CN117965813A - Real-time fluorescence quantitative PCR primer and kit for detecting tomato brown wrinkle fruit virus and application thereof - Google Patents
Real-time fluorescence quantitative PCR primer and kit for detecting tomato brown wrinkle fruit virus and application thereof Download PDFInfo
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Abstract
The invention provides a real-time fluorescent quantitative PCR primer and a kit for detecting tomato brown wrinkle fruit viruses and application thereof, and relates to the technical field of plant virus biological detection. The invention designs a specific primer based on a ToBRFV CP conserved region of a gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 2. The invention adopts the real-time fluorescent quantitative PCR primer to detect the tomato brown wrinkle fruit virus, has the characteristics of strong specificity, high sensitivity and good repeatability, and has better detection effect on samples with low toxin carrying capacity. The invention can be directly used for quantitative monitoring in the field ToBRFV, and provides powerful support for early diagnosis, monitoring and early warning of diseases.
Description
Technical Field
The invention belongs to the technical field of biological detection of plant viruses, and particularly relates to a real-time fluorescent quantitative PCR primer and a kit for detecting tomato brown wrinkle fruit viruses and application thereof.
Background
Tomato brown wrinkle fruit virus (Tomato brown rugosefruitvirus, toBRFV), which mainly endangers the vegetable crops tomato (Solanum lycopersicum) and Capsicum annuum l, belongs to the family baculoviridae (VIRGAVIRIDAE), genus tobamoviruses (Tobamovirus). Tomato brown wrinkle fruit virus is positive-strand single-stranded RNA virus, genome full length about 6392nt, can code 4 proteins, including 126kDa replicase, 183kDa replicase, 30kDa mobile protein and 17.5kDa coat protein.
ToBRFV has short production time, no targeted control agent and high-resistance varieties are available at present, and once plants are infected by the virus, the incidence rate is extremely high, so that the crop yield and the fruit quality are affected. Especially in greenhouse (or greenhouse), if ToBRFV is not controlled effectively, the serious condition of the whole greenhouse (or whole greenhouse) plant is likely to happen. In addition, tomato susceptible plants often appear asymptomatic in early seedling stages. Thus, early diagnosis is particularly important for controlling ToBRFV disease spread.
Currently, commonly used diagnostic or detection methods include serological, molecular biological, and biological assays. Biological detection method judges whether infection is caused by tomato phenotype symptom, and the method has certain disadvantages due to subjective observation and uncertainty caused by symptom; the detection of ToBRFV by serological method is easy to cross react with tobacco mosaic virus and tomato mosaic virus; the molecular biological method has obviously improved sensitivity and detection speed, and can be used for quantification. The real-time fluorescent quantitative PCR technology has the characteristics of stronger specificity, high automation degree, no EB pollution and the like, is a development trend of rapid molecular diagnosis, and has important significance for early and rapid detection of the tomato brown wrinkle fruit virus.
Disclosure of Invention
In view of the above, the invention aims to provide a real-time fluorescent quantitative PCR primer for detecting tomato brown wrinkle fruit virus, a kit and application thereof, and the real-time fluorescent quantitative PCR primer has the characteristics of strong specificity, high sensitivity and good repeatability; can perform quantitative detection and has better detection effect on samples with low toxin carrying capacity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a real-time fluorescence quantitative PCR primer for detecting tomato brown wrinkle fruit virus, which comprises an upstream primer ToBRFV-q1F and a downstream primer ToBRFV-q1R, wherein the nucleotide sequence of the upstream primer ToBRFV-q1F is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer ToBRFV-q1R is shown as SEQ ID No. 2.
The invention also provides a kit for detecting the tomato brown wrinkle fruit virus, which comprises the primer.
Preferably, the kit further comprises a positive quality control and a negative quality control.
More preferably, the positive quality control is a pEASY-T1 vector plasmid containing tomato brown wrinkle fruit virus.
The invention also provides a real-time fluorescence quantitative detection method of the tomato brown wrinkle fruit virus, which comprises the following steps:
s1, extracting total RNA of a disease-sensitive plant;
s2, performing reverse transcription by taking total RNA of the infected plant as a template to synthesize cDNA;
S3, carrying out PCR amplification by using the synthesized cDNA as a template and utilizing the primer to obtain a PCR amplification product;
S4, connecting the obtained PCR amplification product to a vector after purification, transferring into competent cells, screening positive clones, extracting plasmids to obtain ToBRFV plasmid standard substances, and preparing a standard curve according to gradient dilution;
S5, carrying out real-time fluorescence quantitative PCR detection by using the primer by taking cDNA of the sample to be detected as a template, judging whether the sample to be detected carries ToBRFV according to an amplification curve and a cycle threshold, and substituting the cycle threshold into a standard curve equation to calculate the toxin content of the sample to be detected.
Preferably, the reaction system for real-time fluorescence quantitative PCR detection comprises 0.8-1.2 mu L of template, 0.4 mu L of 8-12 mu m/L upstream and downstream primers, 2X SYBR qPCRMix 8-12 mu L of each, and ddH 2 O8-8.5 mu L of total volume of 20 mu L.
Preferably, the reaction procedure for the fluorescent quantitative PCR detection is 95℃for 30s;95℃10s,60℃30s,40 cycles.
Preferably, the plasmid is extracted and sequenced without error, and its concentration is determined using a spectrophotometer by the formula:
C＝A/B×6.02×1014，
plasmid copy number was calculated and used as ToBRFV plasmid standard.
The invention also provides application of the primer in preparation of a product for detecting the tomato brown wrinkle fruit virus.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides a real-time fluorescence quantitative PCR primer for detecting tomato brown wrinkle fruit virus, which designs a specific primer based on a ToBRFV CP gene conserved region, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 2. The invention adopts real-time fluorescence quantitative PCR primer tomato brown wrinkle fruit virus, has the characteristics of strong specificity, high sensitivity and good repeatability, has better detection effect on samples with low virus carrying capacity, and has the concentration of 1.0 multiplied by 10 3 copies/mu l of detectable positive plasmid, and the sensitivity is 100 times of that of common PCR. The invention can be directly used for quantitative monitoring in the field ToBRFV, and provides powerful support for early diagnosis, monitoring and early warning of diseases.
Drawings
FIG. 1 is a standard qRT-PCR curve of ToBRFV plasmid standard;
FIG. 2 shows the qRT-PCR dissolution profile of ToBRFV plasmid standards;
FIG. 3 shows a general RT-PCR sensitivity test, wherein M is 2000bp DNAmarker;1 to 9 are plasmid standard substances with the concentration of 1.0X10 9～1.0×101 copies/. Mu.l in sequence;
FIG. 4 shows qRT-PCR sensitivity detection, wherein 1 to 7 are plasmid standard substances with concentration of 1.0X10 9～1.0×103 copies/. Mu.l in sequence;
FIG. 5 shows the specificity of qRT-PCR detection primers of the present invention, wherein FIG. 1 shows ToMV positive samples; 2 is a TMV positive sample; 3 is a TSWV positive sample; 4 is ToBRFV positive sample;
FIG. 6 is a schematic diagram of a common RT-PCR detection electrophoresis of 11 tomato samples, wherein M is a 2000bp DNA marker;1-11 are 11 tomato samples collected in the field; negative is a negative control; positive is ToBRFV positive control;
FIG. 7 shows qRT-PCR amplification curves of 11 tomato samples, wherein 1-11 are 11 tomato samples collected in the field; 12 is a plasmid standard at a concentration of 1.0X10 6 copies/. Mu.l.
Detailed Description
The invention provides a real-time fluorescence quantitative PCR primer for detecting tomato brown wrinkle fruit virus, which is characterized in that a specific primer pair is designed based on a ToBRFV CP gene conservation region and comprises an upstream primer ToBRFV-q1F and a downstream primer ToBRFV-q1R, wherein the nucleotide sequence of the upstream primer ToBRFV-q1F is GATGACGCAACGGTGGCTAT, as shown in SEQ ID No.1, and the nucleotide sequence of the downstream primer ToBRFV-q1R is CAAGATGCAGGTGCAGAGGA, as shown in SEQ ID No. 2.
The invention also provides a kit for detecting the tomato brown wrinkle fruit virus, which comprises the primer.
In the invention, the kit further comprises a positive quality control product and a negative quality control product; the positive quality control is preferably pEASY-T1 vector plasmid containing tomato brown wrinkle fruit virus.
The invention also provides a real-time fluorescence quantitative detection method of the tomato brown wrinkle fruit virus, which comprises the following steps:
s1, extracting total RNA of a disease-sensitive plant;
s2, performing reverse transcription by taking total RNA of the infected plant as a template to synthesize cDNA;
S3, carrying out PCR amplification by using the synthesized cDNA as a template and utilizing a specific primer to obtain a PCR amplification product;
S4, connecting the obtained PCR amplification product to a vector after purification, transferring into competent cells, screening positive clones, extracting plasmids to obtain ToBRFV plasmid standard substances, and preparing a standard curve according to gradient dilution;
S5, carrying out real-time fluorescence quantitative PCR detection by using the primer by taking cDNA of the sample to be detected as a template, judging whether the sample to be detected carries ToBRFV according to an amplification curve and a cycle threshold, and substituting the cycle threshold into a standard curve equation to calculate the toxin content of the sample to be detected.
In the invention, the reaction system for real-time fluorescence quantitative PCR detection preferably comprises 0.8-1.2 mu L of template, 0.4 mu L of 8-12 mu m/L upstream and downstream primers, 2X SYBR qPCRMix-12 mu L of each, and ddH 2 O8-8.5 mu L of the total volume of 20 mu L; more preferably, 1. Mu.l of template, 0.4. Mu.l of each of 10 μm/L of upstream and downstream primers, 10. Mu.l of 2 XSYBR qPCR Mix, and 8.2. Mu.l of ddH 2 O, and a total volume of 20. Mu.l are included.
In the present invention, the reaction procedure for the fluorescent quantitative PCR detection is preferably 95℃for 30s;95℃10s,60℃30s,40 cycles.
In the present invention, the plasmid was extracted and sequenced, and its concentration was measured using a spectrophotometer by the formula: c=a/b×6.02×10 14, plasmid copy number was calculated and used as ToBRFV plasmid standard. In the formula, C is copies/. Mu.l, A is plasmid concentration ng/. Mu.l, and B is plasmid DNA molecular weight.
The invention also provides application of the primer in preparation of a product for detecting the tomato brown wrinkle fruit virus.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
No specific technique or condition is identified in the examples, which follow the techniques or conditions described in the literature in this field, or follow the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, are all commercially available products.
Example 1
1. Primer design and Synthesis
Referring to ToBRFV genomic sequences (OM 305070.1) in NCBI database, fluorescent quantitative primers ToBRFV-q1F/ToBRFV-q1R were designed using the conserved region of the CP gene, and amplified fragments were 134bp in size. The conventional PCR primer ToBRFV-F/ToBRFV-R with the length of 354bp is designed in the small subunit region of replicase (RdRP), and the sequence is shown in the table 1:
TABLE 1 primer information
RNA extraction
100Mg of the infected plant material was added to the RNase-removed centrifuge tube, liquid nitrogen was added, the plant material was ground with a grinding rod, and the total RNA was extracted in the subsequent step according to the Trizol (Tiangen Biochemical Co., ltd.).
3. Reverse transcription synthesis of cDNA
After removal of genomic DNA from 5. Mu.g total RNA sample, first strand cDNA was synthesized using HiScript II 1st Strand cDNA Synthesis Kit (Nanjinouzan medical science, inc.). The reverse transcription experimental procedure is as follows: (1) RNA template denaturation Random hexamers (50 ng/. Mu.l) 1. Mu.l, 5. Mu.g total RNA, RNase-free ddH 2 O were supplemented to 12. Mu.l, reaction conditions: placing on ice at 65deg.C for 5min for 2min; (2) Genomic DNA was removed, the mixture of the previous step, 4X gDNAwiperMix. Mu.l, reaction conditions: 42 ℃ for 2min; (3) cDNA synthesis reaction, 10X RTMix. Mu.l of the mixture of the previous step, 2. Mu.l of HiScript II Enzyme Mix, and reaction conditions: 25 ℃ for 5min;50 ℃ for 45min;85 ℃ for 2min.
4. Cloning of the Gene of interest
The 20. Mu.l RT-PCR reaction system comprises 1. Mu.l cDNA, 1. Mu.l each of 10 μm/L upstream and downstream primers, 10. Mu.l 2 XTaq Mastermix (Nanjinozan medical science, inc.), and ddH 2 O7. Mu.l. The reaction procedure is: 95 ℃ for 5min;95 ℃ for 30s,53 ℃ for 30s,72 ℃ for 30s,35 cycles; 7min at 72 ℃.
After the RT-PCR products are purified and recovered, the concentration is measured by a spectrophotometer, then the products are connected by a pEASY-T1 carrier (Beijing full gold biotechnology Co., ltd.) and transformed into escherichia coli competent cells (Beijing full gold biotechnology Co., ltd.), 500 μl of LB culture medium is added, shaking culture is carried out for 1h at 37 ℃ and 200rpm/min, 350 μl of supernatant is removed by centrifugation, 60 μl of culture solution is coated on an LB solid plate containing 50 μg/ml of ampicillin, after 8 hours of culture at 37 ℃, bacterial colony PCR is carried out by picking, colonies conforming to the expected fragment size are placed in an LB liquid culture medium containing ampicillin, shaking culture is carried out separately, plasmid extraction is completed by using Ai Kerui plasmid extraction kit, plasmids are detected by agarose gel electrophoresis, PCR identification is carried out by using a universal primer M13F, M R, 20 μl of plasmid sample conforming to the product fragment size is sent to the engineering (Shanghai) Co., ltd for sequencing, and the rest plasmid sample is stored in a refrigerator at-20 ℃ for later use.
5. Preparation of plasmid Standard
After the plasmid sample is sequenced without error, the concentration of the plasmid sample is determined by using a spectrophotometer, and the concentration is calculated by the formula: c=a/b×6.02×10 14, plasmid copy number was calculated and used as ToBRFV plasmid standard. In the formula, C is copies/. Mu.l, A is plasmid concentration ng/. Mu.l, and B is plasmid DNA molecular weight.
6. Standard Curve establishment
Plasmid standards were diluted according to a 10-fold concentration gradient and qRT-PCR was performed using an ABI 7500 real-time fluorescent quantitative PCR apparatus with 5 plasmid samples with a final concentration of 1.0X10 4～1.0×108 copies/. Mu.l as templates, each sample being repeated three times. The instrument automatically generates a standard curve. The 20. Mu. lqRT-PCR reaction system included 1. Mu.l of plasmid template, 0.4. Mu.l of each of 10. Mu.m/L upstream and downstream primers, 2X SYBR qPCRMix. Mu.l, and ddH 2 O8.2. Mu.l. The reaction procedure is: 95 ℃ for 30s;95℃10s,60℃30s,40 cycles.
7. Standard curve and dissolution curve
Real-time fluorescent quantitative PCR was performed on 5 plasmid samples with final concentration of 1.0 x 10 4～1.0×108 copies/. Mu.l as template, the instrument automatically generated a standard curve with amplification efficiency of 102% and R 2 =0.993 (fig. 1). A good linear relationship between the circulation threshold and plasmid concentration was demonstrated, and the dissolution profile had a single peak at 79.6℃C (FIG. 2).
Example 2 real-time fluorescence quantitative primer sensitivity
Using the specific primer qPCR primer of the present invention, ordinary PCR and qRT-PCR were performed on 9 plasmid samples at a concentration of 1.0X10 1～1.0×109 copies/. Mu.l, respectively, and it was found from FIG. 3 that the amplified band was weak at a plasmid concentration of 1.0X10 5 copies/. Mu.l, so that the lowest detectable positive plasmid concentration of ordinary PCR was 1.0X10 5 copies/. Mu.l, and the lowest detectable positive plasmid concentration of qRT-PCR was 1.0X10 3 copies/. Mu.l (FIG. 4), and thus the sensitivity of qRT-PCR was 100 times that of ordinary PCR.
Example 3 real-time fluorescent quantitative primer specificity
The qRT-PCR primer detects tomato mosaic virus (ToMV), tobacco Mosaic Virus (TMV) and Tomato Spotted Wilt Virus (TSWV) without amplification curves, and ToBRFV positive samples have better amplification curves (figure 5), which shows that the primer specificity is better.
Example 4 application of real-time fluorescence quantitative primer
11 Samples of suspected tomato infection ToBRFV were collected in the field, and the results of the common RT-PCR detection showed that 9 samples of 11 tomato samples showed a single band, were positive samples, had a fragment size of about 354bp, were negative for samples No. 6 and No. 11, and had no target band for the negative control (FIG. 6). After sequencing, the sequences were submitted to NCBI system for BLAST alignment with a similarity of 99% to ToBRFV sequences (OR 593752.1), confirming that positive tomato samples were infected with ToBRFV. Wherein, the primers used in the common RT-PCR are SEQ ID No.3 and SEQ ID No.4.
The real-time fluorescence quantitative PCR detection results show that the amplification curves of 11 samples are good (FIG. 7), the sample toxicity rate is 100%, the Ct value is between 17.67 and 19.84, and the virus content of the samples is between 4.56 multiplied by 10 7～2.11×108 copies/. Mu.l (Table 1). Therefore, two samples which are negative in the common RT-PCR can be detected by a qRT-PCR method with higher sensitivity, and the real-time fluorescent quantitative PCR is suitable for detecting field low-toxicity samples and has higher detection rate.
TABLE 111 results of qRT-PCR detection of tomato samples
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. The real-time fluorescence quantitative PCR primer for detecting the tomato brown wrinkle fruit virus is characterized by comprising an upstream primer ToBRFV-q1F and a downstream primer ToBRFV-q1R, wherein the nucleotide sequence of the upstream primer ToBRFV-q1F is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer ToBRFV-q1R is shown as SEQ ID No. 2.
2. A kit for detecting tomato brown wrinkle fruit virus, comprising the primer of claim 1.
3. The kit of claim 2, further comprising a positive quality control and a negative quality control.
4. The kit according to claim 3, wherein the positive quality control is a pEASY-T1 vector plasmid containing tomato brown wrinkle fruit virus.
5. The real-time fluorescence quantitative detection method for the tomato brown wrinkle fruit virus is characterized by comprising the following steps of:
s1, extracting total RNA of a disease-sensitive plant;
s2, performing reverse transcription by taking total RNA of the infected plant as a template to synthesize cDNA;
s3, carrying out PCR amplification by using the synthesized cDNA as a template and using the primer of claim 1 to obtain a PCR amplification product;
S4, connecting the obtained PCR amplification product to a vector after purification, transferring into competent cells, screening positive clones, extracting plasmids to obtain ToBRFV plasmid standard substances, and preparing a standard curve according to gradient dilution;
S5, carrying out real-time fluorescence quantitative PCR detection by using cDNA of the sample to be detected as a template and using the primer of claim 1, judging whether the sample to be detected carries ToBRFV according to an amplification curve and a cycle threshold, and substituting the cycle threshold into a standard curve equation to calculate the toxin content of the sample to be detected.
6. The method according to claim 5, wherein the reaction system for real-time fluorescent quantitative PCR detection comprises 0.8-1.2. Mu.l of template, 0.4. Mu.l of 8-12. Mu.m/L upstream and downstream primers, 8-12. Mu.l of 2 XSYBR qPCR Mix, 8-8.5. Mu.l of ddH 2 O, and 20. Mu.l of total volume.
7. The method according to claim 5, wherein the fluorescent quantitative PCR detection is performed at 95℃for 30 seconds; 95℃10s,60℃30s,40 cycles.
8. The method according to claim 5, wherein the plasmid is extracted and sequenced, and the concentration is determined by spectrophotometry, by the formula:
C＝A/B×6.02×1014，
plasmid copy number was calculated and used as ToBRFV plasmid standard.
9. Use of the primer of claim 1 for the preparation of a product for detecting tomato brown wrinkle fruit virus.
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