CN110412028B - Counting method of insect particle viruses - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 54
- 241000700605 Viruses Species 0.000 title claims abstract description 46
- 239000002245 particle Substances 0.000 title claims abstract description 41
- 241000238631 Hexapoda Species 0.000 title claims abstract description 32
- 239000012128 staining reagent Substances 0.000 claims abstract description 18
- 238000007865 diluting Methods 0.000 claims abstract description 10
- 238000010186 staining Methods 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 32
- 241000701451 unidentified granulovirus Species 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 229960000583 acetic acid Drugs 0.000 claims description 16
- 239000012362 glacial acetic acid Substances 0.000 claims description 16
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 claims description 14
- 238000010790 dilution Methods 0.000 claims description 14
- 239000012895 dilution Substances 0.000 claims description 14
- 230000003287 optical effect Effects 0.000 claims description 14
- 241000500437 Plutella xylostella Species 0.000 claims description 13
- 238000004043 dyeing Methods 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 241001635274 Cydia pomonella Species 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- 241000537222 Betabaculovirus Species 0.000 claims description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 claims description 3
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims description 2
- 239000012153 distilled water Substances 0.000 claims description 2
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims description 2
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 27
- 238000001514 detection method Methods 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 241000607479 Yersinia pestis Species 0.000 abstract description 3
- 239000000575 pesticide Substances 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 14
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- 238000004458 analytical method Methods 0.000 description 4
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- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000002917 insecticide Substances 0.000 description 2
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- 238000012986 modification Methods 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241000701366 unidentified nuclear polyhedrosis viruses Species 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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- Molecular Biology (AREA)
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Abstract
The invention provides a method for counting insect particle viruses, which comprises the following steps: 1) diluting the insect particle virus sample by 2-1000 times by using a staining reagent and staining the insect particle virus sample; 2) diluting the sample dyed in the step 1) by 2-1000 times by using a decolorizing reagent and decolorizing the sample; 3) counting the sample decolorized in the step 2) by using a bacteria counting plate, and calculating the particle concentration of the insect particle virus according to a formula. Compared with the prior art, the method provided by the invention is convenient to operate, easy to judge and accurate in result. Not only can quantitatively detect the insect particle body virus, but also can improve the accuracy and the reliability of detection. The method provided by the invention is simple to operate, and the used instrument is simple and easy, so that the method can be widely popularized and applied to the fields of pesticide production, agricultural pest control and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a quantitative detection method of viruses, in particular to a counting method of insect particle viruses.
Background
The insect granulosis virus belongs to the genus Beta baculovirus (Beta baculoviral) of the family baculovirus, exists in the form of protein inclusion bodies, each virus inclusion body contains 1 virus particle, and the virus inclusion bodies are in oblong granular shapes, are about 300-511nm long and are about 119-350nm wide. The infectivity of the virus is mainly determined by the integrity of DNA and protein capsid, and the virion is coated with granular protein in compact crystal lattice to form an inclusion body of the virus. The virus inclusion bodies can be processed into environment-friendly and safe biological insecticides for controlling host insects.
In the preparation of biological insecticide, the content measurement of virus inclusion body is always the key index for controlling product quality. However, inclusion bodies of insect granulosis virus are significantly smaller than nuclear polyhedrosis virus and cannot be counted under optical microscope in haemocytometer plates. The method of counting using an electron microscope and the molecular biological method based on fluorescence quantification are complicated in operation, require special equipment, and are expensive in apparatus. Methods for determining virus concentration by bioassay, methods for determining virus concentration based on insect mortality are time consuming and laborious. With spectrophotometry, the results are unstable and the error is large due to the formation of heterogeneous solutions. That is, the current counting method for the insect granulosis virus in the book body part in actual production generally has the problems of complicated operation, long culture time, poor result repeatability or excessive dependence on expensive instruments.
Therefore, a simple and rapid method for counting the insect particle viruses with low experience dependence and good repeatability is needed at present.
Disclosure of Invention
Therefore, the invention aims to provide a method for counting insect particle body viruses, which comprises the steps of sample pretreatment of the insect particle body viruses and counting by using a bacterial counting plate. The counting method provided by the invention is convenient to operate, easy to judge, accurate in result and capable of being widely popularized and applied to the fields of pesticide production, agricultural pest control and the like.
The invention comprises the following technical scheme:
a method of enumerating insect granulosis viruses, the method comprising the steps of:
1) diluting the insect particle virus sample by 2-1000 times by using a staining reagent and staining the insect particle virus sample;
2) diluting the sample dyed in the step 1) by 2-1000 times by using a decolorizing reagent and decolorizing the sample;
3) counting the sample decolorized in the step 2) by using a bacteria counting plate, and calculating the particle concentration of the insect particle virus according to a formula.
Preferably, the insect particle virus is a diamondback moth particle virus or codling moth particle virus
Preferably, in step 1), the staining reagent is selected from trypan blue, methylene blue eosin or coomassie brilliant blue G250.
More preferably, in step 1), the staining reagent is coomassie brilliant blue G250; further preferably, the concentration of the Coomassie brilliant blue G250 is 0.1-10G/L; even more preferably, the concentration of the Coomassie brilliant blue G250 is 0.5-5G/L; most preferably, the concentration of the Coomassie Brilliant blue G250 is 1G/L;
preferably, in the step 1), the staining comprises treating the insect granulosis virus sample with a staining reagent at 95-100 ℃ for at least 5 min; preferably, the staining reagent treatment time is at least 5min, at least 6min, at least 7min, at least 8min, at least 9min, at least 10 min; more preferably, the staining reagent treatment time is at least 10 min;
preferably, in step 1), the dilution factor is 2-100, more preferably 2-50, and most preferably 10.
Preferably, in step 2), the decolorizing agent is selected from distilled water, glacial acetic acid or a mixed solution of methanol, glacial acetic acid and water;
preferably, the decolorizing agent is a mixed solution of methanol, glacial acetic acid and water;
more preferably, the volume ratio of methanol to glacial acetic acid to water in the mixed solution is (1-5): (1-5): (1-10);
further preferably, the volume ratio of methanol, glacial acetic acid and water in the mixed solution is 1: (1-10);
still further preferably, the volume ratio of methanol, glacial acetic acid and water in the mixed solution is 1: 1: 8.
preferably, in step 2), the decolorizing agent is diluted for 1-10 minutes. .
Preferably, in step 2), the decolorizing agent is diluted 2-100 times, more preferably 2-50 times, and most preferably 10 times.
Preferably, in step 3), the counting uses an optical microscope, more preferably a phase contrast optical microscope;
preferably, in the step 3), the magnification of the microscope is 4-20 times of that of the eyepiece and 40-100 times of that of the objective; more preferably 20 times the eyepiece and 63 times the objective lens.
Calculating the formula:
cell counting plate formula of 16 × 25 cells: cell number/ml is 100 cells/100 × 400 × 10000 × dilution times;
1. cell counting plate calculation formula of 25 × 16 grids: cell number/ml 80 cell number/80 × 400 × 10000 × dilution factor.
The inventors of the present invention have found that, in the conventional method, it is necessary to determine whether or not microscopic particles are virus particles in counting, and the dependency of experience is very large, depending on actual experience. By using the method of the invention, the virus sample is treated by using the staining reagent with specific concentration, and the virus particles are colored after being diluted by specific times by using the decolorizing reagent, so that the judgment is easy, and the insect particle virus sample can be directly and quantitatively detected under an optical microscope.
Compared with the prior art, the invention has the following advantages:
1) the method provided by the invention is convenient to operate, easy to judge and accurate in result.
2) The method provided by the invention not only can quantitatively detect the insect granule virus, but also can improve the accuracy and reliability of detection.
3) The method provided by the invention is simple to operate, and the used instrument is simple and easy, so that the method can be widely popularized and applied to the fields of pesticide production, agricultural pest control and the like.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a photograph of a sample of a diamondback moth granulosis virus treated with a Coomassie brilliant blue G250 staining reagent and diluted with a decolorizing reagent and counted under a microscope using a bacterial counting plate, and it can be seen from FIG. 1 that the sample of the diamondback moth granulosis virus treated with the method of the present invention is more easily observed under the microscope;
FIG. 2 is a picture of the untreated sample of the diamond back moth granulosis virus counted under a microscope using a bacterial counting plate, and it can be seen from FIG. 2 that the untreated sample of the diamond back moth granulosis virus is not easily observed under the microscope;
FIG. 3 is a photograph of a sample of Plutella xylostella granulosis virus stained with a staining reagent, diluted with water and counted under a microscope using a bacterial counting plate, and it can be seen from FIG. 3 that the sample in the photograph is not as clearly visible as the sample diluted with a destaining reagent.
FIG. 4 is a picture of using a bacterial counting plate to count under a microscope after a codling moth granulosis virus sample is treated by using the method of the present invention, and it can be seen from FIG. 4 that the codling moth granulosis virus sample treated by using the method of the present invention is more clearly visible under the microscope.
Detailed Description
The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a process are given, but the scope of the present invention is not limited to the following embodiments.
Unless otherwise specified, the reagents used in the present invention are either laboratory or analytical grade and are commercially available.
Example 1 method for enumerating sample of Plutella xylostella granulosis Virus
1) Taking the concentration of 1 × 1012Sample 1 of the H.plutella xylostella granulosis virus OB/ml was diluted 10-fold with the staining reagent of the present invention, i.e., 0.1ml of the stock solution sample was taken, and 0.9ml of the staining reagent was added. The dyeing reagent comprises the following formula: coomassie brilliant blue G250 concentration of 1G/L, 0.22 μm membrane filtration. Sample 1 three sample replicates were selected.
Then, the sample was left at 100 ℃ for 10min and cooled to room temperature.
2) Diluting the sample treated in the step 1) by 100 times by using a decolorizing reagent and counting. The decolorizing reagent is methanol: glacial acetic acid: water in a volume ratio of 1: 1: 8.
3) counting under optical microscope
A bacterial counting plate was used, and the thickness of the cell was 0.02. mu.m.
Phase contrast optical microscope, 20 times eyepiece, 40 times objective or 63 times objective.
The sample concentration was calculated by the following formula
Calculating a formula according to the dilution times and the concentration of the sample to be detected:
the concentration (OB/ml) of the stock solution sample is 80 sum of the number of small square virus particles multiplied by 5 multiplied by 50000 multiplied by dilution factor
1.4 analysis of results
The diamondback moth granulosis virus sample is clearly seen under a microscope after being dyed and diluted by a decolorizing reagent (figure 1), the visibility difference from a non-dyed sample is larger (figure 2), and the visibility of a sample diluted by water is smaller (figure 3). after the virus particles are dyed by the dyeing agent of the method, the counting concentration of the decolorizing reagent is 6.5 × 1011OB/ml, no staining and no use of decolorizing agent the concentration of the counting (estimated) was 1.23 × 1011OB/ml-1.5×1011OB/ml, determined using a Guavaaesycyte microcapillary cell analyzer, was 3.86 × 1010OB/ml. This is due to the lack of staining and the absence of counting with decolorizing agents, due to the viral particle diameter of only 200nm, whereas the counting chamber height of the bacterial counting plate is 0.1mm, with a difference of nearly 200 times, and due to errors caused by imaging factors such as focusing. When the capillary cell analyzer is used for measurement, a sample needs to be filtered to remove impurities, and the capillary cell analyzer automatically identifies virus particle clusters which are adhered together and exceed a preset size, so that the final concentration of particle measurement is low.
Example 2 quantitative detection of Cydia pomonella particle Virus samples
1) Taking the concentration of 1 × 1012OB/ml of codling moth granulosis virus sample (sample 2), the dyeing reagent of the invention dilutes the sample 2 by 2 times, namely 0.1ml of stock solution sample is taken, and 0.1ml of the dyeing reagent is added. The dyeing reagent comprises the following formula: coomassie brilliant blue G250 concentration of 5G/L, 0.22 μm membrane filtration. Sample 2 three sample replicates were taken.
Then, the sample was left at 95 ℃ for 5min and cooled to room temperature.
2) Diluting the sample treated in the step 1) by 1000 times by using a decolorizing reagent and counting. The decolorizing reagent is methanol: glacial acetic acid: water in a volume ratio of 1: 1: 10.
3) counting under optical microscope
A bacterial counting plate was used, and the thickness of the cell was 0.02. mu.m.
Phase contrast optical microscope, 20 times eyepiece, 40 times objective or 63 times objective.
The sample concentration was calculated by the following formula
Calculating a formula according to the dilution times and the concentration of the sample to be detected:
the concentration (OB/ml) of the stock solution sample is 80 sum of the number of small square virus particles multiplied by 5 multiplied by 50000 multiplied by dilution factor
1.4 analysis of results
The codling moth granulosis virus sample is dyed, and the dyed virus particles can be clearly seen under a microscope after being diluted by using a decolorizing reagent (figure 4). The counting concentration of 7.0 × 10 is counted without using a dyeing method10OB/ml, lower than concentration after dyeing 2.1 × 1011OB/ml-2.6×1011OB/ml。
Example 3 method for enumerating sample of Plutella xylostella granulosis Virus
1) Taking the concentration of 1 × 1012Sample 1 of the plutella xylostella granulosis virus OB/ml was diluted 1000-fold with the staining reagent of the present invention, i.e., 0.1ml of the stock solution sample was taken, and 99.9ml of the staining reagent was added. The dyeing reagent comprises the following formula: trypan blue concentration was 0.1g/L, and 0.22 μm membrane filtration was performed. Sample 1 three sample replicates were selected.
Then, the sample was left at 95 ℃ for 6min and cooled to room temperature.
2) Diluting the sample treated in the step 1) by 2 times with a decolorizing reagent and counting. The decolorizing reagent is methanol: glacial acetic acid: water in a volume ratio of 1: 1: 1.
3) counting under optical microscope
A bacterial counting plate was used, and the thickness of the cell was 0.02. mu.m.
Phase contrast optical microscope, 20 times eyepiece, 40 times objective or 63 times objective.
The sample concentration was calculated by the following formula
Calculating a formula according to the dilution times and the concentration of the sample to be detected:
the concentration (OB/ml) of the stock solution sample is 80 sum of the number of small square virus particles multiplied by 5 multiplied by 50000 multiplied by dilution factor
1.4 analysis of results
The diamondback moth granulosis virus sample is clearly visible under a microscope after being dyed and diluted by a decolorizing reagent, and the visibility difference of the sample with the sample without dyeing is larger.
Example 4 quantitative detection of Cydia pomonella particle Virus samples
1) Taking the concentration of 1 × 1012OB/ml of Cydia pomonella particle virus sample (sample 2), the dyeing reagent of the invention dilutes sample 2 by 50 times, namely 0.1ml of stock solution sample is taken, and 4.9ml of the dyeing reagent is added. The dyeing reagent comprises the following formula: coomassie brilliant blue G250 concentration of 0.5G/L, 0.22 μm membrane filtration. Sample 2 three sample replicates were taken.
Then, the sample was left at 95 ℃ for 5min and cooled to room temperature.
2) Diluting the sample treated in the step 1) by 10 times with a decolorizing reagent and counting. The decolorizing reagent is methanol: glacial acetic acid: water in a volume ratio of 1: 1: 10.
3) counting under optical microscope
A bacterial counting plate was used, and the thickness of the cell was 0.02. mu.m.
Phase contrast optical microscope, 20 times eyepiece, 40 times objective or 63 times objective.
The sample concentration was calculated by the following formula
Calculating a formula according to the dilution times and the concentration of the sample to be detected:
the concentration (OB/ml) of the stock solution sample is 80 sum of the number of small square virus particles multiplied by 5 multiplied by 50000 multiplied by dilution factor
1.4 analysis of results
The codling moth granulosis virus sample is clearly visible under a microscope after being dyed and diluted by a decolorizing reagent.
Finally, the above embodiments are only used for illustrating the technical features of the present invention, and modifications or equivalent substitutions made by those skilled in the art based on the technical features of the present invention should be covered by the claims of the present invention without departing from the spirit and scope of the present invention.
Claims (24)
1. A method of enumerating insect granulosis viruses, the method comprising the steps of:
1) diluting the insect granulosis virus sample by 2-1000 times by using trypan blue, methylene blue eosin or Coomassie brilliant blue G250, and dyeing the insect granulosis virus sample;
2) diluting the sample dyed in the step 1) by 2-1000 times by using distilled water, glacial acetic acid or a mixed solution of methanol, glacial acetic acid and water, and decoloring;
3) counting the sample decolorized in the step 2) by using a bacteria counting plate, and calculating the particle concentration of the insect particle virus according to a formula.
2. The method of claim 1, wherein the insect particle virus is a diamondback moth particle virus or codling moth particle virus.
3. The method of claim 1, wherein in step 1), the staining reagent is coomassie brilliant blue G250.
4. The method according to claim 3, wherein the concentration of Coomassie Brilliant blue G250 is 0.1-10G/L.
5. The method according to claim 3, wherein the concentration of the Coomassie Brilliant blue G250 is 0.5-5G/L.
6. The method of claim 3, wherein the concentration of the Coomassie Brilliant blue G250 is 1G/L.
7. The method according to claim 1, wherein in step 1), the staining comprises treating the insect granulosis virus sample with a staining reagent at 95-100 ℃ for at least 5 min.
8. The method of claim 1, wherein in step 1), the staining comprises treating the insect granulosis virus sample with a staining reagent at 95-100 ℃ for at least 6min, at least 7min, at least 8min, at least 9min, or at least 10 min.
9. The method according to claim 1, wherein in step 1), the staining comprises treating the insect granulosis virus sample with a staining reagent at 95-100 ℃ for at least 10 min.
10. The method according to claim 1, wherein in step 1), the dilution factor is 2-100.
11. The method according to claim 1, wherein in step 1), the dilution factor is 2-50.
12. The method according to claim 1, wherein in step 1), the dilution factor is 10-fold.
13. The method according to claim 1, wherein, in step 2), the decolorizing agent is a mixed solution of methanol, glacial acetic acid, and water.
14. The method according to claim 13, wherein the volume ratio of methanol to glacial acetic acid to water in the mixed solution is (1-5): (1-5): (1-10).
15. The method of claim 13, wherein the volume ratio of methanol, glacial acetic acid, and water in the mixed solution is 1: (1-10).
16. The method of claim 13, wherein the volume ratio of methanol, glacial acetic acid, and water in the mixed solution is 1: 1: 8.
17. the method according to claim 1, wherein, in step 2), the decolorizing agent is diluted for 1-10 minutes.
18. The method according to claim 1, wherein, in step 2), the decolorizing agent is diluted 2-100 times.
19. The method according to claim 1, wherein, in step 2), the decolorizing agent is diluted 2-50 times.
20. The method according to claim 1, wherein, in step 2), the decolorizing agent is diluted by a factor of 10.
21. The method of claim 1, wherein in step 3), the counting uses an optical microscope.
22. The method according to claim 1, wherein in step 3) the counting uses a phase contrast optical microscope.
23. The method according to claim 21 or 22, wherein in step 3) the microscope has a magnification of 4-20 times for the eyepiece and 40-100 times for the objective.
24. The method according to claim 21 or 22, wherein in step 3) the microscope is at a magnification of 20 times eyepiece and 63 times objective.
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| CN101889583A (en) * | 2010-08-31 | 2010-11-24 | 中国科学院武汉病毒研究所 | Anti-Cydia pomonella biological pesticide and preparation method thereof |
| CN103820398A (en) * | 2014-02-17 | 2014-05-28 | 齐鲁动物保健品有限公司 | Mink enteritis virus recombinant subunit vaccine and preparation method thereof |
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| WO2004030631A2 (en) * | 2002-10-01 | 2004-04-15 | Chiron Corporation | Anti-cancer and anti-infectious disease compositions and methods for using same |
| CN101566579A (en) * | 2009-05-27 | 2009-10-28 | 扬州大学 | Dyeing counting method for detecting plaques of recombinant fowl pox virus live vaccine |
| CN103122353B (en) * | 2012-09-27 | 2014-11-12 | 华中农业大学 | Porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as preparation method and application thereof |
| CN103529205B (en) * | 2013-10-29 | 2015-06-17 | 中美华世通生物医药科技（武汉）有限公司 | Quantitative determination method for titer of recombinant insect baculovirus |
| CN106857680B (en) * | 2017-03-16 | 2020-05-05 | 河南省济源白云实业有限公司 | Granular virus dry suspending agent and preparation method thereof |
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| CN101889583A (en) * | 2010-08-31 | 2010-11-24 | 中国科学院武汉病毒研究所 | Anti-Cydia pomonella biological pesticide and preparation method thereof |
| CN103820398A (en) * | 2014-02-17 | 2014-05-28 | 齐鲁动物保健品有限公司 | Mink enteritis virus recombinant subunit vaccine and preparation method thereof |
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| 关于昆虫包含体病毒染色方法的探讨;王雄;《河北农业大学学报》;19851231;第8卷(第4期);第109-116页 * |
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