CN105886662A - Method and special primer for rapidly breeding ST (swine testis) sensitive cell lines of hog cholera virus lapinized virus strain (C strain) - Google Patents
Method and special primer for rapidly breeding ST (swine testis) sensitive cell lines of hog cholera virus lapinized virus strain (C strain) Download PDFInfo
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Abstract
The invention discloses a method for rapidly breeding ST (swine testis) sensitive cell lines of a hog cholera lapinized virus strain (C strain) by virtue of a reverse transcription PCR (RT-PCR) (reverse transcription-polymerase chain reaction) technology and a real-time fluorescent quantitative RT-PCR technology. The method comprises the following steps: 1) acquiring ST monoclonal cells; 2) inoculating the hog cholera lapinized virus strain (C strain); 3) extracting RNAs of various batches of hog cholera lapinized virus liquid (virus liquid) which are continuously harvested, and determining that ST monoclonal cell lines can continuously infect the hog cholera lapinized virus strain (C strain) by virtue of the RT-PCR technology; and 4) determining that the hog cholera lapinized virus strain of which the number of copies is 10<6> copies/mL or above are the ST sensitive cell lines of the hog cholera lapinized virus strain (C strain) by virtue of the real-time fluorescent quantitative RT-PCR technology. With the application of the method disclosed by the invention, the sensitive cell lines can be rapidly provided for the production of hog cholera lapinized virus vaccines, so that a vaccine production cycle is shortened and vaccine cost is reduced.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly relate to one RT-PCR technology (i.e. reverse transcription PCR)
Method with real-time fluorescence quantitative RT-PCR quick breeding fever virus lapinized Chinese Strain (C strain) ST sensitive cell line.
Background technology
Swine fever be the one caused by swine fever virus (Classical swine fever virus, CSFV) acute,
Hot, hyperinfection and the deadly infectious disease of lethal, be commonly called as rinderpest, in one's early years also known as hog cholera.This disease is to send out
Life is most, endanger the widest pig infectious disease maximum, popular.China in Recent Years Died Of Disease pig number accounts for raises the 8 of sum
%-10%, wherein 1/3rd are caused by swine fever.Swine fever is classified as A class by OIE (OIE)
One of 16 kinds of Notifiable diseases, swine fever is also set to a class deadly infectious disease by China, belongs to that harm is serious, needs are taked
One of the severeest animal epidemic forcing to prevent, control and put out.
Swine fever is dispersed throughout the whole world, has high degree in contact infectiousness.At present, vaccination is the weight of prevention and control swine fever
Means, many countries and regions are wanted successively to announce to have eliminated swine fever.China's hog cholera lapinised virus vaccine has with it and lures
Raw immunoreation is fast, and without residual virulence, immune swine, boar, piglets can be resisted the features such as the attack of swine fever virulent strain,
Become the most safely and effectively attenuated vaccine generally acknowledged in the world.
The swine Fever Vaccine being currently used for immunity mainly has four kinds: spleen drenches Seedling, breast rabbit Seedling, cell vaccine and connection Seedling.These four
The swine fever strain of vaccine be all China be fever virus lapinized Chinese Strain (be called for short C strain).Swine fever spleen drenches Seedling and breast rabbit Seedling is rabbit
Source tissue's Seedling, is that the living tissue utilizing into rabbit and breast rabbit produces preparation.Drench Seedling due to swine fever spleen and breast rabbit Seedling uses dynamic
The immune organ of thing is made, although immune effect is preferable, but cost is high, easily pollutes, and yields poorly, poor between batch
Different greatly, and after inoculation, stress is big.And swine fever cell vaccine to have production cost low, it is adaptable to batch production,
The features such as differences between batches are little, and side reaction is low increasingly come into one's own.But due in swine fever cell vaccine production process,
Cell produces poison weak effect, and virus titer is relatively low, causes the immune effect of vaccine finished product to drench Seedling not as good as swine fever spleen.Therefore,
Screen the cell line sensitive to fever virus lapinized Chinese Strain, the production of swine fever cell vaccine is had reality improving virus titer
Meaning, and economic benefit can be brought.
Summary of the invention
It is an object of the invention to provide one RT-PCR technology and real-time fluorescence quantitative RT-PCR quick breeding pig
The method of pestilence Lapinized strain (C strain) ST (Pig testicular cell) sensitive cell line, to overcome existing swine fever cell epidemic disease
The defect that present in Seedling production, immune effect is undesirable.
The method of quick breeding fever virus lapinized Chinese Strain provided by the present invention (C strain) ST sensitive cell line, mainly wraps
Include following steps:
1) ST monoclonal cell is obtained；
2) inoculation fever virus lapinized Chinese Strain (C strain)；
3) extract the RNA of each batch hog cholera lapinised virus liquid (virus liquid) of results continuously, enter by RT-PCR technology
Row Preliminary Identification, determines that ST monoclonal cell system can persistent infection hog cholera lapinised virus (C strain)；
4) with real-time fluorescence quantitative RT-PCR detection by quantitative hog cholera lapinised virus liquid, to determine hog cholera lapinised virus
The copy number of strain (C strain), fever virus lapinized Chinese Strain (C strain) can persistent infection cell, and determine the 3rd
The copy number of batch and the later virus liquid of batch results is able to maintain that 106Copy (copies)/mL's is pig
Pestilence Lapinized strain (C strain) ST sensitive cell line.
In the above-mentioned methods, described step 1) preparation method of ST monoclonal cell is: will pollute without exogenous virus
ST cell is at 37 DEG C, 5%CO2Incubator is cultivated 48h, by 0.2% (mass/volume, W/V, unit g/100mL)
Trypsinization become the single cell being dispersed in after carry out cell counting, then with for 15% (volume/volume, V/V,
Unit/mL/100mL) to be diluted to cell concentration be 10-100cells/mL for the DMEM culture medium of serum, add with pipettor
Sample comprises only the hole of a cell, every day to 96 orifice plates, every hole 100 μ L, basis of microscopic observation, record labelling
Observe, after cell quantity increases, more progressively amplification culture, obtain ST monoclonal cell.
Described step 2) in inoculation fever virus lapinized Chinese Strain (C strain) method be: by fever virus lapinized Chinese Strain (C
Strain) inoculation ST monoclonal cell, at 37 DEG C, 5%CO2Cultivating in incubator, every 48h gathers in the crops virus liquid, continuously
Gathering in the crops 8 batch venom, it is to be checked that each batch virus liquid is placed in-80 DEG C of short-term preservations.
Described step 3) in RT-PCR authentication method, it may include following steps:
3.1) total serum IgE of hog cholera lapinised virus liquid (virus liquid) is extracted；
3.2) total serum IgE with virus liquid synthesizes its cDNA for template reverse transcription；
3.3) RT-PCR detection: with cDNA as template, carry out PCR expansion under the guiding of RT-PCR specific primer
Increasing, reaction carries out 1% agarose gel electrophoresis to pcr amplification product, if a length of 383bp can be amplified after terminating
Purpose fragment, then testing result is positive, otherwise be feminine gender.
Described step 3.2) in be that the RNA Reverse Transcription box extracted is carried out two-step method reverse transcription, the first step
Reaction system is: Oligo dT Primer (50uM) 1 μ L, dNTP Mixture (10mM each) 1 μ L, Template
RNA 2 μ L, Rnase free ddH2O 6 μ L, reaction condition is: 65 DEG C of 5min, cools down rapidly；Second step is anti-
The system is answered to be: Template RNA and Primer Mixture (product of the first step) 10 μ L, 5*PrimeScript
II Buffer 4 μ L, Rnase inhibitor (40U/ μ L) 0.5 μ L, PrineScript II RTase
(200U/ μ L) 1 μ L, Rnase free H204.5 μ L, reaction condition is: mixing, 42 DEG C of 30-60min,
95 DEG C of 5min, cooled on ice.
Described step 3.3) in for RT-PCR identify specific primer, be with fever virus lapinized Chinese Strain (C strain)
Full-length genome specific conservative region in hold 54-436bp (SEQ ID NO:1 in sequence table) from 5 ' be right
As design, purpose sheet segment length 383bp, in order to detect ST monoclonal cell whether can persistent infection swine fever rabbitization weak
Strain (C strain).
Described step 3.3) in the forward primer identified for RT-PCR have in sequence table shown in SEQ ID NO:2
Nucleotide sequence, downstream primer has the nucleotide sequence shown in SEQ ID NO:3 in sequence table.
Described step 3.3) in RT-PCR reaction system be: template cDNA 1 μ L, 2 × Taq PCR Master MIX
12.5 μ L, forward primer (10 μMs/μ L) 1 μ L, downstream primer (10 μMs/μ L) 1 μ L, add Rnase free
H2O to 25 μ L；The reaction condition of described RT-PCR is: first 94 DEG C of denaturations, 5min；Then 94 DEG C of degeneration 30s,
56 DEG C-64 DEG C annealing 20s, 72 DEG C extend 30s, totally 35 circulations；Last 72 DEG C extend 10min.
Described step 4) in real-time fluorescence quantitative RT-PCR detection method, it may include following steps:
4.1) Criterion curve: using swine fever virus (C strain) as standard substance, gradient dilution becomes 1 × 108、1×107、
1×106、1×105、1×104、1×103、1×102、1×101Copies/ μ L, with the standard substance of variable concentrations
As template, under the guiding of primer and TaqMan probe, carry out real-time fluorescence quantitative PCR detection, after detection terminates,
With the concentration Log value (X-axis) of each standard substance, its corresponding Ct value (Y-axis) is mapped, draw standard curve；
4.2) extract hog cholera lapinised virus liquid (virus liquid) geneome RNA, with extract geneome RNA as mould
Plate, carries out the detection of swine fever virus (C strain) real-time fluorescence quantitative RT-PCR under the guiding of primer and TaqMan probe；
4.3) according to change and the intensity of fluorescence signal, the swine fever virus (C strain) in testing sample is carried out qualitative,
Detection by quantitative, occurs that amplified fluorescence curve shows in sample containing swine fever virus, does not occurs that amplified fluorescence curve shows sample
Product do not contain swine fever virus, further according to fluorescence signal intensity and the step 1 of sample) in standard curve, draw and treat
The copy number of swine fever virus contained in test sample product.
Described step 4.1) and 4.2) in for real-time fluorescence quantitative PCR detection primer and TaqMan probe, be
To hold the 151-243bp to be from 5 ' in the specific conservative region of the full-length genome of fever virus lapinized Chinese Strain (C strain)
Object designs, purpose sheet segment length 93bp (SEQ ID NO:4 in sequence table), in order to determine the copy number of virus
(purpose fragment 93bp of real-time fluorescence quantitative PCR amplification is a part for step 3.3RT-PCR amplified production).
Described step 4.1) and 4.2) in for the nucleotides sequence of forward primer of real-time fluorescence quantitative RT-PCR detection
Arrange as shown in SED ID NO:4 in sequence table, SEQ ID NO:5 in the nucleotide sequence of downstream primer such as sequence table
Shown in.
Described step 4.1) and 4.2) in for real-time fluorescence quantitative RT-PCR detection TaqMan probe, its core
Nucleotide sequence is as shown in SEQ ID NO:6 in sequence table；Described probe is through fluorescently-labeled, its 5 ' end labelling
Reporter fluorescence group, 3 ' ends is had to be marked with quenching fluorescence group；Described reporter fluorescence group is FAM, and described fluorescence is quenched
The group that goes out is TAMRA；For preventing PCR to be extended when expanding, phosphatizing treatment is held in the 3 ' of described probe.
Described step 4.1) and 4.2) in the reaction system of real-time fluorescence quantitative RT-PCR be: template ribonucleic acid 2 μ L,
2 × RT-PCR buffer 12.5 μ L, forward primer (20 μMs/μ L) 0.5 μ L (10ng), downstream primer
(20 μMs/μ L) 0.5 μ L (10ng), Mix enzyme 0.5 μ L, HS enzyme 0.5 μ L, TaqMan probe (20 μMs
/ μ L) 0.5 μ L (10ng), add Rnase free H2O to 25 μ L；The reaction condition of real-time fluorescence quantitative PCR
For: first 52 DEG C of 15min, 95 DEG C of 5s；Then 94 DEG C of 5s, 58 DEG C of 40s, totally 45 circulations.
Another object of the present invention is to provide in quick breeding fever virus lapinized Chinese Strain (C strain) ST sensitive cells system, method
Primer special, including:
Step 3) in T-PCR technology carry out the forward primer of Preliminary Identification, it has SEQ ID NO:2 in sequence table
Shown nucleotide sequence；Downstream primer, it has the nucleotide sequence shown in SEQ ID NO:3 in sequence table；
Or
Step 4) in the forward primer of real-time fluorescence quantitative RT-PCR detection by quantitative, its nucleotide sequence such as sequence
In table shown in SED ID NO:5；Downstream primer, its nucleotide sequence is as shown in SEQ ID NO:6 in sequence table；
TaqMan probe, its nucleotide sequence is as shown in SEQ ID NO:7 in sequence table.
The invention provides one RT-PCR technology and real-time fluorescence quantitative RT-PCR quick breeding swine fever rabbitization
The method of low virulent strain (C strain) ST (Pig testicular cell) sensitive cell line.The present invention can be quickly the weak poison of swine fever
The production of vaccine provides sensitive cell line, shortens the production of vaccine cycle and reduces vaccine cost, solving pig now
Pestilence cell vaccine is due to the undesirable difficult problem of the low immune effect of vaccine caused of swine fever virus titre.
Compared with prior art, the method have the advantages that
1, the ST sensitive cell line of present invention screening can persistent infection fever virus lapinized Chinese Strain (C strain), it is possible to even
Continuous Multiple harvests hog cholera lapinised virus liquid, can directly use in production process；
2, the method for the screening ST sensitive cell line that the present invention sets up is, after cell carries out monoclonal, first to use RT-PCR
Technology carries out Preliminary Identification, determine ST monoclonal cell system can persistent infection hog cholera lapinised virus (C strain), then use
Real-Time Fluorescent Quantitative PCR Technique detection by quantitative hog cholera lapinised virus liquid, can more accelerate than conventional immunofluorescence method
Speed ground selection-breeding swine fever ST sensitive cell line；
3, the ST sensitive cell line of present invention screening is the strain (fever virus lapinized Chinese Strain (C used for swine Fever Vaccine
Strain)), it is possible to directly apply to swine Fever Vaccine and produce；
4, the ST cell line of present invention screening is sensitive to swine fever virus, and can high titre persistent infection swine fever virus,
Be conducive to overcoming current swine fever virus cell to cultivate the difficult problem that titre is low, the production of vaccine cycle can be shortened and reduce vaccine
This, the production to swine Fever Vaccine has realistic meaning.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 is C4 strain single ST cell growth conditions in 96 orifice plates
Fig. 2 is E11 strain single ST cell growth conditions in 96 orifice plates
Fig. 3 is the real-time fluorescence quantitative RT-PCR amplification curve of standard substance
Fig. 4 is the standard curve of real-time fluorescence quantitative RT-PCR
Fig. 5 is that D3 strain, C4 strain, C8 strain, D5 strain, E10 strain, E11 strain, F9 strain and H9 strain swine fever rabbitization are weak
The real time fluorescent quantitative testing result of strain (C strain) copy number
Detailed description of the invention
In following embodiment, method therefor is conventional method if no special instructions, and concrete steps can be found in:
" Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W.,
Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring
Harbor)。
Described percent concentration be if no special instructions mass/mass (W/W, unit g/100g) percent concentration,
Mass/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/100mL) percentage ratio
Concentration.
The acquirement approach of the various biomaterials described in embodiment is only to provide a kind of approach of acquisition of testing to reach
To specifically disclosed purpose, the restriction to biological material source of the present invention should not become.It is true that used biology
The source of material is widely, any keeps on the right side of the law and the biomaterial that can obtain of moral ethics can be according to reality
Execute the prompting in example and replace use.
The primer and TaqMan probe are by the synthesis of company of precious biological engineering (Dalian) company limited.
Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and concrete
Operating process, embodiment will assist in and understands the present invention, but protection scope of the present invention is not limited to following embodiment.
Embodiment, quick breeding fever virus lapinized Chinese Strain (C strain) ST sensitive cell line
Present invention RT-PCR technology and real-time fluorescence quantitative RT-PCR quick breeding fever virus lapinized Chinese Strain (C
Strain) method of ST (Pig testicular cell) sensitive cell line, comprise the following steps:
1) ST monoclonal cell is obtained
By the ST cell (purchased from American Type Culture Collecti (ATCC)) that pollutes without exogenous virus at 37 DEG C, 5%CO2
Incubator is cultivated 48h, becomes single with the trypsinization of 0.2% (mass/volume, W/V, unit g/100mL)
Cell counting is carried out, then with 15% (volume/volume, V/V, Unit/mL/100mL) serum after the cell being dispersed in
It is 10-100 cell (cells)/mL (generally, trypsinization that DMEM culture medium is diluted to cell concentration
Liquid and serum addition only need to mark ratio), with pipettor sample-adding to 96 orifice plates, every hole 100 μ L, show
Micro-Microscopic observation, record labelling comprise only the hole of a cell, are respectively labeled as D3 strain, C4 strain, C8 strain, D5
Strain, E10 strain, E11 strain, F9 strain and H9 strain, observe every day, wherein, and C4 strain, E11 strain single ST cell
Growth conditions is as shown in Figure 1 and Figure 2.Cell quantity increase after progressively amplification culture, first amplification culture to 24 orifice plates,
Amplification culture is to 6 orifice plates again, finally the ST cell of clone is seeded to 25cm2In Tissue Culture Flask, DMEM is trained
The serum addition supported in base changes 10% (volume/volume, V/V, Unit/mL/100mL) into, carries out cell routine training
Support, obtain ST monoclonal cell.
2) inoculation fever virus lapinized Chinese Strain (C strain)
D3 strain, C4 strain, C8 strain, D5 strain, E10 strain, E11 strain, F9 strain and H9 strain ST monoclonal cell are expanded
Increase and cultivate to 75cm2In Tissue Culture Flask, all by 1% (volume/volume, V/V, Unit/mL/100mL) virus quantity
Inoculation fever virus lapinized Chinese Strain (C strain, purchased from China Veterinery Drug Inspection Office), at 37 DEG C, 5%CO2Incubator
Middle cultivation, every 48h gathers in the crops virus liquid, and (swine fever virus does not cause cytopathy to results 8 batches, can receive continuously continuously
Obtaining venom, each receipts are a batch).
3) extract the RNA of 8 batch virus liquids of results continuously, carry out Preliminary Identification by RT-PCR technology, determine
ST monoclonal cell system can persistent infection hog cholera lapinised virus (C strain), concrete grammar comprises the following steps:
3.1) total serum IgE of virus liquid is extracted
The virus liquid sampling gathering in the crops each batch, carries with the TaKaRaRNAiso Reagent test kit of TAKARA company
Take total serum IgE, specifically comprise the following steps that
3.1.1 pipettor draws 200 μ L virus liquids, adds 1mL RNAiso Reagent, mix homogeneously.
3.1.2 in the homogenate lysate of above-mentioned steps 3.1.1, add chloroform (1/5 body of RNAiso Reagent
Accumulated amount), cover tightly centrifugal lid, (chloroform boiling point is low, volatile, should carefully be centrifuged lid and dash forward during vibration in firmly concussion
So flick).After fully emulsified solution is milky white shape (no phase separation phenomenon), then room temperature stands 5 minutes.
3.1.312,000g, 4 DEG C centrifugal 15 minutes.
3.1.4 carefully taking out centrifuge tube from centrifuge, Aspirate supernatant is transferred in the centrifuge tube that another is new.
3.1.5 in supernatant, isopyknic isopropanol is added, after the centrifuge tube that turns upside down fully mixes, at 15-30
10 minutes are stood at DEG C.
3.1.612,000g, 4 DEG C centrifugal 10 minutes.Typically after centrifugal, bottom test tube, there will be precipitation.
3.1.7RNA the careful supernatant discarded of cleaning precipitated, adds the ethanol lmL of 75% lentamente along centrifugal tube wall
(being sure not to touch precipitation), turn upside down washing centrifuge tube tube wall gently, and 12,000g, 4 DEG C careful after centrifugal 5 minutes
Discard ethanol.
3.1.8RNA dissolving drying at room temperature precipitates 2-5 minute, adds appropriate RNasefree water dissolution precipitation,
Precipitation can be blown and beaten gently with liquid-transfering gun if desired, save backup after RNA precipitate is completely dissolved.
3.2 synthesize its cDNA with the RNA of virus liquid for template reverse transcription
The RNA Reverse Transcription box extracted is carried out two-step method reverse transcription, and first step reaction system is: Oligo dT
Primer (50uM) 1 μ L, dNTP Mixture (10mM each) 1 μ L, Template RNA 2 μ L, Rnase
free ddH2O 6 μ L, reaction condition is: 65 DEG C of 5min, cools down rapidly；Second step reaction system is: Template
RNA and Primer Mixture (product of the first step) 10 μ L, 5*PrimeScript II Buffer 4 μ L,
Rnase inhibitor (40U/ μ L) 0.5 μ L, PrineScript II RTase (200U/ μ L) 1 μ L, Rnase
free H204.5 μ L, reaction condition is: mixing, 42 DEG C of 30-60min, 95 DEG C of 5min, cooled on ice.
3.3RT-PCR detection
The gene order of the hog cholera lapinised virus (C strain) on retrieval Genbank, with the method for bioinformatics, warp
Cross comparison analysis, find the specific conservative region of hog cholera lapinised virus (C strain) full-length genome, with from 5 ' ends the
(383bp, SEQ ID NO:1 in sequence table) is object in 54-436bp region, with software Primer Express2.0
It is designed for the primer special that RT-PCR identifies, in order to detect ST monoclonal cell whether can persistent infection swine fever rabbit
Changing low virulent strain (C strain), primer sequence is as follows:
The specific primer of RT-PCR qualification hog cholera lapinised virus (C strain):
Forward primer (Forward primer): SEQ ID NO in 5'-ATTTGGTTCAGGGCCTCC-3'(sequence table:
2)
Downstream primer (Reverse primer): SEQ ID NO in 5'-TCCACTCCCATTGGTTTT-3'(sequence table:
3)。
With cDNA as template, under the guiding of RT-PCR primer special, carry out RT-PCR amplification, RT-PCR reactant
System is: template cDNA 1 μ L, 2 × Taq PCR Master MIX 12.5 μ L, forward primer (10 μMs/μ L)
1 μ L, downstream primer (10 μMs/μ L) 1 μ L, add Rnase free H2O to 25 μ L；Described RT-PCR's
Reaction condition is: first 94 DEG C of denaturations, 5min；Then 94 DEG C of degeneration 30s, 56 DEG C-64 DEG C annealing 20s, 72 DEG C
Extend 30s, totally 35 circulations；Last 72 DEG C extend 10min.
Reaction carries out 1% agarose gel electrophoresis to RT-PCR amplified production, if can amplify a length of after terminating
The purpose fragment of 383bp, then testing result is positive, otherwise is negative.
Through RT-PCR Preliminary Identification, determine gained D3 strain, C4 strain, C8 strain, D5 strain, E10 strain, E11 strain, F9
Strain and H9 strain ST monoclonal cell system can persistent infection hog cholera lapinised virus (C strain).
4) with real-time fluorescence quantitative RT-PCR detection by quantitative virus liquid, to determine the copy number of virus.Virus energy
Reach persistent infection cell, and the copy number gathering in the crops venom in the 3rd batch and later batch is able to maintain that 106
Copies/mL's is that (swine fever virus infection cell has adaptation rank to fever virus lapinized Chinese Strain (C strain) ST sensitive cell line
Section, front two batch venom poison valencys are relatively low), concrete grammar comprises the following steps:
For primer and the TaqMan probe of real-time fluorescence quantitative RT-PCR detection, it is with hog cholera lapinised virus (C strain)
In the specific conservative region of full-length genome from 5 ' hold 151-243bp (long 93bp, SEQ ID NO in sequence table:
4) it is the part of step 3.3RT-PCR amplified production 383bp (fragment of 93bp mesh be) of object designs, uses
To determine the copy number of virus, the sequence of primer and TaqMan probe is as follows:
Forward primer (Forward primer): SEQ ID NO:5 in 5'-CCCTGGGTGGTCTAAG-3'(sequence table)
Downstream primer (Reverse primer): SEQ ID NO:6 in 5'-CATGCCCTCGTCCAC-3'(sequence table)
TaqMan probe: 5 '-(FAM) CCTGAGTACAGGACAGTCGTCAGTAGTT (TAMRA)-3 ' (SEQ in sequence table
ID NO:7).
Described TaqMan probe is through fluorescently-labeled, and its 5 ' end is marked with reporter fluorescence group, 3 ' end labellings
There is quenching fluorescence group；Described reporter fluorescence group is FAM, and described fluorescent quenching group is TAMRA；For preventing PCR
Being extended during amplification, phosphatizing treatment is held in the 3 ' of described probe.
4.1 Criterion curves: using swine fever virus (C strain) as standard substance, gradient dilution becomes 1 × 108、1×107、
1×106、1×105、1×104、1×103、1×102、1×101Copy (copies)/μ L, each sample does
2 repetitions, using the standard substance of variable concentrations as template, carry out the most glimmering under the guiding of primer and TaqMan probe
Fluorescent Quantitative PCR detects.
Described step 4.1) in the reaction system of real-time fluorescence quantitative RT-PCR be: template ribonucleic acid 2 μ L, 2 × RT-PCR
Buffer 12.5 μ L, forward primer (20 μMs/μ L) 0.5 μ L (10ng), downstream primer (20 μMs/μ L)
0.5 μ L (10ng), Mix enzyme 0.5 μ L, HS enzyme 0.5 μ L, TaqMan probe (20 μMs/μ L) 0.5 μ L
(10ng) Rnase free H, is added2O to 25 μ L；The reaction condition of real-time fluorescence quantitative RT-PCR is: first 52
DEG C 15min, 95 DEG C of 5s；Then 94 DEG C of 5s, 58 DEG C of 40s, totally 45 circulations.
The amplification curve of standard substance (curve the most corresponding 1 × 10 from left to right as shown in Figure 38、1×107、1×106、
1×105、1×104、1×103、1×102、1×101The standard substance of copy (copies)/μ L concentration), standard
Product amplification curve is smooth serpentine curve.After detection terminates, the concentration Log value (X-axis) with each standard substance is right
Its corresponding Ct value (Y-axis) is mapped, and draws standard curve, and (abscissa is Ct value to standard curve such as Fig. 4, vertical seat
It is designated as copy number) shown in, R2=0.999, error is little, standard curve the linear equation obtained is:
Y=-3.394x+40.812.
The geneome RNA of each strain virus liquid of 4.2 8 batches extracting results, with the geneome RNA extracted be
Template, carries out real-time fluorescence quantitative RT-PCR detection under the guiding of primer and TaqMan probe.
Described step 4.2) in the reaction system of real-time fluorescence quantitative RT-PCR be: template ribonucleic acid 2 μ L, 2 × RT-PCR
Buffer 12.5 μ L, forward primer (20 μMs/μ L) 0.5 μ L (10ng), downstream primer (20 μMs/μ L)
0.5 μ L (10ng), Mix enzyme 0.5 μ L, HS enzyme 0.5 μ L, TaqMan probe (20 μMs/μ L) 0.5 μ L
(10ng) Rnase free H, is added2O to 25 μ L；The reaction condition of real-time fluorescence quantitative PCR is: first 52 DEG C of 15min,
95℃5s；Then 94 DEG C of 5s, 58 DEG C of 40s, totally 45 circulations.
4.3, according to the change of fluorescence signal and intensity, carry out qualitative and quantitative analysis to testing sample, fluorescence occur
Amplification curve shows in sample containing swine fever virus (C strain), does not occurs amplified fluorescence curve to show in sample and does not contains
Swine fever virus (C strain), further according to fluorescence signal intensity and the step 4.1 of sample) in standard curve, draw and treat
The copy number of swine fever virus (C strain) contained in test sample product, real-time fluorescence quantitative RT-PCR testing result is shown in Table 1.
Result is as shown in table 1 and Fig. 5, and in gained 8 strain ST monoclonal cell system, C4 and E11 two strain cell is received
The 3rd batch obtained is able to maintain that 10 to the 8th batch venom copy number6Copies/mL, and peak period venom copy
Number is up to 107Copies/mL, produces poison and measures apparently higher than other cell strain, belong to fever virus lapinized Chinese Strain (C strain) ST
Sensitive cell line, can be applicable to the production of swine Fever Vaccine.
Table 1 swine fever virus (C strain) real-time fluorescence quantitative RT-PCR testing result
Claims (10)
1. the method for quick breeding fever virus lapinized Chinese Strain (C strain) ST sensitive cell line, mainly comprises the steps that
1) ST monoclonal cell is obtained；
2) inoculation fever virus lapinized Chinese Strain (C strain)；
3) extract the RNA of each batch hog cholera lapinised virus liquid (virus liquid) of results continuously, enter by RT-PCR technology
Row Preliminary Identification, determines that ST monoclonal cell system can persistent infection hog cholera lapinised virus (C strain)；
4) with real-time fluorescence quantitative RT-PCR detection by quantitative hog cholera lapinised virus liquid, to determine hog cholera lapinised virus
The copy number of strain (C strain), determine fever virus lapinized Chinese Strain (C strain) can persistent infection cell, and the 3rd
The copy number of batch and the later virus liquid of batch results is able to maintain that 106The determination of copy (copies)/mL
For fever virus lapinized Chinese Strain (C strain) ST sensitive cell line.
The side of quick breeding fever virus lapinized Chinese Strain the most according to claim 1 (C strain) ST sensitive cell line
Method, it is characterised in that: described step 1) preparation method of ST monoclonal cell is: the ST that will pollute without exogenous virus
Cell is at 37 DEG C, 5%CO2Incubator is cultivated 48h, by 0.2% (mass/volume, W/V, unit g/100mL)
Trypsinization become the single cell being dispersed in after carry out cell counting, then with for 15% (volume/volume, V/V,
Unit/mL/100mL) to be diluted to cell concentration be 10-100cells/mL for the DMEM culture medium of serum, add with pipettor
Sample comprises only the hole of a cell, every day to 96 orifice plates, every hole 100 μ L, basis of microscopic observation, record labelling
Observe, after cell quantity increases, more progressively amplification culture, obtain ST monoclonal cell.
Quick breeding fever virus lapinized Chinese Strain the most according to claim 1 and 2 (C strain) ST sensitive cell line
Method, it is characterised in that: described step 2) in inoculation fever virus lapinized Chinese Strain (C strain) method be: by pig
Pestilence Lapinized strain (C strain) inoculation ST monoclonal cell, at 37 DEG C, 5%CO2Incubator is cultivated, every 48h
Results virus liquid, continuously 8 batch venom of results, it is to be checked that each batch virus liquid is placed in-80 DEG C of short-term preservations.
4. sensitive thin according to quick breeding fever virus lapinized Chinese Strain (C strain) ST described in claim 1 or 2 or 3
Born of the same parents system method, it is characterised in that: described step 3) in RT-PCR authentication method, it may include following steps:
3.1) extract hog cholera lapinised virus liquid (virus liquid) total serum IgE；
3.2) total serum IgE with virus liquid synthesizes its cDNA for template reverse transcription；
3.3) RT-PCR detection: with cDNA as template, carry out PCR expansion under the guiding of RT-PCR specific primer
Increasing, reaction carries out 1% agarose gel electrophoresis to pcr amplification product, if a length of 383bp can be amplified after terminating
Purpose fragment, then testing result is positive, otherwise be feminine gender.
The side of quick breeding fever virus lapinized Chinese Strain the most according to claim 4 (C strain) ST sensitive cell line
Method, it is characterised in that: described step 3.2) in be that the RNA Reverse Transcription box extracted is carried out two-step method is anti-
Transcribing, first step reaction system is: Ol igo dT Primer (50uM) 1 μ L, dNTP Mixture (10mM each)
1 μ L, Template RNA 2 μ L, Rnase free ddH2O 6 μ L, reaction condition is: 65 DEG C of 5min, fast
Quickly cooling is but；Second step reaction system is: Template RNA and Primer Mixture (product of the first step) 10 μ L,
5*PrimeScript II Buffer 4 μ L, Rnase inhibitor (40U/ μ L) 0.5 μ L, PrineScript
II RTase (200U/ μ L) 1 μ L, Rnase free H204.5 μ L, reaction condition is: mixing, 42 DEG C of 30-60min,
95 DEG C of 5min, cooled on ice.
6. according to quick breeding fever virus lapinized Chinese Strain (C strain) the ST sensitive cell line described in claim 4 or 5
Method, it is characterised in that: described step 3.3) in for RT-PCR identify forward primer have in sequence table
Nucleotide sequence shown in SEQ ID NO:2, downstream primer has the nucleoside shown in SEQ ID NO:3 in sequence table
Acid sequence.
7. sensitive thin according to quick breeding fever virus lapinized Chinese Strain (C strain) ST described in claim 4 or 5 or 6
Born of the same parents system method, it is characterised in that: described step 3.3) in RT-PCR reaction system be: template cDNA 1 μ L,
2 × Taq PCR Master MIX 12.5 μ L, forward primer (10 μMs/μ L) 1 μ L, downstream primer (10 μMs/μ L)
1 μ L, adds Rnase free H2O to 25 μ L；The reaction condition of described RT-PCR is: first 94 DEG C of denaturations, 5min；
Then 94 DEG C of degeneration 30s, 56 DEG C-64 DEG C annealing 20s, 72 DEG C extend 30s, totally 35 circulations；Last 72 DEG C
Extend 10min.
8. sensitive thin according to arbitrary described quick breeding fever virus lapinized Chinese Strain (C strain) ST of claim 1 to 7
Born of the same parents system method, it is characterised in that: described step 4) in real-time fluorescence quantitative RT-PCR detection method, including with
Lower step:
4.1) Criterion curve: using swine fever virus (C strain) as standard substance, gradient dilution becomes 1 × 108、1×107、
1×106、1×105、1×104、1×103、1×102、1×101Copies/ μ L, with the standard substance of variable concentrations
As template, under the guiding of primer and TaqMan probe, carry out real-time fluorescence quantitative PCR detection, after detection terminates,
With the concentration Log value (X-axis) of each standard substance, its corresponding Ct value (Y-axis) is mapped, draw standard curve；
4.2) extract hog cholera lapinised virus liquid (virus liquid) geneome RNA, with extract geneome RNA as mould
Plate, carries out the real-time fluorescence quantitative RT-PCR inspection of swine fever virus (C strain) under the guiding of primer and TaqMan probe
Survey；
4.3) according to change and the intensity of fluorescence signal, the swine fever virus (C strain) in testing sample is carried out qualitative,
Detection by quantitative, occurs that amplified fluorescence curve shows in sample containing swine fever virus, does not occurs that amplified fluorescence curve shows sample
Product do not contain swine fever virus, further according to fluorescence signal intensity and the step 1 of sample) in standard curve, draw and treat
The copy number of swine fever virus contained in test sample product.
The side of quick breeding fever virus lapinized Chinese Strain the most according to claim 8 (C strain) ST sensitive cell line
Method, it is characterised in that: described step 4.1) and 4.2) in for real-time fluorescence quantitative RT-PCR detection upstream draw
The nucleotide sequence of thing is as shown in SED ID NO:5 in sequence table, in the nucleotide sequence of downstream primer such as sequence table
Shown in SEQ ID NO:6；The nucleotide sequence of TaqMan probe is as shown in SEQ ID NO:7 in sequence table, described
Probe is through fluorescently-labeled, and its 5 ' end is marked with reporter fluorescence group (such as FAM), and 3 ' ends are marked with cancellation
(such as TAMRA, phosphatizing treatment is held in the 3 ' of described probe to fluorophor；
Described step 4.1) and 4.2) in the reaction system of real-time fluorescence quantitative RT-PCR be: template ribonucleic acid 2 μ L,
2 × RT-PCR buffer 12.5 μ L, forward primer (20 μMs/μ L) 0.5 μ L (10ng), downstream primer
(20 μMs/μ L) 0.5 μ L (10ng), Mix enzyme 0.5 μ L, HS enzyme 0.5 μ L, TaqMan probe (20 μMs
/ μ L) 0.5 μ L (10ng), add Rnase free H2O to 25 μ L；The reaction of real-time fluorescence quantitative RT-PCR
Condition is: first 52 DEG C of 15min, 95 DEG C of 5s；Then 94 DEG C of 5s, 58 DEG C of 40s, totally 45 circulations.
10. in quick breeding fever virus lapinized Chinese Strain (C strain) the ST sensitive cells system, method described in claim 1-9
Primer special, including:
Step 3) in T-PCR technology carry out the forward primer of Preliminary Identification, it has SEQ ID NO:2 in sequence table
Shown nucleotide sequence；Downstream primer, it has the nucleotide sequence shown in SEQ ID NO:3 in sequence table；
Or
Step 4) in the forward primer of real-time fluorescence quantitative RT-PCR detection by quantitative, its nucleotide sequence such as sequence
In table shown in SED ID NO:5；Downstream primer, its nucleotide sequence is as shown in SEQ ID NO:6 in sequence table；
TaqMan probe, its nucleotide sequence is as shown in SEQ ID NO:7 in sequence table.
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