CN114480740B - Targeting sequencing library construction and detection method suitable for 15 plant quarantine viruses - Google Patents

Targeting sequencing library construction and detection method suitable for 15 plant quarantine viruses Download PDF

Info

Publication number
CN114480740B
CN114480740B CN202210150996.9A CN202210150996A CN114480740B CN 114480740 B CN114480740 B CN 114480740B CN 202210150996 A CN202210150996 A CN 202210150996A CN 114480740 B CN114480740 B CN 114480740B
Authority
CN
China
Prior art keywords
virus
plant
viruses
rna
quarantine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210150996.9A
Other languages
Chinese (zh)
Other versions
CN114480740A (en
Inventor
毛凌峰
孙凯
尹传林
俞晓平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Boyi Technology Co ltd
Original Assignee
Hangzhou Boyi Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Boyi Technology Co ltd filed Critical Hangzhou Boyi Technology Co ltd
Priority to CN202210150996.9A priority Critical patent/CN114480740B/en
Publication of CN114480740A publication Critical patent/CN114480740A/en
Application granted granted Critical
Publication of CN114480740B publication Critical patent/CN114480740B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The application provides a targeted sequencing library construction and detection method suitable for 15 plant quarantine viruses, designs a specific primer pool aiming at the 15 plant quarantine viruses, combines a specific multiple reverse transcription technology with a nanopore technology, and establishes a targeted capturing sequencing method aiming at the 15 plant quarantine viruses, so that the method has the advantages of high sensitivity, high flux and low cost, can realize the single library construction sequencing detection of the 15 plant quarantine viruses, and can solve the problems of low detection flux and single detection target existing in the current plant virus quarantine.

Description

Targeting sequencing library construction and detection method suitable for 15 plant quarantine viruses
Technical Field
The application relates to the field of plant virus quarantine, in particular to a targeted sequencing library construction and detection method suitable for 15 plant quarantine viruses.
Background
Plant viruses are considered "plant cancers" that lead to low yields of crops and threaten human food safety, thereby causing a series of social problems. With the continuous development of international agricultural product trade in China, the invasion risk of quarantine plant viruses is increased year by year, and the agricultural production safety in China is seriously threatened, so that the establishment of a rapid quarantine plant virus detection technology and a monitoring system is an important precondition for guaranteeing the agricultural safety production. At present, quarantine work of plant viruses has been generally carried out in units of various harbors, universities and universities of various provinces and municipalities, academy of agricultural sciences and the like.
At present, the traditional method for detecting the plant virus comprises serological detection, electron microscope observation, PCR or RT-PCR, chip hybridization and the like, but the method for detecting the plant virus has a plurality of defects, such as inaccurate judgment easily caused by electron microscope observation, and the nucleic acid amplification technology is limited by the design of multiple amplification primers and the efficiency of the serological technology is limited, so that the popularization and the application of various detection methods in practical work are greatly limited.
Over the past decade, the development of sequencing technology has been rapid, and sequencing technology has revolutionized the field of molecular biology from the first generation Sanger method to the second generation sequencing technology (Next-generation sequencing, NGS). The sequencing technology is also suitable for rapid detection of plant viruses, and currently mainstream plant virus detection based on deep sequencing mainly adopts NGS technology.
However, the virus detection based on NGS sequencing technology still faces many problems, which are mainly manifested in the following schemes: 1. NGS sequencers belong to a large-scale instrument on equipment, and are expensive. Requiring a specialized laboratory or corporate platform; 2. in experimental operation, sequencing library preparation is complex and complicated in steps, and requires professional experimental operators. 3. NGS experiment operations and sequencing cycles were 2-3 days in sequencing procedure and detection cycle. 4. In sequencing data quality control and data analysis, NGS relies on PCR amplification and therefore has a certain sequencing preference, resulting in incomplete genome coverage and because NGS sequencing is not 1Kb long, viral genomes need to be assembled, and to a certain extent the results are extremely dependent on software and algorithm optimization and improvement.
Nanopore sequencing technology is a single molecule sequencing technology that performs base detection based on the change in potential difference as a nucleic acid molecule passes through a nanopore. Unlike existing sequencing techniques, nanopore sequencing techniques do not have a DNA synthesis step, and are currently the only techniques that can directly sequence nucleic acid molecules. Meanwhile, the nanopore sequencing also has the advantages of overlong reading length, portability, real-time sequencing, high-throughput multi-platform and other sequencing platforms. But the reagent cost is 5-10 times higher than that of the second generation sequencing, and the technology is difficult to popularize in practice due to the lack of a bioinformatic analysis procedure aiming at plant quarantine viruses, so that further improvement is needed. In addition, the plant quarantine virus genome types in China are various, some are RNA viruses and some are DNA viruses, and the RNA viruses comprise RNA viruses containing PolyA tails and RNA viruses without PolyA tails. At present, different database building schemes are generally needed for different types of plant viruses, so that the cost and difficulty for simultaneously detecting different types of plant viruses at a time are increased.
Disclosure of Invention
The application aims to provide a targeted sequencing library construction and detection method suitable for 15 plant quarantine viruses, which is characterized in that the characteristic that all plant viruses can generate RNA is utilized to construct a plant quarantine virus specific reverse transcription primer pool, and a specific multiple reverse transcription technology is combined with a nanopore technology to construct a targeted capturing sequencing method aiming at 15 plant quarantine viruses, so that the method has the advantages of high sensitivity, high flux and low cost, can realize the aim of detecting 15 plant quarantine viruses by single library construction sequencing, and can solve the problems of low detection flux and single detection aim existing in the current plant virus quarantine.
In order to achieve the above purpose, the technical scheme provides a targeted sequencing library building method suitable for 15 plant quarantine viruses, which comprises the following steps:
extracting RNA of a plant sample, and performing reverse transcription on the RNA of the plant sample by utilizing a specific primer pool to synthesize virus cDNA, wherein the specific primer pool comprises primer sequences shown in SEQ ID NO.1-SEQ ID NO. 30;
carrying out PCR amplification on different viruses according to conserved sequences at two ends of the virus cDNA to obtain enriched double-stranded DNA;
and quantifying and diluting the enriched double-stranded DNA, and performing adaptor ligation.
The scheme can carry out sequencing and library establishment aiming at the following 15 plant quarantine viruses, wherein the 15 plant quarantine viruses comprise: arabis mosaic virus, bean pod mottle virus, cocoa clades virus, carnation ringspot virus, maize chlorosis dwarf virus, maize chlorotic mottle virus, oat mosaic virus, potato broomcorn virus, potato a virus, potato V virus, potato yellow dwarf virus, southern bean mosaic virus, sugarcane streak virus, tobacco ringspot virus, and wheat streak mosaic virus.
Arabis mosaic virus (ArMV), bean Pod Mottle Virus (BPMV), cocoa ringworm virus (CSSV), carnation ringspot virus (CRSV), maize Chlorosis Dwarf Virus (MCDV), maize Chlorotic Mottle Virus (MCMV), oat Mosaic Virus (OMV), potato broomcirus (PMTV), potato a virus (PVA), potato V Virus (PVV), potato Yellow Dwarf Virus (PYDV), southern Bean Mosaic Virus (SBMV), sugarcane Streak Virus (SSV), tobacco ringspot virus (TRSV), and Wheat Streak Mosaic Virus (WSMV).
15 plant quarantine virus directory and classification are shown in the following table one
List-one quarantine virus directory and classification
The specific primer pool of the scheme aims at 15 virus sequences, and the specific primer sequences in the specific pool primer pool are shown in the following table II:
and (II) table: primer sequences in specific primer pools
The scheme is characterized in that virus nucleic acid is extracted and enriched, a sequencing library is built for the virus nucleic acid by using corresponding reagents, and a programmed bioinformatics analysis program is built, so that the purpose of detecting and analyzing whether plant samples contain plant quarantine viruses in real time is achieved.
In the "extract plant sample RNA" step, plant sample RNA is extracted using a plant RNA extraction kit. The type of the plant RNA extraction kit can be selected fromOther plant RNA extraction kits can also be used for the RNA Mini Kit.
In one embodiment of the present protocol, a plant sample to be tested is taken, added with liquid nitrogen and ground to a powder, using a plant RNA extraction kitExtracting total RNA of a plant sample by using an RNA Mini Kit, performing electrophoresis and nanodrop detection, and adjusting the final concentration of the RNA of the plant sample to 250ng/ul.
In the "reverse transcription of plant sample RNA into viral cDNA Using specific primer pools" step, the SSP primer of the nanopore sequencing kit is used in combination, such that the 5' end of each viral cDNA is ligated. In some embodiments, the 5' end of the cDNA may be ligated using SSP primers in the nanopore sequencing kit PCR-cDNA Barcoding Kit (SQK-PCB 109).
The reverse transcription system of this scheme can employ a Therom Maxima H Minus Reverse Transcriptase reverse transcription system, the first reverse transcription system is shown in Table III below:
TABLE III first reverse transcription System
Placing on ice after heat preservation at 65 ℃ for 5 minutes, adding a second reverse transcription system to the de-rotated RNA placed on ice, heat preservation at 42 ℃ for 2 minutes, and adding Maxima H Minus Reverse Transcriptase ul into the system; preserving heat at 62 ℃ for 90 minutes; preserving the temperature at 85 ℃ for 5 minutes; placed on ice. The second reaction system after cDNA synthesis is shown in Table IV below:
table IV second reverse transcription System
In other words, during the reverse transcription process: RNA solution of plant sample, specific primer pool, dNTP mix and DEPC H 2 O was mixed and incubated at 65℃for 5 minutes and then placed on ice, followed by 5X Reverse Transcriptase Buffer, RNase Inhibitor, 10uM SSP, DEPC H2O to the above-described unwound RNA, 42℃for 2 minutes and then Maxima H Minus Reverse Transcriptase ul of water was added to the system, 62℃for 90 minutes, 85℃for 5 minutes and placed on ice.
Since the amount of cDNA obtained by reverse transcription is relatively small, it is necessary to enrich the cDNA. In the step of carrying out PCR amplification on different viruses according to conserved sequences at two ends of virus cDNA to obtain enriched double-stranded DNA, the method utilizes a barcode primer (LWB 1-12) to carry out PCR amplification on different samples to obtain enriched double-stranded DNA.
In one embodiment, PCR amplification of different cDNAs is performed using, but not limited to, longAmptaq mix polymerase from New England Biolabs, using the barcode primers (LWB 1-12) in the cDNA-PCR Barcoding Kit (SQK-PCB 109 withQK-PBK 004) kit, in combination with the conserved sequences at both ends of the cDNA.
The PCR amplification system and conditions in this scheme are as follows:
the PCR system is shown in Table five:
table five PCR System
The conditions for the PCR reaction cycle were as follows:
TABLE six PCR reaction cycle conditions
1ul NEB Exonuclease 1 is added into the PCR tube after the PCR reaction is finished to remove RNA in the PCR system, and the reaction conditions are as follows: after reaction at 37℃for 15 minutes, the reaction was carried out at 80℃for 15 minutes.
In the step of quantifying and diluting enriched double-stranded DNA of the present scheme, the refinement comprises the following steps:
a. preparing AMPure XP beads to mediate, and uniformly mixing;
b. mix every 4 tubes of PCR solution together into a 1.5ml centrifuge tube;
c. oscillating suspension AMPure XP beads;
d. 160ul beads were added to the PCR mixture;
e. rotating in the hybridization furnace for 5min at room temperature;
f. treating the incubated solution by a magnetic rack, discarding the supernatant, adding 200ul of 70% ethanol solution, and washing twice (the tube is immobilized on the rack);
g. airing;
12ul of water is dissolved, the magnetic beads are separated by rotating in a hybridization furnace at room temperature for 10min, and the supernatant after the treatment of a magnetic rack is transferred into a 1.5ml centrifuge tube.
In an example of this protocol, enriched double stranded DNA is purified to a nucleic acid concentration of 100ng/ul that meets the conditions of an OD260/280 of 1.8-2.1 and an OD260/230 of 2.2-2.5.
In the "make linker ligation" step, 200ng of library product was pipetted, diluted to 11ul with water, 1ul of RAP was added, mixed well, and room temperature for 5min.
As shown in fig. 1, in a second aspect, the present solution provides a detection method suitable for 15 plant quarantine viruses:
extracting RNA of a plant sample, and performing reverse transcription on the RNA of the plant sample by utilizing a specific primer pool to synthesize virus cDNA, wherein the specific primer pool comprises primer sequences shown in SEQ ID NO.1-SEQ ID NO. 30;
carrying out PCR amplification on different viruses according to conserved sequences at two ends of the virus cDNA to obtain enriched double-stranded DNA;
quantifying and diluting the enriched double-stranded DNA, and performing adaptor connection to obtain a nucleic acid sample library;
performing on-machine real-time detection on the nucleic acid sample library to obtain nanopore sequencing data;
and grouping the nanopore sequencing data and comparing the nanopore sequencing data with a standard virus database to determine a plant quarantine virus sequence.
Other steps are as described in the scheme of the first aspect, except for on-machine detection and nanopore sequencing data processing.
The method for detecting the nucleic acid sample library in real time and acquiring the nanopore sequencing data comprises the following steps:
1. sequencing Buffer (SQB), loading Beads (LB), flush teather (FLT) and Flush Buffer (FB) were thawed at room temperature and the tubes were placed on ice after thawing was completed.
2. Vortex mixing Sequencing Buffer (SQB) and Flush Buffer (FB), centrifugation and placement on ice.
3. Centrifuging the Flush test (FLT) tube, blowing and mixing, and placing on ice.
4. And opening the cover of the nanopore sequencing device, and sliding the starting cover of the flow cell clockwise to enable the starting cover to be visible.
5. The SpotON chip is started and loaded.
6. After opening the filling port, it is checked whether there are small bubbles under the cap. A small amount is withdrawn to clear any bubbles:
a P1000. Mu.l pipette was set to 200ul
Inserting the gun head into the filling opening
The dial is turned until the dial displays 220-230. Mu.l, or until a small amount of buffer is seen to enter the pipette tip position.
7. Preparing a chip perfusion mixture: mu.l of thawed and mixed Flush teathers (FLT) were added directly to the thawed and mixed Flush Buffer (FB) tube and mixed by up and down blowing.
8. 800. Mu.l of the perfusion mixture was loaded into the flow-through cell through the perfusion port, avoiding the introduction of air bubbles. And 5 minutes.
9. The contents of Loading Beads (LB) were thoroughly mixed with a pipette.
At a new pipe, the preparation library is loaded with the following table seven:
table seven preparation library component
10. The SpotON sample port cover is gently lifted to make the SpotON sample port accessible.
11. 200 μl of priming mix was loaded into the chip through the priming port (not the SpotON sample port) avoiding the introduction of bubbles.
12. Mu.l of sample was added to the flow cell in a drop-wise manner through the Spoton sample port. Before the next drop is added, please ensure that each drop flows into a port.
13. The SpotON sample port cap was gently fitted back to ensure that the plug entered the SpotON port, the oil port was closed, and then the valve cap was fitted back.
14. The sequence was run on-machine using a MinION sequencer and Dell Precision T3630 workstation (Intel i7-8700CPU/32G MEM/1.92T SSD/P2200 GPU) at room temperature.
In the step of "grouping nanopore sequencing data and comparing with standard virus database to determine plant quarantine virus sequence", a local standard virus database is first established, the nanopore sequencing data is used to debark the database by Guppy software, and BlastN (parameter e -5 To e -10 ) Compared with the local database, blastN (parameter e -5 To e -10 ) And compared with a local database, the virus nucleic acid sequence in the plant sample is accurately obtained.
Compared with the prior art, the technical scheme has the following characteristics and beneficial effects:
the method can realize the detection of the plant quarantine virus of 15 in single database-building sequencing detection, reduce the cost and difficulty of simultaneously detecting different types of plant viruses in a single time, realize the detection real-time analysis with high detection efficiency, achieve the effect of rapid and on-site detection, and can be applied to the on-site rapid detection of the plant quarantine virus by quarantine departments.
Drawings
Fig. 1 is a flow chart of a detection method suitable for 15 plant quarantine viruses according to the present scheme.
FIG. 2 is a graph showing the number of nucleic acid sequences of viral origin at various concentrations of viral nucleic acid.
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products available commercially without the manufacturer's attention.
The following examples and experimental examples relate to instruments comprising: a MinION sequencer, a high-speed centrifuge, a Qubit3.0, a metal bath, a pipettor, a magnetic rack and the like.
The reagents involved include: plant nucleic acid extraction kitRNA Mini Kit), ONT library Kit: cDNA-PCR Barcoding Kit (SQK-PCB 109 withSQK-PBK 004), AMPure XP purified magnetic beads, qubitTM detection kit, ONT sequencing chip, etc.
Example one design of diseased tomatoes to test the feasibility and sensitivity of the protocol
15 virus standard RNA nucleic acid sequences are obtained through a T7 in vitro synthesis mode, wherein the 15 viruses are respectively: arabis mosaic virus (ArMV), bean Pod Mottle Virus (BPMV), cocoa ringworm virus (CSSV), carnation ringspot virus (CRSV), maize Chlorosis Dwarf Virus (MCDV), maize Chlorosis Mottle Virus (MCMV), oat Mosaic Virus (OMV), potato broomcirus (PMTV), potato a virus (PVA), potato V Virus (PVV), potato Yellow Dwarf Virus (PYDV), southern Bean Mosaic Virus (SBMV), sugarcane Streak Virus (SSV), tobacco ringspot virus (TRSV) and Wheat Streak Mosaic Virus (WSMV) are tested by mixing them in proportion with healthy tomato total RNA at different concentrations in plant samples to assess the feasibility and sensitivity of the method using established plant quarantine virus targeted sequencing techniques.
1. In vitro transcription: the DNA of 15 virus standard RNA nucleic acid sequences with T7 promoter is subjected to in vitro transcription reaction. The template DNA concentration was diluted to 50 ng/. Mu.l, and 15 viruses were subjected to in vitro transcription using TaKaRa T7 in vitro transcription kit, and incubated at 42℃for 2 hours, and the reaction system was as shown in Table eight below:
table eight reaction System
2. RNA purification
a. 1-2. Mu.l of DNase I (10U/. Mu.l, taKaRa) was added to the reverse transcription RNA obtained in step 1, and the temperature was maintained at 37℃for 15-20 min, the purpose of which was to delete the template DNA.
b. The system is filled with water to 200 mu l, transferred to a new 1.5mL centrifuge tube, 200 mu l of saturated phenol/chlorophosphoric acid (1:1, RNA mass) is added, and the system is refrigerated at 4 ℃ for centrifugation at 10000-12000 r/min for 3min.
c. Discarding the precipitate of the product after centrifugation in the step (2), sucking the upper water phase after centrifugation into a new centrifuge tube, adding 600 μl (540 μl isopropanol+60 μl sodium acetate with total volume of 1/10), mixing the mixture upside down, and standing at-20deg.C for 30min.
d. Taking out the centrifuge tube in the step (3) in a refrigerator, freezing the centrifuge at 4 ℃, and centrifuging for 15min at 10000-12000 r/min.
e. And (3) discarding the supernatant of the liquid after the centrifugation in the step (4), adding 700 mu l of 75% ethanol, and centrifuging at 4 ℃ for 5min by using a refrigerated centrifuge of 10000-12000 r/min. After removal, the supernatant was discarded and this step was repeated.
f. Discarding the supernatant after the centrifugation in the step (5), freezing the supernatant in a centrifuge at 4 ℃ for 10000r/min, and carrying out air separation for 2-3 min. Taking out after centrifugation, sucking redundant supernatant with a gun head, opening a cover of the centrifuge tube, airing, and removing redundant ethanol.
g. And (3) adding 20 mu l of sterile double distilled water into the tube dried in the step (6) to dissolve RNA, thereby obtaining RNA with higher purity.
3. Healthy plant RNA extraction (tomato)
The application usesRNA Mini Kit plant RNA extraction Kit.
a. Fresh tomato leaves, about 0.1g, are sheared and placed into a 1.5mL centrifuge tube, a proper amount of small steel balls for grinding are added, and the mixture is placed into liquid nitrogen for quick freezing for 3min.
b. And (3) putting the plant sample frozen in the step (1) into a vibration crusher, crushing for about 150s at 70Hz, and obtaining the plant sample without massive tissues.
1mL RL+20. Mu.l DTT (50X) was prepared as lysates, 500. Mu.l each sample was added, gently swirled to mix roar, frozen in centrifuge at 4℃at 12000r/min, and centrifuged for 5min.
d. Sucking 300 μl of the supernatant after centrifugation in step (3) into a yellow filter column, placing the filter column into a matched centrifuge tube, and centrifuging at 4deg.C in a refrigerated centrifuge at 12000r/min for 1min.
e. Discarding the column, reserving the filtrate, adding 1/2 volume of ethanol into the centrifuge tube in the step (4), lightly blowing and mixing uniformly, transferring into a filter column taken out of an aluminum plate, placing the filter column into a new matched centrifuge tube, freezing the centrifuge at 4 ℃, centrifuging for 12000r/min, centrifuging for 1min, and discarding the filtrate.
f. 500 μl RWA was added to the column after centrifugation in step (5) along the tube wall, refrigerated at 4deg.C, centrifuge 12000r/min, centrifuge for 30s, and discard the filtrate. This step is to remove salt.
DNase I digestion conditions were as follows: 10 XDNase I Buffer, 5. Mu.l; recombinant DNase I,4 μl; free H2O,41 μl.
The total amount of the above system was 50. Mu.l, and the prepared system was put into a filter column, and each sample was 50. Mu.l and allowed to stand at room temperature for 15 minutes. Further 300. Mu.l RWB was added, centrifuged at 12000r/min at 4℃for 30s, and the filtrate was discarded.
h. 600 μl RWB was added to the tube after centrifugation in step (7), refrigerated at 4deg.C, centrifuged at 12000r/min for 30s, and the filtrate was discarded.
I. And (3) putting the centrifuge tube in the step (8) back into a refrigerated centrifuge at the temperature of 4 ℃, centrifuging for 30s at 12000r/min, and discarding the filtrate.
g. Taking out the filter column after centrifugation in the step (9), putting the filter column into a new 1.5mL centrifuge tube, adding 50-200 mu l of Free H2O into the middle of the filter column, standing for 5min at room temperature, freezing the centrifuge at 4 ℃, centrifuging for 2min at 12000r/min, discarding the filter column, and measuring the concentration of the extracted RNA.
4. cDNA Synthesis
The application dilutes the virus with healthy plants to 10%, 5%, 0.5%, 0.05%, 0%,5 different concentrations to verify the lowest concentration detectable.
(1) Preparation of virus samples at different concentrations.
a.10%: virus pool 2.5. Mu.l+50. Mu.l healthy tomato RNA
b.10% Virus 15. Mu.l+15. Mu.l healthy tomato RNA
2 μl+18 μl healthy tomato RNA of 5% virus
d0.5% Virus 2. Mu.l+18. Mu.l healthy tomato RNA
e.0%: only healthy tomato RNA
(2) cDNA Synthesis
Reverse transcription was performed using Therom Maxima H Minus Reverse Transcriptase reverse transcription system RNA, and reverse transcription primers used a virus specific primer pool (RTPmix). The first reaction system is shown in Table nine below:
table nine first reaction System
Preserving the temperature at 65 ℃ for 5 minutes; placing on ice:
table ten second reaction System
(3) Transferring the second reaction system into the unwinding RNA in the previous step, and preserving the temperature at 42 ℃ for 2 minutes; maxima H Minus Reverse Transcriptase 1ul is added into the system; preserving heat at 62 ℃ for 90 minutes; preserving the temperature at 85 ℃ for 5 minutes; placed on ice. The cDNA synthesis was completed.
(4) cDNA double-strand synthesis and amplification, PCR amplification was performed on different samples based on the conserved sequences at both ends of cDNA, using the barcode primer (LWB 1-12) in cDNA-PCRsequencing Kit.
As the PCR amplification, a LongAmptaq mix polymerase from New England Biolabs was used.
The PCR system is shown in Table twelve below:
table twelve PCR system
LWB 8-12 corresponds to 10%, 5%, 0.5%, 0.05%, 0% of the virus sample, respectively.
The PCR reaction cycle was set as follows: 95 ℃ for 30sec; [ 95 ℃ for 15sec;62 ℃,15sec;65 ℃,40sec ] 35 cycles; 65 ℃ for 6min;4 ℃, hold.
1ul NEB Exonuclease 1 was added to the PCR tube after completion of the PCR reaction to remove RNA in the PCR system. The reaction conditions are as follows: 37 ℃ for 15min;80 ℃ for 15min.
(5) dsDNA purification
Pcr products were purified using AMPure XP beads: vibrating and suspending AMPure XP beads, adding 160 μl of AMPure XP beads into the pcr product obtained in the step (4), placing into a hybridization furnace, and rotating at room temperature for 5min. Taking out, placing on a magnetic rack, standing for 2min, after the AMPure XP bead is completely separated from the liquid, discarding the supernatant, adding 200 μl of 70% ethanol prepared in advance, washing twice the AMPureXP bead, uncovering and airing the excessive ethanol, adding 12 μl of ddH2O for dissolving, rotating at room temperature in a hybridization furnace for 10min, separating magnetic beads by the magnetic rack, transferring the supernatant into a new 1.5mL centrifuge tube to obtain dsDNA with higher purity, detecting the double-stranded DNA nucleic acid purified in the step (1) by using Nanodrop, satisfying the OD260/280 as 1.8-2.1 and OD260/230 as 2.2-2.5, quantifying the double-stranded DNA nucleic acid purified in the step (1) by using a Qubit method, and diluting to the nucleic acid concentration of 100 ng/ul.
(6) Connecting joint
200ng of the library product from step (5) was pipetted, diluted to 11. Mu.l with ddH2O, 1. Mu.l of RAP was added, and after mixing, the mixture was allowed to stand at room temperature for 5min.
3. Library loading and nano Kong Shishi sequencing
The detailed steps are as follows:
1. sequencing Buffer (SQB), loading Beads (LB), flush teather (FLT) and one tube of Flush Buffer (FB) were thawed at room temperature and the tubes were placed on ice after thawing was completed.
2. Vortex mixing Sequencing Buffer (SQB), flush Buffer (FB) tubes, and after centrifugation, place on ice.
3. Centrifuging the Flush test (FLT) tube, blowing and mixing, and placing on ice.
4. And opening the cover of the nanopore sequencing device, and sliding the starting cover of the flow cell clockwise to enable the starting cover to be visible.
5. Starting and loading SpotON chip
6. After opening the filling port, it is checked whether there are small bubbles under the cap. A small amount is withdrawn to clear any bubbles:
a P1000. Mu.l pipette was set to 200ul
Inserting the gun head into the filling opening
The dial is turned until the dial displays 220-230. Mu.l, or until a small amount of buffer is seen entering the pipette tip
7. Preparing a chip perfusion mixture: 30 μl of thawed and mixed Flush teather (FLT) was added directly to the tube of thawed and mixed Flush Buffer (FB) and mixed by up and down blowing 8. 800 μl of perfusion mixture was loaded into the flow-through cell through the perfusion port avoiding the introduction of air bubbles. And 5 minutes.
9. The contents of Loading Beads (LB) were thoroughly mixed with a pipette.
At a new pipe, the preparation library is loaded with thirteen of the following tables:
preparation library for thirteen tables
10. The SpotON sample port cover is gently lifted to make the SpotON sample port accessible.
11. 200 μl of priming mix was loaded into the chip through the priming port (not the SpotON sample port) avoiding the introduction of bubbles.
12. Mu.l of sample was added to the flow cell in a drop-wise manner through the Spoton sample port. Before the next drop is added, please ensure that each drop flows into a port.
13. The SpotON sample port cap was gently fitted back to ensure that the plug entered the SpotON port, the oil port was closed, and then the valve cap was fitted back.
14. The sequence was run on-machine using a MinION sequencer and Dell Precision T3630 workstation (Intel i7-8700CPU/32G MEM/1.92T SSD/P2200 GPU) at room temperature.
4. Virus-derived sequence data analysis
Sequencing was performed using a FLO-MIN106 sequencing chip for a total of 5h1m. Sequencing data statistics are shown in Table fourteen, and the number of virus-derived nucleic acid sequences at different concentrations of virus nucleic acid is shown in FIG. 2.
Fourteen detection results
/>
The present application is not limited to the above-mentioned preferred embodiments, and any person who can obtain other various products under the teaching of the present application can make any changes in shape or structure, and all the technical solutions that are the same or similar to the present application fall within the scope of the present application.
SEQUENCE LISTING
<110> Hangzhou cypress bright technologies Co., ltd
<120> targeted sequencing library construction and detection method suitable for 15 plant quarantine viruses
<130> HZPZL1211962
<160> 30
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213> artificial sequence
<400> 1
cttgcctgtc gctctatctt ccarttgtta gtgacc 36
<210> 2
<211> 36
<212> DNA
<213> artificial sequence
<400> 2
cttgcctgtc gctctatctt cagtttctcc acttgg 36
<210> 3
<211> 36
<212> DNA
<213> artificial sequence
<400> 3
cttgcctgtc gctctatctt cgttcaatct gcacaa 36
<210> 4
<211> 35
<212> DNA
<213> artificial sequence
<400> 4
cttgcctgtc gctctatctt cgcacatgct acatt 35
<210> 5
<211> 39
<212> DNA
<213> artificial sequence
<400> 5
cttgcctgtc gctctatctt cttacggcaa cgcgtttcc 39
<210> 6
<211> 39
<212> DNA
<213> artificial sequence
<400> 6
cttgcctgtc gctctatctt caatrtgccc gacaactgc 39
<210> 7
<211> 42
<212> DNA
<213> artificial sequence
<400> 7
cttgcctgtc gctctatctt cctactcttt cacaggctta ag 42
<210> 8
<211> 39
<212> DNA
<213> artificial sequence
<400> 8
cttgcctgtc gctctatctt ctcattgacc aacccactg 39
<210> 9
<211> 36
<212> DNA
<213> artificial sequence
<400> 9
cttgcctgtc gctctatctt caattgtgag agtctc 36
<210> 10
<211> 36
<212> DNA
<213> artificial sequence
<400> 10
cttgcctgtc gctctatctt caggcacatt acattg 36
<210> 11
<211> 36
<212> DNA
<213> artificial sequence
<400> 11
cttgcctgtc gctctatctt cgcttgtgtt gcacta 36
<210> 12
<211> 36
<212> DNA
<213> artificial sequence
<400> 12
cttgcctgtc gctctatctt ctaggacacg gagtac 36
<210> 13
<211> 36
<212> DNA
<213> artificial sequence
<400> 13
cttgcctgtc gctctatctt ctcgaaaatt tcatcg 36
<210> 14
<211> 36
<212> DNA
<213> artificial sequence
<400> 14
cttgcctgtc gctctatctt caatatgcag ggttga 36
<210> 15
<211> 36
<212> DNA
<213> artificial sequence
<400> 15
cttgcctgtc gctctatctt ctaaatcgtc cacctc 36
<210> 16
<211> 36
<212> DNA
<213> artificial sequence
<400> 16
cttgcctgtc gctctatctt cgttcacgag ctcata 36
<210> 17
<211> 36
<212> DNA
<213> artificial sequence
<400> 17
cttgcctgtc gctctatctt cacctgggca tgtatg 36
<210> 18
<211> 36
<212> DNA
<213> artificial sequence
<400> 18
cttgcctgtc gctctatctt cggttgcgyt gaagac 36
<210> 19
<211> 39
<212> DNA
<213> artificial sequence
<400> 19
cttgcctgtc gctctatctt ccgtggcatg tatggttca 39
<210> 20
<211> 37
<212> DNA
<213> artificial sequence
<400> 20
cttgcctgtc gctctatctt caatttctca ccaaacc 37
<210> 21
<211> 36
<212> DNA
<213> artificial sequence
<400> 21
cttgcctgtc gctctatctt cattccaccg gtttag 36
<210> 22
<211> 37
<212> DNA
<213> artificial sequence
<400> 22
cttgcctgtc gctctatctt caacttgttc taatggt 37
<210> 23
<211> 36
<212> DNA
<213> artificial sequence
<400> 23
cttgcctgtc gctctatctt ctagaactcg acccat 36
<210> 24
<211> 36
<212> DNA
<213> artificial sequence
<400> 24
cttgcctgtc gctctatctt crtacgacac gtatag 36
<210> 25
<211> 36
<212> DNA
<213> artificial sequence
<400> 25
cttgcctgtc gctctatctt ccatdaccat gcaccg 36
<210> 26
<211> 37
<212> DNA
<213> artificial sequence
<400> 26
cttgcctgtc gctctatctt caratacaaa cgggcat 37
<210> 27
<211> 35
<212> DNA
<213> artificial sequence
<400> 27
cttgcctgtc gctctatctt ccatgygcat cagga 35
<210> 28
<211> 36
<212> DNA
<213> artificial sequence
<400> 28
cttgcctgtc gctctatctt cygcactaga aaacat 36
<210> 29
<211> 40
<212> DNA
<213> artificial sequence
<400> 29
cttgcctgtc gctctatctt cacaccattg aaactgtgcg 40
<210> 30
<211> 39
<212> DNA
<213> artificial sequence
<400> 30
cttgcctgtc gctctatctt ccacatcatc tgcatcatg 39

Claims (8)

1. The targeted sequencing and library building method suitable for the 15 plant quarantine viruses is characterized by comprising the following steps of: extracting RNA of a plant sample, and performing reverse transcription on the RNA of the plant sample by utilizing a specific primer pool to synthesize virus cDNA, wherein the specific primer pool comprises primer sequences shown in SEQ ID NO.1-SEQ ID NO. 30; carrying out PCR amplification on different viruses according to conserved sequences at two ends of the virus cDNA to obtain enriched double-stranded DNA; quantifying and diluting the enriched double-stranded DNA, and performing adaptor connection; the 15 plant quarantine viruses include: arabis mosaic virus, bean pod mottle virus, cocoa clades virus, carnation ringspot virus, maize chlorosis dwarf virus, maize chlorotic mottle virus, oat mosaic virus, potato broomcorn virus, potato a virus, potato V virus, potato yellow dwarf virus, southern bean mosaic virus, sugarcane streak virus, tobacco ringspot virus, and wheat streak mosaic virus.
2. The method of claim 1, wherein in the step of reverse transcription of plant sample RNA into viral cDNA using a specific primer pool, SSP primers of a nanopore sequencing kit are used in combination such that the 5' end of each viral cDNA is ligated.
3. The method for constructing a library by targeting sequencing of 15 plant quarantine viruses according to claim 1, wherein the biarcode primer carries out PCR amplification on different cDNAs according to conserved sequences at two ends of the cDNAs.
4. The targeted sequencing pooling method for 15 plant quarantine viruses according to claim 1, wherein the PCR cycling conditions are: 95 ℃,30sec, 95 ℃ and 30sec execute 11-18 cycles; performing 11-18 cycles at 62 ℃ for 15sec; executing 11-18 cycles at 65 ℃ for 1 min; performing 2 cycles at 65 ℃ for 6min; and maintained at 4 ℃.
5. The detection method suitable for the 15 plant quarantine viruses is characterized by comprising the following steps of: obtaining a plant sample to be tested; extracting RNA of a plant sample, and performing reverse transcription on the RNA of the plant sample by utilizing a specific primer pool to synthesize virus cDNA, wherein the specific primer pool comprises primer sequences shown in SEQ ID NO.1-SEQ ID NO. 30; carrying out PCR amplification on different viruses according to conserved sequences at two ends of the virus cDNA to obtain enriched double-stranded DNA; quantifying and diluting the enriched double-stranded DNA, and performing adaptor connection to obtain a nucleic acid sample library; performing on-machine real-time detection on the nucleic acid sample library to obtain nanopore sequencing data; grouping the nanopore sequencing data and comparing with a standard virus database to determine a plant quarantine virus sequence, the 15 plant quarantine virus comprising: arabis mosaic virus, bean pod mottle virus, cocoa clades virus, carnation ringspot virus, maize chlorosis dwarf virus, maize chlorotic mottle virus, oat mosaic virus, potato broomcorn virus, potato a virus, potato V virus, potato yellow dwarf virus, southern bean mosaic virus, sugarcane streak virus, tobacco ringspot virus, and wheat streak mosaic virus.
6. The method according to claim 5, wherein in the step of "reverse transcription synthesis of viral cDNA from plant sample RNA using specific primer pool", SSP primer of nanopore sequencing kit is used to make 5' end of each viral cDNA add a linker.
7. The method according to claim 5, wherein the enriched double-stranded DNA is purified to a nucleic acid concentration of 100ng/ul under conditions that the OD260/280 is 1.8-2.1 and the OD260/230 is 2.2-2.5.
8. A kit comprising a pool of specific primers suitable for reverse transcription of 15 plant quarantine virus, wherein the pool of specific primers comprises the primer sequences shown in SEQ ID No.1-SEQ ID No. 30.
CN202210150996.9A 2022-02-18 2022-02-18 Targeting sequencing library construction and detection method suitable for 15 plant quarantine viruses Active CN114480740B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210150996.9A CN114480740B (en) 2022-02-18 2022-02-18 Targeting sequencing library construction and detection method suitable for 15 plant quarantine viruses

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210150996.9A CN114480740B (en) 2022-02-18 2022-02-18 Targeting sequencing library construction and detection method suitable for 15 plant quarantine viruses

Publications (2)

Publication Number Publication Date
CN114480740A CN114480740A (en) 2022-05-13
CN114480740B true CN114480740B (en) 2023-10-24

Family

ID=81482431

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210150996.9A Active CN114480740B (en) 2022-02-18 2022-02-18 Targeting sequencing library construction and detection method suitable for 15 plant quarantine viruses

Country Status (1)

Country Link
CN (1) CN114480740B (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1952649A (en) * 2005-10-19 2007-04-25 中华人民共和国北京出入境检验检疫局 A gene chip, nucleotide sequence and reagent kit for detecting virus of potato family plant
CN110946137A (en) * 2012-10-16 2020-04-03 孟山都技术公司 Methods and compositions for controlling plant viral infections
CN111440846A (en) * 2020-04-09 2020-07-24 江苏先声医学诊断有限公司 Position anchoring bar code system for nanopore sequencing library building
CN112176032A (en) * 2020-10-16 2021-01-05 广州市达瑞生物技术股份有限公司 Primer combination for nanopore sequencing and library building of respiratory pathogens and application thereof
CN113430303A (en) * 2021-06-29 2021-09-24 杭州圣庭医疗科技有限公司 Rapid identification method for 23 respiratory RNA viruses based on nanopore sequencer
CN113637796A (en) * 2021-07-06 2021-11-12 杭州圣庭医疗科技有限公司 Rapid identification method for 8 herpesviruses based on nanopore sequencer

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021243271A2 (en) * 2020-05-29 2021-12-02 Front Range Biosciences, Inc. Methods and compositions for pathogen detection in plants

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1952649A (en) * 2005-10-19 2007-04-25 中华人民共和国北京出入境检验检疫局 A gene chip, nucleotide sequence and reagent kit for detecting virus of potato family plant
CN110946137A (en) * 2012-10-16 2020-04-03 孟山都技术公司 Methods and compositions for controlling plant viral infections
CN111440846A (en) * 2020-04-09 2020-07-24 江苏先声医学诊断有限公司 Position anchoring bar code system for nanopore sequencing library building
CN112176032A (en) * 2020-10-16 2021-01-05 广州市达瑞生物技术股份有限公司 Primer combination for nanopore sequencing and library building of respiratory pathogens and application thereof
CN113430303A (en) * 2021-06-29 2021-09-24 杭州圣庭医疗科技有限公司 Rapid identification method for 23 respiratory RNA viruses based on nanopore sequencer
CN113637796A (en) * 2021-07-06 2021-11-12 杭州圣庭医疗科技有限公司 Rapid identification method for 8 herpesviruses based on nanopore sequencer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Nanopore-Based Complete Genome Sequence of a Sri Lankan Cassava Mosaic Virus (Geminivirus) Strain from Thailand;Ana M. Leiva等;Microbiology;第9卷(第6期);1-3 *
纳米孔测序技术在植物病原检测中的应用与展望;路惠馨等;农业生物技术学报;第29卷(第9期);1817-1824 *

Also Published As

Publication number Publication date
CN114480740A (en) 2022-05-13

Similar Documents

Publication Publication Date Title
CN112359093B (en) Method and kit for preparing and expressing and quantifying free miRNA library in blood
CN109411014A (en) A kind of cyclic method of plant chloroplast full-length genome assembling based on the sequencing of two generations
WO2011146942A1 (en) Methods and kits to analyze microrna by nucleic acid sequencing
CN114480740B (en) Targeting sequencing library construction and detection method suitable for 15 plant quarantine viruses
CN113308565B (en) Molecular marker primer combination for rapidly identifying morphological characters of waxberry leaves and application thereof
CN108642207B (en) Detection method for rapidly and accurately identifying vaccinium plants
CN107075559A (en) The pre-treating method of clinical micro biopsy paraffin-embedded tissue
CN113151542A (en) Development method and application of pinus armandi genome SNP
CN112695133B (en) Method for screening molecular marker related to pear brown skin by using BSA-seq
CN114231602B (en) Method for rapidly measuring and calculating cold demand of peach variety based on qRT-PCR detection method
CN115261504B (en) Molecular marker related to pear pollen abortion traits and screening method thereof
CN114277091B (en) Method for constructing high-quality immune repertoire library
CN110923304B (en) Molecular marker, primer pair and method for identifying sex of ginkgo biloba
CN109609681B (en) Identification method of loblolly pine individual based on chloroplast genome sequence
CN110819734B (en) Apple tree rot fungus LAMP amplification primer and apple tree rot disease detection kit
CN107760796A (en) A kind of SSR molecular marker primer of Rapid identification locust tree polyploid
CN111763668B (en) Sequencing primer group and PCR-based whole genome sequencing method
CN108728579B (en) Method for detecting various plant viruses by adopting direct RT-PCR (reverse transcription-polymerase chain reaction) of plant microtissue
CN102766700B (en) Primer set for testing mud crab bicistronic mRNA virus, test method and rapid diagnosis kit
CN113637783B (en) Old mango mtSSR marker primer developed based on mitochondrial genome sequence and application thereof
CN116536394B (en) Construction method of marine organism single cell transcriptome library
CN112075343B (en) Method for simply, conveniently and effectively detecting existence of label in tobacco gene editing material
CN113151425B (en) Single cell sequencing method for improving accuracy based on key indexes
CN116411127B (en) Molecular marker primer combination for rapidly identifying mature-period characters of peach fruits and application thereof
CN113481319B (en) SSR molecular marker primer for identifying green and crisp plum varieties and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant