maintained at 10 under the conditions of 37℃ and 20 min of reaction. 5 The detection limit for CGMMV (cucumber green mottle mosaic virus) reached 10 copies / μL at 42℃ for 20 min. 4 copies / μL.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A MIRA primer set for detecting cucumber green mottle mosaic virus, characterized in that, The primer set contains an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in any one of SEQ ID NO: 1 to 3, and the nucleotide sequence of the downstream primer is shown in any one of SEQ ID NO: 4 to 6.

2. The MIRA primer set according to claim 1, characterized in that, The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

4.

3. The use of the MIRA primer set according to claim 1 or 2 in the preparation of products for detecting cucumber green mottle mosaic virus.

4. The application according to claim 3, characterized in that, The products are testing reagents and / or testing kits.

5. A MIRA primer-probe combination for detecting cucumber green mottle mosaic virus, characterized in that, It includes the MIRA primer set as described in claim 1 or 2 and the probe with the nucleotide sequence shown in SEQ ID NO:

7.

6. A product for detecting cucumber green mottle mosaic virus, characterized in that, Contains the MIRA primer set as described in claim 1 or 2.

7. The product according to claim 6, characterized in that, It also contains a probe with a nucleotide sequence as shown in SEQ ID NO:

7.

8. The product according to claim 6, characterized in that, It also contains MIRA amplification reaction reagents.

9. A method for detecting cucumber green mottle mosaic virus for non-diagnostic purposes, characterized in that, Includes the following steps: S1. Extract RNA from the sample to be tested and reverse transcribe it into cDNA; S2. Using the cDNA obtained in step S1 as a template, perform MIRA amplification using any one of the products described in claims 6 to 8 to obtain MIRA amplification products; The MIRA amplification temperature is 37–42℃, and the amplification reaction time is 10–20 min; S3. Place the MIRA amplification product obtained in step S2 under blue light for observation. If the MIRA amplification product does not fluoresce under blue light, the sample to be tested does not contain cucumber green mottle mosaic virus; if the MIRA amplification product fluoresces under blue light, the sample to be tested contains cucumber green mottle mosaic virus.

10. A method for detecting cucumber green mottle mosaic virus for non-diagnostic purposes, characterized in that, Includes the following steps: S11. Extract RNA from the sample to be tested and reverse transcribe it into cDNA; S12. Using the cDNA obtained in step S1 as a template, add a biotin-modified biotin to the 5' end of the downstream primer in the MIRA primer set of any of the products described in claims 6 to 8, then perform MIRA amplification, and detect the amplification using a single nucleic acid test strip. If red lines appear on both the control line and the test line of the single nucleic acid test strip, the sample to be tested contains cucumber green mottle mosaic virus; If a red line appears on the control line of a single nucleic acid test strip but not on the test line, the sample to be tested does not contain cucumber green mottle mosaic virus. If no red lines appear on either the control line or the test line of the single nucleic acid test strip, the test is invalid and needs to be repeated.

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