Virus positive control plasmid for strawberry virus-free seedling detection and detection primer sequence

Abstract

The invention discloses a virus positive control plasmid for strawberry virus-free seedling detection. The sequence of the virus positive control plasmid is as follows: AGGC.

Description

Technical Field

[0001] The present invention relates to A01H, to the field of plant regeneration through tissue culture technology, and in particular to a virus positive control plasmid and detection primer sequence for strawberry virus-free seedling detection Background Art

[0002] Strawberry virus has a wide range of harmful effects and is an important disease in strawberry production. The occurrence and harm of strawberry virus has become an urgent problem to be solved in strawberry production in China. Selecting virus-free seedlings for strawberry planting is an important means to solve the problem of strawberry virus. However, the existing strawberry virus-free seedlings cannot achieve true virus removal. The detection cycle of virus-free seedlings is long, and there is no positive control during the detection, resulting in the inability to effectively verify the virus-free effect, further exacerbating the risk of virus transmission in the strawberry industry. Therefore, it is very important to develop a virus positive control plasmid for strawberry virus-free seedling detection.

[0003] Chinese invention patent CN113684319A discloses a specific nucleic acid sequence and amplification primer, kit and method for rapid detection of strawberry mild yellow edge virus, by screening strawberry mild yellow edge virus specific conservative primers and probes, based on the reverse transcription-recombinase polymerase constant temperature amplification method, combined with a test strip to visualize the amplification product results, to achieve simple, rapid and highly sensitive detection of strawberry mild yellow edge virus, but there is no positive control, and the detection accuracy is low. Chinese invention patent CN114280292A discloses a colloidal gold test strip for strawberry mild yellow edge virus and its preparation method and application, the colloidal gold labeled antibody is a polyclonal antibody prepared from strawberry mild yellow edge virus, the detection line T is formed by the polyclonal antibody prepared from strawberry mild yellow edge virus; the quality control line C is formed by mouse anti-rabbit IgG or mouse anti-rabbit IgG solution, the colloidal gold rapid detection test strip is fast and specific, and is convenient for rapid detection of strawberry seedlings, virus-free seedlings, etc., but the preparation steps are complicated, and there is no positive control, and the accuracy is not high. Summary of the invention

[0004] In order to develop a virus positive control plasmid for strawberry virus-free seedling detection, the first aspect of the present invention provides a virus positive control plasmid for strawberry virus-free seedling detection, the sequence of the virus positive control plasmid is as follows:

[0005] AGGC N CA Y GC H CACCA H TGTAC H GTTAGAGA YY TGGTGAAG Y T N AATAAAGTAGA H GT NY T World Cup GGCCCAT H TGGGCC Y T W GATTAGG.

[0006] As a preferred embodiment, the method for preparing the virus positive control plasmid comprises the following steps:

[0007] S1 planted strawberry seedlings and distinguished strawberry plants that showed virus phenotypes from those that did not;

[0008] S2: Mature tissues of strawberry plants showing virus phenotypes and those not showing virus phenotypes were taken to extract total RNA;

[0009] S3 purifies and detects the extracted RNA, sequences it, compares the RNA sequence with the database, and merges it into a sequence chain;

[0010] S4 designs the sequence strand to contain the cDNA sequence template of the subtype virus to obtain a positive control plasmid;

[0011] S5 designed RT-PCR primers for virus detection and verified the accuracy of the positive control plasmid.

[0012] The second aspect of the present invention provides a detection primer, which is a virus detection RT-PCR primer obtained by using the above-mentioned method for preparing the virus positive control plasmid for strawberry virus-free seedling detection.

[0013] As a preferred embodiment, the sequence of the detection primer is as follows:

[0014] SMYEV-FQ:AGGC N CA Y GC H CACCA H TGT

[0015] SMYEV-RQ:CCTAATC S A R GGCCCA D ATGGG.

[0016] As a preferred embodiment, in step S2, the mature tissue is ground into a sample using liquid nitrogen grinding method, and then total RNA is extracted using a kit.

[0017] As a preferred embodiment, the database includes a combination of an mRNA database, an RFam database, and a Repbase database.

[0018] As a preferred embodiment, the virus phenotype exhibited by the strawberry plants in step S1 is strawberry mild yellow edge virus.

[0019] As a preferred embodiment, the RNA purification test extracted in step S3 includes RNA integrity test and RNA purity test.

[0020] As a preferred embodiment, the method is applied to the detection of virus-free strawberry seedlings, and the detection method of virus-free strawberry seedlings comprises the following steps:

[0021] M1 collected tissue samples of virus-free strawberry seedlings;

[0022] M2 extracted total RNA from virus-free strawberry seedling tissue samples;

[0023] M3 reverse transcribes total RNA into cDNA;

[0024] M4 was amplified by PCR using RT-PCR primers, with a virus positive control plasmid as a positive control and a virus-free sample as a negative control;

[0025] M5 was separated by agarose gel electrophoresis, and the band distribution of the PCR amplification product was compared with that of the positive control and the negative control to determine whether the virus was contained.

[0026] As a preferred embodiment, the conditions for PCR amplification are as follows: 90-98°C, 8-13 min; 90-98°C, 10-20 s; 55-65°C, 0.5-1.5 min, and 40 cycles.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The virus positive control plasmid for strawberry virus-free seedling detection of the present invention uses strawberry plants showing a virus phenotype to extract RNA, and the extraction step is simple, which simplifies the preparation steps of the positive control plasmid.

[0029] (2) The virus positive control plasmid for strawberry virus-free seedling detection of the present invention is applied to the detection of strawberry virus-free seedlings, which can accurately evaluate the virus-free effect of strawberry seedlings and improve the detection accuracy of the virus-free effect.

[0030] (3) The virus positive control plasmid for strawberry virus-free seedling detection of the present invention is applied to the detection of strawberry virus-free seedlings, thereby achieving rapid and efficient detection and shortening the detection time.

[0031] (4) The virus positive control plasmid for strawberry virus-free seedling detection described in the present invention can achieve excellent detection of strawberry mild yellow edge virus, has good detection specificity, and can be applied to the detection of various strawberry varieties.

[0032] (5) The virus positive control plasmid for strawberry virus-free seedling detection of the present invention can be used as a positive control and can be applied to large-scale strawberry planting, thus providing technical guarantee for the healthy development of the strawberry industry and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the gel electrophoresis image of strawberry mild yellow edge virus.

[0034] Figure 2 This is the gel electrophoresis image of the negative control sample. DETAILED DESCRIPTION

[0035] Example

[0036] A virus positive control plasmid for strawberry virus-free seedling detection, the sequence of the virus positive control plasmid is as follows:

[0037] AGGC N CA Y GC H CACCA H TGTAC H GTTAGAGA YY TGGTGAAG Y T N AATAAAGTAGA H GT NY T World Cup GGCCCAT H TGGGCC Y T W GATTAGG.

[0038] The preparation method of the virus positive control plasmid comprises the following steps:

[0039] S1 planted strawberry seedlings and distinguished strawberry plants that showed virus phenotypes from those that did not;

[0040] S2: Mature tissues of strawberry plants showing virus phenotypes and those not showing virus phenotypes were taken to extract total RNA;

[0041] S3 purifies and detects the extracted RNA, sequences it, compares the RNA sequence with the database, and merges it into a sequence chain;

[0042] S4 designs the sequence strand to contain the cDNA sequence template of the subtype virus to obtain a positive control plasmid;

[0043] S5 designed RT-PCR primers for virus detection and verified the accuracy of the positive control plasmid.

[0044] The sequences of the virus detection RT-PCR primers are as follows:

[0045] SMYEV-FQ:AGGC N CA Y GC H CACCA H TGT

[0046] SMYEV-RQ:CCTAATC S A R GGCCCA D ATGGG.

[0047] The underlined bases are designed for strawberry mild yellow edge virus subtypes.

[0048] The specific steps of step S1 are as follows:

[0049] Different varieties of strawberry seedlings were planted in inorganic substrates using cultivation bags under artificial climate conditions to determine whether the seedlings themselves carry the virus and avoid secondary infection. Strawberry plants that show virus phenotypes and those that do not show virus phenotypes were screened for subsequent analysis.

[0050] The specific steps of step S2 are as follows:

[0051] Extract total RNA from fully mature tissues (leaves, shortened stems, floral organs, and roots) of strawberry plants that exhibited a viral phenotype in step S1 as a positive control;

[0052] Total RNA was extracted from fully mature tissues (leaves, shortened stems, flower organs, roots) of strawberry plants grown in the same environment in step S1 and showing no virus phenotype, as a negative control.

[0053] The method for extracting total RNA is a kit extraction method, and the specific steps are as follows:

[0054] The kit is the EASYspin plant RNA rapid extraction kit from Adelaide Biotechnology Co., Ltd.

[0055] (1) Grinding samples using liquid nitrogen grinding method: After freezing the samples in liquid nitrogen, use a grinder to grind them. Plant tissues such as stem segments become brittle and easy to break under the low temperature of liquid nitrogen. When grinding with a grinder, the samples can be quickly broken and fine particles can be obtained, which is convenient for subsequent experimental processing;

[0056] (2) Take out 500 μL of lysate RLT, pour it into a 1.5 mL centrifuge tube, add 50 μL of PLANTaid, mix well and set aside;

[0057] (3) After grinding the plant tissue into fine powder in liquid nitrogen, 50 mg of the fine powder was transferred into the above centrifuge tube containing RLT and PLANTaid, and immediately shaken vigorously by hand for 20 seconds to fully lyse;

[0058] (4) Use a pipette tip to mix and swirl to help lysis, which can shear DNA, reduce viscosity and increase yield;

[0059] (5) Centrifuge the lysate at 13,000 rpm for 5 minutes to precipitate insoluble fragments and PLANTaid bound to polysaccharides and polyphenols;

[0060] (6) Transfer the supernatant of the lysate to a new centrifuge tube and add anhydrous ethanol equal to half the volume of the supernatant. If precipitation occurs, mix it immediately by pipetting. Centrifugation is not required.

[0061] (7) Add the mixture to the adsorption column RA in portions (less than 720 μL each time), place the adsorption column RA in a collection tube, and centrifuge at 13,000 rpm for 2 min. Discard the waste liquid to ensure that all the liquid passes through the filter membrane after centrifugation without residue.

[0062] (8) Add 700 μL of deproteinized solution RW1, let it stand at room temperature for 1 min, then centrifuge at 13000 rpm for 30 s, and discard the waste liquid;

[0063] (9) Add 500 μL of rinse solution RW and centrifuge at 13,000 rpm for 30 s. Discard the waste liquid and add 500 μL of rinse solution RW again. Repeat the above steps.

[0064] (10) Place the adsorption column RA back into the empty collection tube and centrifuge at 13,000 rpm for 3 min to remove the rinse solution to prevent the residual ethanol in the rinse solution from inhibiting downstream reactions;

[0065] (11) Take out the adsorption column RA from the collection tube and place it in an RNase-free centrifuge tube. According to the expected RNA yield, add 50 μL of RNase-free water to the middle part of the adsorption membrane, then place it at room temperature for 1 min, and then centrifuge at 12000 rpm for 1 min.

[0066] (12) If the expected RNA yield is greater than 30 μg, add 50 μL of RNase-free water, repeat step (7), and combine the two wash solutions to obtain the extracted RNA.

[0067] (13) The protein was further tested by agarose gel electrophoresis.

[0068] The RNA purification test extracted in step S3 includes RNA integrity test and RNA purity test.

[0069] The specific steps of RNA integrity testing are as follows:

[0070] Take 3 μL of the above RNA sample, add 2 μL of bromophenol blue, mix thoroughly and then load the sample for electrophoresis. If the electrophoresis results show two clear bands, one of which is about twice as wide as the other, corresponding to 28S rRNA and 18S rRNA, respectively, it indicates that the RNA extraction quality is good; if the bands are fuzzy or unclear, it means that the RNA extraction quality does not meet the standard and RNA extraction needs to be repeated.

[0071] The specific steps for RNA purity testing are as follows:

[0072] Take 1 μL RNA sample and drop it onto the probe of the micro-volume UV spectrophotometer. If the displayed spectrum shows a single peak and the A260 / A280 value is between 1.8 and 2.0, it indicates that the RNA is of high purity; if the A260 / A280 value is greater than 2, it indicates that the RNA may contain ethanol that has not evaporated completely; if the A260 / A280 value is less than 1.8, it indicates that the sample may contain proteins or phenols and the RNA sample has been contaminated.

[0073] The specific steps of RNA sequencing are as follows:

[0074] The RNA samples were sent to Novogene for small RNA sequencing. The raw data generated by the sequencing was first processed and cleaned, and then compared with multiple bioinformatics databases to filter out sequences that matched known genes, non-coding RNAs, and repetitive sequences. After data processing, the heat map distribution analysis was used to further identify regions that may be related to the virus. These regions were assembled to generate longer contig sequences (i.e., spliced ​​continuous sequence fragments), which were eventually established as fragments of the strawberry virus for subsequent research and verification.

[0075] The database includes a combination of an mRNA database, an RFam database (for alignment of non-coding RNA families), and a Repbase database (for alignment of repetitive sequences).

[0076] The virus positive control plasmid is applied to the detection of virus-free strawberry seedlings, and the detection method comprises the following steps:

[0077] M1 collected tissue samples of virus-free strawberry seedlings;

[0078] M2 Extraction of total RNA from virus-free strawberry seedling tissue samples: Use an RNA extraction kit to extract total RNA from strawberry plant tissues;

[0079] M3 reverse transcribes total RNA into cDNA;

[0080] M4 performed PCR amplification of cDNA using RT-PCR primers, with virus positive control plasmid as positive control and virus-free sample as negative control;

[0081] M5 was separated by agarose gel electrophoresis, and the band distribution of the PCR amplification product was compared with that of the positive control and the negative control to determine whether the virus was contained.

[0082] The conditions of the PCR amplification are as follows: 95° C., 10 min; 95° C., 15 s; 60° C., 1 min, and 40 cycles.

[0083] The specific steps of step M5 are as follows:

[0084] The PCR products were separated by agarose gel electrophoresis and stained with nucleic acid dye (Ethidium Bromide). The electrophoresis results were observed and the amplified bands of the sample PCR products were compared with those of the positive control sequence to confirm whether the strawberry sample carried the virus. If a band matching the positive control was shown, it was carrying the virus, and a negative sample did not show the corresponding band.

[0085] The RNA extraction kit is the EASYspin plant RNA rapid extraction kit from Adelaide Biotechnology Co., Ltd.

Claims

1. A virus positive control plasmid for strawberry virus-free seedling detection, characterized in that: The sequence of the virus positive control plasmid is as follows: <h2 style=";text-align:left;direction:ltr">AGGC<h2 style=";text-align:left;direction:ltr"> N <h2 style=";text-align:left;direction:ltr"> CA<h2 style=";text-align:left;direction:ltr"> Y <h2 style=";text-align:left;direction:ltr"> GC<h2 style=";text-align:left;direction:ltr"> H <h2 style=";text-align:left;direction:ltr"> CACCA<h2 style=";text-align:left;direction:ltr"> H <h2 style=";text-align:left;direction:ltr"> TGTAC<h2 style=";text-align:left;direction:ltr"> H <h2 style=";text-align:left;direction:ltr"> GTTAGAGA<h2 style=";text-align:left;direction:ltr"> YY <h2 style=";text-align:left;direction:ltr"> TGGTGAAG<h2 style=";text-align:left;direction:ltr"> Y <h2 style=";text-align:left;direction:ltr"> T<h2 style=";text-align:left;direction:ltr"> N <h2 style=";text-align:left;direction:ltr"> AATAAA GTAGA<h2 style=";text-align:left;direction:ltr"> H <h2 style=";text-align:left;direction:ltr"> GT<h2 style=";text-align:left;direction:ltr"> NY <h2 style=";text-align:left;direction:ltr"> T<h2 style=";text-align:left;direction:ltr"> VM <h2 style=";text-align:left;direction:ltr"> GGCCCAT<h2 style=";text-align:left;direction:ltr"> H <h2 style=";text-align:left;direction:ltr"> TGGGCC<h2 style=";text-align:left;direction:ltr"> Y <h2 style=";text-align:left;direction:ltr"> T<h2 style=";text-align:left;direction:ltr"> W <h2 style=";text-align:left;direction:ltr"> GATTAGG.

2. The virus positive control plasmid for strawberry virus-free seedling detection according to claim 1, characterized in that: The preparation method of the virus positive control plasmid comprises the following steps: S1 planted strawberry seedlings and distinguished strawberry plants that showed virus phenotypes from those that did not; S2: Mature tissues of strawberry plants showing virus phenotypes and those not showing virus phenotypes were taken to extract total RNA; S3 purifies and detects the extracted RNA, sequences it, compares the RNA sequence with the database, and merges it into a sequence chain; S4 designs the sequence strand to contain the cDNA sequence template of the subtype virus to obtain a positive control plasmid; S5 designed RT-PCR primers for virus detection and verified the accuracy of the positive control plasmid.

3. A detection primer, characterized in that The virus detection RT-PCR primer is obtained by the preparation method of the virus positive control plasmid for strawberry virus-free seedling detection described in claim 2.

4. The detection primer according to claim 3, characterized in that The sequences of the detection primers are as follows: SMYEV-FQ: AGGC N CA Y GC H CACCA H TGT <h2 style=";text-align:left;direction:ltr">SMYEV-RQ:CCTAATC<h2 style=";text-align:left;direction:ltr"> S <h2 style=";text-align:left;direction:ltr"> A<h2 style=";text-align:left;direction:ltr"> R <h2 style=";text-align:left;direction:ltr"> GGCCCA<h2 style=";text-align:left;direction:ltr"> D <h2 style=";text-align:left;direction:ltr"> ATGGG.

5. The virus positive control plasmid for strawberry virus-free seedling detection according to claim 2, characterized in that, In step S2, the mature tissue is ground into a sample using a liquid nitrogen grinding method, and then total RNA is extracted using a kit.

6. The virus positive control plasmid for strawberry virus-free seedling detection according to claim 2, characterized in that, The database includes a combination of an mRNA database, an RFam database, and a Repbase database.

7. The virus positive control plasmid for strawberry virus-free seedling detection according to claim 2, characterized in that, The virus phenotype exhibited by the strawberry plants in step S1 is strawberry mild yellow edge virus.

8. The virus positive control plasmid for strawberry virus-free seedling detection according to claim 2, characterized in that, The RNA purification test extracted in step S3 includes RNA integrity test and RNA purity test.

9. The virus positive control plasmid for strawberry virus-free seedling detection according to claim 2, characterized in that, Applied to the detection of virus-free strawberry seedlings, the detection method of virus-free strawberry seedlings comprises the following steps: M1 collected tissue samples of virus-free strawberry seedlings; M2 extracted total RNA from virus-free strawberry seedling tissue samples; M3 reverse transcribes total RNA into cDNA; M4 was amplified by PCR using RT-PCR primers, with a virus positive control plasmid as a positive control and a virus-free sample as a negative control; M5 was separated by agarose gel electrophoresis, and the band distribution of the PCR amplification product was compared with that of the positive control and the negative control to determine whether the virus was contained.

10. The virus positive control plasmid for detecting virus-free strawberry seedlings according to claim 9, characterized in that: The conditions of the PCR amplification are as follows: 90-98°C, 8-13 min; 90-98°C, 10-20 s; 55-65°C, 0.5-1.5 min, and 40 cycles.

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