CN104388585A - Dengue hemorrhagic fever virus II nucleic acid molecular characteristic standard sample and preparation method thereof - Google Patents
Dengue hemorrhagic fever virus II nucleic acid molecular characteristic standard sample and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing a dengue hemorrhagic fever virus II nucleic acid molecular characteristic standard sample. The dengue hemorrhagic fever virus II nucleic acid molecular characteristic standard sample is prepared according to the steps of synthesis of sequences, cloning of vectors, connection of target fragments and vectors, plasmid transformation, extraction and freezing dried storage of recombinant plasmids and the like. A standard sample that contains virus characteristic sequence information and is suitably used for analyzing the virus and the content thereof in a sample is prepared according to the method; the standard sample provided by the invention has good homogeneity, high stability and good purity, can be stored for long term, and is prepared according to the simple preparation method; a good standard sample is provided for detection research, medical research and the like of dengue hemorrhagic fever virus II type so as to achieve comparison of results from different laboratories and guarantee the quality control of laboratories; and a standard sample is also provided for fast and accurate detection of dengue hemorrhagic fever virus II type for inspection and quarantine institutions, import and export trade and the like.
Description
Technical field
The present invention relates to a kind of dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample and preparation method thereof, belong to biology field.
Background technology
Singapore hemorrhagic fever is widely current in subtropical and tropical zones, is a kind of wide, that morbidity is many, harm is larger human infectious disease that distributes.In Asia, Pacific Islands and in, many countries such as South America have caused serious threat all.After World War II, also once had popular in Japan.In recent years, in Asia, Africa and the torrid zone of South America and the sickness rate of subtropical zone dengue virus infection be obvious ascendant trend.China, was all found on the ground such as Hainan Island and Guangxi in 1978 in Foshan this disease of Late Cambrian later.
At present, the diagnostic method of dengue hemorrhagic fever virus Type II is primarily of diagnostic techniquess such as virus purification, agarose immunodiffusion technique, molecular biology.In recent years, along with the development of Protocols in Molecular Biology, the advantage that real-time fluorescence PCR technology is quick with it, responsive, specificity is good accounts for very large advantage, cause the concern of numerous investigator, but existing molecular Biological Detection test kit mostly is company and designs positive control according to document, does not have unified standard.Along with the increase of foreign trade, the inspection process time limit shortens, and is difficult to the quality control ensureing laboratory.
Summary of the invention
For the problems referred to above, the present invention develops a kind of dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample, can be used for positive control when characteristic sequence detects, to ensure the tractability of detected result.Concrete technical scheme is as follows.
The preparation method of dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample of the present invention, mainly comprises the steps:
1) synthesize virus sequence, its sequence is as described in Sequence NO.1: and a T7 promotor is increased before every section of sequence;
2) composition sequence flush end be cloned into plasmid and cut qualification and glue purification by enzyme;
3) ligation of object fragment and carrier;
4) by competence Bacterial Transformation plasmid;
5) recombinant plasmid extracts;
6) spectrophotometer method is adopted to carry out quality examination to recombinant plasmid;
7) choose purity and all qualified recombinant plasmid of integrity, solution is mixed, and again measure concentration and purity, then carry out packing freeze-drying, the standard model prepared is placed in-80 DEG C of Refrigerator stores.
The qualification of recombinant plasmid is also comprised: namely from the bacterium liquid that step 4) obtains, get 1-3 bacterium colony in step described in the preparation method of above-mentioned dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample, be inoculated in the LB substratum containing ammonia benzyl and cultivate, extract plasmid, then pcr amplification is carried out, PCR primer carries out agarose gel electrophoresis, screening positive clone.
Determining nucleic acid sequence and sequence comparative analysis is also comprised: the bacterium being about to be accredited as through PCR positive recombinant plasmid is checked order and carries out homology analysis in step described in the preparation method of above-mentioned dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample.
The dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample that the present invention obtains, is the recombinant vectors of the nucleotide sequence containing Sequence NO.1.And described recombinant vectors is plasmid.
Further, above-mentioned steps 2) concrete steps of described clone and glue purification are: sequence flush end are cloned in pUC19 plasmid; Enzyme is cut qualification and is selected
ecor I and
xbai carries out enzyme as the cloning site of plasmid vector and cuts, 37 DEG C, reaction 1-2h, and reaction system is DNA 16 μ L, 10X buffer 2 μ L,
ecorI 1 μ L,
xbai 1 μ L; Digestion products, through 1% agarose gel electrophoresis, cuts required fragment, and glue reclaims purifying.
Further, above-mentioned steps 3) the ligation concrete steps of described object fragment and carrier are: condition is 16 DEG C, reaction 16h, reaction system is: step 2) the purified product 1 μ L that obtains, composition sequence X μ L: the mol ratio of itself and 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) concrete steps of described competence Bacterial Transformation plasmid are: competence bacterium are added in the aseptic Eppendorf pipe of precooling, get the ligation liquid that step 3) obtains, be added in the pipe containing DH5 α competence bacterium, light mixing, leaves standstill 30min, 42 DEG C of heat shock 90s on ice, do not rock pipe, leave standstill 5min on ice, add not containing the LB substratum of ammonia benzyl, 37 DEG C, 225rpm, cultivates 1h and obtains conversion fluid; Get on LB solid medium that conversion fluid coats containing ammonia benzyl, be inverted for 37 DEG C and cultivate 16h.
The present invention is based on the RNA sequence of virus and the basis of cDNA sequence complementation, DNA synthetic technology is utilized to synthesize the nucleic acids characteristic sequence of virus, be cloned in plasmid vector, prepared a kind of standard model comprising virus characteristic sequence information, be suitable for this virus and contents level in analytic sample; Standard model good uniformity provided by the invention, stability is strong, can preserve for a long time, purity is good, and preparation method's process is simple, standard model can be provided for dengue hemorrhagic fever virus Type II detect delay, medical research etc., to realize the result comparison of different experiments room, thus ensure the quality control in laboratory; Simultaneously also for the enterprises such as inspection and quarantine mechanism, foreign trade realize providing standard model to the quick and precisely detection of dengue hemorrhagic fever virus Type II.
Accompanying drawing explanation
Fig. 1 plasmid enzyme restriction electrophorogram, Fig. 2 recombinant plasmid sequencer map, Fig. 3 sequence alignment is reported.
Embodiment
Experiment material: pUC19 plasmid, agarose, restriction enzyme
ecor I and
xbai, T4 Ligase test kit, gel purification kit, DH5 α bacterial strain, LB substratum are purchased from the precious biotechnology company limited in Dalian, and Wizard PlusSV Minipreps DNA Purification System is purchased from promega company.
Embodiment 1: the preparation of standard model
1. composition sequence: utilize software Clone Manager 7.0 to download from GenBank the sequence published, carry out homology analysis and primer coupling, analyze the virus sequence determining to fit to: ATG AAT AAC CAA CGA AAA AAG GCG AGA AGT ACG CCT TTC AAT ATG CTG AAA CGC GAG AGA AAC CGC GTG TCA ACT GTG CAA CAG CTG ACA AAG AGA TTC TCA CTT GGA ATG CTG CAA GGA CGC GGA CCA TTA AAA CTG TTC ATG GCC CTT GTG GCG TTC CTT CGT TTC CTA ACA ATC CCA CCA ACA GCA GGG ATA CTA AAA AGA TGG GGA ACG ATC AAA AAA TCA AAA GCT ATC AAT GTC TTG AGA GGG TTC AGG AA, sequent synthesis authorized company completes, for ease of nucleic acid sequence in-vitro transcription is become RNA, before every section of sequence, increase a T7 promotor, sequence 20bp:TAA TAC GAC TCA CTA TAG GG,
2. composition sequence flush end be cloned into pUC19 plasmid and cut qualification and glue purification by enzyme: sequence clone being entered in pUC19 plasmid, analyzing through sequence restriction enzyme site, selecting
ecor I and
xbai carries out enzyme as the cloning site of plasmid vector and cuts, as shown in Figure 1,37 DEG C, reaction 1-2h, reaction system is as follows:
Digestion products, through 1% agarose gel electrophoresis, cuts required fragment, carries out glue reclaim purifying according to Takara company gel purification kit specification sheets;
3. the ligation of object fragment and carrier
Carry out ligation after glue purification, 16 DEG C, reaction 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, be inverted overnight incubation for 37 DEG C, choose single colony inoculation to spend the night in 37 DEG C of 250rpm shaking culture in 5mL LB substratum, switching 1mL overnight culture enters in 100mL LB liquid nutrient medium cultivates 2-3h in 37 DEG C, makes it to enter logarithmic phase OD
600be about 0.3,4 DEG C, the centrifugal 10min of 4,000g, reclaim bacterium, remove most substratum, add 1 × TSS Buffer of 10mL precooling, resuspended bacterium, be distributed into 200 μ L/ and manage, be placed in-70 DEG C for subsequent use;
5. by competence Bacterial Transformation plasmid: DH5 α competent cell takes out from-70 DEG C of refrigerators, is placed in thawed on ice, gets 100 μ L and is added in the aseptic 1.5mL Eppendorf pipe of precooling.Get the above-mentioned ligation liquid of 10 μ L, be added in the pipe containing DH5 α competence bacterium, gently mix, leave standstill 30min on ice.42 DEG C of heat shock 90s, do not rock pipe.Leave standstill 5min on ice, add 0.9mL not containing the LB substratum of ammonia benzyl, 37 DEG C, 225rpm, cultivate 1h.Get on LB solid medium that 100-300 μ L conversion fluid coats containing ammonia benzyl (100 μ g/mL), be inverted for 37 DEG C and cultivate 16h;
6. the qualification of recombinant plasmid
Get 1-3 bacterium colony, be inoculated in 5mL containing ammonia benzyl LB substratum in, 37 DEG C, 225rpm, cultivate 12h, extract plasmid, and with for 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 Clone Manager 7.0 software carries out sequence homology analysis qualification, and as shown in Figure 3, consistence is 100%;
8. the extraction of recombinant plasmid
Adopt Wizard PlusSV Minipreps DNA Purification System, the bacterium liquid getting 1-10mL incubated overnight moves in 10mL centrifuge tube, the centrifugal 15min of 3,000g, abandons supernatant, being inverted on filter paper makes raffinate stream clean, add the resuspended solution of 250 μ L cell in the bacterial sediment collected gently to mix, complete resuspended bacterium, moves to 1.5mL Eppendorf and manages by re-suspension liquid, add 250 μ L cell pyrolysis liquids more in the tube, mixing 4-6 time of turning upside down; Add 10 μ L alkaline phosphatase enzyme solution, mixing 4-6 time of turning upside down, room temperature leaves standstill 5min; Add 350 μ L neutralizers, mixing 4-6 time of turning upside down, the centrifugal 10min of room temperature 12,000g; Centrifugal column puts into collection tube, centrifugal supernatant is transferred in centrifugal column, room temperature 12, the centrifugal 1min of 000g, discards centrifugal rear liquid, is reentered into by centrifugal column in collection tube, the washing lotion that 750 μ L have added ethanol is added centrifugal column, the centrifugal 1min of room temperature 12,000g, discards centrifugal rear liquid, add 250 μ L washing lotions and repeat above-mentioned steps, the centrifugal 2min of room temperature 12,000g, is transferred in the Eppendorf pipe of an aseptic 1.5ml by centrifugal column, add the rnase-free deionized water of 20-30 μ L sterilizing, place 2-3min, the centrifugal 1min of room temperature 12,000g; Discard centrifugal column, reclaim the plasmid DNA of purifying ,-20 DEG C of preservations;
9. quality examination
Adopt the absorbancy of spectrophotometer method test dna under 260 nm and 280 nm, to evaluate the quality of DNA.The content that the results are shown in Table 1, DNA recombinant plasmid is 2.051 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 all qualified recombinant plasmid of integrity, solution is mixed, measure concentration and purity again, adjustment strength of solution is worth 10.000 μ g/mL, packing freeze-drying (condition: sample is positioned over pre-freeze 24h in-20 DEG C of refrigerators is carried out by 2 μ g/ pipes, ensure that sample is thoroughly freezing, after pre-freeze is terminated, fast sample is placed in the sample tray of Freeze Drying Equipment (SIM company FD5508 type), keep vacuum tightness between 30 ~ 40 mtorr, vacuum-drying 24h, is prepared into lyophilized powder sample.), the standard model prepared is placed in-80 DEG C of Refrigerator stores.
Embodiment 2: the uniformity testing of standard model
Randomly draw sample according to random sampling and carry out analysis of Uniformity:
1. homogeneity bottle
Randomly draw 1 bottle, after adding 200 μ L deionized waters, get three parts, every part of 50 μ L, are numbered 101-103, survey three times, the results are shown in Table 2 for every part; Three groups of qualitative datas are done variance analysis, and quality, without significant difference, meets uniformity requirement, and standard deviation is 0.0006.Purity, all about 1.80, meets purity requirement.
Homogeneity test result (mass unit: μ g) in table 2 bottle
2. homogeneity bottle
Extract 20 bottles 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; 20 groups of quality and purity data are done variance analysis, and variance is neat,
p=0.96>0.05, content, without significant difference, meets uniformity requirement, and its standard deviation is 0.0007.Purity, all about 1.80, meets purity requirement.
Homogeneity test result (mass unit: μ g) between table 3 bottle
Embodiment 3: the stability test of standard model
Randomly draw sample according to random sampling and carry out stability analysis:
1. short-term stability
Randomly draw freeze-drying sample respectively at-20 DEG C, 0 DEG C, 4 DEG C, 25 DEG C, under 37 DEG C of conditions, place 2 weeks.Place 8 bottles at each temperature, totally 40 bottles, totally 40 bottles, number from 301-350, survey 3 inferior qualities for every bottle.With reference to GB/T 15000.3-2008 to the statistical study requirement of sample stability, SPSS 20.0 software is adopted to carry out variance analysis to data;
5 groups of data (table 4) are done variance analysis, obvious significant difference is had between group, compare between each group further, 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 with 4 DEG C three groups with 25 DEG C, 37 DEG C two groups phase mutual significant differences.
Table 4 short-term stability detected result (mass unit: ug)
2. permanent stability
Randomly draw a collection of 45 bottles be first placed on-80 DEG C at, according to the timed interval take out under-20 DEG C of conditions place 12,9,6,3,1 month.9 bottles are placed under each time point.Synchronos method is adopted to measure concentration.With reference to GB/T 15000.3-2008 to the statistical study requirement of sample stability, SPSS 20.0 software is adopted to carry out variance analysis.
5 groups of data (table 5) are done variance analysis, without significant difference between group, meets permanent stability requirement.1 year sample quality standard of appraisal deviation (STDEV)=0.002 is placed at-20 DEG C.
Table 5
permanent stability experimental result (mass unit: ug)
Continued 5
Embodiment 4: the definite value of standard model
1. cooperate definite value
The work of standard model definite value has been participated in jointly by 7 laboratories.Ultraviolet spectrophotometry is adopted to carry out cooperation definite value.Provide 1 bottle of sample for every, point measurement in three days, measures 1 every day, amounts to 3 times.Value data is in table 6, and laboratory averaged measurements is in table 7.
Table 6 laboratory measurements table look-up (mass unit: ug)
Table 7 laboratory average measurements
2. uncertainty combination
The component of uncertainty comprises ununiformity error u1+ stability error between bottle (-20 DEG C/1 year) u2+ replicate measurement error u3, cooperation definite value error u4.Overall uncertainty equals the evolution of each partial uncertainty sum of squares.
The partial uncertainty source of standard model is in table 8.Composite Seismogram is in table 9.
As calculated, expanded uncertainty U=0.032 μ g, k=2.
The uncertain component value of table 8
Table 9 Composite Seismogram
Definite value: 2.05 μ g, expanded uncertainty U=0.032 μ g, k=2.
Definite value result shows: the reagent that 1, preparation adopts meets requirement; 2, blending process is reasonable; 3, testing method meets the demands.
The above; be only the present invention's preferably embodiment; but protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to according to technical scheme of the present invention and inventive concept thereof and replace or change, all should be encompassed within protection scope of the present invention.
SEQUENCE LISTING
<110> Xue Fang
<120> dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample and preparation method thereof
<130> 2014
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 257
<212> DNA
<213> Sequence NO.1
<400> 1
atgaataacc aacgaaaaaa ggcgagaagt acgcctttca atatgctgaa acgcgagaga 60
aaccgcgtgt caactgtgca acagctgaca aagagattct cacttggaat gctgcaagga 120
cgcggaccat taaaactgtt catggccctt gtggcgttcc ttcgtttcct aacaatccca 180
ccaacagcag ggatactaaa aagatgggga acgatcaaaa aatcaaaagc tatcaatgtc 240
ttgagagggt tcaggaa 257
<210> 2
<211> 20
<212> DNA
<213> T7
<400> 2
taatacgact cactataggg 20
Claims (8)
1. the preparation method of dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample, is characterized in that, comprise the steps:
1) synthesize virus sequence, its sequence is as described in Sequence NO.1: and a T7 promotor is increased before every section of sequence;
2) composition sequence flush end be cloned into plasmid and cut qualification and glue purification by enzyme;
3) ligation of object fragment and carrier;
4) by competence Bacterial Transformation plasmid;
5) recombinant plasmid extracts;
6) spectrophotometer method is adopted to carry out quality examination to recombinant plasmid;
7) choose purity and all qualified recombinant plasmid of integrity, solution is mixed, and again measure concentration and purity, then carry out packing freeze-drying, the standard model prepared is placed in-80 DEG C of Refrigerator stores.
2. the preparation method of dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample according to claim 1, it is characterized in that, described step also comprises the qualification of recombinant plasmid: from the bacterium liquid that step 4) obtains, get 1-3 bacterium colony, be inoculated in the LB substratum containing ammonia benzyl and cultivate, extract plasmid, carry out pcr amplification, PCR primer carries out agarose gel electrophoresis, screening positive clone.
3. the preparation method of dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample according to claim 2, it is characterized in that, described step also comprises determining nucleic acid sequence and sequence comparative analysis: checked order by the bacterium being accredited as positive recombinant plasmid through PCR and carry out homology analysis.
4. the dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample prepared by method described in claim 1, the recombinant vectors of the nucleotide sequence namely containing Sequence NO.1.
5. dengue hemorrhagic fever virus Type II nucleic acid molecule characteristic standard sample according to claim 4, it is characterized in that, described recombinant vectors is plasmid.
6. the preparation method of standard model according to claim 1, is characterized in that, step 2) concrete steps of described clone and purifying are: sequence flush end are cloned in pUC19 plasmid; Enzyme is cut qualification and is selected
ecor I and
xbai carries out enzyme as the cloning site of plasmid vector and cuts, 37 DEG C, reaction 1-2h, and reaction system is DNA 16 μ L, 10X buffer 2 μ L,
ecor I 1 μ L,
xbai 1 μ L; Digestion products, through 1% agarose gel electrophoresis, cuts required fragment, and glue reclaims purifying.
7. the preparation method of standard model according to claim 1, it is characterized in that, described in step 3), the ligation concrete steps of object fragment and carrier are: condition is 16 DEG C, reaction 16h, reaction system is: step 2) the purified product 1 μ L that obtains, composition sequence X μ L: the mol ratio of itself and carrier is 3 ~ 20:1, T4 Ligase 1 μ L, 10X Ligase solution 1 μ L, adds deionized water polishing to 10 μ L.
8. the preparation method of standard model according to claim 1, is characterized in that, described in step 4), the concrete steps of competence Bacterial Transformation plasmid are: be added to by competence bacterium in the aseptic Eppendorf pipe of precooling, get the ligation liquid that step 3) obtains, be added in the pipe containing DH5 α competence bacterium, gently mix, leave standstill 30min on ice, 42 DEG C of heat shock 90s, do not rock pipe, leave standstill 5min on ice, add not containing the LB substratum of ammonia benzyl, 37 DEG C, 225rpm, cultivates 1h and obtains conversion fluid; Get on LB solid medium that conversion fluid coats containing ammonia benzyl, be inverted for 37 DEG C and cultivate 16h.
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Non-Patent Citations (3)
| Title |
|---|
| MÁRCIA GONÇALVES DE等: "Dengue virus detection by using reverse transcription-polymerase chain reaction in saliva and progeny of experimentally infected Aedes albopictus from Brazil", 《MEM INST OSWALDO CRUZ》 * |
| 张锦海等: "体外转录法制备登革热1-4型病毒RNA片段", 《解放军预防医学杂志》 * |
| 朱玉兰: "登革热病毒RNA实时荧光定量RT-PCR检测方法的建立", 《热带医学杂志》 * |
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