Multiplex RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and kit for detecting important blueberry viruses

Abstract

The invention belongs to the technical field of plant virus molecular detection, particularly discloses a multiplex RT-PCR method and a kit for detecting important blueberry viruses, and particularly provides a multiplex RT-PCR detection primer for the blueberry viruses, and the sequence of the primer is shown as SEQ ID NO.1-12. The technical scheme provided by the invention is wide in detection range, high in specificity, high in specificity and high in sensitivity. The kit can be used for simultaneously detecting blueberry scorching virus, blueberry shock virus standard strains and variants thereof, tomato fruit ring spot virus, blueberry necrotic mottle virus and apple mosaic virus, solves the problem that the variants and different virus strains cannot be detected by a traditional ELISA method, covers main blueberry viruses and trans-boundary infected key virus types, and is high in specificity and high in sensitivity. The sensitivity is high.

Description

Technical Field

[0001] This invention belongs to the field of plant virus molecular detection technology, specifically relating to a multiplex RT-PCR method and a dedicated kit for the simultaneous detection of multiple viruses in blueberries. Background Technology

[0002] Blueberries, as a high-value economic crop, are widely cultivated globally. Yunnan's blueberry industry ranks among the top in China in terms of area, yield, and output value, and has become an important force in the global blueberry landscape. However, viral infections are a significant factor restricting the development of the blueberry industry. Among them, Blueberry Scorch Virus (BlScV), Blueberry Shock Virus (BlShV) and its variants, Blueberry Necrotic Ring Blotch Virus (BNRBV), and Apple Mosaic Virus (ApMV) are the four most serious viruses affecting blueberry production. In addition, Tomato Zonatespot Virus (TZSV), as a cross-species virus, has been found to infect blueberry plants in recent years, causing ring spots on the fruit and a decline in quality, further exacerbating the risk of losses for the blueberry industry. Plants infected with blueberry scorch virus cannot recover and must be completely eradicated. Plants infected with blueberry shock virus may recover gradually over time, but will significantly affect fruit yield and quality. Plants infected with blueberry ringspot virus will show leaf mottling and necrosis, leading to plant death in severe cases. Plants infected with apple mosaic virus will show mosaic and mottling symptoms, affecting fruit appearance and commercial value. Plants infected with tomato fruit ringspot virus will show systemic mottling and necrosis symptoms, leading to plant death in severe cases.

[0003] Currently, the standard method for detecting blueberry viruses is enzyme-linked immunosorbent assay (ELISA), but this method has significant limitations. Recent studies have revealed new variants of blueberry scorch virus and blueberry shock virus, and there is also a risk of cross-species transmission of tomato fruit ring spot virus. Traditional ELISA methods cannot effectively identify these variants and different strains, resulting in some blueberry plants exhibiting obvious symptoms testing negative, causing significant uncertainty for growers and increasing the risk of virus spread.

[0004] Polymerase chain reaction (PCR) technology is widely used in virus detection due to its high specificity and sensitivity. Multiplex RT-PCR, in particular, can simultaneously amplify specific fragments of multiple target viruses in the same reaction system, enabling simultaneous detection of multiple viruses and offering advantages such as high efficiency, speed, and low cost. While existing technologies include multiplex RT-PCR detection methods for various viruses in crops such as tomatoes, multiplex detection techniques for major blueberry viruses (especially those containing variants) and cross-species viruses have not yet been reported. Therefore, designing a multiplex RT-PCR detection scheme capable of simultaneously and accurately detecting multiple viruses is of great significance for early warning, precise control, and ensuring the healthy development of the blueberry industry. Summary of the Invention

[0005] This invention aims to address the problems of existing blueberry virus detection technologies, such as their inability to effectively detect variant strains, different virus strains, and low detection efficiency. This invention provides a multiplex RT-PCR method and kit for the simultaneous detection of multiple major blueberry viruses, enabling rapid, accurate, and simultaneous detection of important viruses in the blueberry seedling production process.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a primer for multiplex RT-PCR detection of blueberry virus, the primer sequences of which are shown in SEQ ID NO.1 to 12.

[0008] Furthermore, the present invention provides the application of the primers in the preparation of blueberry virus detection and / or diagnostic products.

[0009] In one embodiment, the present invention provides a blueberry virus detection kit, the kit containing the aforementioned primers.

[0010] Furthermore, the present invention provides the application of the kit in the detection of blueberry viruses, including BlScV, BlShV and their variants, TZSV, BNRBV and ApMV.

[0011] Furthermore, the present invention provides a multiplex RT-PCR method for detecting important blueberry viruses, wherein the method uses the primers or the kits described above for detection.

[0012] Furthermore, the method described in this invention involves extracting RNA from the sample to be tested, obtaining cDNA through reverse transcription, performing PCR amplification using the primers described above, and determining the presence or absence of the virus based on the size of the PCR product bands.

[0013] Furthermore, the viruses detected in the method of the present invention include BlScV, BlShV and their variants, TZSV, BNRBV and ApMV.

[0014] The beneficial effects achieved by this invention are as follows:

[0015] 1. Wide detection range: It can simultaneously detect blueberry scorch virus, blueberry shock virus standard strain and its variants, tomato fruit ring spot virus, blueberry necrosis mottle virus, and apple mosaic virus, solving the problem that traditional ELISA methods cannot detect variants and different virus strains, and covering the main blueberry viruses and key virus types that cross-border infection.

[0016] 2. High specificity: The primers are designed based on conserved gene regions of each virus. BLAST verification shows no non-specific binding, and the amplification products of each primer pair have significant differences in length (286bp, 452bp, 638bp, 825bp, 708bp, 900bp), which can be clearly distinguished by electrophoresis, avoiding misjudgment caused by cross-reaction and primer dimers.

[0017] 3. High sensitivity: This method can detect as low as 0.05 pg of plasmid template and 0.5 ng of total RNA from blueberry plant samples, which is far higher than the detection sensitivity of traditional ELISA methods. It can achieve early detection of the virus and provide sufficient time for prevention and control.

[0018] 4. High efficiency and speed: Using multiplex RT-PCR technology, multiple viruses can be detected simultaneously in the same reaction system. The entire detection process (from sample processing to result analysis) only takes 4-5 hours. Compared with traditional single virus detection methods, it significantly shortens the detection time and reduces the detection cost, making it suitable for large-scale sample screening.

[0019] 5. Easy to use: The kit provides a complete set of reagents required for testing, with clear steps and no need for complex professional equipment, making it easy for grassroots testing institutions and growers to use, and facilitating the promotion and application of virus prevention and control technologies. Detailed Implementation

[0020] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. For the testing methods, purchased goods, unless otherwise specified, shall be used under conventional conditions or conditions recommended by the manufacturer. Unless otherwise defined herein, the scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials described herein may also be used in the practice and testing of this disclosure.

[0021] Download blueberry scorch virus (accessions: MH411658.1, DQ266467.1, HQ690963.1, KP232979.1), blueberry shock virus standard strains (accessions: KY927866.1, LC781633.1) and variants (accession: NC_022251.1) from GenBank, and different geographical strains of tomato fruit ring spot virus (accession: The whole genome sequences of MZ404610.1, NC_010489.1, and EF552433.1, and the whole genome sequences of different strains of blueberry necrosis ringspot virus (accession number: JN651148.1) and apple mosaic virus (accession number: AF174584.1) were obtained. Sequence alignment was performed using DNAstar software to determine the conserved regions of each virus (BlScV: capsid protein gene; BlShV and its variants: nucleocapsid protein gene; TZSV: N gene; BN...). For RBV, the CP gene was selected; for ApMV, the CP gene was selected. Based on primer design principles (length 18-30bp, GC content 40%-60%, Tm value 55-65℃, Tm value difference between forward and reverse primers ≤2℃, to avoid primer dimer and hairpin structure formation), 6 pairs of specific primers were designed and screened, targeting BlScV, BlShV standard strain, BlShV mutant strain, TZSV, BNRBV, and ApMV, respectively. The amplification product lengths of each primer pair are significantly different, which facilitates differentiation and identification after electrophoresis.

[0022] The specific primer sequences are as follows:

[0023] (1) Primer pairs against blueberry scorch virus (BlScV):

[0024] Upstream primer BlScV-F: 5'-GCTGCTGAAGATGGTGGTGA-3' (sequence listing SEQ ID NO.1), 20bp in length, GC content 55%, Tm value 60.2℃;

[0025] Downstream primer BlScV-R: 5'-TCGTCGTTGGTGATGATGGT-3' (sequence listing SEQ ID NO.2), 20bp in length, 55% GC content, Tm value 60.0℃;

[0026] The amplification product is 286 bp in length.

[0027] (2) Primer pair against the standard strain of blueberry shock virus (BlShV-S):

[0028] Upstream primer BlShV-SF: 5'-ACGACGACGAAGAAGAAGCC-3' (sequence listing SEQ ID NO.3), 20bp in length, GC content 55%, Tm value 59.8℃;

[0029] Downstream primer BlShV-SR: 5'-GCGGCGATGATGATGTTGTT-3' (sequence listing SEQ ID NO.4), 20bp in length, GC content 55%, Tm value 59.6℃;

[0030] The amplified product is 452 bp in length.

[0031] (3) Primer pairs targeting the blueberry shock virus variant (BlShV-V):

[0032] Upstream primer BlShV-VF: 5'-GAAGAAGCCGCCGAAGATGT-3' (SEQ ID NO.5), 20bp in length, GC content 55%, Tm value 60.1℃;

[0033] Downstream primer BlShV-VR: 5'-TTGTTGTTGCGGCGGATGAT-3', (SEQ ID NO.6), 20bp in length, 55% GC content, Tm value 59.7℃; amplified product length 638bp.

[0034] (4) Primer pairs targeting Tomato Fruit Ring Spot Virus (TZSV):

[0035] Upstream primer TZSV-F: 5'-CGGCGAGAAGAAGAAGAAGC-3' (sequence listing SEQ ID NO.7), 20bp in length, GC content 55%, Tm value 59.7℃;

[0036] Downstream primer TZSV-R: 5'-GCGGCGTTGTTGTTGATGAT-3' (SEQ ID NO.8), 20bp in length, GC content 55%, Tm value 59.5℃;

[0037] The amplified product is 825 bp in length.

[0038] (5) Primer pairs against blueberry necrosis ringspot virus (BNRSV):

[0039] Upstream primer BNRBV-F: 5'-CGGTCCAATGTTAGCCCAGT-3' (sequence listing SEQ ID NO.9), 20bp in length, GC content 55%, Tm value 60.04℃;

[0040] Downstream primer BNRBV-R: 5'-GTGGTCGGGTCACTTGCATA-3' (sequence listing SEQ ID NO.10), 20bp in length, GC content 55%, Tm value 60.04℃;

[0041] The amplification product is 708 bp in length.

[0042] (6) Primer pairs against apple mosaic virus (ApMV):

[0043] The upstream primer ApMV-F: 5'-ATGGCTATCCATGCGACGAG-3' (sequence listing SEQ ID NO.11), 20 bp in length, GC content 55%, Tm value 59.9℃;

[0044] Downstream primer ApMV-R: 5'-TTCATACACGTCCCTCCGTG-3' (sequence listing SEQ ID NO.12), 20bp in length, GC content 55%, Tm value 59.7℃;

[0045] The amplification product is 900 bp in length.

[0046] The above six primer pairs were verified by NCBI Primer-BLAST and showed no non-specific binding. They could only specifically amplify the gene fragments of the corresponding target viruses, and there was no interaction between the primers, so primer dimers would not be formed.

[0047] 2. Multiplex RT-PCR detection method

[0048] The method includes the following steps:

[0049] (1) Extraction of total RNA from samples: leaf or shoot tissues of blueberry plants to be tested were taken and total RNA was extracted using the Trizol method. The RNA was then detected by agarose gel electrophoresis and Nanodrop to ensure good integrity and purity (A260 / A280 was 1.8-2.0).

[0050] (2) cDNA synthesis: Using extracted total RNA as a template, cDNA was synthesized by reverse transcription using random primers (10 μmol / L). The 20 μL reverse transcription reaction system consisted of: 4 μL 5× reverse transcription buffer, 2 μL dNTP mixture (10 mmol / L), 0.5 μL RNase inhibitor (40 U / μL), 1 μL reverse transcriptase (200 U / μL), 1 μL random primers (10 μmol / L), 2 μL total RNA template, and 9.5 μL enzyme-free water. The reaction procedure was: annealing at 25℃ for 10 min, reverse transcription at 42℃ for 60 min, inactivation at 68-72℃ for 15 min, and storage at 4℃ for later use.

[0051] (3) Multiplex RT-PCR amplification: Using the above cDNA as a template, add the above 6 pairs of specific primers and RT-PCR reaction mixture to construct a 25μL reaction system: 12.5μL of 2×Taq PCR Master Mix, 0.5μL of each upstream primer (10μmol / L), 0.5μL of each downstream primer (10μmol / L), 3μL of cDNA template, and 3.5μL of enzyme-free water.

[0052] (4) Reaction procedure: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 59.6℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; 72℃ final extension for 10 min; store at 4℃.

[0053] (5) Result detection and analysis: Take 10 μL of PCR amplification product and perform electrophoresis on a 1.5% agarose gel at 120V for 30 min. After EB staining, observe the results in a gel imaging system. If a 286bp band appears, it indicates that the sample is infected with blueberry scorch virus (BlScV); if a 452bp band appears, it indicates that the sample is infected with the standard strain of blueberry shock virus (BlShV-S); if a 638bp band appears, it indicates that the sample is infected with the variant strain of blueberry shock virus (BlShV-V); if an 825bp band appears, it indicates that the sample is infected with tomato fruit ring spot virus (TZSV); if a 708bp band appears, it indicates that the sample is infected with blueberry necrosis mottle virus (BNRBV); if a 900bp band appears, it indicates that the sample is infected with apple mosaic virus (ApMV); if multiple bands appear simultaneously, it indicates that the sample is mixed with the corresponding virus; if no bands appear, it indicates that the sample is not infected with the above 6 viruses.

[0054] 3. Test kit

[0055] (1) Specific primer mixture: containing 12 primers shown in SEQ ID NO.1-SEQ ID NO.12, each primer having a concentration of 10 μmol / L;

[0056] (2) Reverse transcription reagent kit: including 5× reverse transcription buffer, dNTP mixture (10 mmol / L), RNase inhibitor (40 U / μL), reverse transcriptase (200 U / μL), 6mer random primer (10 μmol / L);

[0057] (3) Controls: Positive control (a mixture of cDNA containing BlScV, BlShV-S, BlShV-V, TZSV, BNRBV, and ApMV), negative control (cDNA from virus-free blueberry plants), and blank control (enzyme-free water).

[0058] (4) Auxiliary reagents: RNA extraction reagent (Trizol reagent, chloroform, isopropanol, 75% ethanol), agarose, EB staining solution, 1×TAE electrophoresis buffer.

[0059] 4. Specificity verification of TZSV, BNRSV, ApMV, BlScV, BlShV-S, and BlShV-V

[0060] (1) Verification of experimental materials:

[0061] ① Positive control samples: Blueberry plant tissues infected with a single virus as verified by sequencing, including TZSV positive strain, BNRBV positive strain, ApMV positive strain, BlScV positive strain, BlShV-S positive strain, and BlShV-V positive strain; and blueberry test-tube seedling tissues artificially mixed with the above 5 viruses.

[0062] ② Negative control sample: tissue from virus-free healthy blueberry plants (verified by both traditional ELISA method and gene sequencing).

[0063] ③ Interference verification samples: tomato plants infected with tomato spotted wilt virus and tomato chlorosis virus.

[0064] ④ Actual test samples: A total of 100 samples of root tips, buds, seedlings and test-tube seedlings were collected from 4 blueberry planting bases and tissue culture rooms, covering different growth stages and cultivation environments.

[0065] (2) Reagents and primers

[0066] ① The primers were synthesized by Shanghai Sangon Biotech. The specific primers SEQ ID NO. 1 to 12 are as follows:

[0067] SEQ ID NO.1:

[0068] BlScV-F: 5'-GCTGCTGAAGATGGTGGTGA-3'

[0069] SEQ ID NO.2:

[0070] BlScV-R: 5'-TCGTCGTTGGTGATGATGGT-3'

[0071] SEQ ID NO.3:

[0072] BlShV-SF: 5'-ACGACGACGAAGAAGAAGCC-3'

[0073] SEQ ID NO.4:

[0074] BlShV-SR: 5'-GCGGCGATGATGATGTTGTT-3'

[0075] SEQ ID NO.5:

[0076] BlShV-VF: 5'-GAAGAAGCCGCCGAAGATGT-3'

[0077] SEQ ID NO.6:

[0078] BlShV-VR: 5'-TTGTTGTTGCGGCGGATGAT-3'

[0079] SEQ ID NO.7:

[0080] TZSV-F: 5'-CGGCGAGAAGAAGAAGAAGC-3'

[0081] SEQ ID NO.8:

[0082] TZSV-R: 5'-GCGGCGTTGTTGTTGATGAT-3'

[0083] SEQ ID NO.9:

[0084] BNRBV-F: 5'-CGGTCCAATGTTAGCCCAGT-3'

[0085] SEQ ID NO.10:

[0086] BNRBV-R: 5'-GTGGTCGGGTCACTTGCATA-3'

[0087] SEQ ID NO.11:

[0088] ApMV-F: 5'-ATGGCTATCCATGCGACGAG-3'

[0089] SEQ ID NO.12:

[0090] ApMV-R: 5'-TTCATACACGTCCCTCCGTG-3'

[0091] ② Reagents: Plant total RNA extraction kit, RT-PCR reverse transcription kit (containing 5×RT MasterMix and 6mer random primers), 2×Taq PCR Mix, agarose, EB staining solution, and 1×TAE electrophoresis buffer, all of which were commercially available analytical grade or biological reagent grade.

[0092] (3) Verification methods and procedures

[0093] ① Sample pretreatment and nucleic acid extraction

[0094] Take 2g of each sample tissue and extract total plant RNA using a plant total RNA extraction kit for later use.

[0095] ② cDNA synthesis

[0096] A 10 μL reaction system was constructed using an RT-PCR reverse transcription kit: 2.5 μL of crude nucleic acid extract, 2 μL of 5×RTMasterMix, 0.5 μL of 6-mer random primers, and deionized water to a final volume of 10 μL. The reaction procedure was: incubation at 25°C for 10 min (primer annealing), reverse transcription at 42°C for 45 min (cDNA synthesis), inactivation at 95°C for 2 min (termination of reverse transcriptase activity), and storage at 4°C for later use.

[0097] ③ Multiplex RT-PCR amplification

[0098] Construct a 25 μL amplification system: 4 μL cDNA template, 1 μL mixed primer solution, 12.5 μL 2×Taq PCR Mix, and deionized water to a final volume of 25 μL. Reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; final extension at 72℃ for 10 min; incubation at 4℃.

[0099] ④ Result detection and judgment

[0100] Take 10 μL of PCR amplification product and electrophoresis it in a 1.5% agarose gel (voltage 120V, time 30min). After EB staining, observe the bands using a gel imaging system.

[0101] The presence of a 286bp band indicates infection with blueberry scorch virus (BlScV); a 452bp band indicates infection with the standard strain of blueberry shock virus (BlShV-S); a 638bp band indicates infection with a variant strain of blueberry shock virus (BlShV-V); an 825bp band indicates infection with tomato fruit ring spot virus (TZSV); a 708bp band indicates infection with blueberry necrosis mottle virus (BNRBV); a 900bp band indicates infection with apple mosaic virus (ApMV); the presence of multiple bands indicates a mixed infection with the corresponding viruses; and the absence of any bands indicates that the sample is not infected with any of the above six viruses.

[0102] Nine templates were used as amplification templates, and single RT-PCR amplification was performed using primer pairs TZSV-F / TZSV-R, BNRBV-F / BNRBV-R, ApMV-F / ApMV-R, BlScV-F / BlScV-R, BlShV-SF / BlShV-SR, and BlShV-VF / BlShV-VR. The reaction volume was 20 μL: 10 μL 2×Taq PCR Master Mix, 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), 2 μL cDNA template, and 2 μL enzyme-free water. The amplification program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 59.6℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; final extension at 72℃ for 10 min; storage at 4℃. After amplification, 10 μL of the product was taken for detection by 1.5% agarose gel electrophoresis.

[0103] (a) Verification Content and Results

[0104] 1. Specificity verification

[0105] Validation of single virus positive samples: Amplification was performed using cDNA from 6 single virus positive samples as templates. The results showed that only the corresponding target virus showed specific bands. The bands were single and clear, without any extraneous bands, and no primer dimers were found.

[0106] Verification of mixed viral samples: Five viral positive cDNA templates were artificially mixed and amplified. Five specific bands appeared simultaneously. The bands were well separated and did not interfere with each other. They could be clearly distinguished by electrophoresis.

[0107] Interference sample verification: Using tomato TSWV and ToCV positive sample cDNA as templates, no specific bands appeared after amplification, proving that the primers had no cross-reactivity with the blueberry target virus and had good specificity.

[0108] (II) Sensitivity Verification

[0109] Six viral-positive cDNA templates were serially diluted 10-fold (10-10). 0 ~10 -5 Each dilution was replicated in triplicate. The results showed that the limit of detection for TZSV, BNRBV, ApMV, BlScV, BlShV, and BlShV-V was 0.1 pg / μL, which is much higher than that of the traditional ELISA method (limit of detection 10 pg / μL), and can meet the detection needs of early viral infection (stage before symptoms appear).

[0110] (III) Actual sample testing and verification

[0111] Results of testing 100 actual samples: A total of 36 positive samples were detected, including 10 positive for TZSV, 5 positive for BNRBV, 3 positive for BlScV, 8 positive for BlScV, 4 positive for BlShV-V, and 6 mixed infections, with an overall positive rate of 36%.

[0112] Results verification: Gene sequencing was performed on all positive samples, and the sequencing results showed ≥98% homology with the standard sequences of each virus; negative samples were retested using the traditional ELISA method, and the results were consistent, proving that the test results were accurate and reliable.

[0113] Tissue compatibility: The positive rates of root tips, buds, seedlings, and test-tube seedlings were 32.1.0%, 31.8%, 33.3%, and 35.7%, respectively. All tissue samples could be stably amplified without interference from the tissue matrix, making them suitable for detection throughout the entire blueberry cultivation process.

[0114] (iv) Repeatability verification

[0115] Ten positive samples (including single and mixed infections) were selected and tested three times at different times by three researchers. The test results were completely consistent, with no significant difference in band brightness, proving that the method has good repeatability and high operational stability.

Claims

1. Primers for blueberry virus multiplex RT-PCR detection, characterized in that, The primer sequences are shown in SEQ ID NO.1 to 12.

2. The use of the primers according to claim 1 in the preparation of blueberry virus detection and / or diagnostic products.

3. A blueberry virus detection kit, characterized in that, The kit contains the primers as described in claim 1.

4. The application of the kit according to claim 3 in blueberry virus detection, characterized in that, The viruses include BlScV, BlShV and their variants, TZSV, BNRBV and ApMV.

5. A multiplex RT-PCR method for detecting important blueberry viruses, characterized in that, The detection was performed using the primers described in claim 1 or the kit described in claim 3.

6. The method according to claim 5, characterized in that, RNA was extracted from the sample to be tested, and cDNA was obtained by reverse transcription. Then, PCR amplification was performed using the primers described in claim 1. The presence or absence of the virus was determined based on the size of the bands in the PCR product.

7. The method according to claim 5 or 6, characterized in that, The viruses include BlScV, BlShV and their variants, TZSV, BNRBV and ApMV.