CN114807450A - Primer probe for identifying flammulina velutipes brown virus - Google Patents
Primer probe for identifying flammulina velutipes brown virus Download PDFInfo
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- CN114807450A CN114807450A CN202210622113.XA CN202210622113A CN114807450A CN 114807450 A CN114807450 A CN 114807450A CN 202210622113 A CN202210622113 A CN 202210622113A CN 114807450 A CN114807450 A CN 114807450A
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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
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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
- C12Q1/68—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
Abstract
The invention discloses a primer probe for identifying flammulina velutipes browning virus, which consists of a primer pair shown in SEQ ID No.1 and SEQ ID No.2 and a probe shown in SEQ ID No. 3. The invention also discloses an identification method of the flammulina velutipes browning virus. The primer probe for identifying the flammulina velutipes browning virus provides technical support for early screening of flammulina velutipes strains without browning virus in production and identification of flammulina velutipes fruiting body virus in the harvest period.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer probe for identifying flammulina velutipes browning viruses.
Background
In the industrialized production of flammulina velutipes, disease prevention and control are technical difficulties in the cultivation process. The browning virus can cause the growth of the hyphae of the flammulina velutipes to be slowed down, the quality and the yield of the fruiting bodies to be reduced, and the enterprise benefit is seriously influenced. Therefore, virus detection and identification are carried out on the main-cultivated needle mushrooms, virus-carrying strains are detected in time, and the method has important significance for optimizing market germplasm resources, evaluating strain quality, ensuring the quality of the picked needle mushrooms and the like.
However, the detection and identification of the brown virus of the needle mushroom are still difficult points in the industry at present and are also important points for restricting the development of the industry. The currently reported detection method for the flammulina velutipes browning virus is a common PCR method generally, and the method is long in time consumption, insensitive in detection result and easy to generate false positive or false negative.
Therefore, research and development of a new method for identifying flammulina velutipes virus is required.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer probe for identifying brown virus aiming at needle mushroom hypha and fruiting body and an identification method thereof aiming at the defects of the prior art.
The invention aims to provide a primer probe for identifying flammulina velutipes browning viruses, which consists of a primer pair shown in SEQ ID No.1 and SEQ ID No.2 and a probe shown in SEQ ID No. 3;
wherein the primer pair and the probe sequence are designed aiming at the coat protein CP1 gene (NCBI accession number AB465309) of the FvBV of the flammulina velutipes Browning virus (Browning virus);
specifically, FAM fluorescent group is added at the 5 'end of the probe sequence SEQ ID No.3, and BHQ quenching group is added at the 3' end.
The invention also aims to provide a kit for identifying the flammulina velutipes browning virus, which comprises the primer pair shown in SEQ ID No.1 and SEQ ID No.2 and a probe shown in SEQ ID No. 3;
wherein, FAM fluorescent group is added at the 5 'end of the probe sequence SEQ ID No.3, and BHQ quenching group is added at the 3' end.
Preferably, the kit is for:
and identifying or assisting in identifying the virus to be detected as the flammulina velutipes browning virus.
Another object of the present invention is to provide a method for identifying a brown virus of needle mushroom, comprising the steps of:
(1) preparing a needle mushroom coarse extract;
(2) extracting virus RNA from the needle mushroom crude extract by using a nucleic acid extraction reagent;
(3) adding primer pairs shown in SEQ ID No.1 and SEQ ID No.2 and a probe combination shown in SEQ ID No.3 to perform a two-step method qRT-PCR nucleic acid amplification reaction, wherein the final concentration of the primers is 0.2 mu M, and the final concentration of the probe is 0.2 mu M;
(4) judging whether the virus is flammulina velutipes browning virus or not according to the Ct value;
in the step (4), the judgment that the Ct value is less than 38 is positive;
wherein the reaction conditions of the two-step method qRT-PCR nucleic acid amplification reaction are as follows: the first step is as follows: 94 ℃ for 5 minutes; the second step is at 94 deg.C for 5 seconds, at 60 deg.C for 30 seconds, and for 40 cycles.
The preparation method of the needle mushroom coarse extract comprises the following steps of:
putting about 3g of fruiting bodies or mycelia of fresh needle mushrooms in a mortar, fully grinding by using liquid nitrogen, and grinding sample powder
Transferring the powder into a 50ml centrifuge tube, quickly adding 11ml of 0.1M phosphate buffer solution into the centrifuge tube, and fully and uniformly mixing;
② fully centrifuging the homogenate for 60min at 4 ℃ and 8000 g;
③ taking about 10ml of the centrifuged supernatant, transferring the supernatant into a new 50ml centrifuge tube, adding 2ml of 1.2M NaCl (final concentration: 0.15M) and 4ml of 40% PEG8000 aqueous solution (0.4g/ml, final concentration: 10% W/V), mixing completely, placing in a refrigerator at 4 ℃, and standing overnight.
Fourthly, centrifuging the overnight treated sample for 50min at the temperature of 4 ℃ and the weight of 8000g, discarding the supernatant, and reversely buckling the centrifugal tube on the absorbent paper to absorb the redundant liquid.
Fifthly, adding 200ul TES buffer solution (10mM Tris-HCl,1mM EDTA,0.15M NaCl, pH7.0) into the precipitate, and suspending the precipitate.
Sixthly, transferring the mixed solution obtained in the fifth step into a new 2ml centrifuge tube, carrying out centrifugation for 10min at the temperature of 4 ℃ and 13000 g. Transferring the supernatant into a new 1.5ml centrifuge tube to obtain the needle mushroom crude extract, and directly detecting virus dsRNA or storing the needle mushroom crude extract at-20 ℃ for later use.
The invention designs 2 primers and 1 probe aiming at a conserved region of a coat protein CP1 gene of flammulina velutipes browning virus FvBV, and simultaneously discloses a treatment of a sample to be detected, a qRT-PCR reaction system, reaction conditions and a result judgment standard. The primer probe for identifying the flammulina velutipes browning virus and the identification method thereof provided by the invention can realize the detection of most flammulina velutipes sample mycelium and fruiting body browning viruses supplied in the market of China, are simple to operate and convenient to apply, and provide a feasible technical method for quickly and accurately detecting the flammulina velutipes browning virus.
Compared with the prior art, the invention has the following beneficial effects:
the method for identifying the flammulina velutipes browning virus provided by the invention adopts a probe method for fluorescent quantitation (a pair of primers and a probe), and a probe sequence is designed between the two primers. Compared with the prior art, the method needs less templates, is simpler and more convenient to operate, and more importantly, has high detection sensitivity.
The method disclosed by the invention can be used for detecting the virus carried in the needle mushroom fruiting body and detecting the virus in the hypha stage, can be used for timely finding whether the virus is carried in the hypha or not, and can be used for timely removing the strain carrying the virus before the strain enters production, so that the economic loss in production is reduced. Provides a technical method for screening strains without flammulina velutipes browning viruses in early production and identifying flammulina velutipes sporophyte viruses in the harvesting stage.
Drawings
FIG. 1 shows the lysis curve of the amplification of the needle mushroom browning virus CP1 gene by using a probe method qRT-PCR.
FIG. 2 shows viral particles observed by a transmission electron microscope.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1: design and synthesis of needle mushroom browning virus CP1 gene primer and probe
Screening the extracted alternative primers according to the following requirements: 1) the position of the probe is close to that of the same-strand primer, and the size of a PCR product is between 100bp and 150 bp; 2) the GC content of the primers and probes is between 25% and 75%; 3) the length of the primer is about 20bp, the Tm value is between 58 ℃ and 60 ℃, the length of the probe is between 25bp and 30bp, and the Tm value is 5 ℃ to 10 ℃ higher than that of a primer; 4) polyN is less than or equal to 4 bp; 5) hairpin is less than or equal to 4 bp; 6) the coverage rate is more than 90 percent; 7) BLAST screening is carried out, and the specificity score is larger than L multiplied by 0.4; 8) the base G should be avoided at the 5' end of the probe.
The primer and probe sequences shown in table 1 below were obtained:
TABLE 1 sequences of primers and probes
In the two primers, SEQ ID No.1 is complementary with a forward sequence of a needle mushroom browning virus RdRp gene, SEQ ID No.2 is complementary with a reverse sequence thereof, and a probe sequence SEQ ID No.3 is complementary with the forward sequence.
FAM fluorescent group is added at the 5 'end of the probe sequence SEQ ID No.3, and BHQ quenching group is added at the 3' end.
The primer, the probe and the groups at the two ends of the probe are all synthesized by Shanghai biological engineering Co.
Example 2: detection of browning virus FvBV in flammulina velutipes pileus
1 preparation of crude extract of flammulina velutipes pileus
Taking about 3g of pileus of fresh needle mushrooms (commonly sold in markets) in a mortar, fully grinding by using liquid nitrogen, transferring the ground sample powder into a 50ml centrifuge tube, quickly adding 11ml of 0.1M phosphate buffer solution into the centrifuge tube, and fully and uniformly mixing;
② fully centrifuging the homogenate for 60min at 4 ℃ and 8000 g;
③ taking about 10ml of the centrifuged supernatant, transferring the supernatant into a new 50ml centrifuge tube, adding 2ml of 1.2M NaCl (final concentration: 0.15M) and 4ml of 40% PEG8000 aqueous solution (0.4g/ml, final concentration: 10% W/V), mixing completely, placing in a refrigerator at 4 ℃, and standing overnight.
Fourthly, fully centrifuging the overnight treated sample, wherein the centrifugation condition is 4 ℃, 8000g and 50min, discarding the supernatant, and reversely buckling the centrifuge tube on absorbent paper to absorb the redundant liquid.
Fifthly, adding 200ul TES buffer solution (10mM Tris-HCl,1mM EDTA,0.15M NaCl, pH7.0) into the precipitate, and suspending the precipitate.
Sixthly, transferring the mixed solution obtained in the fifth step to a new 2ml centrifuge tube, carrying out centrifugation at 13000g for 10min at 4 ℃. Transferring the supernatant into a new 1.5ml centrifuge tube to obtain the needle mushroom pileus crude extract, dividing the needle mushroom pileus crude extract into three parts in parallel to be used as samples to be detected, and carrying out subsequent virus dsRNA extraction and detection.
And setting a negative control and a positive control at the same time, wherein the negative control is TES buffer solution, the positive control is obtained by operating the needle mushroom strain F-4891 according to the steps, and the strain F-4891 is stored in edible fungus institute of academy of agricultural sciences of Shanghai city with the storage number of F-4891. The detection of the viral component of FvBV in the F-4891 mycelium was disclosed in the literature (Chinese agronomy report 2015, 31(6): 234-.
Respectively carrying out the following steps on a sample to be detected, a negative control and a positive control:
2 extraction of viral RNA
Extraction of Viral RNA was performed in a clean bench according to the instructions of the QIAmp Viral RNA Mini Kit (Qiagen).
3 Probe qRT-PCR reaction
1) Architecture configuration
Use of
II Probe qPCR SuperMix UDG (Beijing Quanjin Biotechnology Co., Ltd., Code # AQ712) kit, the configuration system is shown in the following Table 2:
TABLE 2qRT-PCR reaction System
2) qRT-PCR reaction: the reaction tube of the above reaction system was placed in a fluorescent quantitative PCR apparatus, and the reaction procedure was as shown in Table 3 below:
TABLE 3 qRT-PCR reaction cycle number
4 determination of results
And (3) judging the result under the condition that the yin-yang comparison result is true, namely the negative comparison has no Ct value, the positive comparison has the Ct value, and the positive comparison can be judged to be positive under the condition that the Ct value is less than 38 in general. The following table 4 shows the Ct values of the positive control, the sample to be tested and the negative control. As can be seen from Table 4, the three Ct values (Ct1, Ct2, Ct3) of the sample to be detected are all much less than 38, indicating that the brown virus is detected in the sample.
TABLE 4 Ct value results for sample detection
5 Observation of Virus particles by Transmission Electron microscope
And (3) committing an electron microscope laboratory of an analytical testing center of Shanghai transportation university to perform transmission electron microscope observation on the flammulina velutipes crude extract of the three groups of samples so as to verify the result of qRT-PCR. The results in FIG. 2 show that under a transmission electron microscope, browning virus particles can be clearly seen in the positive control and the sample to be detected.
Sequence listing
<110> Shanghai city academy of agricultural sciences
<120> primer probe for identifying flammulina velutipes browning virus
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gctccgctcc tggacaaatc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aacagtctac acgccctcaa ccctcgc 27
Claims (6)
1. A primer probe for identifying flammulina velutipes browning viruses comprises a primer pair shown in SEQ ID No.1 and SEQ ID No.2 and a probe shown in SEQ ID No. 3.
2. The primer probe for identifying flammulina velutipes brown virus according to claim 1, wherein FAM fluorescent group is added to the 5 'end of the probe sequence SEQ ID No.3, and BHQ quenching group is added to the 3' end.
3. A kit for identifying flammulina velutipes browning viruses comprises primer pairs shown in SEQ ID No.1 and SEQ ID No.2 and a probe shown in SEQ ID No. 3.
4. The needle mushroom browning virus identification kit according to claim 3, wherein the probe sequence SEQ ID No.3 has FAM fluorescent group added to the 5 'end and BHQ quenching group added to the 3' end.
5. An identification method of flammulina velutipes browning virus, which comprises the following steps:
(1) preparing a needle mushroom coarse extract;
(2) extracting virus RNA from the needle mushroom crude extract by using a nucleic acid extraction reagent;
(3) adding primer pairs shown in SEQ ID No.1 and SEQ ID No.2 and a probe combination shown in SEQ ID No.3 to perform a two-step method qRT-PCR nucleic acid amplification reaction, wherein the final concentration of the primers is 0.2 mu M, and the final concentration of the probe is 0.2 mu M;
(4) and judging whether the virus is the flammulina velutipes browning virus or not according to the Ct value.
6. The method for identifying flammulina velutipes browning virus of claim 5, wherein the reaction conditions of the two-step qRT-PCR nucleic acid amplification reaction are as follows: the first step is as follows: 5 minutes at 94 ℃; the second step is at 94 deg.C for 5 seconds, at 60 deg.C for 30 seconds, and for 40 cycles.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101134979A (en) * | 2007-04-29 | 2008-03-05 | 上海市农业科学院食用菌研究所 | Mushroom bacterial standard detecting method |
| WO2021016453A1 (en) * | 2019-07-23 | 2021-01-28 | University Of Rochester | Targeted rna cleavage with crispr-cas |
| CN112779230A (en) * | 2020-12-31 | 2021-05-11 | 上海市农业科学院 | LeLac11 of lentinus edodes laccase and application thereof in improving stress tolerance of microorganisms |
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- 2022-06-02 CN CN202210622113.XA patent/CN114807450A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101134979A (en) * | 2007-04-29 | 2008-03-05 | 上海市农业科学院食用菌研究所 | Mushroom bacterial standard detecting method |
| WO2021016453A1 (en) * | 2019-07-23 | 2021-01-28 | University Of Rochester | Targeted rna cleavage with crispr-cas |
| CN112779230A (en) * | 2020-12-31 | 2021-05-11 | 上海市农业科学院 | LeLac11 of lentinus edodes laccase and application thereof in improving stress tolerance of microorganisms |
Non-Patent Citations (2)
| Title |
|---|
| YUMI MAGAE等: "Characterization of a mycovirus associated with the brown discoloration of edible mushroom,Flammulina velutipes", 《VIROLOGY JOURNAL》 * |
| 谢明勇等: "《食品安全导论 第2版》", 31 August 2016 * |
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