CN101921877A - Method for quickly measuring viral titer of rabies viruses by using cell plaques - Google Patents
Method for quickly measuring viral titer of rabies viruses by using cell plaques Download PDFInfo
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- CN101921877A CN101921877A CN 201010273194 CN201010273194A CN101921877A CN 101921877 A CN101921877 A CN 101921877A CN 201010273194 CN201010273194 CN 201010273194 CN 201010273194 A CN201010273194 A CN 201010273194A CN 101921877 A CN101921877 A CN 101921877A
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- rabies virus
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Abstract
The invention provides a method for quickly measuring viral titer of rabies viruses by using cell plaques. The method comprises the following steps of: (1) preparing a methylcellulose covered culture medium; (2) preparing cell suspension; (3) diluting rabies virus solution to be measured to obtain gradient concentration rabies virus solution; (4) adding the cell suspension and the rabies virus solution into cell culture holes of a cell culture plate in a volume ratio of 9:1, and placing the cell culture plate into a CO2 incubator for culture; (5) removing the supernate from the cell culture plate, adding the methylcellulose covered culture medium into the culture holes, and placing the cell culture plate into the CO2 incubator for continuous culture; and (6) performing crystal violet coloration, observing the number of plaques in each culture hole of the cell culture plate, and calculating the viral titer of the rabies virus solution to be measured by using the plaque number. The measurement method has the advantages of simple operation, quickness, accuracy, strong controllability and little lot error, and shortens the inspection period of the viral titer of the rabies viruses.
Description
Technical field
The present invention relates to the measuring method of rabies virus poison valency, especially relate to the method that a kind of cell plaques is measured rabies virus poison valency.
Background technology
At present, the measuring method of rabies virus poison valency generally adopts mouse medium lethal dose (LD50).The LD50 measuring method need be used living animal, and the individual difference of living animal will be brought very big influence to detected result; And the LD50 mensuration cycle is long, and the ability result of determination will certainly prolong the inspection of semifinished product cycle in the production of vaccine like this after 14 days.
In addition, the cell plaques method also is a kind of effective ways that rabies virus poison valency is measured.Behind the virus infected cell, by the restriction of semisolid or solid dielectric, the virus of release can only be by the cell of initial infection to circumferential expansion.Through several proliferating cycles, just form a limitation lesion region, promptly viral plaque.Theoretically, plaque is exactly that a virion by initial cells infected forms, thereby can measure malicious valency by the virion counting.Flow in medium by the virion that prevents from media such as methylcellulose gum or agar to discharge, so that plaque is controlled in the suitable scope.For ease of visual inspection, dyeings such as Viola crystallina commonly used, the viable cell layer is dyed intense violet color, because sick cell absorbing dye not, circular non-staining transparent region is a plaque unit in the cell plate.The titre of viral suspension can be represented with every milliliter of plaque forming unit (PFU/ml).In actually operating, need the suitable medium of configuration, control inoculation condition and reaction conditions effectively and could obtain reliable measurement result.
Reported that both at home and abroad rabies virus plaque experimental period is 5～10, the time that plaque forms is still longer.In addition, the Plaque determination method exists requirement for experiment condition strictness, operation steps complexity, plaque is difficult for shortcomings such as formation maybe can not form, reproducibility difference, does not form the detection method of standard up till now yet.
Summary of the invention
In view of this, main purpose of the present invention is to provide the method for quickly measuring viral titer of rabies viruses by using cell plaques, may further comprise the steps:
1) methylcellulose gum is mixed with the mass ratio of cell maintenance medium according to 1: 99～2: 98, methylcellulose gum is fully dissolved, obtain mass percent concentration and be 1%～2% methylcellulose gum and cover substratum;
2) after digestion was in the cell of logarithmic phase, with the cell growth medium dilution, obtaining cell density was 1 * 10
5Cells/ml～5 * 10
5The cell suspension of cells/ml;
3) use cell maintenance medium with 10 times of rabies virus liquid that serial dilution is to be determined, obtain 10
4～10
8Times serial dilution gradient concentration rabies virus liquid;
4) with step 2) described cell suspension and the described rabies virus liquid of step 3), join with 9: 1 ratio of volume ratio in the cell cultures hole of Tissue Culture Plate, get step 2 simultaneously) described cell suspension and the described cell maintenance medium of step 1), join with 9: 1 ratio of volume ratio in the cell cultures hole of Tissue Culture Plate as negative control, place 36 ℃～38 ℃, CO
2Cultivate 6～12h in the incubator, form cellular layer behind the cell attachment, discard the supernatant liquor in the described Tissue Culture Plate, wash cell with phosphate buffered saline buffer;
5) in the cell cultures hole, add the described methylcellulose gum of step 1) and cover substratum 1ml～2ml,, place 33 ℃～36 ℃, CO as tectum
2After continuing in the incubator to cultivate 72～96h, add staining fluid dyeing, the plaque number of each culture hole of observation of cell culture plate is with the malicious valency of plaque number calculating rabies virus liquid to be determined.
Preferably, methylcellulose gum of the present invention covers the HEPES that contains 50～80mmol/L in the substratum.
Preferably, cell maintenance medium of the present invention is the MEM that contains the bovine serum of 50～100 μ g/ml DEAE-Dextran and 3% (v/v).
Preferably, cell growth medium of the present invention is the MEM that contains the bovine serum of 50～100 μ g/ml DEAE-Dextran and 6% (v/v).
Preferably, CO of the present invention
2CO in the incubator
2Volume content is 2%～10%.Preferably, staining fluid of the present invention be in Viola crystallina, 0.01%～0.1% (w/v) toluylene red of 0.5%～1.5% (w/v) or 0.5%～5% (w/v) iodine liquid any.
Technique effect
The method of cell plaques rapid determination rabies virus of the present invention adopts monolayer cell to cultivate, and has viral plaque is reacted peculiar susceptibility, repeatability and specificity.
Secondly, cell maintenance medium, the cell growth medium of the present invention's employing can promote virus and cell absorption.And the cell growth medium of viral plaque with keep in the liquid, all added DEAE-Dextran, also add the HEPES of 50～80mmol/L in the methylcellulose gum culture medium solution, therefore can accelerate the formation of plaque greatly, shortened the cycle that rabies virus detects.
At last, it is sterilization methylcellulose gum powder directly to be dissolved in keep in the liquid that methylcellulose gum covers the substratum compound method, and the preparation method is simple.
In sum, contain the HEPES of 50～80mmol/L in the methylcellulose gum culture medium solution, can accelerate the formation of plaque; It is short that this method continues the cycle, result of determination with the naked eye on the 3rd～4, and special, responsive, good reproducibility, easy and simple to handle, economy is easy to promote; Solve rabies virus because of there not being the problem that obvious cytopathy is difficult for mensuration, had certain using value; Both can be used for virus clone, also can be used for the mensuration of virus and the detection of neutralizing antibody, and brought bigger convenience for the research of rabies virus.
Description of drawings
Fig. 1 is the visual inspection figure of plaque;
Fig. 2 is the microphotograph of plaque.
Embodiment
The ratio that the present invention has tested methylcellulose gum and cell maintenance medium adopts 1: 99～2: 98 mass ratio, preferably, uses 1: 99 mass ratio, obtains mass percent concentration and be 1% methylcellulose gum covering substratum.
The present invention has tested and added cell suspension and rabies virus liquid, CO in the cell cultures hole
2Cultivate 6～12h in the incubator, form cellular layer behind the cell attachment, preferably, after cell attachment forms the monolayer cell layer, the detection step below continuing.
The present invention has tested and has used 50～80mmol/L HEPES in the methylcellulose gum substratum, preferably, used the HEPES of 50mmol/L in the embodiment of the invention, and the cultivation initial stage the preparation tectum in directly the adding 50mmol/L HEPES, the step that not only simplifies the operation can also make the plaque formation time further shift to an earlier date simultaneously.
The present invention has tested and has added 50～100 μ g/ml DEAE-Dextran in cell growth medium and the cell maintenance medium, also can promote plaque to form, and shortens and observes detection time.
Viola crystallina, 0.01%～0.1% (w/v) toluylene red or three kinds of different staining agents of 0.5%～5% (w/v) iodine liquid that the present invention has tested 0.5%～1.5% (w/v) dye to the cell of cultivating 3～4, all can form clear plaque, the Viola crystallina of preferred 0.5%～1.5% (w/v).
In the embodiment of the invention, virus strain is Flury-LEP strain (China Veterinery Drug Inspection Office, bacterial classification number: AV2012), the method that the virus that other this areas are commonly used such as Flury-HEP strain, ERA strain, CTN-1 strain and aG strain etc. also can be used for the present invention is carried out viral level and is measured.
In the embodiment of the invention, passage cell is BHK-21 cell (a young hamster kidney passage cell), and the method that the passage cell that other this areas are commonly used such as Vero cell (African green monkey kidney cell), PK-2A cell (porcine kidney cell), Hamster Kidney cell (hamster kidney cell) and CEF cell (chick embryo fibroblast) etc. also can be used for the present invention is carried out viral level and measured.
For making the present invention easier to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in to limit the scope of the invention that NM concrete experimental technique in the following example carries out according to the normal experiment method usually.
Embodiment 1
Preparation MEM basal liquid: get 9.6g MEM and be dissolved in the 1000ml water, add 2.2g NaHCO then
3, and adjust pH value to 7.2 with the HCl of 1mol/L or NaOH, and adding DEAE-Dextran to make its concentration be 80 μ g/ml, as 1 * MEM basal liquid, the MEM substratum of increase serum is not as basal liquid after the sterile filtration.In 1 * MEM basal liquid, add the bovine serum of 3% volume percent as cell maintenance medium; In 1 * MEM basal liquid, add the bovine serum of 6% volume percent as cell growth medium; Getting part keeps liquid to add final concentration is that the HEPES of 50mmol/L is standby.
The method of this cell plaques rapid determination rabies virus may further comprise the steps:
(1) sterilized methylcellulose gum is mixed according to 1: 99 mass ratio with the liquid of keeping that contains 50mmol/L HEPES, place more than 3 days for 4 ℃, methylcellulose gum is fully dissolved, obtain mass percent concentration and be 1% methocel solution, as tectum; Face time spent preheating in 37 ℃ of water-baths;
(2) digestion is in the BHK-21 cell of logarithmic phase, dilutes the BHK-21 cell that is in logarithmic phase with growth media, and making cell density is 2.5 * 10
5Cells/ml obtains the BHK-21 cell suspension;
(3) with keeping liquid with 10
1, 10
2, 10
3, 10
4, 10
5, 10
6, 10
7, 10
8Times serial dilution rabies virus liquid to be determined obtains gradient concentration rabies virus liquid;
(4) described cell suspension is joined in the Tissue Culture Plate by the 0.9ml/ hole;
(5) described gradient concentration rabies virus liquid is joined in the Tissue Culture Plate that contains cell suspension that obtains in the step (4) by the 0.1ml/ hole, negative control is set simultaneously, place CO
2Cultivate CO in the incubator
2The CO of incubator
2Volume content is 5%, and temperature is 37 ℃;
Behind (6) 6～12h cell attachments, discarding the supernatant liquor in the described Tissue Culture Plate, is that 0.01mol/L, pH value are 7.2 sterilization PBS flushing one time with volumetric molar concentration, comes off fully up to dead cell; The mass percent concentration that every then hole adding step (1) is prepared is 1% methylcellulose gum covering substratum 1ml, as tectum, places CO
2Continue in the incubator to cultivate CO
2The CO of incubator
2Volume content is 5%, and temperature is 35 ℃;
(7) discard tectum behind the 96h, and be that 0.01mol/L, pH value are 7.2 sterilization PBS flushing three times, until methylcellulose gum is rinsed well with concentration; Every then hole adds the crystal violet solution of 1ml, 1% mass percent, behind the dyeing 20min, slowly washes 15min with tap water under the room temperature condition (25 ℃), rinses well until staining fluid, dries the plaque number of each culture hole of visual inspection Tissue Culture Plate;
(8) negative control does not have the Tissue Culture Plate that plaque occurs, and is judged to effective plate, according to the consumption of the rabies virus liquid to be determined of the plaque number of each culture hole of effective plate, inoculation and the malicious valency that extension rate calculates rabies virus liquid to be determined.
Be respectively shown in the arrow among Fig. 1 and Fig. 2 and observe the viral plaque of observing with microscopically, the extent of dilution of B1 among Fig. 1～B5 correspondence is respectively rabies virus 1 * 10 to be measured
4, 1 * 10
5, 1 * 10
6, 1 * 10
7, 1 * 10
8Extension rate, repeat in 4 holes of each extent of dilution.4 pitting spot numbers of B3 are respectively: 15, the plaque number average in 16,12,10,4 holes within 8～30 scopes, so B3 institute corresponding 1 * 10
6Be the suitableeest extension rate, as previously mentioned, v (the viral volume of every hole inoculation) is 0.1ml, calculates according to formula:
A(PFU/ml)＝(15+16+12+10)/4×10
6/0.1＝1.33×10
8(PFU/ml)
Measuring method of the present invention is simple to operate, controllability is strong, batch between error little, criticize an error less than log10 according to experimental result
0.2, can realize that the stdn of rabies virus poison valency is measured, and shorten half-finished round of visits in the Rabies Vaccine production.
The method of calculation of error are with same rabies virus liquid to be checked between batch, packing in a small amount, and very low temperature is preserved, and divides 10 tests to detect this malicious sample to be checked, and the result of 10 detections takes the logarithm, and calculates the numerical value that obtains behind the overall standard deviation of 10 samples.
Same viral liquid duplicate test 10 times, assay is represented to be respectively with logarithm: 8.33,8.42,8.18,8.24,8.02,8.15,8.35,8.08,8.33,8.27, the calculated population sample standard deviation is 0.12, is log10
0.12
The above only is preferred embodiment of the present invention, and is in order to restriction the present invention, within the spirit and principles in the present invention not all, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the method for a quickly measuring viral titer of rabies viruses by using cell plaques is characterized in that, may further comprise the steps:
1) methylcellulose gum is mixed with the mass ratio of cell maintenance medium according to 1: 99～2: 98, methylcellulose gum is fully dissolved, obtain mass percent concentration and be 1%～2% methylcellulose gum and cover substratum;
2) after digestion was in the cell of logarithmic phase, with the cell growth medium dilution, obtaining cell density was 1 * 10
5Cells/ml～5 * 10
5The cell suspension of cells/ml;
3) use cell maintenance medium with 10 times of rabies virus liquid that serial dilution is to be determined, obtain 10
4～10
8Times serial dilution gradient concentration rabies virus liquid;
4) with step 2) described cell suspension and the described rabies virus liquid of step 3), join with 9: 1 ratio of volume ratio in the cell cultures hole of Tissue Culture Plate, get step 2 simultaneously) described cell suspension and the described cell maintenance medium of step 1), join with 9: 1 ratio of volume ratio in the cell cultures hole of Tissue Culture Plate as negative control, place CO
2Cultivate 6～12h in the incubator, form cell monolayer behind the cell attachment, discard the supernatant liquor in the described Tissue Culture Plate, wash cell with phosphate buffered saline buffer;
5) in the cell cultures hole, add the described methylcellulose gum of step 1) and cover substratum 1ml～2ml,, place CO as tectum
2After continue cultivating 72～96h in the incubator, add staining fluid dyeing, the plaque number of each culture hole of observation of cell culture plate calculates the malicious valency of rabies virus liquid to be determined with the plaque number.
2. the method for cell plaques rapid determination rabies virus according to claim 1 is characterized in that, methylcellulose gum described in the step 1) covers the HEPES that contains 50～80mmol/L in the substratum.
3. the method for cell plaques rapid determination rabies virus according to claim 1 is characterized in that step 2) described in cell be BHK-21 cell, Vero cell, PK-2A cell, Hamster Kidney cell and CEF cell.
4. the method for cell plaques rapid determination rabies virus according to claim 1 is characterized in that: the cell maintenance medium step 1), 3) is the MEM that contains the bovine serum of 50～100 μ g/mlDEAE-Dextran and 3% volume percent.
5. the method for cell plaques rapid determination rabies virus according to claim 1 is characterized in that step 2) described cell growth medium is the MEM that contains the bovine serum of 50～100 μ g/ml DEAE-Dextran and 6% volume percent.
6. the method for cell plaques rapid determination rabies virus according to claim 1 is characterized in that, forms monolayer cell behind the cell attachment in the cell cultures hole in the step 4).
7. the method for cell plaques rapid determination rabies virus according to claim 1 is characterized in that, step 4), 5) in described CO
2CO in the incubator
2Volume content is 2%～10%.
8. the method for cell plaques rapid determination rabies virus according to claim 1 is characterized in that, culture temperature described in the step 4) is 36 ℃～38 ℃.
9. the method for cell plaques rapid determination rabies virus according to claim 1 is characterized in that, culture temperature described in the step 5) is 33 ℃～36 ℃.
10. the method for cell plaques rapid determination rabies virus according to claim 1 is characterized in that, the staining agent in the step 5) is Viola crystallina, 0.01%～0.1%w/v toluylene red or the 0.5%～5%w/v iodine liquid of 0.5%～1.5%w/v.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010273194.4A CN101921877B (en) | 2010-05-31 | 2010-09-02 | Method for quickly measuring viral titer of rabies viruses by using cell plaques |
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|---|---|---|---|
| CN201010186192.1 | 2010-05-31 | ||
| CN201010186192A CN101831509A (en) | 2010-05-31 | 2010-05-31 | Method for titrating rabies viruses quickly with cell plaques |
| CN201010273194.4A CN101921877B (en) | 2010-05-31 | 2010-09-02 | Method for quickly measuring viral titer of rabies viruses by using cell plaques |
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| CN101921877A true CN101921877A (en) | 2010-12-22 |
| CN101921877B CN101921877B (en) | 2014-04-16 |
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| CN201010273194.4A Active CN101921877B (en) | 2010-05-31 | 2010-09-02 | Method for quickly measuring viral titer of rabies viruses by using cell plaques |
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| CN114264530B (en) * | 2021-12-30 | 2023-05-05 | 重庆市畜牧科学院 | Virus plaque determination method based on Avicel and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4514497A (en) * | 1983-12-30 | 1985-04-30 | Novagene, Ltd. | Modified live pseudorabies viruses |
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2010
- 2010-05-31 CN CN201010186192A patent/CN101831509A/en active Pending
- 2010-09-02 CN CN201010273194.4A patent/CN101921877B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4514497A (en) * | 1983-12-30 | 1985-04-30 | Novagene, Ltd. | Modified live pseudorabies viruses |
| US4514497B1 (en) * | 1983-12-30 | 1998-02-24 | Novagene Inc | Modified live pseudorabies viruses |
Non-Patent Citations (5)
| Title |
|---|
| 《中国病毒学》 19940331 邵益斌等 应用快速HEPES蚀斑法检测狂犬病毒 70-73 2 第9卷, 第1期 2 * |
| 《兽医大学学报》 19920930 岳军明等 鹿狂犬病病毒8202株在BHK21细胞上蚀斑形成方法的建立 300-301 1,3-10 第12卷, 第3期 2 * |
| 《兽医大学学报》 19920930 岳军明等 鹿狂犬病病毒8202株在BHK21细胞上蚀斑形成方法的建立 300-301 2 第12卷, 第3期 2 * |
| 《黑龙江畜牧兽医》 19931231 岳军明等 甲基纤维素在狂犬病毒蚀斑中的应用 29-30 1,3-10 , 第3期 2 * |
| 《黑龙江畜牧兽医》 19931231 岳军明等 甲基纤维素在狂犬病毒蚀斑中的应用 29-30 2 , 第3期 2 * |
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| Publication number | Publication date |
|---|---|
| CN101921877B (en) | 2014-04-16 |
| CN101831509A (en) | 2010-09-15 |
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