CN106290861A - A kind of detection method of exogenous avian leukosis virus - Google Patents
A kind of detection method of exogenous avian leukosis virus Download PDFInfo
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- CN106290861A CN106290861A CN201610635653.6A CN201610635653A CN106290861A CN 106290861 A CN106290861 A CN 106290861A CN 201610635653 A CN201610635653 A CN 201610635653A CN 106290861 A CN106290861 A CN 106290861A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
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Abstract
The invention discloses the detection method of a kind of exogenous avian leukosis virus, it is to carry out digesting, diluting by well-grown DF1 cell, passes in Tissue Culture Plate, 37 DEG C, 5%CO2Cultivate 25 35min, during to the cell of Tissue Culture Plate bottom deposit 45 55%, to the blood plasma of the most adherent addition in the every hole of Tissue Culture Plate chicken to be detected, stock solution in Tissue Culture Plate is discarded after overnight incubation, add the maintenance liquid containing 1% hyclone, continue to cultivate 78 days, culture is frozen；Defrosting culture, blows and beats mixing gently, adds in P27 antigen-reactive plate, detects P27 antigen, it is determined that whether chicken to be detected infects exogenous avian leukosis virus.Comparing traditional method, the inventive method can detect higher positive rate to same batch sample, and the S/P average detected is higher, and difference is the most notable；And there is equal stability, accuracy, and higher susceptiveness.
Description
Technical field
The invention belongs to microorganism separation detection technique field, more particularly it relates to an the white blood of exogenous fowl
The detection method of virus.
Background technology
Avian leukosis (Avian leukosis, AL) is by avian leukosis virus (Avian leukosis virus, ALV)
Cause based on the general name of the kinds of tumors disease of hematopoetic cell malignancies hypertrophy.ALV can infect all kinds of chicken group, has vertical biography
Broadcast, horizontal transmission and the circulation way such as insecticide, syringe needle, vaccine pollution, wherein vertical transmission is main infection mode.The most still
Without effective vaccine can prevention and control this is sick, also without effective drug treatment, the ALV of kind of chicken house can only be cut off by control techniques and pass
Dye source, controls this sick from source.
Mainly carry out fowl by means such as detection meconium, cloacal swab, Ovum Gallus domesticus album and virus purification white
Disorders of blood purifies.The mode utilizing chick embryo fibroblast system (DF1 cell) to carry out virus purification can distinguish the inside/outside source property white blood of fowl
Virus, the method is also presently the most effective purification method.The primary operational of existing virus isolation procedure (traditional method)
Step is as follows: 1) by well-grown DF1 passage in Tissue Culture Flask or culture plate, is placed in 37 DEG C, 5%CO2Training
Supporting in case and cultivate, cultivation to DF1 cell grows to 70-80% monolayer；2) aseptic collection chicken anticoagulation, centrifuging and taking blood plasma, by blood
Slurry is inoculated in the DF1 cell growing into 70-80% monolayer in right amount, is placed in 37 DEG C, 5%CO2Incubator is hatched 2 hours, change
Become the maintenance liquid containing 1% hyclone, continue to be placed in 37 DEG C, 5%CO2Incubator is cultivated；3) culture in step 2 is trained
Support after 7-8 days, by culture freeze thawing, then detect P27 antigen by ELISA kit description.
But, in tradition virus isolation procedure, treat that DF1 cell grows to 70-80% monolayer and just inoculates blood plasma, now thin
Born of the same parents are in good condition, highly stable, add the difficulty of Virus entry cell.Virus participates in adherent, the growth of DF1 cell the most completely
And fission process, and the most only hatched 2 hours and just changed liquid, the unimpinged cell of fractionated viral and irreproducible, lead
The positive rate causing detection P27 antigen is relatively low.Additionally, tradition virus isolation procedure operation complexity, cell monolayer need to be cultivated and hatch
Etc. step, technology is difficult to grasp.
Summary of the invention
Based on this, in order to overcome the defect of above-mentioned prior art, the invention provides a kind of new exogenous avian leukosis
The detection method of virus.The method can detect the exogenous ALV in chicken anticoagulation more accurately, more delicately.
In order to realize foregoing invention purpose, this invention takes techniques below scheme:
The detection method of a kind of exogenous avian leukosis virus, comprises the following steps:
(1), carry out well-grown DF1 cell digesting, being diluted to carefully with the DMEM culture fluid containing 10% hyclone
Born of the same parents' density is (1-1.04) × 105Individual/ml, passes in 24 porocyte culture plates, 37 DEG C, 5%CO2Cultivate 25-35min, to carefully
During the cell of born of the same parents culture plate bottom deposit 45-55%, to the blood plasma of the most adherent addition in the every hole chicken to be detected of Tissue Culture Plate,
Overnight incubation；
(2), discarding stock solution in Tissue Culture Plate, every hole adds the DMEM containing 1% hyclone and maintains liquid, continues to cultivate 7-
8 days, culture is placed in-20 DEG C frozen；
(3), the culture of step (2) is thawed, blow and beat mixing gently, draw 150ul culture the most respectively standby in 96 holes
In model, Zai Yong 12 road liquid-transfering gun is drawn 100ul culture and is added in P27 antigen-reactive plate, by ELISA kit (IDEXX)
Description detection P27 antigen, it is determined that whether chicken to be detected infects exogenous avian leukosis virus.
Wherein in some embodiments, described in step (1), the passage after dilution cultivates 30-35min, trains to cell
Support the cell of plate bottom deposit 50-55%, add the blood plasma of chicken to be detected.
Wherein in some embodiments, in chicken plasma volume to be detected and Tissue Culture Plate that in step (1), every hole adds
The volume ratio of culture is 1:20.
Wherein in some embodiments, described in step (1) and (2), the condition in incubator is: 37 DEG C, 5%CO2。
Wherein in some embodiments, described in step (1), the preparation method of the blood plasma of chicken to be detected is: by aseptic collection
The whole blood of chicken to be detected, adherent joins in the sodium heparin anticoagulant that mass percent is 1 ‰, and frozen centrifugation obtains blood plasma.
Wherein in some embodiments, above-mentioned frozen centrifugation condition is 4 DEG C, 10000rpm, centrifugal 25s.
Wherein in some embodiments, culture described in step (3) joins the method for P27 antigen-reactive plate and is: first
Drawing 150ul culture respectively in 96 holes in model, Zai Yong 12 road liquid-transfering gun is drawn 100ul and is added in P27 antigen-reactive plate.
Compared with prior art, the present invention has a following remarkable result:
1, the present invention detects the method for exogenous avian leukosis virus is by suitable concentration by well-grown DF1 cell
Pass in Tissue Culture Plate, only cultivate 25-35min and just inoculate blood plasma, the next day just change the maintenance liquid containing 1% hyclone into,
Virus take part in adherent, growth and the fission process of DF1 cell.Comparing traditional method, the inventive method is to same batch sample
Can detect higher positive rate, the S/P average detected is higher (difference is extremely notable, P 0.01)；And there is equal stablizing
Property, accuracy, and higher susceptiveness；
2, the culture of 150ul freeze thawing is charged first to 96 holes by the method detecting exogenous avian leukosis virus of the present invention
In standby model, then drawing 100ul with 12 road liquid-transfering guns and be added in P27 antigen-reactive plate, this operation shortens sample and joins
The time of P27 antigen-reactive plate, it is ensured that the concordance of different example reaction times.
Accompanying drawing explanation
Fig. 1 is to using the embodiment of the present invention 1 method P27 antigen S/P value with traditional method to carry out correlation analysis
Result figure.
Detailed description of the invention
The present invention is described in detail below in conjunction with the drawings and specific embodiments.
The experiment material related in following example if no special instructions, derives from commercially available, is adopted in following example
The routine operation that is well known to the skilled person of operation.
The detection method of the exogenous avian leukosis virus of embodiment 1
Certain 1 age in days is infected with standard strain ALV-J NX0101 strain (strain is given by poultry research department of Agricultural University Of South China)
Huang Yu kind chicken 61, is randomly divided into 2 groups, first group 30, second group 31, raises by kind of a fowl raising standard.At the 6th week
Gather the anticoagulation of first group of chicken, the anticoagulation of second group of chicken of collection in the 8th week, enter by traditional method and the present embodiment method respectively
Row Viral diagnosis.Comprise the following steps:
1, the anticoagulant collection of chicken and process
(1), preparing concentration is the sodium heparin anticoagulant of 1 ‰: add in 500ml distilled water molten by 0.5g heparin sodium powder
Solve, 121 DEG C of high pressure 30min, cooling.
(2), being dispensed into by anticoagulant in the 1.5ml centrifuge tube of high pressure, 100ul/ manages, and is placed in sampling box, and 96 pipes/
Box (numbering: 1-96), be placed in 4 DEG C of refrigerators standby.
(3), insulation sampler bag or bubble chamber: sampling box is put in sampler bag, put into several ice bag to keep low simultaneously
Temperature (ice bag is The more the better).
(4), sterile blood sampling: use 2ml asepsis injector, every chicken to gather the whole blood of more than 0.7ml, pull out syringe needle (necessary
Pull out syringe needle, in order to avoid haemolysis), adherent join in the centrifuge tube adding anticoagulant, shake up.Record the wing number of chicken simultaneously, be centrifuged
Pipe number and box number (necessary one_to_one corresponding).The blood adopted is put in low temperature samples bag, delivers to laboratory.
(5), centrifugal: will to be placed in tabletop refrigerated centrifuge containing anticoagulation centrifuge tube, 4 DEG C, 10000rpm, centrifugal 25s.
(6), separated plasma: with the most autoclaved rifle head, absorption 150ul blood plasma is in 96 porocyte plates in order, does
Good record.
2, DF1 cell and inoculation are prepared
(1), carry out well-grown DF1 cell digesting, being diluted to carefully with the DMEM culture fluid containing 10% hyclone
Born of the same parents' density is (1-1.04) × 105Individual/ml, passes on (every hole 1ml), 37 DEG C, 5%CO in 24 porocyte culture plates2Cultivate.
(2), the cell treated in above-mentioned steps cultivates 25-35min, to Tissue Culture Plate bottom deposit about 45-55%
During cell, the blood plasma of the most adherent addition in every hole 50ul chicken to be detected, 37 DEG C, 5%CO in Tissue Culture Plate2Incubator is trained
Support overnight.
In the conventional way (1 day in advance by well-grown DF1 passage in 24 porocyte culture plates, be placed in 37 DEG C,
5%CO2Incubator is cultivated to cell and grow to 70-80% monolayer, every the most adherent addition in hole in 24 porocyte culture plates
50ul blood plasma, 37 DEG C, 5%CO2Incubator is cultivated) as comparison.
3, liquid is changed
Overnight incubation after the blood plasma of reception detection chicken, changes liquid next day.Discard stock solution in cell plates, add containing 1% tire cattle
The DMEM of serum maintains liquid, is placed in 37 DEG C, 5%CO2Incubator continues cultivate culture to be placed in-20 DEG C of refrigerators in 7-8 days and freeze
Deposit.During this, every day observes, and makes a record (must assure that after cultivating 8 days, cell is the most dead).
In the conventional way (reception detection chicken blood plasma be placed on 37 DEG C, 5%CO2Incubator is hatched 2 hours, change into
DMEM containing 1% hyclone maintains liquid, continues to be placed in 37 DEG C, 5%CO2Incubator is cultivated 7-8 days) as comparison.
4, the detection of ALV-J P27 antigen
The culture of above-mentioned steps 3 is thawed, blows and beats mixing gently, draw 150ul respectively in 96 holes in model, then
Draw 100ul with 12 road liquid-transfering guns to be added in P27 antigen-reactive plate, detect P27 by the description of ELISA kit (IDEXX)
Antigen, reads data result of determination by microplate reader.When the S/P value of sample is more than 0.2, i.e. judge that this sample is as the positive.
5, result
First group of the 6th week blood sampling testing result of P27 antigen after virus purification is as shown in table 1.
1 first group of the 6th week blood sampling testing result of P27 antigen after virus purification of table
As shown in table 1,30 1 age in days Huang plumage kind chicken infection ALV-J are after 6 weeks, and the anticoagulation of collection uses traditional method respectively
Virus purification is carried out, through the detection of ALV-J P27 antigen with the inventive method.The P27 antigen positive that the inventive method detects
Rate is 80.00% (24/30), the positive rate that traditional technique in measuring goes out only 66.67% (20/30).
Second group of the 8th week blood sampling testing result of P27 antigen after virus purification is as shown in table 2.
2 second groups of the 8th week blood sampling testing results of P27 antigen after virus purification of table
As shown in table 2, the P27 antigen positive rate that the method for the embodiment of the present invention 1 detects is 67.74% (21/31), passes
The positive rate that system method detects only has 61.29% (19/31).
To sum up repeat result of the test to show, to same batch sample, the positive rate detected by the method for the embodiment of the present invention 1
Higher than the positive rate that traditional technique in measuring goes out.
Test example 1 embodiment of the present invention 1 detects the correlation analysis of P27 antigen S/P value with traditional detection method
Utilize JMP8 software, detection method and the S/ of traditional detection method detection P27 antigen to the embodiment of the present invention 1
P value carries out correlation analysis, and result is as shown in Figure 1.
Fig. 1 result shows, the method S/P average (1.70710) of the embodiment of the present invention 1 is higher than traditional method S/P average
, and difference extremely notable (P=0.0073 0.01), the i.e. method of the embodiment of the present invention 1 is sensitiveer than traditional method (1.11072).
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
1. the detection method of an exogenous avian leukosis virus, it is characterised in that comprise the following steps:
(1), carry out well-grown DF1 cell digesting, to be diluted to cell with the DMEM culture fluid containing 10% hyclone close
Degree is (1-1.04) × 105Individual/ml, passes on cultivation 25-35min in Tissue Culture Plate, to Tissue Culture Plate bottom deposit 45-
55% cell time, to the blood plasma of the most adherent addition in the every hole chicken to be detected of Tissue Culture Plate, overnight incubation；
(2), discarding stock solution in Tissue Culture Plate, every hole adds the DMEM containing 1% hyclone and maintains liquid, continues to cultivate 7-8 days,
Culture is placed in-20 DEG C frozen；
(3), the culture of step (2) is thawed, blow and beat mixing gently, add in P27 antigen-reactive plate, detect P27 antigen, sentence
Whether fixed chicken to be detected infects exogenous avian leukosis virus.
The detection method of exogenous avian leukosis virus the most according to claim 1, it is characterised in that institute in step (1)
State the passage after dilution and cultivate 30-35min, to the cell of Tissue Culture Plate bottom deposit 50-55%, to Tissue Culture Plate
The blood plasma of the most adherent addition in every hole chicken to be detected.
The detection method of exogenous avian leukosis virus the most according to claim 1, it is characterised in that every in step (1)
The volume ratio of rear cell culture is 1:20 to the chicken plasma volume to be detected that hole adds with Tissue Culture Plate.
The detection method of exogenous avian leukosis virus the most according to claim 1, it is characterised in that institute in step (1)
The preparation method of the blood plasma stating chicken to be detected is: by the whole blood of chicken to be detected for aseptic collection, and the adherent mass percent that joins is
In the sodium heparin anticoagulant of 1 ‰, frozen centrifugation, obtain blood plasma.
The detection method of exogenous avian leukosis virus the most according to claim 4, it is characterised in that institute in step (1)
Stating frozen centrifugation condition is 4 DEG C, 10000rpm, centrifugal 25s.
The detection method of exogenous avian leukosis virus the most according to claim 1, it is characterised in that institute in step (3)
State cell culture and add to the method for P27 antigen-reactive plate and be: draw 150ul culture the most respectively in 96 holes in model, then
Draw 100ul with 12 road liquid-transfering guns to be added in P27 antigen-reactive plate.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107541487A (en) * | 2017-09-05 | 2018-01-05 | 佛山市高明区新广农牧有限公司 | A kind of separatory cell culture processes of DF 1 of avian leukosis virus |
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| CN107541487A (en) * | 2017-09-05 | 2018-01-05 | 佛山市高明区新广农牧有限公司 | A kind of separatory cell culture processes of DF 1 of avian leukosis virus |
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Address after: 527400 9 East Causeway North Road, Xinxing County new town, Yunfu, Guangdong Patentee after: Winson food group Limited by Share Ltd Address before: 527400 9 East Causeway North Road, Xinxing County new town, Yunfu, Guangdong Patentee before: Guangdong Wens Foodstuff Group Co., Ltd. |