CN107881260A

CN107881260A - Bovine viral diarrhea virus carries out the method for separating identification in a kind of cow's serum

Info

Publication number: CN107881260A
Application number: CN201711145762.0A
Authority: CN
Inventors: 朱礼倩; 朱国强; 黄丽媛
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2017-11-17
Filing date: 2017-11-17
Publication date: 2018-04-06

Abstract

The present invention provides a kind of method for carrying out separating identification to bovine viral diarrhea virus in cow's serum, and process is as follows：By MDBK cells and cow's serum sample centrifugal treating after blind passage three times, then viral RNA is extracted, RNA reverse transcriptions are further synthesized into full-length cDNA using kit；Enter the separation identification that bovine viral diarrhea virus in blood serum sample is completed in performing PCR reaction.The method of the present invention can be handled batch samples simultaneously, not only increase operating efficiency, virus is easier to accurately detect after amplification cultivation, and accuracy rate is improved, and is separated BVDV for clinical sample and commercialization serum batch sample and is each provided with practicable separation method.

Description

Bovine viral diarrhea virus carries out the method for separating identification in a kind of cow's serum

Technical field

The present invention relates to technical field of animal virology, and in particular to bovine viral diarrhea virus is carried out in a kind of cow's serum The method for separating identification.

Background technology

BVDV (bovine viral diarrhea virus) belongs to flaviviridae pestivirus.The virus is single strand plus RNA virus, base Because of group total length about 12.3Kb, including ORFs (openreading frame, ORF), a 5 ' non-translational region (5' Untranslated region, 5 '-UTR) and 3 ' noncoding regions (3'untranslated region, 3 '-NCR).It is based on BVDV5 '-UTR sequence it is well-conserved, BVDV can be divided into two genotype of BVDV-1 and BVDV-2, between them Antigenicity and Genetic diffeerence are larger.In addition to 5 '-UTR, gene point can also be carried out according to Npro and E2 gene order Type, its result are basically identical with classifying according to 5 '-UTR sequence.

BVDV viruses, can be with many animals of infected pigs, sheep etc. 40 in addition to infected cattle, and can cause morbidity, acute sense Persistent infection (Persistent infection, PI) can be switched to after dye, PI affected animals with poison, toxin expelling, promote this throughout one's life The propagation of virus.Have determined that BVDV persistent infections can occur for 8 kinds of animals at present.Terpstra and in 1997 Wensvoort reports the case that BVDV persistent infections occur for pig first.PI oxen are the important infections sources of pig BVDV infection, if The serum of PI oxen prepares cell vaccine, and it is the important channel for causing BVDV to propagate to be used in the BVDV susceptible animals such as pig, ox and sheep.

Use commercialization ELISA antigen detection kits can with examination BVDV PI oxen, but the kit depend on into Mouth product, it is expensive.Viral inactivation treatment need to be passed through in the cow's serum for producing with being used for cell culture in scientific research, if viral Inactivation can thoroughly not cause disseminating for virus.The BVDV ELISA detection kits of commercialization cannot be distinguished by the virus of inactivation and have Infective virus.

The content of the invention

In view of the shortcomings of the prior art, present invention offer is a kind of carries out separation mirror to bovine viral diarrhea virus in cow's serum Fixed method, there is infective virus available for detection, the diagnosis and research for bovine viral diarrhoea provide foundation.

To achieve the above object, present invention offer is a kind of carries out separating identification to bovine viral diarrhea virus in cow's serum Method, detailed process are as follows：

Step 1) first round blind passage：After MDBK cells and cow's serum sample are acted on, remove half serum and add nothing The culture medium of serum continues to cultivate, and cell culture is carried out into freeze thawing after the completion of culture；

Step 2) second takes turns blind passage：After cell culture obtained by MDBK cells and step 1) is acted on, add and contain 2% The culture medium of horse serum continues to cultivate, and cell culture is carried out into freeze thawing after the completion of culture；

Step 3) third round blind passage：MDBK cells and cell culture obtained by step 2) are acted on, after discarding supernatant Add the DMEM culture mediums containing 2% horse serum to continue to cultivate, cell culture is subjected to freeze thawing after the completion of culture；

Step 4) centrifugal treating：Cell culture obtained by step 3) retains supernatant after centrifuging twice；

Step 5) RNA is extracted：After the supernatant of step 4) adds TRIzol LS Reagent and piping and druming effect, chlorine is added Imitative mixing effect, then through centrifuging to obtain supernatant liquor；Isopropyl alcohol liquid is abandoned in centrifugation after the effect of supernatant liquor and isopropanol, add alcohol after Continuous centrifugation abandons alcohol liquid and obtains RNA；

Step 6) synthesizes full-length cDNA：Step 5) RNA reverse transcriptions are synthesized into viral genome using kit cDNA；

Step 7) PCR reacts：CDNA obtained by step 6) is entered into performing PCR reaction and completes bovine viral diarrhea in blood serum sample The separation identification of poison.

Further, the blood sample or cell vaccine ox blood final proof that the cow's serum sample gathers at oxtail vein Deng.Preferably, serum is drawn after the blood sample coagulation gathered at the oxtail vein, ox blood final proof is obtained after serum is centrifuged Product.Preferably, the cell vaccine generally refers to the cow's serum of commercialization with ox blood final proof.

Further, the MDBK cells in the step 1) are incubated overnight, and cover with 80% or so.

Further, in the step 1) MDBK cells and cow's serum sample in 5%CO₂In cell culture incubator, effect 4 is small When.

Further, the culture medium of serum-free is the DMEM culture mediums of serum-free in the step 1), and addition is removal Half serum amount.

Further, the time for continuing culture in the step 1) is 48~56 hours.

Further, frozen-thaw process is as follows in the step 1)：The cell culture of step 1) is after -20 DEG C freeze overnight Room temperature is placed, and is blown and beaten repeatedly after defrosting.

Further, the MDBK cells in the step 2) are incubated overnight, and cover with 80% or so.

Further, in the step 2) cell culture obtained by MDBK cells and step 1) in 5%CO₂Cell culture incubator It is interior, act on 2 hours.

Further, culture is continued in the step 2) in 5%CO₂In cell culture incubator, the time is 48~56 hours.

Further, frozen-thaw process is as follows in the step 2)：The cell culture of step 2) is after -20 DEG C freeze overnight Room temperature is placed, and is blown and beaten repeatedly after defrosting.

Further, MDBK cell pellet overnight cultures are covered with to after 80% in the step 3), are removed culture medium, are added step Rapid 2) gained cell culture, plasma-free DMEM medium are in 5%CO₂In cell culture incubator, act on 2 hours.

Further, culture is continued in the step 3) in 5%CO₂In cell culture incubator, the time is 48~56 hours.It is excellent Elect as 48 hours.

Further, frozen-thaw process is as follows in the step 3)：The cell culture of step 3) is after -20 DEG C freeze overnight Room temperature is placed, and is blown and beaten repeatedly after defrosting.

Further, centrifugation refers to twice in the step 4)：First through centrifuge 5000rpm/min, 4 DEG C centrifuge 5min, Remove cell fragment；Then again by centrifuge 12000rpm/min, 4 DEG C of centrifugation 10min.

Further, TRIzol LS Reagent are added in the step 5) and piping and druming effect refers to add TRIzol LS Reagent simultaneously blows and beats room temperature effect 5min.

Further, chloroform mixing effect is added in the step 5) and refers to add room temperature effect 10min after chloroform mixes.

Further, centrifuged in the step 5) supernatant liquor refers to centrifuge 13000rpm/min, 4 DEG C of centrifugations 15min obtains supernatant liquor.

Further, isopropyl alcohol liquid is abandoned in centrifugation to the step 5) after the effect of clear liquid and isopropanol at the middle and upper levels, add alcohol after It is continuous centrifugation abandon alcohol liquid obtain RNA detailed process it is as follows：Supernatant liquor and centrifuge after isopropanol room temperature effect 10min 13000rpm/min, 4 DEG C of centrifugation 15min abandon isopropyl alcohol liquid, add 75% alcohol and continue centrifuge 8000rpm/min, 4 DEG C of centrifugations 5min abandons alcohol liquid, adds deionized water dissolving and obtains RNA.

Further, kit is Thermoscript in the step 6)^TMRT-PCR kit；Specifically synthesize viral base Because group cDNA processes are as follows：

A) reaction system 1：Random hexamers:1μL；10mMdNTP mix:2μL；RNA:8μL；Cumulative volume：12μL； After 65 DEG C of effect 5min, it is immediately placed on ice；

B) reaction system 2:5×buffer:4μL；DTT(0.1M):1μL；RNaseOUT:1μL； ThermoTranscript^RT:1μL；Water:1μL；Cumulative volume：8μL；

C) reaction system 1 and reaction system 2 are acted on into 10min, 55 DEG C of effects 50min, 85 DEG C of effect 5min in 25 DEG C.

Further, the primer of PCR courses of reaction is as follows in the step 7)：

Sense primer：CATGCCCATAGTAGGAC；

Anti-sense primer:CCATGTGCCATGTACAG.

Further, the reaction system of PCR courses of reaction is as follows in the step 7)：10X Extaq buffer:2.5 μL；dNTP:1.5μL；Sense primer:0.5μL；Anti-sense primer:0.5μL；cDNA:1μL；Extaq:0.4μL；ddH₂O:18.6μ L；Cumulative volume：25μL.Preferably, it is 10 μM that deionized water diluted concentration is used before the sense primer and anti-sense primer use.

Further, the reaction condition of PCR courses of reaction is as follows in the step 7)：94℃ 5min；94℃ 30s, 54 DEG C of 30s, 72 DEG C of 25s, totally 30 circulations；72℃ 10min.

Further, detection process is as follows after the completion of PCR reactions in the step 7)：Using 2% Ago-Gel, by 8 μ Loading after LPCR products and 2 μ 5 × loading of L buffer mixing, the μ L/ holes of DL2000DNA Marker loadings 5, electrophoresis After 30min, after being dyed using EB, detected through gel imager.

Beneficial effect of the present invention：

A kind of method for carrying out separating identification to bovine viral diarrhea virus in cow's serum proposed by the invention, passes through profit Expanded step by step with 96 orifice plates, 24 orifice plates and 6 orifice plates, i.e., by three-wheel blind passage, then extract viral RNA, through synthesis virus Detection is carried out using RT-PCR realize that BVDV separation differentiates after genome cDNA.This method can be entered to batch samples simultaneously Row processing, operating efficiency is not only increased, virus is easier to accurately detect after amplification cultivation, so as to improve accuracy rate；Together When ensure virus integrality, it is time saving and energy saving, be clinical sample and commodity beneficial to the timely diagnosis and research of bovine viral diarrhoea Change serum batch sample separation BVDV and be each provided with practicable separation method, this method is simple to operation, economical and efficient, can Realize that batch sample detects, BVDV can be efficiently separated after blind passage three times, substantially reduces the time of virus purification.

Brief description of the drawings

The PCR primer electrophoresis result figure of Fig. 1 difference samples, in figure：M:DL2000 marker ,-:Negative control, #1-3: 1-3 samples ,+：Positive control, wherein #1 samples are BVDV feminine genders, and No. #2-3 is BVDV positives.

Embodiment

In order to be better understood from the present invention, it is described further with reference to embodiment and accompanying drawing.Need to illustrate , in order that the present invention is more succinct, experimental method, step and condition known to one of ordinary skill in the art etc. can press Routine operation, do not record in detail in the present invention.Illustrate this so that Suburb of Zhengzhou cattle farm samples progress virus purification detection as an example The embodiment of scheme.

Embodiment 1

The collection and processing of step (1) clinical serum sample

1. in October, 2017, from 3 parts of Zhengzhou suburban dairy village collection suspected case blood sample, method is such as Under：Taken a blood sample using through aseptic process, disposable 5ml syringes at tail vein 3ml, emit closed injection using injection needle rapidly Syringe needle, syringe is placed horizontally in the foam reserve tank of cleaning, normal temperature preserves to transport carries out subsequent operation (spring and autumn in laboratory The Winter Solstice can transport storage 3 days at normal temperatures, and summer, which needs to be preserved with ice, transports).

2. after blood clotting, the sterile working in super-clean bench, serum is drawn with pipettor, be transferred to 1.5ml sterilizings Centrifuge tube in.

3. centrifuging 3min with 4 DEG C of centrifuge 12000rpm, supernatant (serum) is transferred to the centrifugation of another 1.5ml sterilizings In pipe.

Step (2) carries out first round blind passage using 96 orifice plates

1. being inoculated with MDBK cells (bovine kidney cells) in 96 orifice plates, 80% is covered with after being incubated overnight.

2. 200 μ L blood serum samples are taken to add in 96 orifice plates respectively from each blood serum sample of step (1), every piece of 96 orifice plates In A1, A2 and A3 hole, wherein access culture medium in A4 holes does negative control.

3. 96 orifice plates in above-mentioned steps are placed in 5%CO₂In cell culture incubator, cultivate 4 hours.

4. sucking 100 μ L serum (leaving 100 μ L) from the A1-A4 holes of 96 orifice plates in order, pay attention to that per hole shifting will be changed Liquid pipette tips, the DMEM culture mediums that 100 μ L serum-frees are then added per hole continue to cultivate 48-56 hours.

Frozen 5. Tissue Culture Plate is placed in -20 DEG C of refrigerators, next day takes out 96 orifice plate room temperatures and placed, and waits culture to thaw Afterwards, blown and beaten repeatedly with liquid-transfering gun, promote cell cracking and releasing virus.

Step (3) carries out the second viral blind passage of wheel using 24 orifice plates

1. 24 orifice plates are inoculated with into MDBK cells, covered with after being incubated overnight to 80%.

2. the cell culture after freeze thawing in step (2) is blown and beaten repeatedly, 24 are all moved into according to corresponding flag sequence In the corresponding hole of orifice plate, per the μ L of pore volume about 200.

3. it is placed in 5%CO₂In cell culture incubator, culture acts on 2 hours, supernatant discarding, pays attention to that per hole pipette tips will be changed.

4. add the culture medium containing 2% horse serum, 500 μ L/ holes.

5. it is placed in 5%CO₂In cell culture incubator, continue to cultivate 48-56 hours.

Frozen 6. Tissue Culture Plate is placed in -20 DEG C of refrigerators, next day takes out Tissue Culture Plate room temperature and placed, and waits culture After defrosting, blown and beaten repeatedly with liquid-transfering gun, promote cell cracking and virus release.

Step (4) carries out third round virus blind passage using 6 orifice plates

1. filling 6 orifice plates inoculation MDBK cells, covered with after being incubated overnight to 80%.

2. discarding culture medium in 6 orifice plates, the cell culture in 24 orifice plates of step (3) is accessed into phase according to flag sequence In 6 orifice plates answered, poison 100 μ L of amount are met per hole, then add 500 μ L plasma-free DMEM mediums per hole.

3. it is placed in 5%CO₂In cell culture incubator, culture acts on 2 hours.

4. discarding supernatant (supernatant is culture medium and comes from the culture in 24 orifice plates), add containing 2% horse serum DMEM culture mediums, 1.5mL/ holes.

5. it is placed in 5%CO₂In cell culture incubator, cultivate 48 hours, attention observation cell state during this.

Frozen 6. Tissue Culture Plate is placed in -20 DEG C of refrigerators, next day takes out Tissue Culture Plate room temperature and placed, and waits culture After defrosting, blown and beaten repeatedly with liquid-transfering gun, promote cell cracking and releasing virus.

7. through centrifuge 5000rpm/min, 4 DEG C of centrifugation 5min, cell fragment is removed, then by 12000rpm/ Min, 4 DEG C of centrifugation 10min, takes supernatant to retain and carries out follow-up test.

The extraction of step (5) virus genome RNA

1. taking the supernatant of 250 μ L above-mentioned steps (4), TRIzol LS Reagent (Ambion, Cat are added: 10296010), 750 μ L/ samples, room temperature acts on 5min after piping and druming.

2. after adding the mixing of 0.2mL chloroforms, room temperature effect 10min.

3. with centrifuge 13000rpm, 4 DEG C of centrifugation 15min, liquid is divided into three layers, and RNA is contained on upper strata, 45 degree inclinations from, Upper liquid is drawn, is careful not to suck in intermediate layer.

4. the supernatant on upper strata is transferred in new 1.5mL centrifuge tubes, isopropanol 0.5mL/ samples, room temperature effect are added 10min is to precipitate RNA.

5. with centrifuge 13000rpm, 4 DEG C of centrifugation 15min, supernatant discarding,

6. toward precipitation in add 75% alcohol, addition be 1mL/ samples, gently vortex after, 4 DEG C of 8000rpm continue from Heart 5min.

7. after discarding alcohol, drying precipitated RNA.

8. after becoming water white transparency by milky Deng precipitation, adding 10 μ L deionized waters, RNA is dissolved.

Step (6) utilizes Thermoscript^TMRT-PCR kit (Invitrogen, catalogue#11146-024) Reverse transcription synthesizes full-length cDNA

1. add reagent according to following component：

Random hexamers:1μL

10mMdNTP mix:2μL

RNA:8μL

Above-mentioned cumulative volume is 12 μ L, and mentioned reagent dosage is a reaction system.

2. after 65 DEG C of effect 5min, it is immediately placed on ice.

3. preparing cDNA synthesis Mix simultaneously, system composition is as follows

5×buffer:4μL

DTT(0.1M):1μL

RNaseOUT:1μL

ThermoTranscript^RT:1μL

Water:1μL

Above-mentioned cumulative volume is 8 μ L

4. reacted as follows：

25 DEG C of effects 10min, 55 DEG C of effects 50min, 85 DEG C of effect 5min.

5. carry out next step PCR reactions after taking out or -20 DEG C save backup.

Step (7) PCR reacts

1. PCR reactions primer used is as follows：

Sense primer BVDVprimer1：CATGCCCATAGTAGGAC

Anti-sense primer BVDVprimer2:CCATGTGCCATGTACAG.

Primer deionized water is diluted to concentration as 10 μM.

2. each PCR reaction systems are as follows：

10×Extaq buffer:2.5μL

dNTP:1.5μL

BVDVprimer1:0.5μL

BVDVprimer2:0.5μL

cDNA:1μL

Extaq:0.4μL

ddH₂O:18.6μL

Cumulative volume：25μL

3. PCR reaction conditions are as follows：

I:94℃5min,

II:94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 25s, totally 30 circulations

III：72℃10min

The detection of step (8) PCR primer

Using 2% Ago-Gel, loading after 8 μ L PCR primers and 2 μ 5 × loading of L buffer are mixed, The μ L/ holes of 100bp DNAMarker loadings 5, after electrophoresis 30min, after being dyed using EB, through gel imager particular bands, PCR productions The band of thing is that 250bp maker bands approach in 281bp, with DL2000marker, and positive control is exactly BVDV, is illustrated and pre- Phase stripe size is consistent, and concrete outcome is as shown in Figure 1.Illustrate that the isolation and identification method of the present invention can be used in identification cow's serum having The BVDV of infectious, this can provide strong guarantee for animal vaccine production with the examinations polluted of BVDV in serum, to face The diagnosis of bovine viral diarrhoea and research offer foundation in bed production.

So far, although those skilled in the art will appreciate that detailed herein have shown and described the exemplary of the present invention Embodiment, still, without departing from the spirit and scope of the present invention, still can directly it be determined according to present disclosure Or derive many other variations or modifications for meeting the principle of the invention.Therefore, the scope of the invention should be understood and defined as Cover other all these variations or modifications.

Claims

A kind of 1. method for carrying out separating identification to bovine viral diarrhea virus in cow's serum, it is characterised in that detailed process is such as Under：

Step 1) first round blind passage：After MDBK cells and cow's serum sample are acted on, remove half serum and add serum-free Culture medium continue to cultivate, cell culture is subjected to freeze thawing after the completion of culture；

Step 2) second takes turns blind passage：After cell culture obtained by MDBK cells and step 1) is acted on, addition contains 2% horse blood Clear culture medium continues to cultivate, and cell culture is carried out into freeze thawing after the completion of culture；

Step 3) third round blind passage：MDBK cells and cell culture obtained by step 2) are acted on, added after discarding supernatant DMEM culture mediums containing 2% horse serum continue to cultivate, and cell culture is carried out into freeze thawing after the completion of culture；

Step 4) centrifugal treating：Cell culture obtained by step 3) retains supernatant after centrifuging twice；

Step 5) RNA is extracted：After the supernatant of step 4) adds TRIzol LS Reagent and piping and druming effect, add chloroform and mix Even effect, then through centrifuging to obtain supernatant liquor；Isopropyl alcohol liquid is abandoned in centrifugation after the effect of supernatant liquor and isopropanol, add alcohol continue from The heart abandons alcohol liquid and obtains RNA；

Step 6) synthesizes full-length cDNA：Step 5) RNA reverse transcriptions are synthesized into full-length cDNA using kit；

Step 7) PCR reacts：CDNA obtained by step 6) is entered into performing PCR reaction and completes bovine viral diarrhea virus in blood serum sample Separation identification.
2. bovine viral diarrhea virus carries out the method for separating identification in cow's serum according to claim 1, its feature exists In the blood sample or cell vaccine ox blood final proof that the cow's serum sample gathers at oxtail vein.
3. the method according to claim 1 for carrying out separating identification to bovine viral diarrhea virus in cow's serum, its feature It is, the MDBK cells in the step 1) are incubated overnight, and cover with 80%；MDBK cells and ox blood in the step 1) Final proof product are in 5%CO₂In cell culture incubator, act on 4 hours；

The culture medium of serum-free is the DMEM culture mediums of serum-free in the step 1), and addition is the half serum of removal Amount；

The time for continuing culture in the step 1) is 48~56 hours；

Frozen-thaw process is as follows in the step 1)：Room temperature is placed after the cell culture of step 1) freezes overnight in -20 DEG C, is thawed Blow and beat repeatedly afterwards.
4. the method according to claim 1 for carrying out separating identification to bovine viral diarrhea virus in cow's serum, its feature It is, the MDBK cells in the step 2) are incubated overnight, and cover with 80%；MDBK cells and step in the step 2) 1) gained cell culture is in 5%CO₂In cell culture incubator, act on 2 hours；

Continue culture in the step 2) in 5%CO₂In cell culture incubator, the time is 48~56 hours；

Frozen-thaw process is as follows in the step 2)：Room temperature is placed after the cell culture of step 2) freezes overnight in -20 DEG C, solution Blown and beaten repeatedly after jelly.
5. bovine viral diarrhea virus carries out the method for separating identification in cow's serum according to claim 1, its feature exists In, in the step 3) culture of MDBK cell pellet overnights cover with to after 80%, remove culture medium, add cell training obtained by step 2) Thing, plasma-free DMEM medium are supported in 5%CO₂In cell culture incubator, act on 2 hours；

Continue culture in the step 3) in 5%CO₂In cell culture incubator, the time is 48~56 hours；

Frozen-thaw process is as follows in the step 3)：Room temperature is placed after the cell culture of step 3) freezes overnight in -20 DEG C, is thawed Blow and beat repeatedly afterwards.
6. the method according to claim 1 for carrying out separating identification to bovine viral diarrhea virus in cow's serum, its feature It is, centrifugation refers to twice in the step 4)：First through centrifuge 5000rpm/min, 4 DEG C of centrifugation 5min, cell fragment is removed； Then again by centrifuge 12000rpm/min, 4 DEG C of centrifugation 10min.
7. bovine viral diarrhea virus carries out the method for separating identification in cow's serum according to claim 1, its feature exists In addition TRIzol LS Reagent and piping and druming, which act on, in the step 5) refers to add TRIzol LS Reagent and blow and beat Room temperature acts on 5min；

Chloroform mixing effect is added in the step 5) and refers to add room temperature effect 10min after chloroform mixes；

Centrifuged in the step 5) supernatant liquor refers to that with centrifuge 13000rpm/min, it is clear that 4 DEG C of centrifugation 15min obtain upper strata Liquid；

Clear liquid abandons isopropyl alcohol liquid to the step 5) with centrifugation after isopropanol effect at the middle and upper levels, and addition alcohol continues centrifugation and abandons alcohol liquid The detailed process for obtaining RNA is as follows：Supernatant liquor and centrifuge 13000rpm/min after isopropanol room temperature effect 10min, 4 DEG C of centrifugations 15min abandons isopropyl alcohol liquid, adds 75% alcohol and continues centrifuge 8000rpm/min, 4 DEG C centrifuge 5min and abandon alcohol liquid, and addition is gone Ionized water dissolves to obtain RNA.
8. bovine viral diarrhea virus carries out the method for separating identification in cow's serum according to claim 1, its feature exists In, in the step 6), kit Thermoscript^TMRT-PCR kit；

In the step 6), specific synthesis full-length cDNA process is as follows：

A) reaction system 1：Random hexamers:1μL；10mMdNTP mix:2μL；RNA:8μL；Cumulative volume：12μL；65℃ After acting on 5min, it is immediately placed on ice；

B) reaction system 2:5×buffer:4μL；DTT(0.1M):1μL；RNaseOUT:1μL；ThermoTranscript^RT:1μ L；Water:1μL；Cumulative volume：8μL；

C) reaction system 1 and reaction system 2 are acted on into 10min, 55 DEG C of effects 50min, 85 DEG C of effect 5min in 25 DEG C.
9. bovine viral diarrhea virus carries out the method for separating identification in cow's serum according to claim 1, its feature exists In the primer of PCR courses of reaction is as follows in the step 7)：

Sense primer：CATGCCCATAGTAGGAC；

Anti-sense primer:CCATGTGCCATGTACAG；

Reaction system is as follows：10X Extaq buffer:2.5μL；dNTP:1.5μL；Sense primer:0.5μL；Anti-sense primer: 0.5μL；cDNA:1μL；Extaq:0.4μL；ddH₂O:18.6μL；Cumulative volume：25μL；

Reaction condition is as follows：94℃5min；94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 25s, totally 30 circulations；72℃10min.
10. the method according to claim 1 for carrying out separating identification to bovine viral diarrhea virus in cow's serum, its feature It is, detection process is as follows after the completion of PCR reactions in the step 7)：Using 2% Ago-Gel, by 8 μ L PCR primers and 2 Loading after μ 5 × loading of L buffer mixing, the μ L/ holes of DL2000 DNA Marker loadings 5, after electrophoresis 30min, using EB After dyeing, detected through gel imager.