Method for carrying out specific detection and absolute quantification on to-be-detected plant sample suspected to be infected with CYRSV (Cytochrome Respiratory Syndrome Virus)

Abstract

The invention discloses a method for performing specific detection and absolute quantification on a to-be-detected plant sample suspected to be infected with CYRSV (Cytochrome Respiratory Syndrome Virus). The method comprises a specific detection step and a quantitative detection step. The CYRSV in a plant sample can be accurately and rapidly identified through the specific detection step; the CYRSV in a sample can be accurately identified and quantified within 4-5 hours in the quantitative detection step, and the method not only saves time, but also saves cost, simplifies experimental steps, is beneficial to taking corresponding remedial measures before plant diseases, and prevents huge economic loss caused by wide spreading of the CYRSV among crops.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and further to the field of plant virus detection technology, and specifically relates to a method for specific detection and absolute quantification of a plant sample suspected of being infected with CYRSV. Background Art

[0002] Chilli yellow ringspot virus (CYRSV) is a member of the genus Othotosporivius, family Tospoviridae, order Buryavirales, and belongs to the evolutionary branch of Watermelon silver mottle Orthotospovirus (WSMoV). It can cause yellow ringspot on pepper and tomato fruits. CYRSV has a similar particle morphology and genome structure to other Othotosporivius viruses. It is a spherical virus with a diameter of about 80-120 nm, consisting of a spherical envelope and large (L), medium (M), and small (S) single-stranded RNA molecular fragments. The L-RNA is 8917 nt long and belongs to negative-sense RNA. Its complementary strand encodes RNA-dependent RNA polymerase (RdRp), which is responsible for replicating and transcribing the viral genome. M-RNA is a double-sense RNA with a length of 4781 nt. Its viral strand encodes nonstructural movement protein (NSm), and its complementary strand encodes two structural glycoproteins (Gn / Gc) that can modify the surface of viral particles. S-RNA is also a double-sense RNA with a length of 3428 nt. The viral strand mainly encodes other nonstructural proteins (NSs), and its complementary strand encodes nucleocapsid protein. In nature, the transmission of CYRSV between plants is mainly carried out by vector insects (such as thrips) through persistent reproduction.

[0003] CYRSV was first discovered, identified and named in Yunnan in 2020. In recent years, it has been common in tomatoes in Yunnan, harming many tomato varieties. The Tomato spotted wilt virus (TSWV), which belongs to the same genus, has been listed as one of the ten most harmful plant viruses in the world due to its wide host range and the huge economic losses it causes. Although CYRSV was discovered in recent years, more and more researchers have begun to pay attention to and study its pathogenicity and infection mechanism. At present, the most important link in the prevention and control of plant viral diseases is the detection and monitoring of plant viruses.

[0004] At present, there are many technologies for plant virus detection, such as serological ELISA detection technology, electron microscopy technology, PCR and other molecular biology technologies. With the continuous improvement of detection requirements and standards, it is increasingly difficult for current conventional detection technologies to meet research requirements. For example, traditional ELISA detection technology, coupled with antibody coating, incubation and other steps, usually takes 1-2 days. And this serological ELISA requires specific viral protein antibodies for detection, and the cost of antibody preparation is often expensive. Although the electron microscope-negative staining technology only takes 0.5-1 hour from sample to result, it can only observe the morphology of the virus, and it is difficult to determine the virus species, and the transmission electron microscope equipment is expensive. Traditional PCR detection technology generally takes 7-8 hours from sample to test result. The use of virus-specific primers can identify the type of virus, but due to its low sensitivity, it is easy to fail to detect samples with very low virus content and result in false negatives. The real-time fluorescence quantitative PCR (RT-qPCR) technology is a quantitative tool with higher sensitivity, lower sample usage, and more specific virus detection than conventional PCR technology. This method only takes 5-6 hours from sample extraction to result analysis, with low detection cost, large detection sample throughput, and highly repeatable and accurate quantitative data. RT-qPCR technology is divided into relative quantification and absolute quantification. Relative quantification is to detect the amount of target gene and internal reference gene in the sample, and then analyze the relative expression of target gene by fixed formula. Absolute quantification is to construct a standard of target gene, calculate the copy number of standard, construct a standard curve, and then quantify the gene copy number of target gene to be detected in the sample. Absolute quantification can not only qualitatively characterize genes, but also quantitatively characterize genes, which is more in line with the determination of virus content in samples. This technology has been widely used in the study of gene function expression and regulation, as well as the quantification of the relative content or absolute copy number of specific nucleic acids in different hosts or pathogens. This technology has shown absolute advantages in virus detection in plant tissues. Summary of the invention

[0005] The object of the present invention is to provide a method for specific detection and absolute quantification of a plant sample suspected of being infected with CYRSV.

[0006] The object of the present invention is achieved by including specific detection and quantitative detection steps, specifically including: A. Specificity detection: 1) Design the N gene-specific primers CYRSV-NF / CYRSV-NR of pepper yellow ringspot virus, and the amplified fragment size is 828 bp; The CYRSV-NF / CYRSV-NR primer sequence is: CYRSV-NF:5'-TCACACTTCCAGAGAAGAACTTGGT-3', CYRSV-NR:5'-ATGTCTAACGTTAAGCAACTT-3'; 2) To identify plants infected with CYRSV as positive samples, extract total RNA and reverse transcribe it into positive sample cDNA; 3) Using the cDNA as a template, amplify the CYRSV N gene-specific fragment with the N gene-specific primers CYRSV-NF / CYRSV-NR, and detect the PCR product by agarose gel; if the target band of the amplified product is 828 bp, the sample to be tested carries CYRSV; if there is no target band at the 828 bp position, the sample to be tested does not carry CYRSV; B. Quantitative detection: 1) Preparation of CYRSV viral plasmid standards: Plants infected with CYRSV were identified as positive samples, total RNA was extracted, reverse transcribed into positive sample cDNA, and the cDNA was used as a template to amplify the CYRSV N gene specific fragment with the designed CYRSV specific primers CYRSV-qN-F / CYRSV-qN-R. The specific bands were detected and recovered by agarose gel, cloned into a conventional T cloning vector (pMD18-T), and sequenced to verify that the plasmid standard was correctly obtained; The CYRSV-qN-F / CYRSV-qN-R primer sequence is: CYRSV-qN-F:5'-GCGGTACTGCAGATGTTGAA-3' CYRSV-qN-R:5'-GGTCCAATCTTCTGGTCCAA-3'; 2) Establishment of CYRSV standard curve: Plasmid standards were diluted in a gradient manner, and CYRSV-qN-F / CYRSV-qN-R were used as fluorescent quantitative primers to perform fluorescent quantitative PCR on plasmid standards of different concentrations to obtain the CT values ​​of plasmid standards of each concentration; a standard curve was established with the logarithmic value of the viral copy number corresponding to each concentration of plasmid standards as the abscissa and the CT value as the ordinate; The CYRSV-qN-F / CYRSV-qN-R fluorescence quantitative primer sequence is: CYRSV-qN-F:5'-GCGGTACTGCAGATGTTGAA-3' CYRSV-qN-R:5'-GGTCCAATCTTCTGGTCCAA-3'; 3) Absolute quantification of CYRSV in the tested plant samples: Extracting total RNA of the plant sample to be tested, and reversely transcribing it into cDNA of the sample to be tested; using the cDNA as a template, and using CYRSV-qN-F / CYRSV-qN-R primers to perform fluorescence quantitative PCR to obtain the CT value of the plant sample to be tested; The CYRSV standard curve was established with the CT value of the standard as the horizontal axis and the corresponding copy number as the vertical axis. The CT value of the sample to be tested was inserted into the above CYRSV standard curve to calculate the copy number of CYRSV in the sample to be tested.

[0007] The specific operations are as follows: (1) Specific identification and detection of CYRSV in plant samples: The N gene of CYRSV is the nucleocapsid protein encoding gene of pepper yellow ringspot virus. The amplification of the N gene can identify whether the sample is infected with CYRSV. We designed the N gene-specific primers CYRSV-NF / CYRSV-NR of the virus, and the amplified fragment size is 828bp. To determine that the plants infected with CYRSV are positive samples, total RNA is extracted and reverse transcribed into positive sample cDNA; using the cDNA as a template, the primers CYRSV-NF / CYRSV-NR are used to amplify the CYRSV N gene-specific fragment; the PCR product is detected by agarose gel. If the target band of the amplified product is 828bp, the sample to be tested carries CYRSV; if there is no target band at the 828bp position, the sample to be tested does not carry CYRSV. The primer sequences of CYRSV-NF / CYRSV-NR are: CYRSV-NF:5'-TCACACTTCCAGAGAAGAACTTGGT-3' CYRSV-NR:5'-ATGTCTAACGTTAAGCAACTT-3'.

[0008] The reverse transcription system and reaction conditions are as follows: Random 6 mers primer (50 μM) 1 μL, dNTP mix 1 μL, total RNA (2 μg) 4 μL, ddH2O 4 μL, total volume 10 μL, 65 degrees for 5 min, and store at 4 degrees for the next step; the above reaction solution 10 μL, 5x RT Buffer 4 μL, Rnase inhibitor 0.5 μL, EvoRtase 1 μL, ddH2O 4.5 μL, total volume 20 μL, 30 degrees for 10 min, 42 degrees for 30 min, 95 degrees for 5 min, and store at 4 degrees for use. The general PCR system is as follows: cDNA template (diluted 10 times) 2 μL, 2x Taq Mix 12.5 μL, 10μM upstream and downstream primers 1 μL each, ddH2O 8.5 μL, total volume 25 μL. PCR reaction conditions: pre-denaturation at 94°C for 30 s, denaturation at 98°C for 10 s, annealing at 54°C for 30 s, extension at 72°C for 1 min, 35 cycles, and final extension at 72°C for 2 min.

[0009] (2) Quantitative detection of CYRSV in plant samples: 1. Preparation of CYRSV viral plasmid standards: Plants infected with CYRSV were identified as positive samples, total RNA was extracted, and reverse transcribed into positive sample cDNA; the cDNA was used as a template to amplify the CYRSV N gene specific fragment with the designed CYRSV specific primers CYRSV-qN-F / CYRSV-qN-R; the specific bands were detected and recovered by agarose gel, cloned into a conventional T cloning vector (pMD18-T) and sequenced to verify the correctness, and the plasmid standard was obtained; the concentration of the plasmid standard was measured, and the copy number was calculated according to the formula: copy number (Copies / μL) = A×6.02×10 23 / B (A is the plasmid concentration g / μL, B is the plasmid DNA molecular weight g / moL), and the viral copy number of the plasmid standard is obtained; the sequence of the above CYRSV primer CYRSV-qN-F / CYRSV-qN-R is: CYRSV-qN-F:5'-GCGGTACTGCAGATGTTGAA-3', CYRSV-qN-R:5'-GGTCCAATCTTCTGGTCCAA-3'.

[0010] The amplified fragment is 224 bp. The reverse transcription system and reaction conditions are the same as above.

[0011] 2. Establishment of CYRSV standard curve: The plasmid standard was diluted in a gradient manner, and CYRSV-qN-F / CYRSV-qN-R was used as a fluorescent quantitative primer. Fluorescent quantitative PCR was performed on plasmid standards of different concentrations to obtain the CT value of each concentration of the plasmid standard; the logarithmic value of the viral copy number corresponding to each concentration of the plasmid standard was used as the abscissa, and the CT value was used as the ordinate to establish a standard curve; the sequence of the CYRSV primer CYRSV-qN-F / CYRSV-qN-R is: CYRSV-qN-F:5'-GCGGTACTGCAGATGTTGAA-3', CYRSV-qN-R:5'-GGTCCAATCTTCTGGTCCAA-3'.

[0012] The reverse transcription system is: Random 6 mers primer (50 μM) 0.5 μL, 5x RT Buffer 2 μL, Evo-MLV 0.5 μL, total RNA (1 μg) 2 μL, ddH2O 5 μL. The reverse transcription system is: 37 degrees for 15 min, 85 degrees for 5 s, and 4 degrees for storage. The fluorescence quantitative PCR system is: cDNA template (diluted 5 times) 1 μL, 2x Taq mix SYBR Green 5 μL, 10μM upstream and downstream primers 0.5 μL each, ddH2O 3 μL, total volume 10 μL. Fluorescence quantitative PCR reaction conditions: 95 degrees pre-denaturation for 30 s, 95 degrees denaturation for 50 s, 60 degrees annealing for 30 s, 40 cycles, and increase the melting curve.

[0013] 3. Absolute quantification of CYRSV in the tested plant samples: The total RNA of the plant sample to be tested is extracted and reverse transcribed into cDNA of the sample to be tested; the cDNA is used as a template and the CYRSV-qN-F / CYRSV-qN-R primers are used for fluorescence quantitative PCR, and the reverse transcription and PCR systems and reaction conditions are the same as above to obtain the CT value of the sample to be tested. The CT value of the standard is used as the horizontal axis and the corresponding copy number is used as the vertical axis to establish a CYRSV standard curve, and the CT value of the sample to be tested is substituted into the above CYRSV standard curve to calculate the copy number of CYRSV in the sample to be tested.

[0014] The present invention establishes a method for specific detection and absolute quantification of CYRSV, (1) specific identification and detection of CYRSV in plant samples: the PCR method for qualitative detection of CYRSV can specifically detect CYRSV in samples within 5-6 hours. The method has strong specificity and high accuracy, and can identify TSWV with high similarity. (2) Quantification of CYRSV copy number in plant samples: CYRSV standard plasmid construction and standard curve establishment; obtaining cDNA of the sample to be tested, and performing fluorescence quantitative PCR with CYRSV-qN-F / CYRSV-qN-R primers to obtain the CT value of the sample to be tested; substituting the CT value of the sample to be tested into the standard curve to calculate the copy number of pepper yellow ringspot virus in the sample to be tested. The method has high accuracy, high sensitivity, strong specificity, good repeatability, etc., and can accurately quantify CYRSV in the sample within 5-6 hours, which not only saves time and simplifies experimental steps, but also helps to take corresponding remedial measures before the plant becomes ill, and prevent the huge economic losses caused by the widespread spread of CYRSV among crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of PCR amplification of CYRSV-specific primers at different annealing temperatures (50°C, 52°C, 54°C, 56°C, 58°C, 59°C, and 60°C) are shown. NC is a negative control using water as a template, and the rest are positive samples of tomato fruits carrying CYRSV artificially prepared in our laboratory; Figure 1 It was shown that the target band of CYRSV could be detected at annealing temperatures of 54°C, 56°C and 60°C, and the band was brightest at 54°C, while the target band was relatively weak at 56°C; therefore, for the CYRSV specific primers of the present invention, 54°C was the most suitable annealing temperature for PCR detection; Figure 2 CYRSV-specific primer detection for samples suspected of CYRSV infection; In the gel on the left, samples 1 to 15 are suspected tomato fruit samples infected by CYRSV, samples 16 to 18 are symptom-free control samples, and NC is a negative control of water; the gel on the right shows PCR of samples 1, 4, and 16 using TSWV, TZSV, and CYRSV specific primers, respectively. The positive is the TSWV positive control, and the TSWV target band is 777 bp; CYRSV was detected in all samples suspected of CYRSV infection. Samples No. 1, No. 4 and No. 16 were selected for TSWV and TZSV detection. No TSWV and TZSV of the same genus as CYRSV were detected in No. 1 and No. 4, and these three viruses were not detected in No. 16 asymptomatic control. This shows that the CYRSV-specific primers CYRSV-NF / CYRSV-NR designed by us can detect CYRSV efficiently, accurately and specifically. Figure 3 For specific detection of CYRSV in tomato fruits and pepper leaves; Samples 1-11 in the left gel are susceptible tomato fruits, and samples 1-12 in the right gel are susceptible pepper leaves; tomato samples 1-11 were all detected to carry CYRSV; pepper leaf samples 1-5 were not detected with CYRSV, while samples 6-12 were detected with obvious CYRSV target bands, among which the weak CYRSV band of sample 6 may be related to the virus content itself; no obvious primer dimers were present in the amplification of different samples by CYRSV primers, indicating that the CYRSV-specific primers of the present invention can efficiently and specifically detect CYRSV in samples; Figure 4 Schematic diagram of the standard curve established for CYRSV standards; Figure 5 It is a schematic diagram of the amplification curve after the standard is serially diluted; The amplification curves of the four replicate samples overlapped or had a small gap in distance during the fluorescence quantitative PCR test, indicating that the CYRSV absolute quantitative method had good repeatability; Figure 6 is the melting curve of the fluorescent quantitative PCR of primers CYRSV-qN-F / CYRSV-qN-R; The melting curves of the samples have high overlap and present a single peak, and the peak value is about 80 degrees, indicating that the primers CYRSV-qN-F / CYRSV-qN-R involved in the present invention have good specificity for fluorescence quantification, and the PCR product is specific without nonspecific amplification. Figure 7 Analysis of viral copy number in CYRSV-infected tomatoes; No. 085 was the symptom-free control group, and Nos. 081-084 were tomato samples with different degrees of susceptibility. The CYRSV copy numbers calculated for each sample were plotted and statistically analyzed. The CYRSV copy number was almost undetectable in the symptom-free control group, while different accumulation amounts of CYRSV were detected in Nos. 081-084; Figure 8 Schematic diagram of the standard curve of CYRSV using reagents from different manufacturers; The upper figure is the standard curve made with domestic reagents, and the lower figure is the standard curve made with imported reagents. There is a good linear relationship between the CT value and the concentration of the plasmid standard. Fig. 9 Schematic diagram of amplification curves of standards using reagents from different manufacturers; Fig.10 Schematic diagram of the melting curve of primers CYRSV-qN-F / CYRSV-qN-R under reagents from different manufacturers; Fig.11 Schematic diagram of the copy number of CYRSV in the same sample using reagents from different manufacturers. DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with the embodiments, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0017] The method for specific detection and absolute quantification of a plant sample suspected of being infected with CYRSV of the present invention comprises specific detection and quantitative detection steps, specifically comprising: A. Specificity detection: 1) Design the N gene-specific primers CYRSV-NF / CYRSV-NR of pepper wild ringspot virus, and the amplified fragment size is 828 bp; The CYRSV-NF / CYRSV-NR primer sequence is: CYRSV-NF:5'-TCACACTTCCAGAGAAGAACTTGGT-3', CYRSV-NR:5'-ATGTCTAACGTTAAGCAACTT-3'; 2) To identify plants infected with CYRSV as positive samples, extract total RNA and reverse transcribe it into positive sample cDNA; 3) Using the cDNA as a template, amplify the CYRSV N gene-specific fragment with the N gene-specific primers CYRSV-NF / CYRSV-NR, and detect the PCR product by agarose gel; if the target band of the amplified product is 828 bp, the sample to be tested carries CYRSV; if there is no target band at the 828 bp position, the sample to be tested does not carry CYRSV; B. Quantitative detection: 1) Preparation of CYRSV viral plasmid standards: Plants infected with CYRSV were identified as positive samples, total RNA was extracted, reverse transcribed into positive sample cDNA, and the cDNA was used as a template to amplify the CYRSV N gene specific fragment with the designed CYRSV specific primers CYRSV-qN-F / CYRSV-qN-R. The specific bands were detected and recovered by agarose gel, cloned into a conventional T cloning vector (pMD18-T), and sequenced to verify that the plasmid standard was correctly obtained; The CYRSV-qN-F / CYRSV-qN-R primer sequence is: CYRSV-qN-F:5'-GCGGTACTGCAGATGTTGAA-3' CYRSV-qN-R:5'-GGTCCAATCTTCTGGTCCAA-3'; 2) Establishment of CYRSV standard curve: Plasmid standards were diluted in a gradient manner, and CYRSV-qN-F / CYRSV-qN-R were used as fluorescent quantitative primers to perform fluorescent quantitative PCR on plasmid standards of different concentrations to obtain the CT values ​​of plasmid standards of each concentration; a standard curve was established with the logarithmic value of the viral copy number corresponding to each concentration of plasmid standards as the abscissa and the CT value as the ordinate; The CYRSV-qN-F / CYRSV-qN-R fluorescence quantitative primer sequence is: CYRSV-qN-F:5'-GCGGTACTGCAGATGTTGAA-3' CYRSV-qN-R:5'-GGTCCAATCTTCTGGTCCAA-3'; 3) Absolute quantification of CYRSV in the tested plant samples: Extracting total RNA of the plant sample to be tested, and reversely transcribing it into cDNA of the sample to be tested; using the cDNA as a template, and using CYRSV-qN-F / CYRSV-qN-R primers to perform fluorescence quantitative PCR to obtain the CT value of the plant sample to be tested; The CYRSV standard curve was established with the CT value of the standard as the horizontal axis and the corresponding copy number as the vertical axis. The CT value of the sample to be tested was inserted into the above CYRSV standard curve to calculate the copy number of CYRSV in the sample to be tested.

[0018] The reverse transcription system and reaction conditions in step A 2) and step B 1) are as follows: Random 6 mers primer (50 μM) 1 μL, dNTP mix 1 μL, total RNA (2 μg) 4 μL, ddH2O 4 μL, total volume 10 μL, 65 degrees for 5 min, and store at 4 degrees for the next step; 10 μL of the above reaction solution, 4 μL of 5x RT Buffer, 0.5 μL of Rnase inhibitor, 1 μL of EvoRtase, 4.5 μL of ddH2O, total volume 20 μL, 30 degrees for 10 min, 42 degrees for 30 min, 95 degrees for 5 min, and store at 4 degrees for use.

[0019] The reaction system for amplification in step A3) is: 2 μL cDNA template (diluted 10 times), 12.5 μL 2x Taq Mix, 1 μL each of 10 μM upstream and downstream primers, 8.5 μL ddH2O, and a total volume of 25 μL.

[0020] The reaction conditions for amplification in step A3) are: pre-denaturation at 4°C for 30 s, denaturation at 98°C for 10 s, annealing at 54°C for 30 s, extension at 72°C for 1 min, 35 cycles, and final extension at 72°C for 2 min.

[0021] The fluorescence quantitative PCR system in step B is: 1 μL of cDNA template (diluted 5 times), 5 μL of 2x Taq mix SYBR Green, 0.5 μL of 10 μM upstream and downstream primers, 3 μL of ddH2O, and a total volume of 10 μL.

[0022] The reaction conditions of fluorescence quantitative PCR in step B were: pre-denaturation at 95 degrees for 30 s, denaturation at 95 degrees for 50 s, annealing at 60 degrees for 30 s, 40 cycles, and adding a melting curve.

[0023] The present invention is further described below with specific implementation cases: Main reagents: SteadyPure Plant RNA extraction kit, Evo M-MLV Reverse transcription kit (forqPCR), Evo M-MLV Plus cDNA Synthesis Kit, SYBR Green Pro Taq HS Premix qPCR Kit (with ROX), 2x Accurate Taq The premixed solution (including dye) was purchased from Hunan Aikerui Biotechnology Co., Ltd.; DL5000 DNA marker and pMD TM18-T Vector Cloning Kit was purchased from Bao Biotechnology (Dalian) Co., Ltd. Main instruments: ABI7500 fluorescence quantitative PCR instrument was purchased from ABI, USA; S1000 ordinary PCR instrument was purchased from Bio-Rad, USA; low-temperature refrigerated centrifuge and NanoDrop2000 micro-nucleic acid analyzer were purchased from Thermo Fisher Scientific.

[0024] 1. Specific detection of CYRSV Design of CYRSV specific primers: Based on the N sequence of the CYRSV nucleocapsid protein encoding gene obtained in our laboratory earlier, the first 25 bp at the 5' end of the sequence was directly used as the upstream primer, and the reverse sequence of the last 21 bp at the 3' end of the sequence was directly used as the downstream primer. The length of the amplified target fragment was 828 bp, and the primer sequence was: CYRSV-NF:5'-TCACACTTCCAGAGAAGAACTTGGT-3', CYRSV-NR:5'-ATGTCTAACGTTAAGCAACTT-3'.

[0025] Specific PCR detection of CYRSV: (1) According to SteadyPure Extract total RNA from the sample according to the instructions of the plant RNA extraction kit. (2) Evo M-MLV Plus cDNA synthesis kit instructions: 2ug of total RNA per sample was reverse transcribed into cDNA. (3) Using the cDNA as template and CYRSV-NF / CYRSV-NR as primers, 2x Accurate TaqThe premix (containing dye) was used for PCR amplification. The PCR amplification system was: cDNA template (diluted 10 times) 2 μL, 2x Taq Mix 12.5 μL, 10 μM CYRSV-NF 1 μL, 10 μM CYRSV-NR 1 μL, ddH2O 8.5 μL, total volume 25 μL. PCR reaction conditions: 94 degrees pre-denaturation for 30 s, 98 degrees denaturation for 10 s, 54 degrees annealing for 30 s, 72 degrees extension for 1 min, 35 cycles, and finally 72 degrees extension for 2 min. (4) Prepare 1% agarose gel, take 5ul of PCR product and load it on 1% agarose gel, and run electrophoresis at 120V in TBE electrophoresis buffer for 30 min. The reverse transcription system and reaction conditions are as follows: Random 6 mersprimer (50 μM) 1 μL, dNTP mix 1 μL, total RNA (2 μg) 4 μL, ddH2O 4 μL, total volume 10 μL, 65 degrees for 5 min, and store at 4 degrees for the next step; the above reaction solution 10 μL, 5x RT Buffer 4 μL, Rnase inhibitor 0.5 μL, EvoRtase 1 μL, ddH2O 4.5 μL, total volume 20 μL, 30 degrees for 10 min, 42 degrees for 30 min, 95 degrees for 5 min, and store at 4 degrees for use. The general PCR system is as follows: cDNA template (diluted 10 times) 2 μL, 2x Taq Mix 12.5 μL, 10 μM upstream and downstream primers 1 μL each, ddH2O 8.5 μL, total volume 25 μL. PCR reaction conditions: 94 degrees pre-denaturation for 30 seconds, 98 degrees denaturation for 10 seconds, 54 degrees annealing for 30 seconds, 72 degrees extension for 1 minute, 35 cycles, and finally 72 degrees extension for 2 minutes. Under the condition of 54 degrees annealing temperature for PCR amplification, the amplification efficiency is the highest, the target band is the brightest, and the specificity is the best. Figure 1 As shown. The above method can specifically identify CYRSV and is highly specialized. It has no reaction to Tomato Spotted Wilt Virus (TSWV) which is highly similar to CYRSV and belongs to the same genus. Figure 2 shown.

[0026] 2. Quantitative Detection of CYRSV Design of CYRSV-specific fluorescent quantitative PCR primers: Based on the N gene sequence of CYRSV, the upstream and downstream primers of fluorescent quantitative PCR were designed using Primer3Plus online primer design software. The amplification length was 224 bp, and the primer sequences were: CYRSV-qN-F:5'-GCGGTACTGCAGATGTTGAA-3', CYRSV-qN-R: 5'-GGTCCAATCTTCTGGTCCAA-3', all primers were synthesized by Shanghai Bioengineering.

[0027] Construction of CYRSV standard plasmid and preparation of standard products: (1) Tomato fruits infected with pepper yellow ringspot virus CYRSV were used as experimental materials. The materials were collected in Yangjie Town, Yuanmou County, Chuxiong City, Yunnan Province. SteadyPure Extract total RNA from the sample according to the instructions of the plant RNA extraction kit. (2) Evo M-MLV Plus cDNA Synthesis Kit Instructions: 2ug of total RNA per sample was reverse transcribed into cDNA. (3) Using the cDNA as template and CYRSV-qN-F / CYRSV-qN-R as primers, 2x Accurate Taq The premix (containing dye) was used for PCR amplification. The PCR amplification system was: cDNA template (diluted 10 times) 2 μL, 2x Taq Mix 12.5 μL, 10 μM CYRSV-qN-F 1 μL, 10 μM CYRSV-qN-R 1 μL, ddH2O 8.5 μL, total volume 25 μL. PCR reaction conditions: 94 degrees pre-denaturation for 30 s, 98 degrees denaturation for 10 s, 54 degrees annealing for 30 s, 72 degrees extension for 1 min, 35 cycles, and finally 72 degrees extension for 2 min. (4) Prepare 1% agarose gel, take 5uL of PCR product and load it on 1% agarose gel, and run electrophoresis at 120V in TBE electrophoresis buffer for 30 min. The electrophoresis results showed that the target band with the CYRSV sample PCR product was at the 224 bp position, and the band was single. (5) After the target band from the previous step is recovered and purified, it is cloned into pMD TM The positive clones were obtained by ampicillin resistance screening. The positive plasmids were extracted from the bacterial solution containing the positive clones according to the instructions of the Axgen plasmid extraction kit. The positive plasmids with correct sequencing were stored at -20 degrees for later use.

[0028] CYRSV standard plasmid construction and standard preparation: The standard plasmid concentration is 298.46 ng / μL, and the DNA molecular weight of the standard plasmid = (2692+224) x 660 = 1924560 g / moL. According to the copy number calculation formula: copy number (Copies / μL) = A × 6.02 × 10 23 / B (A is the plasmid concentration g / μL, B is the plasmid DNA molecular weight g / moL), the copy number of the plasmid standard is 0.94x10 11 .

[0029] CYRSV standard curve establishment and absolute quantitative PCR method: (1) Use DEPC water to dilute the standard sample 10 times in a gradient manner. The copy number of the diluted standard sample is: 0.94x10 9 , 0.94x10 8 , 0.94x10 7 , 0.94x10 6 , 0.94x10 5 , 0.94x10 4 , 0.94x10 3 , 0.94x10 2 , using 8 standards with different copy numbers as templates. (2) Extract total RNA from the sample to be tested according to the previous method, and Evo M-MLV Reverse transcription kit (forqPCR) reverse transcription system to obtain cDNA template: Random 6mers primer (50 μM) 0.5 μL, 5xRT Buffer 2 μL, Evo-MLV 0.5 μL, total RNA (1 μg) 2 μL, ddH2O 5 μL. Reverse transcription system: 37 degrees for 15 min, 85 degrees for 5 s, 4 degrees for storage. (3) CYRSV-qN-F / CYRSV-qN-R were used as fluorescent quantitative PCR primers, according to SYBR Green Pro Taq HS premixed qPCR kit (including ROX) was used for fluorescence quantitative PCR reaction. The fluorescence quantitative PCR reaction system was: cDNA template (diluted 5 times) 1 μL, 2x Taq mixSYBR Green 5 μL, 10uM upstream and downstream primers 0.5 μL each, ddH2O 3 μL, total volume 10 μL. The conditions were: 95 degrees pre-denaturation for 30 s, 95 degrees denaturation for 50 s, 60 degrees annealing for 30 s, 40 cycles, and a melting curve was added. The fluorescence quantitative PCR system in the present invention is 10 μL, which saves half of the system compared to the commonly used 20 μL system, and also saves half of the experimental cost.

[0030] Calculation of CYRSV copy number in sample: Establish CYRSV standard curve with CT value of standard as horizontal coordinate and corresponding copy number as vertical coordinate. Substitute CT value of the sample to be tested into CYRSV standard curve to calculate the copy number of CYRSV in the sample to be tested.

[0031] Example 1 Specific PCR detection of CYRSV in tomato fruits and pepper leaves (1) Sample materials were collected in Yangjie Town, Yuanmou County, Chuxiong City, Yunnan Province. SteadyPure Extract total RNA from samples according to the instructions of the plant RNA extraction kit. (2) Evo M-MLV Plus cDNA synthesis kit instructions: Reverse transcribe 2ug of total RNA per sample into cDNA. (3) Use the cDNA as a template and CYRSV-NF / CYRSV-NR as primers for PCR amplification. The PCR amplification system is: cDNA template (diluted 10 times) 2 μL, 2x Taq Mix 12.5 μL, 10 μMCYRSV-NF 1 μL, 10 μM CYRSV-NR 1 μL, ddH2O 8.5 μL, total volume 25 μL. PCR reaction conditions: pre-denaturation at 94 degrees for 30 s, denaturation at 98 degrees for 10 s, annealing at 54 degrees for 30 s, extension at 72°C for 1 min, 35 cycles, and finally extension at 72 degrees for 2 min. (4) Prepare 1% agarose gel, take 5uL of PCR product and load it on 1% agarose gel, and run electrophoresis at 120v in TBE electrophoresis buffer for 30 min. The results are shown in the figure. Figure 3 The electrophoresis results showed that the target band of the PCR product of the sample with CYRSV was at the position of 828bp, with a single band and no primer dimer, while the sample without CYRSV had no target band.

[0032] Example 2 Determination of CYRSV copy number in tomato fruit The extraction of total RNA from the sample and the reverse transcription of cDNA refer to the above specific implementation method. The CYRSV plasmid standard obtained by the above method and CYRSV-qN-F / CYRSV-qN-R primers were used for fluorescence quantitative PCR, and the steps were the same as above. The sample CT value was substituted into the standard curve to obtain the copy number of each sample. The standard curve is as follows Figure 4 As shown, the standard was diluted 10-fold to form 8 concentration gradients (0.94 x 10 9 , 0.94 x 10 8 , 0.94 x 10 7 , 0.94 x 10 6 , 0.94 x 10 5 , 0.94 x 10 4 , 0.94 x10 3 , 0.94 x 10 2 ), the corresponding CT values ​​were 8.619, 12.183, 15.230, 18.293, 22.006, 24.199, 27.106, and 30.397. Using DEPC water as a template as a negative control, the average CT value was 35.632. There was a good linear relationship between the CT value and the concentration of the plasmid standard, with a slope of -3.0676 and a coefficient of R 2=0.9979, the amplification efficiency is 100%, and the standard curve equation is y =-3.0676 x +36.544. The standard amplification curve is as follows Figure 5 As shown in the figure, the standard was well diluted into different multiples, and the amplification curves of the four replicate samples overlapped or had a small distance difference, indicating that the CYRSV absolute quantitative method of the present invention has good repeatability. The melting curve of the fluorescent quantitative PCR primer CYRSV-qN-F / CYRSV-qN-R is shown in the figure. Figure 6 As shown in the figure, the melting curves of each sample have high overlap, present a single peak, and the peak value is around 80 degrees, indicating that the primers have good specificity for fluorescence quantification, the PCR product is specific, and no non-specific amplification occurs. Substitute the CT value of the sample to be tested into the above CYRSV standard curve to calculate the copy number of CYRSV in the sample to be tested. The copy number statistics of the sample to be tested are as follows: Figure 7 shown.

[0033] Example 3 Determination of CYRSV copy number in tomato leaves using fluorescent quantitative PCR reagents from different manufacturers Two fluorescent quantitative PCR reagents produced by different manufacturers were selected, one was an imported product from QIAGEN, with a product number of 208054, and the other was SYBR Green from Hunan Aikerui Bioengineering Co., Ltd., a domestic company. Pro Taq HS premixed qPCR kit (including ROX), catalog number AG11718. Except for the fluorescence quantitative PCR step, refer to the corresponding instructions, and follow the above steps for the rest of the method. The standard curve is as follows Figure 8 As shown in the figure above, the slope of the standard curve of the domestic reagent is -3.0118, and the coefficient R 2 =0.9954, the amplification efficiency is 100%, and the standard curve equation is y =-3.0118 x +35.307. The slope of the standard curve of the imported reagent in the figure below is -3.0642, and the coefficient R 2 =0.9996, the amplification efficiency is 100%, and the standard curve equation is y =-3.0642 x +34.621. The amplification curves of the standard products under different manufacturers' reagents are as follows Fig. 9 As shown, the standard was diluted 10-fold to form 8 concentration gradients (0.94 x10 9 , 0.94 x 10 8 , 0.94 x 10 7 , 0.94 x 10 6 , 0.94 x 10 5, 0.94 x 10 4 , 0.94 x 10 3 , 0.94 x10 2 ), the average CT values ​​corresponding to the domestic reagent standards in the upper figure are 8.390, 11.485, 14.262, 17.369, 20.392, 22.521, 25.897, and 30.266, and the CT values ​​corresponding to the imported reagent standards in the lower figure are 7.251, 10.233, 13.245, 16.227, 19.299, 22.202, 25.521, and 28.825. During the fluorescence quantitative PCR test, the amplification curves of the three replicate samples overlapped or had a small distance difference, indicating that the CYRSV absolute quantification method has good repeatability. The melting curves of the primers CYRSV-qN-F / CYRSV-qN-R under reagents from different manufacturers are as follows Fig.10 As shown. The melting curves of the samples under different manufacturers' reagents have high overlap and present a single peak, and the peak value is around 80 degrees, indicating that the primers CYRSV-qN-F / CYRSV-qN-R involved in the present invention have good specificity for fluorescence quantification, and the PCR product is specific without non-specific amplification. Fig.11 As shown, tomato leaves inoculated with CYRSV at different time periods were used as experimental samples, and the CYRSV accumulation in the samples was analyzed using the domestic reagents in the upper figure and the imported reagents in the lower figure. The results showed that the accumulation trend of CYRSV was similar under reagents from different manufacturers, indicating that the absolute quantitative plasmid standard, fluorescent quantitative primers and detection method of the present invention are stable and highly repeatable.

Claims

1. A method for specific detection and absolute quantification of a plant sample suspected of being infected with CYRSV, characterized in that: It includes specific detection and quantitative detection steps, including: A. Specificity detection: 1) Design the N gene-specific primers CYRSV-NF / CYRSV-NR of pepper wild ringspot virus, and the amplified fragment size is 828 bp; The CYRSV-NF / CYRSV-NR primer sequence is: CYRSV-NF:5'-TCACACTTCCAGAGAAGAACTTGGT-3', CYRSV-NR:5'-ATGTCTAACGTTAAGCAACTT-3'; 2) To identify plants infected with CYRSV as positive samples, extract total RNA and reverse transcribe it into positive sample cDNA; 3) Using the cDNA as a template, amplify the CYRSV N gene-specific fragment with the N gene-specific primers CYRSV-NF / CYRSV-NR, and detect the PCR product by agarose gel; if the target band of the amplified product is 828 bp, the sample to be tested carries CYRSV; if there is no target band at the 828 bp position, the sample to be tested does not carry CYRSV; B. Quantitative detection: 1) Preparation of CYRSV viral plasmid standards: Plants infected with CYRSV were identified as positive samples, total RNA was extracted, reverse transcribed into positive sample cDNA, the cDNA was used as a template, and the designed CYRSV-specific primers CYRSV-qN-F / CYRSV-qN-R were used to amplify the CYRSV N gene specific fragment, and the specific band was detected and recovered by agarose gel, cloned into a conventional T cloning vector, and sequenced to verify that the plasmid standard was correctly obtained; The CYRSV-qN-F / CYRSV-qN-R primer sequence is: CYRSV-qN-F:5'-GCGGTACTGCAGATGTTGAA-3' CYRSV-qN-R:5'-GGTCCAATCTTCTGGTCCAA-3'; 2) Establishment of CYRSV standard curve: Plasmid standards were diluted in a gradient manner, and CYRSV-qN-F / CYRSV-qN-R were used as fluorescent quantitative primers to perform fluorescent quantitative PCR on plasmid standards of different concentrations to obtain the CT values ​​of plasmid standards of each concentration; a standard curve was established with the logarithmic value of the viral copy number corresponding to each concentration of plasmid standards as the abscissa and the CT value as the ordinate; The CYRSV-qN-F / CYRSV-qN-R fluorescence quantitative primer sequence is: CYRSV-qN-F:5'-GCGGTACTGCAGATGTTGAA-3' CYRSV-qN-R:5'-GGTCCAATCTTCTGGTCCAA-3'; 3) Absolute quantification of CYRSV in the tested plant samples: Extracting total RNA of the plant sample to be tested, and reversely transcribing it into cDNA of the sample to be tested; using the cDNA as a template, and using CYRSV-qN-F / CYRSV-qN-R primers to perform fluorescence quantitative PCR to obtain the CT value of the plant sample to be tested; The CYRSV standard curve was established with the CT value of the standard as the horizontal axis and the corresponding copy number as the vertical axis. The CT value of the sample to be tested was inserted into the above CYRSV standard curve to calculate the copy number of CYRSV in the sample to be tested.

2. The method for specific detection and absolute quantification of a plant sample suspected of being infected with CYRSV according to claim 1, characterized in that: The reverse transcription system and reaction conditions in step A 2) and step B 1) are as follows: Random 6 mersprimer (50 μM) 1 μL, dNTP mix 1 μL, total RNA (2 μg) 4 μL, ddH2O 4 μL, total volume 10 μL, 65 degrees for 5 min, and store at 4 degrees for the next step; 10 μL of the above reaction solution, 4 μL of 5x RT Buffer, 0.5 μL of Rnase inhibitor, 1 μL of Evo Rtase, 4.5 μL of ddH2O, total volume 20 μL, 30 degrees for 10 min, 42 degrees for 30 min, 95 degrees for 5 min, and store at 4 degrees for use.

3. The method for specific detection and absolute quantification of a plant sample suspected of being infected with CYRSV according to claim 1, characterized in that: The reaction system for amplification in step A3) is: 2 μL cDNA template (diluted 10 times), 12.5 μL 2x TaqMix, 1 μL each of 10 μM upstream and downstream primers, 8.5 μL ddH2O, and a total volume of 25 μL.

4. The method for specific detection and absolute quantification of a plant sample suspected of being infected with CYRSV according to claim 1, characterized in that: The reaction conditions for amplification in step A3) are: pre-denaturation at 4°C for 30 s, denaturation at 98°C for 10 s, annealing at 54°C for 30 s, extension at 72°C for 1 min, 35 cycles, and final extension at 72°C for 2 min.

5. The method for specific detection and absolute quantification of a plant sample suspected of being infected with CYRSV according to claim 1, characterized in that: The fluorescence quantitative PCR system in step B is: 1 μL of cDNA template (diluted 5 times), 5 μL of 2x Taq mixSYBR Green, 0.5 μL of 10 μM upstream and downstream primers, 3 μL of ddH2O, and a total volume of 10 μL.

6. The method for specific detection and absolute quantification of a plant sample suspected of being infected with CYRSV according to claim 1, characterized in that: The reaction conditions of fluorescence quantitative PCR in step B were: pre-denaturation at 95 degrees for 30 s, denaturation at 95 degrees for 50 s, annealing at 60 degrees for 30 s, 40 cycles, and adding a melting curve.

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