CN104357581B - Ebola virus nucleic acid molecules characteristic standard sample and preparation method thereof - Google Patents
Ebola virus nucleic acid molecules characteristic standard sample and preparation method thereof Download PDFInfo
- Publication number
- CN104357581B CN104357581B CN201410597668.9A CN201410597668A CN104357581B CN 104357581 B CN104357581 B CN 104357581B CN 201410597668 A CN201410597668 A CN 201410597668A CN 104357581 B CN104357581 B CN 104357581B
- Authority
- CN
- China
- Prior art keywords
- standard sample
- sequence
- nucleic acid
- ebola virus
- plasmid
- 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.)
- Expired - Fee Related
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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the preparation method of a kind of Ebola virus nucleic acid molecules characteristic standard sample, preparing Ebola virus nucleic acid molecules characteristic standard sample by steps such as the synthesis of sequence, cloning vector, purpose fragment and the extraction of the connection of carrier, Plastid transformation and recombinant plasmid, lyophilized preservations, the present invention prepares and a kind of comprises virus characteristic sequence information, is suitable to analyze this virus and the standard sample of contents level in sample；The present invention provide standard sample uniformity is good, stability strong, can preserve for a long time, purity is good, and preparation method process is simple, can be that Ebola virus detection research, medical research etc. provide standard sample, to realize the result comparison of different experiments room, thus ensure the quality control in laboratory；The most also realize the quick and precisely detection of Ebola virus is provided standard sample for enterprises such as inspection and quarantine mechanism, foreign trades.
Description
Technical field
The present invention relates to a kind of Ebola virus nucleic acid molecules characteristic standard sample and preparation method thereof, belong to molecular biosciences
Field.
Background technology
On May 14 nineteen ninety-five (lunar calendar April 15), Zaire finds rare infectious disease Ebola.Ebola (Ebola),
It is used to call the generic term that a group belongs to fiber Viraceae Ebola virus subordinate's several viral, may result in Ebola virus
Hemorrhagic fever, suffers from this disease and can cause people in extremely, comprise several symptoms in various degree, change including nausea,vomiting,diarrhea, the colour of skin,
Muscular stiffness, internal hemorrhage, external hemorrhage, fever etc., the infected's symptom be all the Marburg virus of fiber Viraceae extremely
Similar.This virus is with Ebola river name (this state is once called as Zaire) of Africa cd, and this place is close to the most quick-fried
The clan sent out, the Congo is still the location of nearest four outbursts, including being once very popular of in May, 2005.
Ebola virus, bio-safety grade is 4 grades, and (AIDS is 3 grades, and SARS is 3 grades, and the biggest protection of progression is the tightest
Lattice).Virus latency was up to 2 to 21 days, but generally only had 5 days to 10 days.In this virus epidemic situation of Africa outburst in 2014, this
Ebola's epidemic situation the most serious since having record has caused 932 people dead.
At present, the diagnostic method of Ebola virus is mainly by virus purification, agarose immunodiffusion technique, molecular biology
Etc. diagnostic techniques.In recent years, along with the development of Protocols in Molecular Biology, real-time fluorescence PCR technology is quick with it, sensitive, special
Property good advantage account for the biggest advantage, cause the concern of numerous researcher, but existing molecular Biological Detection kit mostly be public
Take charge of and design positive control according to document, there is no unified standard.Along with the increase of foreign trade, the inspection process time limit shortens,
It is difficult to ensure that the quality control in laboratory.
Summary of the invention
For the problems referred to above, the present invention develops a kind of Ebola virus nucleic acid molecules characteristic standard sample, can be used for feature
Positive control during Sequence Detection, to ensure the tractability of testing result.Concrete technical scheme is as follows.
The preparation method of Ebola virus nucleic acid molecules characteristic standard sample of the present invention, mainly comprises the steps:
1) synthesis virus sequence, its sequence is as described in Sequence NO.1: and before every section of sequence, increase a T7 startup
Son；
2) composition sequence flush end is cloned into plasmid and by being digested qualification and glue purification；
3) purpose fragment and the coupled reaction of carrier；
4) by competence Bacterial Transformation plasmid；
5) recombinant plasmid extracts；
6) use spectrophotometer method that recombinant plasmid is carried out quality testing；
7) choose purity and the most qualified recombinant plasmid of integrality, solution mixed, and again measure concentration and
Purity, then carries out packing lyophilized, the standard sample prepared is placed in-80 DEG C of Refrigerator stores.
Step described in the preparation method of above-mentioned Ebola virus nucleic acid molecules characteristic standard sample also includes recombinant plasmid
Qualification: from the bacterium solution that step 4) obtains, i.e. take 1-3 bacterium colony, be inoculated in the LB culture medium containing ammonia benzyl cultivation, extract matter
Grain, then carries out PCR amplification, and PCR primer carries out agarose gel electrophoresis, screening positive clone.
Step described in the preparation method of above-mentioned Ebola virus nucleic acid molecules characteristic standard sample also includes nucleotide sequence
Measure and sequence comparative analysis: the bacterium of positive recombinant plasmid will be accredited as through PCR and check order and carry out homology analysis.
The Ebola virus nucleic acid molecules characteristic standard sample that the present invention is obtained, is containing Sequence NO.1's
The recombinant vector of nucleotide sequence.And described recombinant vector is plasmid.
Further, above-mentioned steps 2) the concretely comprising the following steps of described clone and glue purification: sequence flush end is cloned into pUC19
In plasmid；It is digested and identifies that selection EcoR I and Xba I is digested as the cloning site of plasmid vector, 37 DEG C, react 1-2h,
Reaction system is DNA 16 μ L, 10X buffer 2 μ L, EcoRI 1 μ L, XbaI 1 μ L；Digestion products is through 1% Ago-Gel
Electrophoresis, cuts required fragment, and glue reclaims and purifies.
Further, above-mentioned steps 3) coupled reaction of described purpose fragment and carrier concretely comprises the following steps: condition is 16 DEG C,
Reaction 16h, reaction system is: step 2) the purified product 1 μ L, composition sequence X μ L that obtain: its mol ratio with carrier is 3 ~
20:1, T4 Ligase 1 μ L, 10X Ligase solution 1 μ L, adds deionized water polishing to 10 μ L.
Further, above-mentioned steps 4) the concretely comprising the following steps of described competence Bacterial Transformation plasmid: competence bacterium is added
In the Eppendorf pipe that precooling is aseptic, take the coupled reaction liquid that step 3) obtains, be added to the pipe containing DH5 α competence bacterium
In, gently mix, stand 30min, 42 DEG C of heat shock 90s on ice, do not rock pipe, stand 5min on ice, add the LB training without ammonia benzyl
Supporting base, 37 DEG C, 225rpm, cultivation 1h obtain conversion fluid；Take on the LB solid medium that conversion fluid is coated containing ammonia benzyl, 37 DEG C of inversions
Cultivate 16h.
The present invention is on the basis that RNA sequence based on virus is complementary with cDNA sequence, utilizes DNA synthetic technology
The nucleic acids characteristic sequence of synthesis virus, is cloned in plasmid vector, prepares one and comprises virus characteristic sequence information, fits
In analyzing this virus and the standard sample of contents level in sample；The standard sample uniformity that the present invention provides is good, stability strong,
Can preserve for a long time, purity good, and preparation method process is simple, can be that Ebola virus detects research, medical research etc. and carries
For standard sample, to realize the result comparison of different experiments room, thus ensure the quality control in laboratory；The most also for inspection and quarantine
The enterprise such as mechanism, foreign trade realizes the quick and precisely detection of Ebola virus is provided standard sample.
Accompanying drawing explanation
Fig. 1 plasmid enzyme restriction electrophoretogram, Fig. 2 recombinant plasmid sequencer map, Fig. 3 sequence alignment is reported.
Detailed description of the invention
Experiment material: pUC19 plasmid, agarose, restriction enzyme EcoR I and Xba I, T4 Ligase kit,
Gel purification kit, DH5 α bacterial strain, LB culture medium are purchased from Dalian treasured bioengineering Co., Ltd, Wizard PlusSV
Minipreps DNA Purification System is purchased from promega company.
Embodiment 1: the preparation of standard sample
1. composition sequence: utilize software Clone Manager 7.0 to download the sequence published from GenBank, enter
Row homology analysis and primer coupling, analyze and determine the virus sequence fitted to: CTA CTA CCA CAA TAT CGG AAC
TTT TCT TTC TCA TTG AAA GAG AAA GAG TTG AAT GTA GGT AGA ACC TTC GGA AAA TTG
CCT TAT CCG ACT CGC AAT GTT CAA ACA CTT TGT GAA GCT CTG TTA GCT GAT GGT CTT
GCT AAA GCA TTT CCT AGC AAT ATG ATG GTA GTT ACG GAA CGT GAG CAA AAA GAA AGC
TTA TTG CAT CAA GCA TCA TGG CAC CAC ACA AGT GAT GAT TTT GGT GAA CAT GCC ACA
GTT AGA GGG AGT AGC TTT GTA ACT GAT TTA GAG AAA TAC AAT CTT GCA TTT AGA TAT
GAG TTT ACA GCA CCT TTT ATA GAA TAT TGC AAC CGT TGC TAT GGT GTT AAG AAT GTT
TTT AAT TGG ATG CAT TAT ACA ATC CCA CAG TGT TAT ATG CAT GTC AGT GAT TAT TAT
AAT CCA CCA CAT AAC CTC ACA CTG GAG AAT CGA GAC AAC CCC CCC GAA GGG CCT AGT
TCA TAC AGG GGT CAT ATG GGA GG, sequent synthesis authorized company completes；For ease of nucleic acid sequence is external
It is transcribed into RNA, before every section of sequence, increases a T7 promoter, sequence 20bp:TAA TAC GAC TCA CTA TAG GG；
2. composition sequence flush end is cloned into pUC19 plasmid and by being digested qualification and glue purification: sequence be cloned into
In pUC19 plasmid, analyze through sequence restriction enzyme site, select EcoR I and Xba I to carry out enzyme as the cloning site of plasmid vector
Cut, as it is shown in figure 1,37 DEG C, react 1-2h, reaction system is as follows:
Digestion products, through 1% agarose gel electrophoresis, cuts required fragment, says according to Takara company gel purification kit
Bright book carries out glue and reclaims purifying；
3. the fragment of mesh and the coupled reaction of carrier
Carrying out coupled reaction after glue purification, 16 DEG C, react 16h, system is as follows:
4. the preparation of competence bacterium
The frozen DH5 α bacterium of picking is inoculated in LB culture medium flat plate, is inverted overnight incubation, chooses single colony inoculation and exist for 37 DEG C
In 5mL LB culture medium in 37 DEG C of 250rpm shaken cultivation overnight, switching 1mL overnight culture enters 100mL LB fluid nutrient medium
In in 37 DEG C cultivate 2-3h, feed them into exponential phase OD600Being about 0.3,4 DEG C, 4,000g are centrifuged 10min, reclaim bacterium,
Remove most culture medium, add 1 × TSS Buffer of 10mL precooling, resuspended bacterium, be distributed into 200 μ L/ pipes, be placed in-70 DEG C standby；
5. by competence Bacterial Transformation plasmid: DH5 α competent cell takes out from-70 DEG C of refrigerators, is placed in thawed on ice,
Take 100 μ L to be added in the 1.5mL Eppendorf pipe that precooling is aseptic.Take 10 μ L above-mentioned coupled reaction liquid, be added to experience containing DH5 α
In the pipe of state bacterium, gently mix, stand 30min on ice.42 DEG C of heat shock 90s, do not rock pipe.Stand 5min on ice, add 0.9mL
LB culture medium without ammonia benzyl, 37 DEG C, 225rpm, cultivates 1h.Take 100-300 μ L conversion fluid to coat containing ammonia benzyl (100 μ g/mL)
LB solid medium on, 37 DEG C be inverted cultivate 16h；
6. the qualification of recombinant plasmid
Take 1-3 bacterium colony, be inoculated in the 5mL LB culture medium containing ammonia benzyl, 37 DEG C, 225rpm, cultivate 12h, extract plasmid,
And with as template, carry out PCR amplification with universal primer M13, PCR primer carries out agarose gel electrophoresis, screening positive clone；
7. determining nucleic acid sequence and sequence alignment analysis
The bacterium being accredited as positive recombinant plasmid through PCR send Takara company to check order, as shown in Figure 2；Sequencing result is used
Clone Manager 7.0 software carries out sequence homology analysis qualification, as it is shown on figure 3, uniformity is 100%；
8. the extraction of recombinant plasmid
Use Wizard PlusSV Minipreps DNA Purification System, take 1-10mL incubated overnight
Bacterium solution move in 10mL centrifuge tube, 3,000g are centrifuged 15min, abandon supernatant, are inverted on filter paper and make raffinate stream clean, the bacterium of collection
Body precipitation adds the 250 μ resuspended solution of L cell gently mix, complete resuspended bacterium, re-suspension liquid is moved to 1.5mL Eppendorf
Pipe, then add 250 μ L cell pyrolysis liquids in the tube, mixing 4-6 time of turning upside down；Add 10 μ L alkaline phosphatase enzyme solutions, on
Lower reverse mixing 4-6 time, room temperature stands 5min；Add 350 μ L neutralizers, turn upside down mixing 4-6 time, room temperature 12,000g from
Heart 10min；Centrifugal column is put in collecting pipe, is transferred in centrifugal column by centrifugal supernatant, room temperature 12, and 000g is centrifuged 1min, abandons
Removing centrifugal rear liquid, be reentered in collecting pipe by centrifugal column, the washing lotion that 750 μ L have added ethanol adds centrifugal column, room temperature
12,000g are centrifuged 1min, discard centrifugal rear liquid, add 250 μ L washing lotion repeat the above steps, room temperature 12, and 000g is centrifuged 2min,
Centrifugal column is transferred in the Eppendorf pipe of an aseptic 1.5ml, adds the rnase-free deionized water of 20-30 μ L sterilizing, place
2-3min, room temperature 12,000g is centrifuged 1min；Discard centrifugal column, reclaim the DNA purified ,-20 DEG C of preservations；
9. quality testing
Use spectrophotometer method test dna absorbance under 260 nm and 280 nm, to evaluate the quality of DNA.Knot
Fruit is shown in Table 1, and the content of DNA recombinant plasmid is 2.061 μ g, and purity meets the requirements；
Table 1 recombinant plasmid content and purity testing result (mass unit: ug)
10. sample packing
Choose purity and the most qualified recombinant plasmid of integrality, solution is mixed, measure concentration and pure again
Degree, adjusts solution concentration and is worth 10.000 μ g/mL, carries out the lyophilized (condition: sample is positioned over-20 DEG C of refrigerators of packing by 2 g/ pipes
Middle pre-freeze 24h, it is ensured that sample is the most freezing, after pre-freeze is terminated, is quickly placed on freeze dryer (SIM company FD5508 by sample
Type) sample tray in, keep vacuum between 30～40 mtorr, be vacuum dried 24h, be prepared as freeze-dried powder sample.),
The standard sample prepared is placed in-80 DEG C of Refrigerator stores.
Embodiment 2: the uniformity testing of standard sample
Randomly draw sample according to method of random sampling and carry out analysis of Uniformity:
1. uniformity in bottle
Randomly draw 1 bottle, after adding 200 L deionized waters, take three parts, every part of 50 L, numbered 101-103, every part of survey
Three times, the results are shown in Table 2；Three groups of qualitative datas are done variance analysis, and variance is neat, P=0.58 > 0.05, and quality is without significant difference, symbol
Closing uniformity requirement, standard deviation is 0.0007.Purity all about 1.80, meets purity requirement.
Homogeneity test result (mass unit: μ g) in 2 bottles of table
2. uniformity between bottle
20 bottles are extracted by simple random sampling method, numbering 201-220, survey 3 times for every bottle.Measuring sequence: for the first time, 201 →
220；For the second time, 220 → 201；For the third time, odd number 201 → 219 → even number 202 → 220, the results are shown in Table 3；By 20 groups of quality and
Purity data does variance analysis, and variance is neat, and P=0.92 > 0.05, content, without significant difference, meets uniformity requirement, its standard deviation
It is 0.0007.Purity all about 1.80, meets purity requirement.
Homogeneity test result (mass unit: μ g) between 3 bottles of table
。
Embodiment 3: the stability test of standard sample
Randomly draw sample according to method of random sampling and carry out stability analysis:
1. short-term stability
Randomly draw lyophilized sample respectively at-20 DEG C, 0 DEG C, 4 DEG C, 25 DEG C, under the conditions of 37 DEG C, place 2 weeks.Each temperature
Lower placement 8 bottles, totally 40 bottles, numbers from 301-350, surveys 3 inferior qualities for every bottle by totally 40 bottles.With reference to GB/T 15000.3-2008 to sample
The statistical analysis requirement of product stability, uses SPSS 20.0 software that data carry out variance analysis, and inspection level P < 0.05 is
There is notable difference；
5 groups of data (table 4) are made variance analysis, has obvious significant difference between group, compare between each group further
Relatively, no difference of science of statistics between-20 DEG C, 0 DEG C and 4 DEG C of groups, no difference of science of statistics between 25 DEG C and 37 DEG C, and-20 DEG C, 0 DEG C and 4
DEG C three groups with 25 DEG C, 37 DEG C two groups phase mutual significant differences.
Table 4 short-term stability testing result (mass unit: ug)
2. long-time stability
Randomly draw a collection of 45 bottles be firstly placed on-80 DEG C at, according to time interval take out under the conditions of-20 DEG C place 12,9,
6,3,1 months.9 bottles are placed under each time point.Synchronos method is used to measure concentration.With reference to GB/T 15000.3-2008 to sample
The statistical analysis requirement of stability, uses SPSS 20.0 software to carry out variance analysis, and inspection level P < 0.05 is the poorest for having
Different.
5 groups of data (table 5) are made variance analysis, without significant difference between group, meets long-time stability requirement.Transfer for-20 DEG C
Put 1 year sample quality standard of appraisal deviation (STDEV)=0.028.
Table 5 long-time stability experimental result (mass unit: ug)
Continued 5
Embodiment 4: the definite value of standard sample
1. cooperation definite value
The work of standard sample definite value has been participated in jointly by 7 laboratories.Ultraviolet spectrophotometry is used to carry out cooperation fixed
Value.Provide 1 bottle of sample, points three days measure for every, measure 1 every day, 3 times altogether.Value data is shown in Table 6, and laboratory is averagely surveyed
Amount data are shown in Table 7.
Table 6 laboratory measurements list (mass unit: ug)
Table 7 laboratory average measurements
2. uncertainty combination
The component of uncertainty includes that inhomogeneities error u1+ stability error between bottle (-20 DEG C/1 year) u2+ repeats to survey
Amount error u3, cooperate definite value error u4.Overall uncertainty is equal to the evolution of each partial uncertainty quadratic sum.
The partial uncertainty source of standard sample is shown in Table 8.Composite Seismogram is shown in Table 9.
It is computed, expanded uncertainty U=0.048 μ g, k=2.
The uncertain component value of table 8
Table 9 Composite Seismogram
| Duplicate measurements error | Cooperation definite value error | Composite Seismogram |
| 0.001 | 0.021 | 0.024 |
Definite value: 2.06 μ g, expanded uncertainty U=0.048 μ g, k=2.
Definite value result shows: 1, the reagent that preparation uses meets requirement；2, preparing process is reasonable；3, method of testing is full
Foot requirement.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto,
Any those familiar with the art in the technical scope of present disclosure, according to technical scheme and
Inventive concept equivalent or change in addition, all should contain within protection scope of the present invention.
SEQUENCE LISTING
<110>Xue Fang
<120>Ebola virus nucleic acid molecules characteristic standard sample and preparation method thereof
<130> 2014
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 500
<212> DNA
<213> Sequence NO.1
<400> 1
ctactaccac aatatcggaa cttttctttc tcattgaaag agaaagagtt gaatgtaggt 60
agaaccttcg gaaaattgcc ttatccgact cgcaatgttc aaacactttg tgaagctctg 120
ttagctgatg gtcttgctaa agcatttcct agcaatatga tggtagttac ggaacgtgag 180
caaaaagaaa gcttattgca tcaagcatca tggcaccaca caagtgatga ttttggtgaa 240
catgccacag ttagagggag tagctttgta actgatttag agaaatacaa tcttgcattt 300
agatatgagt ttacagcacc ttttatagaa tattgcaacc gttgctatgg tgttaagaat 360
gtttttaatt ggatgcatta tacaatccca cagtgttata tgcatgtcag tgattattat 420
aatccaccac ataacctcac actggagaat cgagacaacc cccccgaagg gcctagttca 480
tacaggggtc atatgggagg 500
<210> 2
<211> 20
<212> DNA
<213> T7
<400> 2
taatacgact cactataggg 20
Claims (5)
1. the preparation method of Ebola virus nucleic acid molecules characteristic standard sample, it is characterised in that comprise the steps:
1) synthesis virus sequence, its sequence is as described in Sequence NO.1: and before every section of sequence, increase a T7 promoter；
2) composition sequence flush end is cloned into plasmid and by being digested qualification and glue purification, concretely comprises the following steps: by sequence flush end gram
Grand enter in pUC19 plasmid；It is digested and identifies that selection EcoR I and XbaI is digested as the cloning site of plasmid vector, 37 DEG C,
Reaction 1-2h, reaction system is DNA 16 μ L, 10X buffer 2 μ L, EcoR I 1 μ L, XbaI 1 μ L；Digestion products is through 1%
Agarose gel electrophoresis, cuts required fragment, and glue reclaims and purifies；
3) purpose fragment and the coupled reaction of carrier, concretely comprises the following steps: condition is 16 DEG C, reacts 16h, and reaction system is: step
2) the purified product 1 μ L obtained, composition sequence X μ L: it is 3～20:1 with the mol ratio of carrier, T4Ligase 1 μ L, 10X
Ligase solution1 μ L, adds deionized water polishing to 10 μ L；
4) by competence Bacterial Transformation plasmid, concretely comprise the following steps: competence bacterium is added to the Eppendorf pipe that precooling is aseptic
In, take step 3) the coupled reaction liquid that obtains, it is added in the pipe containing DH5 α competence bacterium, gently mixes, stand 30min on ice,
42 DEG C of heat shock 90s, do not rock pipe, stand 5min on ice, add the LB culture medium without ammonia benzyl, and 37 DEG C, 225rpm, cultivation 1h obtain
Conversion fluid；Take on the LB solid medium that conversion fluid is coated containing ammonia benzyl, be inverted for 37 DEG C and cultivate 16h；
5) recombinant plasmid extracts；
6) use spectrophotometer method that recombinant plasmid is carried out quality testing；
7) choose purity and the most qualified recombinant plasmid of integrality, solution mixed, and again measure concentration and purity,
Then carry out packing lyophilized, the standard sample prepared is placed in-80 DEG C of Refrigerator stores.
The preparation method of Ebola virus nucleic acid molecules characteristic standard sample the most according to claim 1, it is characterised in that
Described step also includes the qualification of recombinant plasmid: from step 4) bacterium solution that obtains takes 1-3 bacterium colony, it is inoculated in the LB containing ammonia benzyl
Cultivating in culture medium, extract plasmid, carry out PCR amplification, PCR primer carries out agarose gel electrophoresis, screening positive clone.
The preparation method of Ebola virus nucleic acid molecules characteristic standard sample the most according to claim 2, it is characterised in that
Described step also includes determining nucleic acid sequence and sequence comparative analysis: will be accredited as the bacterium order-checking of positive recombinant plasmid through PCR
And carry out homology analysis.
4. the Ebola virus nucleic acid molecules characteristic standard sample prepared by method described in claim 1, i.e. contains Sequence
The recombinant vector of the nucleotide sequence of NO.1.
Ebola virus nucleic acid molecules characteristic standard sample the most according to claim 4, it is characterised in that described restructuring carries
Body is plasmid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410597668.9A CN104357581B (en) | 2014-10-30 | 2014-10-30 | Ebola virus nucleic acid molecules characteristic standard sample and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410597668.9A CN104357581B (en) | 2014-10-30 | 2014-10-30 | Ebola virus nucleic acid molecules characteristic standard sample and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104357581A CN104357581A (en) | 2015-02-18 |
| CN104357581B true CN104357581B (en) | 2016-08-24 |
Family
ID=52524887
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410597668.9A Expired - Fee Related CN104357581B (en) | 2014-10-30 | 2014-10-30 | Ebola virus nucleic acid molecules characteristic standard sample and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104357581B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190002838A1 (en) * | 2015-06-19 | 2019-01-03 | Seracare Life Sciences, Inc. | Sindbis control virus |
| CN110669793A (en) * | 2019-10-15 | 2020-01-10 | 中国科学院武汉病毒研究所 | Method for preparing lentiviral particle packaging EBOVRNA as EBOV nucleic acid detection positive reference substance |
| CN111850149A (en) * | 2020-08-06 | 2020-10-30 | 广州海关技术中心 | Vibrio parahaemolyticus nucleic acid detection plasmid DNA reference sample, preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140533B (en) * | 2010-12-24 | 2013-10-30 | 中国检验检疫科学研究院 | Marburg and Ebola dual-virus fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and dual-virus detection PCR system |
-
2014
- 2014-10-30 CN CN201410597668.9A patent/CN104357581B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN104357581A (en) | 2015-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Saren et al. | A snapshot of viral evolution from genome analysis of the tectiviridae family | |
| CN104357581B (en) | Ebola virus nucleic acid molecules characteristic standard sample and preparation method thereof | |
| CN103421906B (en) | Composition and method for detecting drug resistance of staphylococcus aureus | |
| CN112301154B (en) | RDA method and kit for rapidly detecting respiratory syncytial virus | |
| CN102234651A (en) | Nucleotide sequence of gene for resistance to Cereal cyst nematode, Heterodera avenae and application thereof | |
| CN111549182A (en) | Probe and kit for detecting SARS-CoV-2 | |
| CN111187855B (en) | RDA method and kit for rapidly detecting Feline Herpesvirus (FHV) | |
| CN111118219B (en) | RDA method and kit for rapidly detecting influenza A virus | |
| CN102719542A (en) | Simultaneous amplification and testing reagent kit for VC (vibrio cholerae) | |
| CN104388584A (en) | Rift Valley fever virus nucleic acid molecule characteristic standard sample and preparation method thereof | |
| CN103146846A (en) | Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit | |
| CN103215289B (en) | Gene sequence a for causing watermelon bisexual flower development and obtaining method thereof | |
| CN104404167A (en) | Chikungunya virus nucleic acid molecule characteristic standard sample and its preparation method | |
| CN104297220A (en) | Detection method and detection device for mercury ions | |
| Swensen et al. | Evolution of actinorhizal host plants and Frankia endosymbionts | |
| CN102250216B (en) | Rana nigromaculata antimicrobial peptide as well as gene and application thereof | |
| CN104388586A (en) | Dengue hemorrhagic fever virus I nucleic acid molecular characteristic standard sample and preparation method thereof | |
| CN104404168A (en) | Crimean-Congo hemorrhagic fever virus nucleic acid molecule characteristic standard sample and its preparation method | |
| CN112301153B (en) | RDA method and kit for rapidly detecting Canine Coronavirus (CCV) | |
| Nakayama et al. | Comparison of g23 gene sequence diversity between Novosphingobium and Sphingomonas phages and phage communities in the floodwater of a Japanese paddy field | |
| CN104388583A (en) | Lassa fever virus nucleic acid molecular characteristic standard sample and preparation method thereof | |
| CN102492702A (en) | Beta-nodavirus genome complete sequence and cloning method thereof | |
| CN104388585A (en) | Dengue hemorrhagic fever virus II nucleic acid molecular characteristic standard sample and preparation method thereof | |
| CN101319190A (en) | Nucleic acid enricher and uses thereof | |
| CN112301156A (en) | RDA method and kit for rapidly detecting human adenovirus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160824 Termination date: 20161030 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |