CN113817875A - Fluorescent quantitative PCR method for detecting tobacco etch virus - Google Patents

Fluorescent quantitative PCR method for detecting tobacco etch virus Download PDF

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Publication number
CN113817875A
CN113817875A CN202111354944.5A CN202111354944A CN113817875A CN 113817875 A CN113817875 A CN 113817875A CN 202111354944 A CN202111354944 A CN 202111354944A CN 113817875 A CN113817875 A CN 113817875A
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plasmid
mul
tev
pcr
primers
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白静科
吴彦辉
苗圃
李淑君
周密
牛龙龙
王甜甜
李成军
李小杰
王京
牛莉莉
刘林州
姚健
王俊
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Tobacco Research Institute Henan Academy Of Agricultural Sciences
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Tobacco Research Institute Henan Academy Of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The invention belongs to the field of tobacco disease control, and particularly comprises the following steps: a fluorescent quantitative PCR method for detecting tobacco etch virus, comprising the following steps: designing and synthesizing primers and probes, extracting total RNA of tobacco and RT-PCR, identifying target genes, preparing plasmid standard products, establishing a standard curve, carrying out a sensitivity experiment of fluorescent quantitative PCR, and obtaining an experiment result; the invention makes up the defects of a serological detection method and a reverse transcription-polymerase chain reaction (RT-PCR) detection method, can detect TEV in a low-abundance virus sample, has higher sensitivity, and can detect a virus sample with less TEV content; the whole test operation is simplified, and time and labor are saved.

Description

Fluorescent quantitative PCR method for detecting tobacco etch virus
Technical Field
The invention belongs to the technical field of tobacco disease control, and particularly relates to a fluorescent quantitative PCR method for detecting tobacco etch virus.
Background
The Tobacco Etch Virus (TEV) belongs to Potyvirus (Potyvirie) family, and members of Potyvirus (Potyvirus) belong to Potyvirus (Potyvirus) family, can infect more than 120 crops in 19 families, and is a worldwide virus disease damaging Tobacco. TEV mainly harms leaves, small spots appear on leaves in the early stage of disease attack, white stripes and lightning-shaped stripes appear after the small spots are enlarged, the small spots develop along veins, and the leaves are full of disease spots in severe cases. In the end stage of onset, the diseased tissue is scorched and shed, although the veins remain, the main veins become scorched streaks, and the branch veins turn black and curled. Due to infection with TEV, tobacco yield and quality are severely reduced. Currently, for detecting TEV, a serological detection method exists in the early stage, but the detection sensitivity is limited, and the molecular biological detection technology can well make up for the defect. The molecular biology detection technology of TEV is commonly known as reverse transcription-polymerase chain reaction (RT-PCR) detection method. Both methods can specifically detect TEV contained in a sample, and have relatively high sensitivity, but the detection of TEV in a low-abundance virus sample is still slightly insufficient, and the virus content in the sample cannot be determined. The real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) technology becomes one of the important technical means for detecting many existing plant viruses due to its advantages of high sensitivity, high specificity, quantifiability and the like.
Disclosure of Invention
To solve the problems set forth in the background art described above. The invention provides a fluorescent quantitative PCR method for detecting tobacco etch virus, which has the characteristic of higher detection sensitivity, can detect a virus sample with less TEV content, and has the advantages of simplified whole test operation, time saving and labor saving.
In order to achieve the purpose, the invention provides the following technical scheme: a fluorescent quantitative PCR method for detecting tobacco etch virus, comprising the following steps:
(1) designing and synthesizing a primer and a probe: according to a Coat Protein (CP) gene conserved sequence (AY 787757.1) of TEV and a HSC70-1 gene (DV 161835) in an NCBI database, Primer Premier 5.0 software is used for designing primers and probes for fluorescent quantitative PCR detection according to a Primer and TaqMan probe design principle, wherein the primers comprise an upstream Primer and a downstream Primer, and the specificity of the designed primers is ensured by comparing the primers and BLAST in NCBI;
(2) extracting total RNA of tobacco and RT-PCR: extracting total RNA of plants by a Trizol method, taking 0.1g of TEV tobacco virus samples, and extracting the total RNA by using a Trizol (Invirtogen) reagent, wherein the specific method refers to a reagent instruction, and healthy tobacco leaves are used as negative control; determining the concentration of the obtained RNA by using an Eppendorf D30 ultraviolet spectrophotometer, and detecting the integrity of the RNA by using 1% agarose gel electrophoresis;
reaction system for first strand cDNA Synthesis: 2 mul of RNA, 1 mul of Random Primer and 7 mul of DEPC-ddH2O 7, firstly, performing conservation at 65 ℃ for 5min, quickly placing on ice for more than 2min, and then adding the following reagents: 5 × RT buffer 5 μ l, RRI 0.5 μ l, M-MLV 1 μ l, dNTPmix 5 μ l, DEPC-ddH2O 3.5 μ l, and reaction conditions: 10min at 30 ℃, 60min at 42 ℃ and 15min at 70 ℃, and storing the obtained cDNA at-20 ℃ for subsequent experiments;
and (3) PCR reaction system: 2, 10 mul of Premix Taq (TaKaRa Taq Version 2.0), 0.5 mul of 10 mul/L upstream and downstream primers respectively, 2 mul of cDNA and 2 mul of ddH2O 7 mul;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles, and finally extension at 72 ℃ for 10 min;
(3) and identifying the target gene: carrying out agarose gel electrophoresis on the PCR product, cutting off a target band, purifying and recovering the gel recovery kit, connecting the gel recovery kit to a pMD18-T Vector, converting the gel recovery kit into competent cells Trans 5 alpha, carrying out overnight culture at 37 ℃, screening through blue white spots, respectively selecting a white single colony and a blue single colony, placing the white single colony and the blue single colony in an LB liquid culture medium containing ampicillin, and carrying out split shaking culture (37 ℃, 220r/min and 15 h), and using a Tiangen plasmid extraction kit to complete the extraction of plasmids;
the plasmid is identified by PCR with pMD18-T Vector universal Primers BcaBEST Sequencing Primers and M13 Primers, and the PCR reaction system is as follows: premix Taq (TaKaRa Taq Version 2.0) 10 μ l, BcaBEST Sequencing Primers (10 μ M)0.5 μ l, M13 Primers (10 μ M)0.5 μ l, plasmid template 2 μ l, ddH2O 7 μ l; PCR reaction procedure: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 60s, 35 cycles, and extension at 72 ℃ for 10 min;
detecting the PCR product by 1% agarose gel electrophoresis, sending the plasmid sample of the expected size target band to biological engineering (Shanghai) corporation for sequencing, and storing the rest plasmid sample at-20 deg.C;
(4) and preparing a plasmid standard product: after the plasmid sample is sequenced and identified, the plasmid sample is subjected to light splittingThe value of plasmid OD260 was determined photometrically by the formula: c = A/B × 6.02 × 1014(wherein A represents the plasmid concentration ng/mul, B represents the plasmid DNA molecular weight, and C represents copies/mul), calculating the plasmid concentration copy number, and using the plasmid concentration copy number as a plasmid standard substance;
(5) and establishing a standard curve: diluting the plasmid standard substance with EASY Dilution by 10 times gradient to obtain TEV final concentration of 3.73 × 10-3.73 × 1099 plasmid samples of copies/mul and the final concentration of internal reference genes are 5.03 x 10-5.03 x 109carrying out RT-qPCR by using 9 plasmid samples of copies/mul and using the diluted plasmid samples as a template by using an ABI 7500 fast real-time fluorescence quantitative PCR instrument, carrying out three times of technical repetition on each concentration, and automatically generating a standard curve by using the instrument;
the reaction system is 20 mul: 10 mul of Probe qPCR Mix (2 x), 0.4 mul of each upstream and downstream primer of 10 mummol/L, 0.8 mul of Probe, 0.2 mul of ROX Reference Dye II (50 x), 2 mul of plasmid template and 6.2 mul of sterilized water;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, PCR reaction at 95 ℃ for 5s, 57 ℃ for 15s, and 72 ℃ for 30s for 45 cycles;
(6) and the sensitivity experiment of fluorescent quantitative PCR: the final concentration of TEV after dilution was 3.73X 101—3.73×109carrying out RT-qPCR and conventional PCR on 9 plasmid standard substances of copies/mu l, and comparing the detection sensitivity of the two methods;
(7) and the experimental result is as follows:
first, establishment of standard curve
The results of the real-time fluorescence quantitative PCR standard curve chart 1 and the graph 2 show that the Ct value has an increasing trend along with the gradual reduction of the positive plasmid template concentration, and the concentration of TEV and the internal reference gene in the plasmid standard substance is 101-109Within the copies/mul range, the linear relation between the Ct value and the concentration of the plasmid standard substance is good, and the detection of the toxicity content of the sample can be carried out by referring to the standard curve according to the Ct value of the sample to be detected;
TEV and internal reference gene TaqMan real-time fluorescence quantitative PCR reaction, wherein each plasmid gradient concentration is subjected to three technical repetitions, the result is shown in figure 3, and the coefficient of variation of the three repetitions is less than 2%, which indicates that the experiment has good repeatability and reliable experiment result;
second, sensitivity test result of fluorescent quantitative PCR
As shown in FIGS. 4 and 5, the TaqMan fluorescent quantitative PCR method for detecting TEV can detect 3.73X 10 of TEV at the lowest in the same sample1copies/mL virus sample, and the conventional RT-PCR detection method can detect 3.73X 10 at the lowest2The virus sample of copies/mL shows that the sensitivity of the TaqMan fluorescent quantitative PCR detection TEV is improved by 10 times compared with the conventional RT-PCR detection.
Preferably, the primers and probes are synthesized by Biotechnology engineering (Shanghai) Inc.:
an upstream primer TEV-F: CAGCCTATGGAGTGAATGAAGA, respectively;
the downstream primer TEV-R: TGAAACTTGCTCCTCACCATC, respectively;
and (3) probe: FAM-TTCTATGCACCACACCA-MGB.
Compared with the prior art, the invention has the beneficial effects that:
the invention makes up the defects of a serological detection method and a reverse transcription-polymerase chain reaction (RT-PCR) detection method, can detect TEV in a low-abundance virus sample, has higher sensitivity, and can detect a virus sample with less TEV content; the whole test operation is simplified, and time and labor are saved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph showing the results of a TEV standard curve test according to the present invention;
FIG. 2 is a diagram showing the results of a standard curve test of reference genes according to the present invention;
FIG. 3 is a diagram showing the experimental results of the real-time fluorescent quantitative PCR reaction of TEV and reference gene according to the present invention;
FIG. 4 is a TaqMan fluorescence quantitative PCR detection result diagram of TEV under the same sample;
FIG. 5 is a diagram showing the results of conventional RT-PCR detection of TEV in the same sample according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
Referring to fig. 1-5, the present invention provides the following technical solutions: a fluorescent quantitative PCR method for detecting tobacco etch virus, comprising the following steps:
(1) designing and synthesizing a primer and a probe: according to a Coat Protein (CP) gene conserved sequence (AY 787757.1) of TEV and a HSC70-1 gene (DV 161835) in an NCBI database, Primer Premier 5.0 software is used for designing primers and probes for fluorescent quantitative PCR detection according to a Primer and TaqMan probe design principle, wherein the primers comprise an upstream Primer and a downstream Primer, and the specificity of the designed primers is ensured by comparing the primers and BLAST in NCBI;
the primers and probes were synthesized by Biotechnology engineering (Shanghai) GmbH:
an upstream primer TEV-F: CAGCCTATGGAGTGAATGAAGA, respectively;
the downstream primer TEV-R: TGAAACTTGCTCCTCACCATC, respectively;
and (3) probe: FAM-TTCTATGCACCACACCA-MGB;
(2) extracting total RNA of tobacco and RT-PCR: extracting total RNA of plants by a Trizol method, taking 0.1g of TEV tobacco virus samples, and extracting the total RNA by using a Trizol (Invirtogen) reagent, wherein the specific method refers to a reagent instruction, and healthy tobacco leaves are used as negative control; determining the concentration of the obtained RNA by using an Eppendorf D30 ultraviolet spectrophotometer, and detecting the integrity of the RNA by using 1% agarose gel electrophoresis;
reaction system for first strand cDNA Synthesis: 2 mul of RNA, 1 mul of Random Primer and 7 mul of DEPC-ddH2O 7, firstly, performing conservation at 65 ℃ for 5min, quickly placing on ice for more than 2min, and then adding the following reagents: 5 × RT buffer 5 μ l, RRI 0.5 μ l, M-MLV 1 μ l, dNTPmix 5 μ l, DEPC-ddH2O 3.5 μ l, and reaction conditions: 10min at 30 ℃, 60min at 42 ℃ and 15min at 70 ℃, and storing the obtained cDNA at-20 ℃ for subsequent experiments;
and (3) PCR reaction system: 2, 10 mul of Premix Taq (TaKaRa Taq Version 2.0), 0.5 mul of 10 mul/L upstream and downstream primers respectively, 2 mul of cDNA and 2 mul of ddH2O 7 mul;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles, and finally extension at 72 ℃ for 10 min;
(3) and identifying the target gene: carrying out agarose gel electrophoresis on the PCR product, cutting off a target band, purifying and recovering the gel recovery kit, connecting the gel recovery kit to a pMD18-T Vector, converting the gel recovery kit into competent cells Trans 5 alpha, carrying out overnight culture at 37 ℃, screening through blue white spots, respectively selecting a white single colony and a blue single colony, placing the white single colony and the blue single colony in an LB liquid culture medium containing ampicillin, and carrying out split shaking culture (37 ℃, 220r/min and 15 h), and using a Tiangen plasmid extraction kit to complete the extraction of plasmids;
the plasmid is identified by PCR with pMD18-T Vector universal Primers BcaBEST Sequencing Primers and M13 Primers, and the PCR reaction system is as follows: premix Taq (TaKaRa Taq Version 2.0) 10 μ l, BcaBEST Sequencing Primers (10 μ M)0.5 μ l, M13 Primers (10 μ M)0.5 μ l, plasmid template 2 μ l, ddH2O 7 μ l; PCR reaction procedure: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 60s, 35 cycles, and extension at 72 ℃ for 10 min;
detecting the PCR product by 1% agarose gel electrophoresis, sending the plasmid sample of the expected size target band to biological engineering (Shanghai) corporation for sequencing, and storing the rest plasmid sample at-20 deg.C;
(4) and preparing a plasmid standard product: after the plasmid sample is sequenced and identified, the value of the plasmid OD260 is determined by a spectrophotometer according to the formula: c = A/B × 6.02 × 1014(wherein A represents the plasmid concentration ng/mul, B represents the plasmid DNA molecular weight, and C represents copies/mul), calculating the plasmid concentration copy number, and using the plasmid concentration copy number as a plasmid standard substance;
(5) and establishing a standard curve: mixing the plasmidsDiluting the standard substance with EASY Dilution by 10 times gradient to obtain TEV final concentration of 3.73 × 10-3.73 × 1099 plasmid samples of copies/mul and the final concentration of internal reference genes are 5.03 x 10-5.03 x 109carrying out RT-qPCR by using 9 plasmid samples of copies/mul and using the diluted plasmid samples as a template by using an ABI 7500 fast real-time fluorescence quantitative PCR instrument, carrying out three times of technical repetition on each concentration, and automatically generating a standard curve by using the instrument;
the reaction system is 20 mul: 10 mul of Probe qPCR Mix (2 x), 0.4 mul of each upstream and downstream primer of 10 mummol/L, 0.8 mul of Probe, 0.2 mul of ROX Reference Dye II (50 x), 2 mul of plasmid template and 6.2 mul of sterilized water;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, PCR reaction at 95 ℃ for 5s, 57 ℃ for 15s, and 72 ℃ for 30s for 45 cycles;
(6) and the sensitivity experiment of fluorescent quantitative PCR: the final concentration of TEV after dilution was 3.73X 101—3.73×109carrying out RT-qPCR and conventional PCR on 9 plasmid standard substances of copies/mu l, and comparing the detection sensitivity of the two methods;
(7) and the experimental result is as follows:
first, establishment of standard curve
The results of the real-time fluorescence quantitative PCR standard curve chart 1 and the graph 2 show that the Ct value has an increasing trend along with the gradual reduction of the positive plasmid template concentration, and the concentration of TEV and the internal reference gene in the plasmid standard substance is 101-109Within the copies/mul range, the linear relation between the Ct value and the concentration of the plasmid standard substance is good, and the detection of the toxicity content of the sample can be carried out by referring to the standard curve according to the Ct value of the sample to be detected;
TEV and internal reference gene TaqMan real-time fluorescence quantitative PCR reaction, wherein each plasmid gradient concentration is subjected to three technical repetitions, the result is shown in figure 3, and the coefficient of variation of the three repetitions is less than 2%, which indicates that the experiment has good repeatability and reliable experiment result;
second, sensitivity test result of fluorescent quantitative PCR
As shown in FIGS. 4 and 5, the TaqMan fluorescent quantitative PCR method for detecting TEV can detect 3.73X 10 of TEV at the lowest in the same sample1copies/mL of viral sample, conventional RT-PCThe R detection method can detect 3.73 multiplied by 10 at the lowest2The virus sample of copies/mL shows that the sensitivity of the TaqMan fluorescent quantitative PCR detection TEV is improved by 10 times compared with the conventional RT-PCR detection.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (2)

1. A fluorescent quantitative PCR method for detecting tobacco etch virus is characterized in that: the method comprises the following steps:
(1) designing and synthesizing a primer and a probe: according to a Coat Protein (CP) gene conserved sequence (AY 787757.1) of TEV and a HSC70-1 gene (DV 161835) in an NCBI database, Primer Premier 5.0 software is used for designing primers and probes for fluorescent quantitative PCR detection according to a Primer and TaqMan probe design principle, wherein the primers comprise an upstream Primer and a downstream Primer, and the specificity of the designed primers is ensured by comparing the primers and BLAST in NCBI;
(2) extracting total RNA of tobacco and RT-PCR: extracting total RNA of plants by a Trizol method, taking 0.1g of TEV tobacco virus samples, and extracting the total RNA by using a Trizol (Invirtogen) reagent, wherein the specific method refers to a reagent instruction, and healthy tobacco leaves are used as negative control; determining the concentration of the obtained RNA by using an Eppendorf D30 ultraviolet spectrophotometer, and detecting the integrity of the RNA by using 1% agarose gel electrophoresis;
reaction system for first strand cDNA Synthesis: 2 mul of RNA, 1 mul of Random Primer and 7 mul of DEPC-ddH2O 7, firstly, performing conservation at 65 ℃ for 5min, quickly placing on ice for more than 2min, and then adding the following reagents: 5 × RT buffer 5 μ l, RRI 0.5 μ l, M-MLV 1 μ l, dNTPmix 5 μ l, DEPC-ddH2O 3.5 μ l, and reaction conditions: 10min at 30 ℃, 60min at 42 ℃ and 15min at 70 ℃, and storing the obtained cDNA at-20 ℃ for subsequent experiments;
and (3) PCR reaction system: 2, 10 mul of Premix Taq (TaKaRa Taq Version 2.0), 0.5 mul of 10 mul/L upstream and downstream primers respectively, 2 mul of cDNA and 2 mul of ddH2O 7 mul;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles, and finally extension at 72 ℃ for 10 min;
(3) and identifying the target gene: carrying out agarose gel electrophoresis on the PCR product, cutting off a target band, purifying and recovering the gel recovery kit, connecting the gel recovery kit to a pMD18-T Vector, converting the gel recovery kit into competent cells Trans 5 alpha, carrying out overnight culture at 37 ℃, screening through blue white spots, respectively selecting a white single colony and a blue single colony, placing the white single colony and the blue single colony in an LB liquid culture medium containing ampicillin, and carrying out split shaking culture (37 ℃, 220r/min and 15 h), and using a Tiangen plasmid extraction kit to complete the extraction of plasmids;
the plasmid is identified by PCR with pMD18-T Vector universal Primers BcaBEST Sequencing Primers and M13 Primers, and the PCR reaction system is as follows: premix Taq (TaKaRa Taq Version 2.0) 10 μ l, BcaBEST Sequencing Primers (10 μ M)0.5 μ l, M13 Primers (10 μ M)0.5 μ l, plasmid template 2 μ l, ddH2O 7 μ l; PCR reaction procedure: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 60s, 35 cycles, and extension at 72 ℃ for 10 min;
detecting the PCR product by 1% agarose gel electrophoresis, sending the plasmid sample of the expected size target band to biological engineering (Shanghai) corporation for sequencing, and storing the rest plasmid sample at-20 deg.C;
(4) and preparing a plasmid standard product: after the plasmid sample is sequenced and identified, the value of the plasmid OD260 is determined by a spectrophotometer according to the formula: c = A/B × 6.02 × 1014(wherein A represents the plasmid concentration ng/mul, B represents the plasmid DNA molecular weight, and C represents copies/mul), calculating the plasmid concentration copy number, and using the plasmid concentration copy number as a plasmid standard substance;
(5) and establishing a standard curve: diluting the plasmid standard substance with EASY Dilution by 10 times gradient to obtain TEV final concentration of 3.73 × 10-respectively3.73×1099 plasmid samples of copies/mul and the final concentration of internal reference genes are 5.03 x 10-5.03 x 109carrying out RT-qPCR by using 9 plasmid samples of copies/mul and using the diluted plasmid samples as a template by using an ABI 7500 fast real-time fluorescence quantitative PCR instrument, carrying out three times of technical repetition on each concentration, and automatically generating a standard curve by using the instrument;
the reaction system is 20 mul: 10 mul of Probe qPCR Mix (2 x), 0.4 mul of each upstream and downstream primer of 10 mummol/L, 0.8 mul of Probe, 0.2 mul of ROX Reference Dye II (50 x), 2 mul of plasmid template and 6.2 mul of sterilized water;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, PCR reaction at 95 ℃ for 5s, 57 ℃ for 15s, and 72 ℃ for 30s for 45 cycles;
(6) and the sensitivity experiment of fluorescent quantitative PCR: the final concentration of TEV after dilution was 3.73X 101—3.73×109carrying out RT-qPCR and conventional PCR on 9 plasmid standard substances of copies/mu l, and comparing the detection sensitivity of the two methods;
(7) and the experimental result is as follows:
first, establishment of standard curve
The results of the real-time fluorescence quantitative PCR standard curve chart 1 and the graph 2 show that the Ct value has an increasing trend along with the gradual reduction of the positive plasmid template concentration, and the concentration of TEV and the internal reference gene in the plasmid standard substance is 101-109Within the copies/mul range, the linear relation between the Ct value and the concentration of the plasmid standard substance is good, and the detection of the toxicity content of the sample can be carried out by referring to the standard curve according to the Ct value of the sample to be detected;
TEV and internal reference gene TaqMan real-time fluorescence quantitative PCR reaction, wherein each plasmid gradient concentration is subjected to three technical repetitions, the result is shown in figure 3, and the coefficient of variation of the three repetitions is less than 2%, which indicates that the experiment has good repeatability and reliable experiment result;
second, sensitivity test result of fluorescent quantitative PCR
As shown in FIGS. 4 and 5, the TaqMan fluorescent quantitative PCR detection method of TEV can detect 3.73X 10 at the lowest1copies/mL virus sample, and the conventional RT-PCR detection method can detect 3.73X 10 at the lowest2Samples of copies/mL virus, indicated TaqMThe sensitivity of an fluorescent quantitative PCR detection of TEV is improved by 10 times compared with the conventional RT-PCR detection.
2. The fluorescent quantitative PCR method for detecting tobacco etch virus according to claim 1, wherein: the primers and probes were synthesized by Biotechnology engineering (Shanghai) GmbH:
an upstream primer TEV-F: CAGCCTATGGAGTGAATGAAGA, respectively;
the downstream primer TEV-R: TGAAACTTGCTCCTCACCATC, respectively;
and (3) probe: FAM-TTCTATGCACCACACCA-MGB.
CN202111354944.5A 2021-11-16 2021-11-16 Fluorescent quantitative PCR method for detecting tobacco etch virus Pending CN113817875A (en)

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