TOCV fluorescence detection probe primer group, detection method, kit and application

Abstract

The invention discloses a TOCV fluorescence detection probe primer group, a detection method, a kit and application, and belongs to the technical field of virus detection. The invention solves the problems that the detection of ToCV in the prior art depends on RT-PCR technology, needs high-end large-scale equipment, consumes long time and is difficult to meet the real-time field monitoring requirement. The invention provides a TOCV fluorescence detection probe primer group which comprises a probe and an upstream primer, wherein the sequence of the upstream primer is shown as SEQ ID No.1; the sequence of the downstream primer is shown as SEQ ID No.2. The invention provides a detection method, a kit and application based on the primer. The tomato chlorosis virus is detected through an MIRA fluorescent rapid detection technology, a specific probe primer group is provided, transcription is not needed, detection including amplification and fluorescent detection can be completed within 25 minutes at the soonest, complex instruments are not needed, 37-43 DEG C constant-temperature detection is achieved, field portable equipment is compatible, direct experiments can be carried out in the field, and the kit is high in sensitivity and high in sensitivity. Screening of virus-carrying plants in a seedling raising period or before dispatching is facilitated, and virus diffusion is blocked.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and specifically relates to a TOCV fluorescent detection probe primer set, a detection method, a kit and applications. Background Art

[0002] Tomato chlorosis virus (ToCV) belongs to the Closteroviridae family and the genus Crinivirus. It has a wide host range, infecting crops in the Solanaceae family, including tomatoes, bell peppers, and potatoes, as well as a variety of plants in the Cruciferae and Cucurbitaceae families. The virus is transmitted by insect vectors, including Bemisia tabaci, Trialeurodes vaporariorum, and Trialeurodes abutilonea. The highly invasive Middle East Asia Minor 1 (MEAM1) and Mediterranean (MED) whiteflies are the primary vectors of ToCV. ToCV has an incubation period, and symptoms only appear three weeks after seedling infection, increasing the risk of seedling transportation and transmission. Infection with ToCV causes interveinal chlorosis, yellowing, thickening and brittleness of leaves, dwarfing of plants, stunted fruit development and reduced quality. In severe cases, the entire plant dies, resulting in a yield reduction of more than 50% or even a total loss of production.

[0003] However, the existing ToCV detection relies on RT-PCR technology, which requires high-end large-scale equipment and is time-consuming, making it difficult to meet the needs of real-time field monitoring. Summary of the Invention

[0004] In view of the problem that the detection of ToCV in the existing technology relies on RT-PCR technology, which requires high-end large-scale equipment and is time-consuming, and is difficult to meet the needs of real-time field monitoring, the present invention provides a TOCV fluorescent detection probe primer set, detection method, kit and application.

[0005] The technical solution adopted in the present invention is as follows:

[0006] A TOCV fluorescence detection probe primer set, comprising:

[0007] Probe:

[0008]

[0009] Upstream primer:

[0010] 5'-TTCAGATTGTATTACACATGGAGGGGTTGAAAG-3';

[0011] Downstream primer:

[0012] 5'-GTCCAAATGCATTGTCCTTCCAAGCGGCGA-3'.

[0013] A TOCV fluorescence detection method based on the TOCV fluorescence detection probe primer set comprises the following steps:

[0014] S1: Extract RNA from the plant to be tested;

[0015] S2: adding the RNA extracted in S1 to a constant temperature rapid amplification system containing the probe primer set to perform a constant temperature rapid amplification reaction;

[0016] S3: Determine whether the tomato chlorosis virus is present based on the fluorescence detection results.

[0017] Preferably, the amplification conditions in S2 include: an amplification temperature of 37-43°C.

[0018] Preferably, the amplification temperature is 37°C.

[0019] Preferably, the fluorescence signal is detected every 30 seconds in S3.

[0020] An application of the TOCV fluorescent detection probe primer set in detecting tomato chlorosis virus.

[0021] A constant temperature rapid amplification kit containing the TOCV fluorescence detection probe primer set.

[0022] Preferably, the total reaction volume of the fluorescence constant temperature rapid amplification kit is 50 μL, the amount of RNA added from the plant to be tested is 5 μL, and the reaction system includes:

[0023] Dry powder reaction tube: 1;

[0024] Dry powder reaction solution: 29.4 μL;

[0025] The upstream primer of claim 1: 2 μL, concentration 10 μM;

[0026] The downstream primer of claim 1: 2 μL, concentration 10 μM;

[0027] The probe primer of claim 1: 0.6 μL, concentration 10 μM;

[0028] ddH2O and RNA template: 13.5 μL

[0029] Start reaction buffer: 2.5 μL

[0030] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0031] 1. The present invention detects tomato chlorosis virus based on the MIRA rapid detection technology and provides fluorescent probes and specific primers. The extracted RNA of the test plant can be directly amplified without transcription, and the detection, including amplification and fluorescence detection, can be completed in as fast as 25 minutes. No complex instruments are required, and a constant temperature of 37-43°C is achieved. The method is compatible with on-site portable equipment and can be used for direct field experiments. This helps screen infected plants during the seedling stage or before transportation, thereby blocking the spread of the virus.

[0032] 2. Since ToCV is an RNA genome, it is extremely prone to mutation and the pathogenicity of different isolates varies significantly. Therefore, the rapid detection technology based on the viral capsid CP gene in the present invention can reduce false negatives caused by genome mutations and provide data support for disease-resistant breeding and regional prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Screening orthogonal amplification profiles for primers;

[0034] Figure 2 Screening of backcross amplification profiles for primers;

[0035] Figure 3 Screening linear amplification profiles for temperature gradients;

[0036] Figure 4 Screening fluorescence profiles for temperature gradients;

[0037] Figure 5 To screen the linear amplification profile for specificity;

[0038] Figure 6 Fluorescence images were screened for specificity;

[0039] Figure 7 is the linear amplification pattern of the lowest detection limit;

[0040] Figure 8 Screening fluorescence patterns for the lowest detection limit;

[0041] Figure 9 Verify the linear amplification profile for multi-virus specificity;

[0042] Figure 10 Fluorescence images were validated for multi-virus specificity. DETAILED DESCRIPTION

[0043] To make the purpose, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of this application.

[0044] Primer design:

[0045] A primer pair was designed based on the conserved region of the DNA sequence corresponding to the Tomato chlorosis virus capsid protein (CPm), ultimately obtaining three upstream primers, three downstream primers, and one pre-modified probe primer. The pre-modified probe primer was modified to obtain the probe, as shown in Table 1:

[0046] Table 1

[0047]

[0048]

[0049] In order to verify the effectiveness of the probe primer set designed by the present invention, the present invention conducted the following experiments:

[0050] RNA extraction

[0051] Sample source: Fresh tomato leaves containing TOCV virus

[0052] Kit: TransZol Up Plus RNA Kit (full gold, with two parts, A and B)

[0053] Extraction results: RNA concentration in each sample is:

[0054] Sample 1: 27.5 ng / μL, A260 / A280: 2.01, A260 / A230: 1.36;

[0055] Sample 2: 19.4 ng / μL, A260 / A280: 1.93, A260 / A230: 1.55;

[0056] Sample 3: 19.1 ng / μL, A260 / A280: 1.9, A260 / A230: 0.91;

[0057] Note: RNA samples were stored in a -80°C freezer.

[0058] (1) Primer screening

[0059] Sample: RNA sample 3; ddH20 (negative control)

[0060] Kit: RNA Constant Temperature Rapid Amplification Kit (Fluorescence Type), Amp Future

[0061] Sample volume: Add 2.5 μL RNA sample 3 to 25 μL reaction system

[0062] Primer + probe combination:

[0063] Combination 1: (1F1R+p)F: 5'-TTCAGATTGTATTACACATGGAGGGTTGAAAG-3'

[0064] R: 5'-GTCCAAATGCATTGTCCTTCCAAGCGGCGA-3'

[0065] P: 5'-TTCCGACCGATTTCACAATACTTCAACAAA[FAMdT][THF]

[0066] [BHQ1dT]GGTATGGGACGACTGC-[3'C3spacer]

[0067] Combination 2: (1F2R+p)F: 5'-TTCAGATTGTATTACACATGGAGGGTTGAAAG-3'

[0068] R: 5'-CTGCCTGGAATTGTTGAGATCTTCGCTAAAGTC-3'

[0069] P: 5'-TTCCGACCGATTTCACAATACTTCAACAAA[FAMdT][THF]

[0070] [BHQ1dT]GGTATGGGACGACTGC-[3'C3spacer]

[0071] Combination 3: (1F3R+p)F: 5'-TTCAGATTGTATTACACATGGAGGGTTGAAAG-3'

[0072] R: 5'-ATGGTCCACGAACCTGCCTGGAATTGTTGAGATC-3'

[0073] P: 5'-TTCCGACCGATTTCACAATACTTCAACAAA[FAMdT][THF]

[0074] [BHQ1dT]GGTATGGGACGACTGC-[3'C3spacer]

[0075] Combination 4: (1F1R+p)F: 5'-TTCAGATTGTATTACACATGGAGGGTTGAAAG-3'

[0076] R: 5'-GTCCAAATGCATTGTCCTTCCAAGCGGCGA-3'

[0077] P: 5'-TTCCGACCGATTTCACAATACTTCAACAAA[FAMdT][THF]

[0078] [BHQ1dT]GGTATGGGACGACTGC-[3'C3spacer]

[0079] Combination 5: (2F1R+p)F: 5'-AGATTGTATTACACATGGAGGGTTGAAAGAGA-3'

[0080] R: 5'-GTCCAAATGCATTGTCCTTCCAAGCGGCGA-3'

[0081] P: 5'-TTCCGACCGATTTCACAATACTTCAACAAA[FAMdT][THF]

[0082] [BHQ1dT]GGTATGGGACGACTGC-[3'C3spacer]

[0083] Combination 6: (3F1R+p)F: 5'-GATTGTATTACACATGGAGGGTTGAAAGAG-3'

[0084] R: 5'-GTCCAAATGCATTGTCCTTCCAAGCGGCGA-3'

[0085] P: 5'-TTCCGACCGATTTCACAATACTTCAACAAA[FAMdT][THF]

[0086] [BHQ1dT]GGTATGGGACGACTGC-[3'C3spacer]

[0087] Amplification procedure: constant temperature metal bath amplification for 20 min, amplification temperature set at 37°C; number of cycles: 40; fluorescence signal collected every 30 s.

[0088] Test results

[0089] The orthogonal experiment results are as follows Figure 1 As shown ( Figure 1Middle: Orange: 1F1R+p; Red: 1F2R+p; Green: 1F3R+p), the backcross results are as follows Figure 2 As shown (orange: 1F1R+p-1; red: 1F1R+p-2; green: 2F1R+p; blue: 3F1R+p): from Figure 1 and Figure 2 It can be seen that the exponential growth period of 1F1R+p is the most significant, indicating that the 1F1R+p combination is the best. Therefore, the probe primer set finally obtained in the present invention is 1F1R+p:

[0090] F (SEQ ID No. 1): 5'-TTCAGATTGTATTACACATGGAGGGGTTGAAAG-3'

[0091] R (SEQ ID No. 2): 5'-GTCCAAATGCATTGTCCTTCCAAGCGGCGA-3'

[0092] P:

[0093]

[0094] (2) Screening of amplification temperature

[0095] Sample: RNA sample 2, concentration 19.4 ng / μL; ddH2O (negative control);

[0096] Kit: RNA Constant Temperature Rapid Amplification Kit (Basic Type)-II, Anpu Future;

[0097] Sample volume: Add 2.5 μL of extracted RNA to the reaction system;

[0098] Reaction system:

[0099] Table 2

[0100]

[0101]

[0102] Amplification procedure: constant temperature metal bath amplification for 20 min, amplification temperature settings 37°C, 38°C, 39°C, 40°C, 41°C, 43°C; number of cycles: 40; fluorescence signal collected every 30 s.

[0103] Experimental results:

[0104] Amplification diagram Figure 3 As shown, Figure 3 N: Negative, negative control; P: Positive, positive sample. Figure 4 As shown, from Figure 3 It can be seen that when the amplification temperature is 37℃, 38℃ and 43℃, the negative control curve is stable, and the positive sample is in an exponential growth period. Figure 4 It can be seen that the fluorescence intensity of the positive sample is also relatively strong when the amplification temperature is 37°C. Therefore, 37°C is determined to be the optimal amplification temperature after comprehensive consideration.

[0105] (3) Specificity detection

[0106] Sample: RNA sample 1, concentration 27.5 ng / μL; ddH2O (negative control)

[0107] Kit: RNA Constant Temperature Rapid Amplification Kit (Basic Type)-Ⅱ, Amp Future

[0108] Experimental conditions: constant temperature metal bath amplification for 20 min, amplification temperature set at 37°C; number of cycles: 40; fluorescence signal collected every 30 s.

[0109] Experimental results: Specificity test results are as follows Figure 5 (N: Negative, negative control; P: Positive, positive sample) and Figure 6 As shown, Figure 5 and Figure 6 The example is the test result of fresh tomato leaves infected with TOCV virus collected in the field; Figure 5 It can be seen that the amplification curve of the fresh tomato leaves infected with TOCV virus collected in the field almost overlaps with the amplification curve of the positive sample, while the negative curve is stable. Figure 6 It can be seen that the amplification curves of fresh tomato leaves infected with TOCV virus collected in the field and the positive samples both have obvious fluorescence, while the negative samples do not, indicating that it has good specificity.

[0110] (4) Minimum detection limit detection

[0111] Sample: RNA sample 2, concentration 19.4 ng / μL; ddH2O (negative control)

[0112] Kit: RNA Constant Temperature Rapid Amplification Kit (Basic Type)-Ⅱ, Amp Future

[0113] Experimental conditions: constant temperature metal bath amplification for 20 min, amplification temperature set at 37°C; number of cycles: 40; fluorescence signal collected every 30 s.

[0114] Experimental results: The minimum detection limit results are as follows Figure 7-8 As shown ( Figure 7 N: Negative, negative control; P: Positive, positive sample), such as Figure 7-8 As shown, diluted to 10 4The curve is stable when the dilution is 10 3 times, that is, the minimum detection limit is 0.0194ng / μL.

[0115] (5) Multi-virus specificity verification

[0116] Samples: positive (RNA sample 1, concentration 27.8 ng / μL), negative (template is ddH2O), TOCV (RNA, concentration 19.8 ng / μL), TYLCV (Tomato yellow leaf curl virus) & TOCV (RNA, concentration 139.72 ng / μL), TOBRFV (Tomato brown wrinkled fruit virus) & TOCV (RNA, concentration 58.48 ng / μL), TSWV (Tomato spotted wilt virus) (RNA, concentration 19.84 ng / μL), TOLCNDV (Tomato leaf curl virus) (DNA, concentration 4.11 ng / μL), TYLCV (Tomato yellow leaf curl virus) (RNA, concentration 6.74 ng / μL).

[0117] Kit: RNA Constant Temperature Rapid Amplification Kit (Basic Type)-Ⅱ, Anpu Future.

[0118] Experimental conditions: constant temperature metal bath amplification for 20 min, amplification temperature set at 37°C; number of cycles: 40; fluorescence signal collected every 30 s.

[0119] Experimental results: The results are as follows Figure 9-10 As shown ( Figure 9 N: Negative, negative control; P: Positive, positive sample), from Figure 9-10 It can be seen that if the sample contains TOCV, the amplification curve will show an obvious exponential growth period and fluorescence will appear, while other virus samples will not show this, indicating that the present invention has good specificity and will not cause false positives in the case of multiple viruses.

[0120] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A TOCV fluorescence detection probe primer set, characterized in that: include: Probe: 5’-TTCCGACCGATTTCACAATACTTCAACAAA[FAMdT] [THF] [BHQ1dT]GGTATGGG ACGACTGC-[3’C3spacer]; Upstream primer: 5'-TTCAGATTGTATTACACATGGAGGGGTTGAAAG-3'; Downstream primer: 5'-GTCCAAATGCATTGTCCTTCCAAGCGGCGA-3'.

2. A TOCV fluorescence detection method based on the TOCV fluorescence detection probe primer set according to claim 1, characterized in that: The following steps are involved: S1: Extract RNA from the plant to be tested; S2: adding the RNA extracted in S1 to a constant temperature rapid amplification system containing the probe primer set according to claim 1 to perform a constant temperature rapid amplification reaction; S3: Determine whether tomato chlorosis virus is present based on the fluorescence detection results.

3. A TOCV fluorescence detection method according to claim 2, characterized in that: The amplification conditions in S2 include: an amplification temperature of 37-43°C.

4. A TOCV fluorescence detection method according to claim 3, characterized in that: The amplification temperature was 37°C.

5. A TOCV fluorescence detection method according to claim 2, characterized in that: In S3, the fluorescence signal was detected every 30 s.

6. Use of the TOCV fluorescent detection probe primer set according to claim 1 in detecting TOCV.

7. A fluorescence isothermal rapid amplification kit containing the TOCV fluorescence detection probe primer set according to claim 1.

8. The fluorescence isothermal rapid amplification kit according to claim 7, characterized in that: The total reaction volume of the fluorescence constant temperature rapid amplification kit is 50 μL, and the amount of RNA added from the plant to be tested is 5 μL. The reaction system includes: Dry powder reaction tube: 1; Dry powder reaction solution: 29.4 μL; The upstream primer of claim 1: 2 μL, concentration 10 μM; The downstream primer of claim 1: 2 μL, concentration 10 μM; The probe primer of claim 1: 0.6 μL, concentration 10 μM; ddH2O and RNA template: 13.5 μL; Start reaction buffer: 2.5 μL.

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