CN112029764B - Extraction method of latent virus genome - Google Patents
Extraction method of latent virus genome Download PDFInfo
- Publication number
- CN112029764B CN112029764B CN202011051065.0A CN202011051065A CN112029764B CN 112029764 B CN112029764 B CN 112029764B CN 202011051065 A CN202011051065 A CN 202011051065A CN 112029764 B CN112029764 B CN 112029764B
- Authority
- CN
- China
- Prior art keywords
- virus
- precipitate
- magnetic
- solution
- supernatant
- 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
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to an extraction method of a latent virus genome, which utilizes the isoelectric point difference of plant virus capsid protein and plant cell protein, the characteristics of pathogenic genetic materials and the requirements of different plant viruses on PEG concentration. The PEG-coated magnetic core is adopted to successfully separate the latent virus genome from mulberry leaves infected with 4 pathogens, and the obtained genome has higher purity and higher concentration through semi-quantitative and quantitative PCR, thus being applicable to PCR experiments and high-throughput sequencing. Solves the problems of low content of the latent virus pathogen in woody plants, few indicated plants and difficult purification.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for extracting a latent virus genome.
Background
The virus often causes huge harm to human beings, and the example that agriculture and forestry plants suffer great losses because of the virus is also inexhaustible, such as rice Douglas virus, potato virus Y, wheat yellow dwarf virus and the like.
The virus is a non-cellular organism, has small form and simple structure, and cannot be observed under an optical microscope, so that the classification and purification of the virus are particularly important for the research of the virus. Viruses cannot be cultured in selective media like other microorganisms, and cannot propagate on media for extended purification like bacteria, so viruses must be extracted from host tissues. Because viruses stimulate plants to attract the media such as insects, the mixed infection rate of several viruses, such as Y virus and S virus of potato, is more than 80% in the field-collected sample infected with the viruses. This also causes difficulties in purification of plant viruses.
At present, the virus separation mainly comprises the following methods:
centrifugation is the earliest method adopted by people, mainly utilizes the density difference of virus particles and cell fragments, but the purified virus impurities are very much, after the high-speed and ultra-high-speed centrifuges are developed, people obtain more satisfactory purification effects, and differential centrifugation and density gradient centrifugation are developed on the basis, but the large-scale devices are very expensive, the requirement on the concentration of the virus is higher, the concentration of 5-10mg/kg is the lowest limit of purification of the method, and multiple experiments are often needed to obtain the purified virus.
The precipitation method mainly uses PEG to precipitate viruses under a certain particle concentration, but the concentration difference of PEG required by different virus types is quite large, for example, 4% of PEG can precipitate tobacco mosaic virus, 11% of PEG can precipitate potato virus S, moreover, a part of non-virus substances can be precipitated by PEG, so that the purified virus contains more impurities, and the PEG precipitation method must be combined with low-speed centrifugation, so that virus particles are easy to polymerize and can not be precipitated, and are lost by centrifugation. Meanwhile, the method needs to use an organic clarifying agent, which is easy to cause harm to operators.
According to the charge method, virus particles can be directly adsorbed on the surface of polyethylene or polystyrene, and then the virus particles can be directly captured by using a polyethylene centrifuge tube, but the method is poor in nonspecific property, and the obtained genome types and the obtained genome amounts are too small.
The immune method uses the specific combination of antigen and antibody, it uses the magnetic bead wrapped by antibody or other medium easy to separate as carrier, and makes the antibody react with antigen to form antigen-antibody-medium complex, and the complex can be directionally moved under the action of magnetic field or other external force capable of separating medium so as to attain the goal of separation, and the obtained virus purity is higher, but said method must know the capsid protein of virus and can prepare polyclonal antibody, and its required period is longer.
The filtration method uses the molecular weight of the viral coat protein to centrifuge and precipitate the virus particles, and then uses a dialysis bag with the Cutoff of 3kDa to carry out dialysis, but the method has the main purpose of obtaining the protein, has longer operation period, only needs 3d dialysis, and has serious degradation of the genome of the virus.
In summary, the prior art has problems of more impurities, insufficient extraction amount or long extraction period of extracted latent virus genome.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for extracting a latent virus genome.
The invention aims to provide an extraction method of a latent virus genome, which comprises the following steps:
step one, sample pretreatment
Collecting mulberry leaves infected with latent viruses, freezing for 7-15 days, and then mixing and grinding the mulberry leaves and PBS buffer solution into slurry according to a ratio of 1g to 5-15mL to obtain a sample solution;
step two, concentration of virus
Centrifuging the sample solution at 3000-10000r, removing precipitate, and collecting supernatant to obtain virus extractive solution;
step three, magnetic bead activation
Placing the magnetic bead solution to be activated on a magnetic rack for 2-3min, and removing the supernatant; the obtained precipitate is activated by PBS buffer solution with pH of 8.0, pH of 7.6, pH of 7.2 and containing 0.25-0.35mM sodium chloride, and finally the supernatant is removed to obtain activated magnetic beads;
step four, combining magnetic beads and virus extracting solution
Mixing activated magnetic beads with virus extract, vibrating and incubating, and removing supernatant after the adsorption of a magnetic rack; adding PBS buffer solution with pH of 7.2 and 0.25-0.35mM sodium chloride into the precipitate, mixing, standing on a magnetic rack for 2-3min, removing supernatant, and collecting the precipitate as magnetic bead-virus complex;
step five, elution of latent virus genome
Eluting the magnetic bead-virus complex by using a buffer solution E containing Tris-HCl, EDTA, 1-mercaptoglycerol and SDS to obtain a virus eluent;
step six, DNA enzyme digestion
Digesting the virus eluent in the step five by DNASE enzyme I to obtain the latent virus genome.
Preferably, in the extraction method of the latent virus genome, in the first step, the freezing temperature is-28 to-18 ℃.
Preferably, in the extraction method of the latent virus genome, in the first step, the concentration of the PBS buffer is 0.1 and M, pH is 7.2.
Preferably, in the third step of the extraction method of the latent virus genome, the magnetic beads are-PEG 2000 magnetic beads.
Preferably, the extraction method of the latent virus genome comprises the following specific steps of PBS buffer solution activation:
adding PBS buffer solution with pH of 8.0 into the precipitate, mixing, placing in a magnetic rack for 2-3min, and removing supernatant; adding PBS buffer solution with pH of 7.6 into the precipitate, mixing, placing in a magnetic rack for 2-3min, and removing supernatant; adding PBS buffer solution with pH of 7.2 into the precipitate, mixing, placing in a magnetic rack for 2-3min, and removing supernatant; adding PBS buffer solution with pH of 7.2 and 0.3mM sodium chloride into the precipitate, mixing, standing in a magnetic rack for 2-3min, and removing supernatant to obtain activated magnetic beads for use.
Preferably, in the extraction method of the latent virus genome, in the fifth step, the composition of the buffer solution E is as follows: 5mM Tris-HCl,0.5mM EDTA,1mL/1000mL 1-mercaptoglycerol, 1g/100mL SDS, ddH2O as solvent, pH 8.5.
Preferably, the extraction method of the latent virus genome comprises the following specific steps of:
adding buffer solution E into the magnetic bead-virus complex, placing in a water bath at 70 ℃ for 5min, then placing in a magnetic rack for adsorption for 5min, sucking the supernatant into a new centrifuge tube, adding 2-3 times of isopropyl alcohol and 10-20 mu L of 5M sodium chloride solution into the supernatant, freezing for 1h, centrifuging for 10min at 4 ℃ and 14000r, washing the precipitate with 70-75% ethanol solution, and finally dissolving with 100 mu L of enzyme-free water to obtain virus eluent.
Preferably, the extraction method of the latent virus genome comprises the following steps:
digesting the virus eluent in the step five by using DNASE enzyme I, and inactivating the DNASE enzyme I by using 70 ℃; adding isopropanol 2-3 times the volume of the digestive juice and 10-20 mu L of 5M sodium chloride solution, freezing for 1h, centrifuging at 4 ℃ and 14000r for 10min, washing the precipitate with 70-75% ethanol, and dissolving with 50 mu L of enzyme-free water to obtain the latent virus genome.
Preferably, the extraction method of the latent virus genome further comprises a PCR detection step:
performing reverse transcription on the latent virus genome obtained in the step six, and then performing PCR detection on a reverse transcription product by using PARTF668/PARTR975, wherein the random primer sequence is 5 '-NNNNNNNN-3';
PARTF668：5’-ACATCGTCTTGGCGATCCTAA-3’
PARTR975：5’-TCTGTATTCGGCAGAACCGTAA-3’。
preferably, the extraction method of the latent virus genome further comprises the step of quantitative PCR:
the quantitative PCR primers were as follows:
YJH CV1 F：5’-GAGTACGAACACACTTGGCC-3’
YJH CV1 R：5’-AGTCTTGGTGTGCAGGGAAT-3’。
compared with the prior art, the extraction method of the latent virus genome provided by the invention has the following beneficial effects:
1. according to isoelectric point difference of plant virus capsid protein and plant cell protein, pathogenic genetic material characteristics and PEG concentration requirements of different plant viruses, the invention provides a method for preparing the plant virus. The characteristic that nonspecific magnetic beads are combined with PEG to precipitate and combine virus particles is utilized for the first time, the PEG-coated magnetic core is adopted, the latent virus genome is successfully separated from complex mulberry leaves infected with 4 pathogens, the obtained genome has higher purity and higher concentration through semi-quantitative and quantitative PCR, the problems of low pathogen content, few indication plants and difficult purification in woody plants are solved, and the obtained genome has higher concentration and proportion and can be used for PCR experiments and high-flux sequencing.
2. The invention is to accelerate the study of the biological characteristics of the mulberry latent virus, and successfully purify the mulberry latent virus genome from a mulberry leaf blade containing 4 pathogens. Comparing the adsorption specificity of the-OH magnetic beads, -COOH magnetic beads, si-HA magnetic beads, polyelectrolyte layer magnetic beads, -PEG2000 magnetic beads, tosylated magnetic beads and pure magnetic cores to different viruses, and optimizing the PH and salt concentration of PBS; and secondly, reducing the influence of host RNA by non-ultralow temperature freezing (-28 to-18 ℃), removing DNA impurities by DNASE enzyme I, removing proteins by boiling and precipitating, and obtaining the mulberry latent virus genome with higher purity. The optimal conditions proposed by the invention are as follows: frozen sample 10d at-28 ℃,1g sample was triturated with 10mL PBS; centrifuging at 5000 rpm for 10min to remove precipitate; the PH of PBS is reduced from 8.0 to 7.2 for 3 times, the influence of the magnetic bead preservation solution on the subsequent latent virus genome, electrophoresis experiments and the like is reduced, and the PEG2000 magnetic beads for activation can be effectively used.
Drawings
FIG. 1 is an electrophoretogram of the latent virus genome extracted after infection with different viruses;
lane 1, wherein, latent virus; lane 2, mulberry nematode polyhedra virus (nematode 6309); lane 3, mulberry geminivirus; lane 4, mulberry 356 small RNA; lane 5, mulberry actin gene; lane 6, mulberry TUB gene; lane 7, negative control for latent virus, distilled water; lane M, DL2000;
FIG. 2 is an electrophoresis diagram of the extracted latent virus genome after infection with different magnetic beads and different viruses;
wherein, A, mulberry geminivirus; b, mulberry actin gene; c, mulberry TUB gene; d, mulberry 356 small RNA; e, transmitting polyhedral viruses by mulberry nematodes; f, latent virus;
lane M, DL2000; lane 1, -OH hydroxyl magnetic beads; lane 2, -COOH carboxyl magnetic beads; lane 3, tosylactated magnetic beads; lane 4, -PEG2000 magnetic beads; lane 5, si-HA magnetic beads; lane 6, pure magnetic core; lanes 7,polyelectrolyte layer magnetic beads; lane 8 positive control, positive control is the product of direct extraction of mulberry leaf RNA from the sample solution of example 1, followed by reverse transcription.
Detailed Description
In order that those skilled in the art will better understand the technical scheme of the present invention, the present invention will be further described with reference to specific embodiments and drawings.
Reagents and methods not specifically identified in the present invention are all of the prior art.
Example 1
A method of extracting a latent viral genome comprising the steps of:
the concentration of PBS buffer used in example 1 was 0.1M.
Step one, sample pretreatment
Mulberry leaves infected simultaneously by four pathogens are used and identified by reverse transcription PCR. The four pathogens are respectively mulberry geminivirus (NCBI gene accession number KP 303687.1), mulberry nematode-transmitted polyhedral virus (NCBI gene accession number NC_ 038767.1), a mulberry picornavirus (mulberry 356 picornavirus, NCBI gene accession number KJ 532933), and mulberry latent virus (NCBI gene accession number MH 282498).
Mulberry leaves infected with latent virus are collected, put into a freezer at-28 ℃ for freezing for 10 days, then 1g of the leaves are put into a clean mortar, 10mL of PBS buffer solution with pH of 7.2 is added for uniform grinding, and a sample solution is obtained.
Step two, concentration of virus
Centrifuging the sample solution at 5000r for 10min, removing part of cell nuclei, chloroplasts and other precipitates, taking the supernatant as virus extract, and placing on ice or a refrigerator at 4 ℃ for standby.
Step three, magnetic bead activation
The types of the magnetic beads adopted in the step are as follows: -OH beads (primi micel, PMC 001), -carboxyl beads (primi micel, PMC 002), si-HA beads (primi micel, PMCSIHA), polyelectrolyte layer beads (Adem-tech, 07010), -PEG2000 beads (primi micel, PMC 003), tosylactated beads (semafor, M280), and pure cores (primi micel, PMC 007). The magnetic rack used in the following steps is Omega, MSD03.
Placing the magnetic bead solution to be activated on a magnetic rack for 2min, and removing the supernatant; the obtained precipitate was activated with PBS buffer containing 0.3mM sodium chloride at pH 8.0, pH 7.6, pH 7.2, and pH 7.2 in this order, and finally the supernatant was removed to obtain activated magnetic beads for use. The specific activation method is as follows:
taking 100 mu L of magnetic bead solution with the concentration of 10mg/mL, loading into a 1.5mL centrifuge tube, placing in a magnetic rack for 2min, and removing the supernatant; adding 1mL of PBS buffer solution with pH of 8.0 into the precipitate, uniformly mixing, placing the mixture in a magnetic rack for 2min, and removing the supernatant; adding 1mL of PBS buffer solution with pH of 7.6 into the precipitate, uniformly mixing, placing the mixture in a magnetic rack for 2min, and removing the supernatant; adding 1mL of PBS buffer solution with pH of 7.2 into the precipitate, uniformly mixing, placing the mixture in a magnetic rack for 2min, and removing the supernatant; 1mL of PBS buffer solution with pH 7.2 and 0.3mM sodium chloride is added into the precipitate, the mixture is placed on a magnetic rack for 2min after being uniformly mixed, and the supernatant is removed to obtain activated magnetic beads for later use.
Fourth, combining magnetic beads and virus extracting solution:
(1) 1mL of the virus extract was added to a 1.5mL magnetic bead centrifuge tube, and incubated at 25℃with 600r shaking for 2h. Removing the supernatant after the magnetic frame adsorbs for 5min;
(2) Adding 1mL of PBS buffer solution with pH of 7.2 and 0.5mM sodium chloride into the precipitate, uniformly mixing, placing the mixture in a magnetic rack for 2-3min, removing the supernatant, repeating the step (2) for 4 times, washing out impurities which are not tightly combined, and finally collecting the precipitate as the magnetic bead-virus complex.
Step five, elution of the latent virus genome:
adding 100 mu L of buffer solution E into the magnetic bead-virus compound in the step four, uniformly mixing, replacing a centrifuge tube, placing in a water bath at 70 ℃ for 5min, placing in a magnetic rack for adsorption for 5min, carefully sucking the supernatant into a new 1.5mL centrifuge tube, adding 2-3 times of isopropyl alcohol and 10-20 mu L of 5M sodium chloride solution, freezing for 1h, centrifuging at 4 ℃ for 10min at 14000r, washing the precipitate with ethanol solution with the volume fraction of 70-75% for 2 times, and dissolving with 100 mu L of enzyme-free water to obtain virus eluent.
Wherein the composition of buffer E is: 5mM Tris-HCl,0.5mM EDTA,1mL/1000mL 1-mercaptoglycerol, 1g/100mL SDS, ddH as solvent 2 O，pH 8.5。
Step six, DNA enzyme digestion:
digesting the virus eluent in the step five by using commercial DNASE enzyme I, and inactivating the DNASE enzyme I for 5min at 70 ℃; adding 2-3 times volume of isopropanol and 10-20 mu L of 5M sodium chloride solution, freezing for 1h, centrifuging at 4 ℃ and 14000r for 10min, washing the precipitate with 70-75% ethanol for 2 times, and dissolving with 50 mu L of enzyme-free water (DNASE, RNASE, PROTEASE) to obtain a sample rich in latent virus genome.
The temperature of DNASE enzyme I digestion is 37 ℃ and the time is 30min, and the system is as follows: 125 mul. 15U of DNase I,1 Xbuffer (Promega, M610A) was included.
Step seven, qualitative PCR detection:
the latent virus genome obtained in step six was reverse transcribed using a reverse transcription kit (Promega, A5001) in which random primers of random 6 were used as primers and the reverse transcription system was carried out according to the instructions (RNA template 4. Mu.L; mgCl) 2 3 μL; the others are all fixed additions to the kit instructions).
Reverse transcription conditions were 70℃for 5min;25 ℃ for 5min;42 ℃ for 60min;70 ℃ for 5min. And (5) ending.
PCR detection was performed on the reverse transcription products (genomes of the individual viruses and Morus alba) using specific primers, the sequences of which are shown in Table 1. The random primer sequence of random 6 is 5'-NNNNNN-3'. The PCR conditions were: 94 ℃ for 3min;30 cycles (94 ℃,30s;52 ℃,35s;72,1 min); 72 ℃ for 5min; preserving at 12 ℃.
TABLE 1 genomic primers for each virus and mulberry
FIG. 1 shows the electrophoresis results of the different extracted genomes, and it can be seen from FIG. 1 that the method of the present invention can be used to extract the high purity mulberry latent virus genome from the mulberry sample infected with 4 pathogens. FIG. 2 is an electrophoresis diagram of the extracted latent virus genome after infection with different magnetic beads and different viruses; the results showed that the effect of the PEG2000 beads was best, but the pure magnetic nuclei in lane 6 of FIG. 2F were also amplified to form bands, but the pure magnetic nuclei had a fine texture, were not easily recovered during the extraction process, and were unstable and therefore discarded.
Step eight, quantitative analysis:
using BIO-RAD CFX Connect TM Fluorescent quantitative PCR detection system. Using reagent PowerGreen PCR Master Mix(Applied/>Cat 4367659) for the different groups in the reverse transcription product obtained in step sevenThe genome was subjected to quantitative PCR analysis.
The primers used for quantitative PCR are shown in Table 2.
TABLE 2 quantitative primers for different genomes
The quantitative PCR reaction system is as follows:
Powergreen PCR Master Mix 12.5.5. Mu.L, 1. Mu.L for the upstream primer, 1. Mu.L for the downstream primer, 1. Mu.L for the internal reference primer, 2. Mu.L for the template, and 25. Mu.L for distilled water.
The procedure used for the quantitative PCR experiments was:
1)95.0℃for 3min
2)95.0℃for 10s
3)55.0℃for 20s
4)72.0℃for 20s
5)75.0℃for 5s
+Plate Read
6)GOTO 2,40more times
7)Melt Curve 65.0to 95.0℃,increment 0.5℃,
0:05+Plate Read
8) Ending
We counted the fluorescence values before and after treatment of the mulberry nematode-transmitted polyhedrosis virus, the mulberry gemini virus, the mulberry 356 picornavirus, the mulberry action, the mulberry TUB with the content of the latent virus remaining unchanged before and after treatment (as an internal reference for quantitative PCR, i.e., assuming that the fluorescence value of the latent virus is unchanged), and the results are shown in Table 3.
Wherein after the treatment: refers to the potentially virus genome-rich samples obtained following the procedure one to seven of example 1.
Before treatment: refers to the sample pretreatment according to the procedure of step one of example 1, the obtained sample solution, the RNA thereof was extracted with an RNA kit, and then a genome sample was obtained with a reverse transcription kit.
As can be seen from the results of Table 3, the sample extracted by the method of example 1 of the present invention has significantly reduced impurities of the mulberry nematode-transmitted polyhedrosis virus, the mulberry geminivirus, the mulberry 356 picornavirus, the mulberry action and the mulberry TUB, and the method of example 1 is illustrated by combining with PCR qualitative detection analysis to ensure the extraction amount of the latent virus.
TABLE 3 quantitative PCR results
It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Sequence listing
<110> Ankang college
<120> a method for extracting latent virus genome
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<400> 1
cctcactgaa gctcctctca ac 22
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<400> 2
acaatttccc gttcagcagt gg 22
<210> 3
<211> 22
<212> DNA
<213> artificial sequence
<400> 3
gatccacgcc atggtcgtta cc 22
<210> 4
<211> 22
<212> DNA
<213> artificial sequence
<400> 4
gtaccagtgc aagaaagcct tg 22
<210> 5
<211> 21
<212> DNA
<213> artificial sequence
<400> 5
acatcgtctt ggcgatccta a 21
<210> 6
<211> 22
<212> DNA
<213> artificial sequence
<400> 6
tctgtattcg gcagaaccgt aa 22
<210> 7
<211> 18
<212> DNA
<213> artificial sequence
<400> 7
cattgtcagg ctgtggat 18
<210> 8
<211> 21
<212> DNA
<213> artificial sequence
<400> 8
ccagaatcct cgtgattata c 21
<210> 9
<211> 20
<212> DNA
<213> artificial sequence
<400> 9
agaggtatga tggccacgca 20
<210> 10
<211> 22
<212> DNA
<213> artificial sequence
<400> 10
gattcattcg cttctgtaca ag 22
<210> 11
<211> 21
<212> DNA
<213> artificial sequence
<400> 11
agaccgccgg agcctcaaat c 21
<210> 12
<211> 23
<212> DNA
<213> artificial sequence
<400> 12
gaacgaaatt gactgtagag aac 23
<210> 13
<211> 20
<212> DNA
<213> artificial sequence
<400> 13
cattcaagcc gttctttctc 20
<210> 14
<211> 20
<212> DNA
<213> artificial sequence
<400> 14
catcaagtgg tcggtgagat 20
<210> 15
<211> 20
<212> DNA
<213> artificial sequence
<400> 15
ccaataatgt gaagtctagc 20
<210> 16
<211> 20
<212> DNA
<213> artificial sequence
<400> 16
tgaactgctc gctgaccctc 20
<210> 17
<211> 20
<212> DNA
<213> artificial sequence
<400> 17
gagtacgaac acacttggcc 20
<210> 18
<211> 20
<212> DNA
<213> artificial sequence
<400> 18
agtcttggtg tgcagggaat 20
<210> 19
<211> 19
<212> DNA
<213> artificial sequence
<400> 19
agttgcctca gagcttgca 19
<210> 20
<211> 20
<212> DNA
<213> artificial sequence
<400> 20
tccactgaag tctcgaaccc 20
<210> 21
<211> 20
<212> DNA
<213> artificial sequence
<400> 21
tccctggcac gcaagtggta 20
<210> 22
<211> 21
<212> DNA
<213> artificial sequence
<400> 22
gtacactatg gaccctggca c 21
<210> 23
<211> 20
<212> DNA
<213> artificial sequence
<400> 23
atccacaatt aaccgccagc 20
<210> 24
<211> 21
<212> DNA
<213> artificial sequence
<400> 24
tgaccgtact catgcctaac a 21
Claims (7)
1. A method of extracting a latent virus genome, comprising:
step one, sample pretreatment
Collecting mulberry leaves infected with latent viruses, freezing for 7-15 days, and then mixing and grinding the mulberry leaves and PBS buffer solution into slurry according to the ratio of 1g to 5-15mL to obtain a sample solution;
step two, concentration of virus
Centrifuging the sample solution at 3000-10000r, removing precipitate, and collecting supernatant to obtain virus extractive solution;
step three, magnetic bead activation
Placing the magnetic bead solution to be activated in a magnetic rack for 2-3min, and removing the supernatant; the obtained precipitate is activated by PBS buffer solution with pH of 8.0, pH of 7.6, pH of 7.2 and containing 0.25-0.35mM sodium chloride, and finally the supernatant is removed to obtain activated magnetic beads;
step four, combining magnetic beads and virus extracting solution
The magnetic beads are PEG2000 magnetic beads;
mixing activated magnetic beads with virus extract, vibrating and incubating, and removing supernatant after the adsorption of a magnetic rack; adding PBS buffer solution with pH of 7.2 and 0.25-0.35mM sodium chloride into the precipitate, mixing, standing on a magnetic rack for 2-3min, removing supernatant, and collecting the precipitate as magnetic bead-virus complex;
step five, elution of latent virus genome
Adding buffer solution E into the magnetic bead-virus compound, placing in a water bath at 70 ℃ for 5min, then placing in a magnetic rack for adsorption for 5min, sucking the supernatant into a new centrifuge tube, adding 2-3 times of isopropyl alcohol and 10-20 mu L of 5M sodium chloride solution into the supernatant, freezing for 1h, centrifuging for 10min at 4 ℃ and 14000r, washing the precipitate with ethanol solution with the volume fraction of 70-75%, and finally dissolving with 100 mu L of enzyme-free water to obtain virus eluent;
step six, DNA enzyme digestion
Digesting the virus eluent in the step five by using DNASE enzyme I, and inactivating the DNASE enzyme I by using 70 ℃; adding isopropanol 2-3 times the volume of the digestive juice and 10-20 mu L of 5M sodium chloride solution, freezing for 1h, centrifuging at 4 ℃ and 14000r for 10min, washing the precipitate with 70-75% ethanol, and dissolving with 50 mu L of enzyme-free water to obtain the latent virus genome.
2. The method according to claim 1, wherein in the first step, the freezing temperature is-28 to-18 ℃.
3. The method of claim 2, wherein in step one, the concentration of the PBS buffer is 0.1 to M, pH to 7.2.
4. The method of claim 1, wherein the PBS buffer activation in step three comprises the specific steps of:
adding PBS buffer solution with pH of 8.0 into the precipitate, mixing, placing in a magnetic rack for 2-3min, and removing supernatant; adding PBS buffer solution with pH of 7.6 into the precipitate, mixing, placing in a magnetic rack for 2-3min, and removing supernatant; adding PBS buffer solution with pH of 7.2 into the precipitate, mixing, placing in a magnetic rack for 2-3min, and removing supernatant; adding PBS buffer solution with pH of 7.2 and 0.25-0.35mM sodium chloride into the precipitate, mixing, standing in a magnetic rack for 2-3min, and removing supernatant to obtain activated magnetic beads for use.
5. The method of claim 1, wherein in step five, the composition of buffer E is: 5mM Tris-HCl,0.5mM EDTA,1mL/1000mL 1-mercaptoglycerol, 1g/100mL SDS, ddH as solvent 2 O，pH 8.5。
6. The method for extracting a latent virus genome according to claim 1, further comprising a PCR detection step of:
performing reverse transcription on the latent virus genome obtained in the step six, and then performing PCR detection on a reverse transcription product by using PARTF668/PARTR975, wherein the random primer sequence is 5'-NNNNNN-3';
PARTF668：5’-ACATCGTCTTGGCGATCCTAA-3’
PARTR975：5’-TCTGTATTCGGCAGAACCGTAA-3’。
7. the method of extracting a latent virus genome according to claim 1, further comprising a quantitative PCR step of:
the quantitative PCR primers were as follows:
YJH CV1 F：5’-GAGTACGAACACACTTGGCC-3’
YJH CV1 R：5’-AGTCTTGGTGTGCAGGGAAT-3’。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011051065.0A CN112029764B (en) | 2020-09-29 | 2020-09-29 | Extraction method of latent virus genome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011051065.0A CN112029764B (en) | 2020-09-29 | 2020-09-29 | Extraction method of latent virus genome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112029764A CN112029764A (en) | 2020-12-04 |
| CN112029764B true CN112029764B (en) | 2023-09-19 |
Family
ID=73573211
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011051065.0A Active CN112029764B (en) | 2020-09-29 | 2020-09-29 | Extraction method of latent virus genome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112029764B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296126A (en) * | 2011-06-01 | 2011-12-28 | 中华人民共和国北京出入境检验检疫局 | Detection method of hop stunt viroid |
| CN104805073A (en) * | 2015-04-21 | 2015-07-29 | 益百尚(北京)生物技术有限责任公司 | Kit for extracting viral genome nucleic acid and use method thereof |
| CN105734055A (en) * | 2016-05-05 | 2016-07-06 | 安康学院 | Method for purifying mulberry phytoplasma genome |
-
2020
- 2020-09-29 CN CN202011051065.0A patent/CN112029764B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296126A (en) * | 2011-06-01 | 2011-12-28 | 中华人民共和国北京出入境检验检疫局 | Detection method of hop stunt viroid |
| CN104805073A (en) * | 2015-04-21 | 2015-07-29 | 益百尚(北京)生物技术有限责任公司 | Kit for extracting viral genome nucleic acid and use method thereof |
| CN105734055A (en) * | 2016-05-05 | 2016-07-06 | 安康学院 | Method for purifying mulberry phytoplasma genome |
Non-Patent Citations (2)
| Title |
|---|
| 周云龙.转基因生物标志物物质研制与应用.中国质检出版社,2014,第6章 基因组DNA的标准物质制备. * |
| 磁珠和蛋白质标签特异性免疫沉淀体系的建立;文莉薇;吴智妹;朱鸿亮;;中国农业科技导报(03);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112029764A (en) | 2020-12-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106574265B (en) | Method for isolating RNA with high yield | |
| CA2012777C (en) | Process for isolating nucleic acid | |
| CN112322612A (en) | Plasmid extraction kit and extraction method | |
| CA2310782A1 (en) | Methods of nucleic acid isolation | |
| CN112391384B (en) | Kit for rapidly extracting genomic DNA of gram-negative bacteria and extraction method | |
| CN102676503A (en) | Method for quickly extracting DNA (deoxyribonucleic acid) | |
| CN116286794A (en) | Extraction reagent combination, extraction kit and extraction method for microbial genome DNA in soil | |
| CN111662959A (en) | High-throughput rapid detection method for fungi | |
| CN105734055B (en) | A kind of purification process for mulberry tree phytoplasma genome | |
| CN112029764B (en) | Extraction method of latent virus genome | |
| CN101086011B (en) | Edible mushroom and plant double-strand RNA virus detection kit and its uses | |
| EP4163371A1 (en) | Nucleic acid extraction method and application | |
| CA2498951A1 (en) | Method for enriching procaryotic dna | |
| Smith et al. | Rapid purification of 70 S RNA from media of cells producing RNA tumor viruses | |
| CN114703173A (en) | Lambda phage DNA extraction kit and extraction method | |
| KR101760726B1 (en) | Method for isolation of metagenomic DNA from animal food with detaching of bacteria and phenol-chloroform | |
| CN109055361A (en) | It is a kind of extract ganoderma lucidum total DNA reagent and its application | |
| CN108866042B (en) | Extraction method of trace RNA | |
| CN113308460A (en) | Kit for extracting bacterial DNA in paraffin section | |
| CN106957842B (en) | Extraction method of BAC clone DNA | |
| CN111235249B (en) | Method for detecting mitochondrial genome open state | |
| CN109652409A (en) | A method of extracting aureus plasmid | |
| CN109022426A (en) | It is a kind of extract Antrodia camphorata total DNA reagent and application | |
| CN108060160A (en) | A kind of fast preparation method of BAC-DNA for FISH probe mark | |
| RU2736224C2 (en) | Method for rapid release of genomic dna for clinical studies |
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 |