Real-time fluorescent quantitative PCR (polymerase chain reaction) method for detecting tobacco pepper vein mottle virus
By designing specific primer pairs and performing real-time quantitative PCR amplification using the real-time quantitative PCR method, the problem of insufficient accuracy and sensitivity in the detection of pepper vein mottle virus in existing technologies has been solved. This has enabled high-sensitivity and specific detection of pepper vein mottle virus, providing technical support for early monitoring and accurate quantification.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-22
- Publication Date
- 2026-03-17
AI Technical Summary
Existing technologies for detecting the vein mottle virus in tobacco and capsicum have limitations in accuracy and sensitivity, making it impossible to achieve early detection and accurate quantification of low-abundance virus samples. Furthermore, conventional molecular biology methods are ineffective in controlling the spread of the virus.
A real-time quantitative PCR method was used, with specific primer pairs designed to extract total RNA and perform reverse transcription. The specific primers were then used for real-time quantitative PCR amplification, and the viral load was quantified using a standard curve, achieving high sensitivity and specificity for the detection of pepper vein mottle virus.
It achieves highly sensitive detection of pepper vein mottle virus, can accurately identify it at low viral loads, has high specificity, can monitor early and accurately quantify, and provides technical support for disease control.
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Figure CN121674631A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of detection technology, specifically relating to a real-time quantitative PCR method for detecting tobacco and capsicum vein mottle virus. Background Technology
[0002] Chilli veinal mottle virus (ChiVMV) is a positive-sense single-stranded RNA virus belonging to the genus *Potatovirus* of the family *Potatovirus*. This virus can cause severe disease, damaging pepper and tobacco production and significantly impacting the yield and quality of these crops. Infecting tobacco with ChiVMV results in symptoms such as circular chlorotic spots, necrotic spots, and systemic necrosis on the leaves, affecting the usability of the tobacco leaves. ChiVMV is primarily transmitted by aphids, but can also be spread through contact, and its spread is rapid.
[0003] Detection of tobacco virus diseases includes biological identification, immunological methods, and molecular biological identification methods. Traditional biological identification methods, such as symptom observation and laboratory culture, while practical, suffer from low accuracy and are labor-intensive, and are gradually being replaced by modern molecular biological techniques. Immunological methods have low sensitivity and specificity, and are often used for detection and identification in the middle and late stages of the disease, failing to provide early monitoring and warning. Conventional molecular biological detection methods include reverse transcriptase-polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). These techniques have high specificity and sensitivity, but are still somewhat insufficient for detecting ChiVMV in low-abundance viral samples and cannot determine the viral load in the sample.
[0004] Early detection, monitoring, and warning are crucial for preventing the spread of plant viral diseases. Therefore, establishing a method that can accurately, sensitively, and rapidly detect pepper vein mottle virus in field plant samples and vector insects is of great significance for the effective control of this virus. Summary of the Invention
[0005] To address the problems existing in the background technology, the present invention provides a real-time quantitative PCR method for detecting tobacco pepper vein mottle virus.
[0006] To achieve the above objectives, the technical solution of the present invention is as follows:
[0007] A real-time quantitative PCR method for detecting vein mottle virus in tobacco capsicum includes the following steps:
[0008] (1) Based on the conserved sequence of the capsid protein CP gene of ChiVMV (pepper vein mottle virus), a specific primer pair was designed and synthesized;
[0009] (2) Extract total RNA from the tobacco samples to be tested and perform reverse transcription to obtain cDNA;
[0010] (3) Using the cDNA as a template, perform real-time quantitative PCR amplification using the specific primer pair;
[0011] (4) Determine whether the sample carries ChiVMV (pepper vein mottle virus) based on the amplification curve, melting curve and Ct value, and quantify the viral load using the standard curve.
[0012] The primer pair includes an upstream primer ChiVMVqF and a downstream primer ChiVMVqR, whose nucleotide sequences are as follows:
[0013] SEQ ID NO.1: 5'-CAACACCAACCATTCACGC-3';
[0014] SEQ ID NO. 2: 5'-CTACAACATGGCACGAGTGATT-3'.
[0015] The reaction system for real-time quantitative PCR is as follows: 10 µL of 2×TB Green Premix Dimer Eraser, 0.6 µL each of 10 µmol / L qPCR upstream and downstream primers, 0.4 µL of 50×ROX Reference Dye II, 2 µL of plasmid template or cDNA template, and sterile water to a total volume of 20 µL.
[0016] The reaction conditions for real-time quantitative PCR were as follows: pre-denaturation at 95℃ for 30s, PCR reaction at 95℃ for 3s, 57℃ for 30s, and 72℃ for 30s, for 40 cycles; the amplification product length was 97 bp.
[0017] The standard curve was established as follows: a recombinant plasmid containing a ChiVMV CP gene-specific fragment was used as a plasmid standard and serially diluted 10-fold to obtain a concentration range of 2.78 × 10⁻⁶. 1 -2.78×10 9 Nine plasmid samples (copies / µL) were used as templates for real-time quantitative PCR, and a standard curve was plotted based on the Ct value versus the logarithm of the template concentration.
[0018] The linear equation of the obtained standard curve is: y = -3.3186x + 40.309, where y represents the cycle threshold (Ct value), x represents the logarithm of the plasmid concentration, the coefficient of determination R2 of the standard curve is 0.9986, and the amplification efficiency is 100.13%.
[0019] The detection method of the present invention has the following beneficial effects:
[0020] (1) It has good specificity and can accurately identify ChiVMV. It has no cross-reaction with various viruses commonly found in tobacco, such as TMV, CMV, and PVY, which can effectively avoid false positive results and ensure the accuracy of detection.
[0021] (2) It has high sensitivity, with a detection limit of 2.78×10¹ copies / μL, which is 10 times higher than that of conventional PCR. It can achieve early and accurate detection of samples with low viral load, thus buying time for disease control.
[0022] (3) It is highly practical. The method of this invention can not only quickly determine the virus-carrying status of field samples, but also accurately quantify the viral load, providing reliable technical support for the epidemic monitoring, disease warning and prevention and control of ChiVMV in the field. Attached Figure Description
[0023] Figure 1 This is an electrophoresis diagram of the PCR amplification products from Example 1;
[0024] Figure 2 Electrophoresis images of the PCR amplification products of 8 white single colonies and 2 blue single colonies from Example 1;
[0025] Figure 3 This is a standard curve of real-time RT-qPCR for pepper vein mottle virus in Example 1;
[0026] Figure 4 This is a graph showing the primer specificity detection results in Example 2;
[0027] Figure 5 This is a graph showing the real-time RT-qPCR sensitivity detection results of pepper vein mottle virus in Example 3;
[0028] Figure 6 This is a graph showing the sensitivity detection results of the conventional PCR method for pepper vein mottle virus in Example 3;
[0029] Figure 7 This is an amplification curve of 14 suspected ChiVMV-infected tobacco samples detected by the RT-qPCR method of the present invention in Example 4;
[0030] Figure 8 This is a comparison of melting curves of 14 suspected ChiVMV-infected tobacco samples from Example 4;
[0031] Figure 9 This is a graph showing the results of routine PCR testing on 14 suspected ChiVMV-infected tobacco samples in Example 4. Detailed Implementation
[0032] The present invention will be further described below with reference to embodiments.
[0033] Unless otherwise specified, the raw materials used in the examples are all commercially available raw materials.
[0034] Materials: Tobacco mosaic virus (TMV), Tobacco cucumber mosaic virus (CMV), Tobacco potato virus Y (PVY), Tobacco etch virus (TEV), Tobacco vein banding mosaic virus (TVBMV), Tomato spotted wilt virus (TSWV), Tomato yellow leaf curl virus (TYLCV), and Chilli veinalmottle virus (ChiVMV) were all kindly provided by the Plant Protection Research Group of the Tobacco Research Institute of Henan Academy of Agricultural Sciences.
[0035] Instruments: ABI 7500 Real-Time PCR System (ABI, USA); Liquid Nitrogen Cryogenic Grinder (JXFSTPRP-24L, Shanghai Jingxin); Eppendorf D30 UV Spectrophotometer; Bio-Rad Gel Doc XR+ Gel Imaging Analysis System; Eppendorf PCR System; Lu Xiangyi TGL-16M High-Speed Refrigerated Centrifuge.
[0036] Reagents: Promega Total RNA Extraction Kit; PrimeScript FAST RT reagent Kit with gDNA Erase Reverse Transcription Kit (Takara); 2×3G Taq Master Mix for PAGE (Red Dye) (Vazyme); Gel Extraction Kit (TaKaRa Mini BEST Agarose GEL DNA Extraction Kit, Takara); 2×HiAmp PCR Master Mix (Dye) (Kangwei Century); Plasmid Extraction Kit (TaKaRaMini BEST Plasmid Purification Kit Ver.4.0, Takara); Quantitative Real-Time Immunoassay Kit (TBGreen Premix DimerEraser™, Takara); pMD TM18-T Vector Cloning Kit (Takara); competent cells (Trans 5α, Beijing TransGen Biotech Co., Ltd.); primer synthesis and sequencing were all performed by Sangon Biotech (Shanghai) Co., Ltd.
[0037] Example 1: A real-time quantitative PCR method for detecting vein mottle virus in tobacco capsicum.
[0038] 1. Primer design and synthesis
[0039] Based on the conserved sequence of the coat protein (CP) gene of the chili vein mottle virus (ChiVMV) isolate from Yunnan tobacco, China (GenBank accession number: JX088636) in the NCBI database, specific primers were designed using Primer Premier 5.0 software according to the primer design principles of real-time quantitative PCR. The specificity of the primers was verified by sequence alignment using the NCBI Primer-BLAST tool.
[0040] The specific primers obtained are: upstream primer ChiVMVqF (SEQ ID NO.1): 5'-CAACACCAACCATTCACGC-3'; downstream primer ChiVMVqR (SEQ ID NO.2): 5'-CTACAACATGGCACGAGTGATT-3';
[0041] The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0042] 2. Total RNA extraction and cDNA synthesis from tobacco
[0043] 0.1g of tobacco leaf samples infected with ChiVMV were collected, and total RNA was extracted using the Promega Total RNA Extraction Kit. Healthy tobacco leaves were used as a negative control. The extraction procedure was performed according to the kit instructions. RNA concentration was measured using an Eppendorf D30 UV spectrophotometer, and RNA integrity was detected by 1% agarose gel electrophoresis. OD was screened. 260 / OD 280 RNA samples with a ratio in the range of 1.8 to 2.1 were used for subsequent cDNA synthesis.
[0044] cDNA was synthesized using the Takara PrimeScript FAST RT reagent Kit with gDNA Erase, following the instructions in the kit's manual. The synthesized cDNA was stored at -20°C for later use.
[0045] 3. PCR amplification and cloning identification of the target gene
[0046] 3.1 PCR amplification
[0047] PCR amplification was performed using the cDNA synthesized in step 2 as a template. The PCR reaction system (20 μL) consisted of: 10 μL of 2×3G Taq MasterMix for PAGE (Red Dye), 0.5 μL each of 10 μmol / L forward and reverse primers, 2 μL of cDNA template, and 7 μL of ddH2O. The reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 57℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 10 min.
[0048] The PCR amplification products were detected by 2% agarose gel electrophoresis, and the results are shown in the figure. Figure 1 (500 markers, 1 ChiVMV positive, 1 healthy tobacco negative), where M: 500bp molecular weight standard; 1: ChiVMV-infected tobacco sample; 2: healthy tobacco sample. Results showed that the positive ChiVMV-infected tobacco sample exhibited a specific single band at 97 bp, as expected.
[0049] 3.2 Cloning of the target gene
[0050] After agarose gel electrophoresis, the target band of the PCR product was excised and purified using a gel extraction kit. The purified target fragment was ligated into the pMD18-T vector and transformed into Trans 5α competent cells. After overnight culture at 37°C, blue-white screening was performed. White single colonies and blue single colonies (negative control) were picked and inoculated into LB liquid medium containing ampicillin, and cultured at 37°C and 220 r / min for 6 h.
[0051] 3.3 Bacterial culture PCR identification and sequencing
[0052] Bacterial PCR identification was performed using universal primers for the pMD18-T vector. The universal primers were RV-M (SEQ ID NO.3): 5'-GAGCGGATAACAATTTCACACAGG-3' and M13-47 (SEQ ID NO.4): 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.
[0053] Bacterial PCR reaction system (20 μL): 10 μL of 2×HiAmp PCR Master Mix (Dye), 0.5 μL each of 10 μmol / L universal primers, 2 μL of bacterial template, and 7 μL of ddH2O; reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; final extension at 72℃ for 10 min.
[0054] The bacterial culture PCR products were detected by agarose gel electrophoresis, and the results are shown in the figure. Figure 2 Where M: 500bp molecular weight standard; 1-8, 8 white single colonies; 9-10, 2 blue single colonies. Electrophoresis results show that the PCR product size of the blue single colony culture is 156bp, and the PCR product size of the white single colony culture is 253bp, both as expected.
[0055] Positive bacterial cultures with amplified products matching the expected size were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. The sequencing results were compared and analyzed by NCBI BLASTn, confirming that the inserted fragment was a ChiVMV CP gene-specific fragment, indicating that the target gene was successfully cloned.
[0056] 4. Preparation of plasmid standards
[0057] Positive bacterial clones with correct sequencing results were selected and inoculated into LB broth containing ampicillin, and cultured overnight at 37°C and 220 rpm. Plasmids were extracted using a Takara extraction kit, following the kit's instructions. The OD of the plasmids was measured using a UV spectrophotometer. 260 The value is calculated using the formula C = A / B × 6.02 × 10. 14 (Where A is the plasmid concentration in ng / μL; B is the molecular weight of the plasmid DNA; C is the plasmid copy number in copies / μL) Calculate the plasmid copy number and use this plasmid as a standard.
[0058] 5. Establishment and validation of real-time quantitative PCR detection system
[0059] 5.1 Establishment of the Standard Curve
[0060] The plasmid standard was serially diluted 10-fold using EASY Dilution II to obtain a final concentration of 2.78 × 10¹–2.78 × 10¹. 9 Nine gradient concentrations of plasmid samples were prepared at 9 copies / μL. Using each gradient dilution of plasmid as a template, RT-qPCR was performed using an ABI 7500 fast real-time quantitative PCR instrument. Each concentration was set to 3 replicates, and the instrument automatically generated a standard curve.
[0061] RT-qPCR reaction system (20 μL): 10 μL TB Green Premix DimerEraser (2×), 0.6 μL each of 10 μmol / L forward and reverse primers, 0.4 μL ROX Reference DyeⅡ (50×), 2 μL plasmid template, and 6.4 μL sterile water;
[0062] Reaction conditions: 95℃ pre-denaturation for 30s; 95℃ denaturation for 3s, 57℃ annealing for 30s, 72℃ extension for 30s, for a total of 40 cycles.
[0063] See results Figure 3 In 2.78×10¹-2.78×10 9 Within the range of copies / μL concentration, the Ct value shows an increasing trend as the concentration of positive plasmid template gradually decreases, and the Ct value has a good linear relationship with the logarithm of the plasmid standard concentration. The slope of the standard curve in the figure is -3.3186, the coefficient of determination (R²) is 0.9986, the amplification efficiency is 100.13%, and the linear equation is y = -3.3186x + 40.309 (where y is the Ct value and x is the logarithm of the plasmid concentration). The Ct value combined with the standard curve can be used to accurately quantify ChiVMV in the sample.
[0064] 5.2 Repeatability Validation
[0065] Nine plasmid standards diluted 10-fold were used as templates for RT-qPCR amplification, with three replicates for each concentration. The repeatability of the method was evaluated by calculating the standard deviation (SD) and coefficient of variation (CV) of the Ct values. The RT-qPCR repeatability test results of plasmid standards at different concentrations are shown in Table 1.
[0066] The results showed that the standard deviation of Ct values in three replicates of each concentration gradient sample was small, and the coefficient of variation was less than 1%. The lowest concentration (2.78×10¹ copies / μL) had the largest coefficient of variation, at 0.4036%, which proves that the real-time fluorescence quantitative PCR method established in this invention has good repeatability and the detection results are stable and reliable.
[0067] Table 1. Results of repeatability testing by real-time quantitative PCR
[0068]
[0069] Example 2 Specificity Detection
[0070] To verify the specificity of the detection method of the present invention, TMV (tobacco mosaic virus), CMV (cucumber mosaic virus), PVY (potato virus Y), TEV (tobacco etch virus), TVBMV (tobacco vein mosaic virus), TSWV (tomato spotted wilt virus), and TYLCV (tomato yellow leaf curl virus) positive cDNA and TYLCV (tomato yellow leaf curl virus) positive DNA were used as templates, and conventional PCR amplification was performed using ChiVMVqF / ChiVMVqR primers; healthy tobacco cDNA was used as a negative control.
[0071] PCR products were detected by 2% agarose gel electrophoresis (electrophoresis conditions: 125V, 30min).
[0072] Test results are shown Figure 4 Where M is the 500bp molecular weight standard; 1 is a tobacco sample infected with TMV; 2 is a tobacco sample infected with CMV; 3 is a tobacco sample infected with PVY; 4 is a tobacco sample infected with TEV; 5 is a tobacco sample infected with TVBMV; 6 is a tobacco sample infected with TSWV; 7 is a tobacco sample infected with ChiVMV; 8 is a tobacco sample infected with TYLCV; and 9 is a healthy tobacco sample.
[0073] The results showed that only the ChiVMV positive sample showed a specific band at 97bp, while no band was found in the other virus samples and the negative control of healthy tobacco. This indicates that the detection method of the present invention has good specificity, can specifically identify ChiVMV, and has no cross-reactivity.
[0074] Example 3 Sensitivity Detection
[0075] The detection sensitivity of the RT-qPCR method of this invention was compared with that of conventional PCR, with a final concentration of 2.78 × 10⁻⁶. 9 Nine serially diluted ChiVMV plasmid standards with a concentration of ~2.78 × 10¹ copies / µl were used as materials, and two detection methods were employed. The results are as follows:
[0076] 1. Sensitivity of RT-qPCR (see...) Figure 5 ): Figure 5 Grouping Explanation: A~I correspond to plasmid standard concentrations of 2.78 × 10⁻⁶. 9 Amplification curves of ~2.78×10¹ copies / µl (curves from left to right are 10...) 9 Plasmid standards at concentration gradients of ~10¹ were used; K was a blank control. Results showed that the ChiVMV detection method based on RT-qPCR could detect viral samples with a minimum concentration of 2.78 × 10¹ copies / μL.
[0077] 2. Sensitivity of conventional PCR (see...) Figure 6 ): Figure 6Grouping Explanation: M represents the 500bp molecular weight standard; 1~9 correspond to plasmid standard concentrations of 2.78×10⁻⁹. 9 ~2.78×10¹ copies / µl; 10 is the blank control. The results show that conventional PCR methods can only detect virus samples with a concentration of 2.78×10² copies / μL.
[0078] As can be seen, the sensitivity of the RT-qPCR detection of ChiVMV in this invention is 10 times higher than that of conventional PCR methods.
[0079] Example 4: Practical application of the detection method of the present invention
[0080] The RT-qPCR method established in Example 1 was used to detect 14 tobacco samples suspected of being infected with ChiVMV (pepper vein mottle virus) collected in the field. The specific operation was as follows: total RNA was extracted from the samples, and virus detection was performed by RT-qPCR technology. The virus-carrying status of the samples was determined based on the amplification curve, melting curve and Ct value, and the viral load of positive samples was calculated in combination with the standard curve.
[0081] Meanwhile, the above samples were subjected to routine PCR testing to verify the accuracy of the RT-qPCR detection results of this invention. The results and analysis are as follows:
[0082] 1. RT-qPCR detection results
[0083] (1) Amplification curve analysis (see) Figure 7 ): Figure 7 Grouping is as follows: A represents the ChiVMV positive control; B-H correspond to field samples 12, 13, 10, 1, 6, 4, and 7; Group I includes uninfected samples (2, 3, 5, 8, 9, 11, and 14), negative controls, and CK blank controls. Results showed that the 7 field samples (12, 13, 10, 1, 6, 4, and 7) corresponding to B-H, and the positive control (A) all exhibited good amplification curves; no amplification was observed in the 7 field samples, negative controls, and blank controls in Group I.
[0084] (2) Melting curve analysis (see) Figure 8 ): Figure 8 Grouping is as follows: Groups A-H represent ChiVMV positive controls and the seven amplified field samples mentioned above; Group I represents unamplified field samples, negative controls, and blank controls. Results showed that groups A-H all exhibited a single melting peak at the same location, indicating good specificity of the amplification products; Group I did not show a single melting peak, further confirming the lack of specific amplification.
[0085] (3) Viral load calculation: The Ct values of the above 7 RT-qPCR positive samples were 24.47, 20.88, 21.83, 20.74, 19.38, 19.45, and 19.64, respectively. Based on the standard curve plotted using ChiVMV plasmid standards, the viral load of each sample was calculated to be 5.93 × 10⁻⁶. 4 7.15×10 5 3.7×10 5 7.88×10 5 2.03×10 6 1.93×10 6 1.69×10 6 copies / μL.
[0086] 2. Routine PCR validation results
[0087] The results of routine PCR testing can be found in the following table. Figure 9 , Figure 9 Grouping Explanation: M represents the 500bp molecular weight standard; 1-14 represent 14 field test samples; 15 represents the ChiVMV positive control; 16 represents the healthy negative control; and 17 represents the blank control. The results showed that samples 1, 4, 6, 7, 10, 12, and 13, as well as the positive control (15), all showed a specific single band at 97bp; samples 2, 3, 5, 8, 9, 11, and 14, as well as the negative control (16) and the blank control (17), did not show any specific bands, which was completely consistent with the RT-qPCR detection results.
[0088] The above analysis shows that 7 out of 14 tobacco samples suspected of being infected with ChiVMV were positive for ChiVMV. Conventional PCR test results confirm that the RT-qPCR detection system established in this invention has good accuracy and specificity and can be used for rapid detection of ChiVMV and quantitative analysis of viral load in field tobacco.
Claims
1. A real-time fluorescent quantitative PCR method for detecting Tobacco pepper vein banding virus, characterized by, The method comprises the following steps: (1) designing and synthesizing a specific primer pair based on the conserved sequence of the coat protein CP gene of the pepper veinal mottle virus ChiVMV; (2) extracting total RNA of a tobacco sample to be tested and performing reverse transcription to obtain cDNA; (3) using the cDNA as a template, performing real-time fluorescent quantitative PCR amplification by using the specific primer pair; (4) judging whether the sample carries the pepper veinal mottle virus ChiVMV according to an amplification curve, a melting curve and a Ct value, and quantifying a virus load by using a standard curve.
2. The real-time fluorescent quantitative PCR method according to claim 1, characterized by, The primer pair comprises an upstream primer ChiVMVqF and a downstream primer ChiVMVqR, and the nucleotide sequences of the primer pair are as follows: SEQ ID NO. 1: 5'-CAACACCAACCATTCACGC-3'; SEQ ID NO. 2: 5'-CTACAACATGGCACGAGTGATT-3'.
3. The real-time fluorescent quantitative PCR method according to claim 1 or 2, characterized by, The length of the amplification product is 97 bp.
4. The real-time fluorescent quantitative PCR method according to claim 1, characterized by, The reaction system of the real-time fluorescent quantitative PCR is as follows: 2x TB Green Premix DimerEraser 10 µL, 10 µmol / L of qPCR upstream primer and downstream primer, each 0.6 µL, 50x ROX Reference Dye II 0.4 µL, plasmid template or cDNA template 2 µL, and sterilized water, which is supplemented to a total volume of 20 µL.
5. The real-time fluorescent quantitative PCR method according to claim 4, characterized by, The reaction condition of the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95℃ for 30 s, PCR reaction at 95℃ for 3 s, at 57℃ for 30 s and at 72℃ for 30 s, and 40 cycles.
6. The real-time fluorescent quantitative PCR method according to claim 1, characterized by, The standard curve is established by 10-fold gradient dilution of recombinant plasmid containing ChiVMV CP gene-specific fragment as plasmid standard, obtaining 9 plasmid samples with concentration range of 2.78×10 1 copies / µL as template for real-time fluorescent quantitative PCR, and the standard curve is plotted according to the Ct value and the logarithmic value of template concentration. 9 copies / µL as template for real-time fluorescent quantitative PCR, and the standard curve is plotted according to the Ct value and the logarithmic value of template concentration.
7. The real-time fluorescent quantitative PCR method according to claim 6, characterized by, The linear equation of the standard curve is y = -3.3186x + 40.309, wherein y represents the cycle threshold value, i.e., the Ct value, and x represents the logarithmic value of the plasmid concentration. The determination coefficient R of the standard curve is 0.9986, and the amplification efficiency is 100.13%. 2 The linear equation of the standard curve is y = -3.3186x + 40.309, wherein y represents the cycle threshold value, i.e., the Ct value, and x represents the logarithmic value of the plasmid concentration. The determination coefficient R of the standard curve is 0.9986, and the amplification efficiency is 100.13%. <