Primer, kit and detection method for detecting tomato brown wrinkled fruit virus based on digital PCR (Polymerase Chain Reaction) technology

Abstract

The invention discloses a primer, a kit and a detection method for detecting tomato brown wrinkled fruit virus based on a digital PCR technology, and relates to the technical field of plant virus biological detection.The primer comprises ToBRFV-CqF and ToBRFV-CqR, the nucleotide sequence of the ToBRFV-CqF is shown as SEQ ID NO: 1, and the nucleotide sequence of the ToBRFV-CqR is shown as SEQ ID NO: 2. The method disclosed by the invention has extremely high sensitivity, specificity and repeatability, can be used for accurately detecting a sample with extremely low virus loading quantity, and can be directly used for quantitatively monitoring the tomato brown wrinkled fruit virus in the field.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection of plant viruses, and particularly relates to a primer, a kit and a detection method for detecting Tomato brown rugose fruit virus based on digital PCR technology. Background Art

[0002] Tomato, as a widely cultivated vegetable crop globally, occupies an important position in the agricultural economy. However, the emergence of Tomato brown rugose fruit virus (ToBRFV) has posed a great threat to the cultivation of vegetables such as tomato and pepper. ToBRFV belongs to the genus Tobamovirus of the family Virgaviridae, and is a positive-sense single-stranded RNA virus. Its transmission speed is rapid and it has spread rapidly worldwide.

[0003] At present, there is no effective control agent for ToBRFV, nor are there high-resistant varieties. Once a plant is infected, the incidence rate is extremely high, which not only seriously affects the crop yield, but also greatly reduces the fruit quality. Especially in the greenhouse or greenhouse planting environment, if not controlled in time, it may lead to the disease of the whole shed or the whole greenhouse plants, causing serious economic losses. More intractably, ToBRFV usually does not show obvious symptoms in the seedling stage, greatly increasing the difficulty of early diagnosis.

[0004] Existing detection methods for ToBRFV have many limitations. The biological detection method relies on observing the phenotypic symptoms of plants, and the detection results are affected by the subjective factors of the observer and the uncertainty of the symptoms, which are neither accurate nor can be quantitatively analyzed; serological methods such as enzyme-linked immunosorbent assay (ELISA) are prone to cross-react with other viruses of the genus Tobamovirus, resulting in false positive results; in molecular biological methods, reverse transcription polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative PCR (qPCR), although they have high sensitivity and specificity and can be quantified, traditional qPCR relies on standard curves and amplification efficiency, cannot achieve absolute quantification, and the detection sensitivity for low-virus-load samples is also insufficient.

[0005] Therefore, it is urgent to develop a method that can detect ToBRFV early, accurately, sensitively and specifically. Based on digital PCR (dPCR) technology, the present invention aims to solve the deficiencies of existing detection technologies and provide key technical support for the early prevention and control of ToBRFV. Summary of the Invention

[0006] The present invention addresses the above problems and provides a primer, a kit and a detection method for detecting Tomato brown rugose fruit virus based on digital PCR technology. By designing specific primers and combining digital PCR technology, the present invention realizes the high-sensitivity and high-specificity detection of ToBRFV, and is applicable to the early diagnosis and quantitative monitoring of field diseases.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a primer for detecting Tomato brown rugose fruit virus based on digital PCR technology. The primer includes ToBRFV-CqF and ToBRFV-CqR. The nucleotide sequence of ToBRFV-CqF is shown in SEQ ID NO: 1, and the nucleotide sequence of ToBRFV-CqR is shown in SEQ ID NO: 2.

[0008] The present invention also provides a kit for detecting Tomato brown rugose fruit virus based on digital PCR technology. The kit includes a positive control product of the PCB301 vector plasmid of Tomato brown rugose fruit virus, a negative control product, and primers as shown in SEQ ID NO: 1 to SEQ ID NO: 2.

[0009] Further, the plasmid copy number of the positive control product is 1.0×10 3 copies / μL.

[0010] The present invention also provides a detection method for detecting Tomato brown rugose fruit virus based on digital PCR technology, which specifically includes the following steps: S1: Extract the total RNA of the diseased plant; S2: Use the total RNA of the plant as a template to perform reverse transcription to synthesize cDNA; S3: Use the synthesized cDNA as a template and use the above primers to perform digital PCR amplification by the dye method; S4: Analyze the amplification result by a digital PCR instrument to determine whether the test sample carries ToBRFV.

[0011] Further, the reaction system for digital PCR detection includes 2 μL of template, 0.8 μL of each of the upstream and downstream primers at 10 μM / L, 10 μL of 2× digital PCR Mix, 0.5 μL of fluorescent dye, 0.4 μL of Taq enzyme, and 5.5 μL of ddH2O, with a total volume of 20 μL.

[0012] Further, the reaction program for digital PCR detection is 95°C for 2 min; 95°C for 15 s, 60°C for 45 s, for 40 cycles.

[0013] Compared with the prior art, the beneficial effects of the present invention are: The present invention provides a primer, a kit, and a detection method for detecting Tomato brown rugose fruit virus based on digital PCR technology. The primer of the present invention has good specificity, and the method of the present invention has high sensitivity. The detection limit for detecting Tomato brown rugose fruit virus is 1.9×10 0copies / μL. When detecting the same sample, the coefficient of variation is not higher than 8%, indicating good repeatability. The detection kit of the present invention is easy to operate, has extremely high sensitivity, specificity, and repeatability, can accurately detect samples with extremely low virus loads, is 100 times more sensitive than qPCR, has a higher positive detection rate compared to qPCR in its detection results, and does not rely on CT values and standard curves to determine whether the result is positive, making it more convenient and suitable for early diagnosis and quantitative monitoring of field diseases. Brief Description of the Drawings

[0014] Figure 1 shows the qPCR sensitivity detection of the present invention. In the figure, 1 to 8 are plasmid standards with concentrations of 1.9×10 6 ~1.9×10 -1 copies / μL in sequence; Figure 2 shows the corresponding cycle thresholds of qPCR detection of plasmid standards with concentrations of 1.9×10 6 ~1.9×10 -1 copies / μL in sequence of the present invention; Figure 3 is the qPCR standard curve of the plasmid standards of the present invention; Figure 4 is the one-dimensional diagram of dPCR sensitivity detection of the present invention. In the figure, the concentrations of plasmid standards are 1.9×10 6 ~1.9×10 -1 copies / μL in sequence; Figure 5 is the one-dimensional diagram of dPCR of 3 repeated plasmid standards of the present invention; Figure 6 is the specific detection of primer dPCR of the present invention. The detected samples are Tomato brown rugose fruit virus (ToBRFV), Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), and Tomato spotted wilt virus (TSWV) respectively; Figure 7 is the RT-PCR detection of ToBRFV virus with primers ToBRFV-2F / ToBRFV-2R of the present invention. Maker: DL10000; 1: positive control; 2: negative control; 3 - 22 are field samples; Figure 8 are the negative and positive quality control products of the present invention; Figure 9 is the one-dimensional diagram of dPCR detection of some samples of the present invention. Detailed Embodiments

[0015] To make the objectives and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0016] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0017] Example 1 1. Primer design and synthesis Referring to the ToBRFV genome sequence (OM305070.1) in the NCBI database, in the conserved region of the CP gene, the fluorescence quantitative PCR primers ToBRFV-CqF / ToBRFV-CqR were designed online using Primer3.0. Their nucleotide sequences are shown in SEQ ID NO:1~SEQ ID NO:2, and the amplified fragment size is 111bp. Conventional PCR primers ToBRFV-1F / ToBRFV-1R were designed in the small subunit region of the replicase (RdRP). Their nucleotide sequences are shown in SEQ ID NO:3~SEQ ID NO:4, with a length of 6420bp and are used for plasmid construction; ToBRFV- 2F / ToBRFV-2R (1534-3733) is used for virus detection, and its nucleotide sequence is shown in SEQID NO:5~SEQ ID NO:6. The sequences are shown in Table 1: Table 1 Primers used in the experiment

[0018] Extraction of total RNA and synthesis of cDNA Collect 0.1g of the diseased part of tomato fruit, grind it into powder with liquid nitrogen, and extract total RNA using the TaKaRa MiniBEST Plant RNA Extraction Kit. Reverse transcription system: 1μL of template RNA, 1μL of Oligo (dT) primers, 0.5μL of dNTP, and make up to 10μL with DEPC water. Denature at 65°C for 5min, place on ice for 2min, and then add the following reagents: 5μL of 5×M-MLV Buffer, 1μL of RTase M-MLV, 0.5μL of RNAase Inhibitor, with a total volume of 10μL, and incubate at 42°C for 1h.

[0019] 3. Cloning of the target gene The primers ToBRFV-1F and ToBRFV-1R were used for RT-PCR amplification. After recovering the target fragment and ligating it with the PCB301 vector (Biosai Biotechnology), the product was transformed into DH5α competent cells (TransGen Biotech Co., Ltd.). After successful transformation, the plasmid was extracted using a plasmid extraction kit (Takara). After agarose gel electrophoresis, the plasmid was sequenced and identified. After successful identification, the plasmid sample was stored in a -20°C refrigerator for subsequent use.

[0020] 4. Preparation of plasmid standards After the plasmid sample was correctly sequenced, the plasmid concentration was measured using a NanoDrop ND-100 nucleic acid and protein analyzer. The copy number (copies / μL) was calculated using the formula: copy number (copies / μL) = (6.02×10 23 ) × (plasmid concentration) / (DNA length × 660). The copy number of the positive plasmid was calculated to be: 1.9×10 9 copies / μL.

[0021] 5. Establishment of qPCR standard curve The plasmid standards were diluted at a 10-fold concentration gradient. Eight plasmid samples with final concentrations of 1.9×10 6 ~1.9×10 -1 copies / μL were used as templates for qPCR detection (ABI Q3), and each sample was repeated 3 times. The 20 μL qPCR reaction system included 1 μL of plasmid template, 0.8 μL of each upstream and downstream primer at 10 μM / L, 10 μL of TB Green Premix Ex Taq II (Takara), 0.4 μL of ROX Reference Dye, and 7 μL of ddH2O. The reaction program was: 95°C for 30 s; 95°C for 10 s, 60°C for 30 s, for a total of 40 cycles.

[0022] 6. Establishment of dPCR detection method The plasmid standards were diluted at a 10-fold concentration gradient. Eight plasmid samples with final concentrations of 1.9×10 6 ~1.9×10 -1 copies / μL were used as templates for dPCR detection. The experimental instrument was a digital PCR instrument (model: S6) from Shenzhen Borui Biotechnology Co., Ltd. The 20 μL dPCR amplification system was used to prepare the reaction solution, including 2 μL of template, 0.8 μL of each upstream and downstream primer at 10 μM / L, 10 μL of 2× digital PCR Mix, 0.5 μL of fluorescent dye, 0.4 μL of Taq enzyme, and 5.5 μL of ddH2O. The reaction program for digital PCR detection was 95°C for 2 min; 95°C for 15 s, 60°C for 45 s, for a total of 40 cycles.

[0023] Figure 1 For qPCR sensitivity detection, in the figure, 1 - 8 are plasmid standards with concentrations of 1.9×10 6 ~1.9×10 -1 copies / μL in sequence; Figure 2 For plasmid standards with concentrations of 1.9×10 6 ~1.9×10 -1 copies / μL in sequence, the corresponding cycle thresholds were detected by qPCR. It can be seen from the figure that when the concentration of the plasmid standard is less than 1.9×10 2 , the cycle threshold is greater than 30, which is an invalid value.

[0024] Figure 3 For the qPCR standard curve of plasmid standards, the accurate and good amplification detection range of the ToBRFV virus by qPCR is 1.9×10 6 copies / μL to 1.9×10 2 copies / μL. The standard curve was plotted within this range, and R 2 =0.9993, showing a good linear relationship.

[0025] Figure 4 For the one - dimensional dPCR sensitivity detection graph, the concentrations of plasmid standards in the figure are 1.9×10 6 ~ 1.9×10 - 1 copies / μL in sequence. Figure 5 For the one - dimensional dPCR graph of 3 replicated plasmid standards.

[0026] Figure 6 For the primer dPCR specificity detection, the tested samples were Tomato brown rugose fruit virus (ToBRFV), Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), and Tomato spotted wilt virus (TSWV). Among them, the detection of ToBRFV was positive, and no other virus samples were detected, indicating good primer specificity.

[0027] Table 2 qPCR and dPCR sensitivity quantitative results of ToBRFV plasmid standards

[0028] Note: No Call means that the concentration is too high, the detection signal is saturated, showing all positive, and the DNA copy number in the reaction system exceeds 1.9×10 4 copies / μL. An invalid value means that the CT value is greater than 30.

[0029] It can be seen from Table 2 that the theoretical concentration of the plasmid standard is lower than 1.9×10 2When the copies / μL is such that the qPCR cycle threshold is greater than 30, it belongs to invalid values, and the coefficient of variation of the detection is between 2% - 4.5%; when the theoretical concentration of the plasmid standard is higher than 1.9×10 3 copies / μL, the concentration exceeds the detection upper limit of dPCR and cannot be detected. The detection lower limit of dPCR can reach as low as 1.9×10 0 copies / μL, which is 100 times more sensitive than qPCR. Although the coefficient of variation shows an increasing trend as the concentration decreases, it does not exceed 10%, indicating good repeatability and stability of the experiment. In summary, in the detection of ToBRFV virus, the qPCR detection method is suitable for the quantitative detection of known diseased samples, and the dPCR method has high sensitivity and is suitable for the quantitative monitoring of early field viruses.

[0030] Example 2 Detection of Field Samples According to the method in Example 1, extract the total RNA of 20 samples, reverse transcribe to synthesize cDNA, and use the synthesized cDNA as a template to perform digital PCR amplification by the dye method using specific primers; the reaction system for digital PCR detection includes 2 μL of template, 0.8 μL each of 10 μM / L upstream and downstream primers, 10 μL of 2× digital PCR Mix, 0.5 μL of fluorescent dye, 0.4 μL of Taq enzyme, and 5.5 μL of ddH2O, with a total volume of 20 μL. The reaction program is preferably 95°C for 2 min; 95°C for 15 s, 60°C for 45 s, for 40 cycles. Use the quality control product provided by this kit to determine the validity of the results. If the detected number of effective droplets (droplets that can detect fluorescent signals are called effective droplets) is greater than 15000, the negative quality control product is detected as negative, and the positive quality control product is detected as positive, then the detection results are determined to be valid. If the number of positive droplets is greater than or equal to 2, then report the sample as positive.

[0031] Figure 7 For the RT-PCR detection of ToBRFV virus with primers ToBRFV-2F / ToBRFV-2R, the detection results are shown in Table 3.

[0032] Figure 8 When using this kit for dPCR detection of field samples, if the negative quality control product is detected as negative and the positive quality control product is detected as positive, then the detection results are determined to be valid.

[0033] Figure 9 For the detection diagram of 4 out of 20 samples using this kit for dPCR detection.

[0034] Table 3 Detection Results of 20 Field Samples for ToBRFV

[0035] Note: + represents virus positive, - represents virus negative. Table 4 Statistical results of ToBRFV detection in 20 field samples

[0036] As can be seen from Table 3, 20 field samples were detected by the digital PCR method provided in this example, and the detection results were 19 positive and 1 negative, with a positive detection rate of 95%. For fluorescence quantitative PCR, 17 were detected positive and 3 negative, with a positive detection rate of 85%, and the positive detection rate of RT-PCR was only 60%. It can be seen that the digital PCR method provided in this example has a higher positive detection rate and higher sensitivity.

[0037] In summary, the kit and method for detecting Tomato brown rugose fruit virus based on digital PCR technology in this example have extremely high sensitivity, specificity and repeatability, and can accurately detect samples with extremely low virus loads. This example can be directly used for quantitative monitoring of Tomato brown rugose fruit virus in the field, providing strong support for early diagnosis and monitoring and early warning of diseases.

[0038] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A primer for detecting Tomato brown rugose fruit virus based on digital PCR technology, characterized in that: The primers include ToBRFV-CqF and ToBRFV-CqR. The nucleotide sequence of ToBRFV-CqF is shown as SEQ ID NO:1, and the nucleotide sequence of ToBRFV-CqR is shown as SEQ ID NO:

2.

2. A kit for detecting Tomato brown rugose fruit virus based on digital PCR technology, characterized in that: The kit includes a positive control product of the PCB301 vector plasmid of tomato brown rugose fruit virus, a negative control product, and the primers described in claim 1.

3. The kit for detecting tomato brown rugose fruit virus based on digital PCR technology according to claim 2, wherein: The copy number of the positive control quality plasmid is 1.0×10 3 copies / μL.

4. A detection method for detecting Tomato brown rugose fruit virus based on digital PCR technology, characterized in that: Specifically, it includes the following steps: S1: Extract the total RNA of the diseased plant; S2: Using the total RNA of the plant as a template, reverse transcribe to synthesize cDNA; S3: Using the synthesized cDNA as a template, perform digital PCR amplification by the dye method using the primers described in claim 1; S4: Analyze the amplification result by a digital PCR instrument to determine whether the test sample carries ToBRFV.

5. The detection method for detecting Tomato brown rugose fruit virus based on digital PCR technology according to claim 4, characterized in that: The reaction system for the digital PCR detection includes 2 μL of template, 0.8 μL each of 10 μM / L upstream and downstream primers, 10 μL of 2× digital PCR Mix, 0.5 μL of fluorescent dye, 0.4 μL of Taq enzyme, and 5.5 μL of ddH2O, with a total volume of 20 μL.

6. The detection method for detecting Tomato brown rugose fruit virus based on digital PCR technology according to claim 4, wherein: The reaction program for the digital PCR detection is 95°C for 2 min; 95°C for 15 s, 60°C for 45 s, for 40 cycles.

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