CN101921877B - 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 PDF

Info

Publication number
CN101921877B
CN101921877B CN201010273194.4A CN201010273194A CN101921877B CN 101921877 B CN101921877 B CN 101921877B CN 201010273194 A CN201010273194 A CN 201010273194A CN 101921877 B CN101921877 B CN 101921877B
Authority
CN
China
Prior art keywords
cell
viral titer
plaques
rabies
rabies virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201010273194.4A
Other languages
Chinese (zh)
Other versions
CN101921877A (en
Inventor
张许科
孙进忠
乔荣岑
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pulaike Biological Engineering Co Ltd
Original Assignee
Pulaike Biological Engineering Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pulaike Biological Engineering Co Ltd filed Critical Pulaike Biological Engineering Co Ltd
Priority to CN201010273194.4A priority Critical patent/CN101921877B/en
Publication of CN101921877A publication Critical patent/CN101921877A/en
Application granted granted Critical
Publication of CN101921877B publication Critical patent/CN101921877B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

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

The method of quickly measuring viral titer of rabies viruses by using cell plaques
Technical field
The present invention relates to the measuring method of rabies virus poison valency, especially relate to a kind of method that 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).LD50 measuring method need to be used living animal, and the individual difference of living animal will be brought very large impact to detected result; And LD50 determination period is long, after 14 days, ability result of determination, will certainly extend the inspection of semifinished product cycle in production of vaccine like this.
In addition, cell plaques method is also a kind of effective ways that rabies virus poison valency is measured.After virus infected cell, by the restriction of semisolid or solid dielectric, the virus of release can only be by the cell infecting at first to circumferential expansion.Through several proliferating cycles, just form a Focal lesion region, i.e. viral plaque.Theoretically, plaque is exactly that a virion by initial cells infected forms, thereby can count the malicious valency of measuring by virion.By the virion that prevents from media such as methylcellulose gum or agar discharging, in medium, flow, to plaque is controlled in suitable scope.For ease of visual inspection, the dyeings such as conventional Viola crystallina, viable cell layer is dyed intense violet color, and due to sick cell absorbing dye not, in cell plate, circular non-staining transparent region is Yi Ge plaque unit.The titre of viral suspension can represent with every milliliter of plaque forming unit (PFU/ml).In actually operating, need to configure suitable medium, effectively control inoculation condition and reaction conditions and could obtain reliable measurement result.
Reported that rabies virus plaque experimental period is 5～10 both at home and abroad, the time of Plaque Formation is still longer.In addition, the existence of Plaque determination method is strict to requirement for experiment condition, operation steps is complicated, plaque is difficult for the shortcomings such as formation maybe can not form, reproducibility is poor, does not form yet up till now the detection method of standard.
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, comprises the following 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 the cell of digestion in logarithmic phase, with cell growth medium dilution, obtaining cell density is 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) by step 2) described cell suspension and step 3) described rabies virus liquid, with the volume ratio ratio of 9: 1, join in the cell cultures hole of Tissue Culture Plate, get step 2 simultaneously) described cell suspension and step 1) described cell maintenance medium, the volume ratio ratio of 9: 1 of usining joins in the cell cultures hole of Tissue Culture Plate as negative control, be placed in 36 ℃～38 ℃, CO 2in incubator, cultivate 6～12h, after cell attachment, form cellular layer, discard the supernatant liquor in described Tissue Culture Plate, with phosphate buffered saline buffer, rinse cell;
5) in cell cultures hole, add step 1) described methylcellulose gum covering substratum 1ml～2ml, as tectum, be placed in 33 ℃～36 ℃, CO 2in incubator, continue to cultivate after 72～96h, 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 plaque number.
Preferably, methylcellulose gum of the present invention covers the HEPES that contains 50～80mmol/L in substratum.
Preferably, the MEM that cell maintenance medium of the present invention is the bovine serum that contains 50～100 μ g/ml DEAE-Dextran and 3% (v/v).
Preferably, cell growth medium of the present invention is the MEM of the bovine serum containing 50～100 μ g/ml DEAE-Dextran and 6% (v/v).
Preferably, CO of the present invention 2cO in 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 Fast Measurement rabies virus of the present invention, adopts monolayer cell culture, has viral plaque is reacted to peculiar susceptibility, repeatability and specificity.
Secondly, cell maintenance medium, the cell growth medium that the present invention adopts can promote virus and cell absorption.And in the cell growth medium and maintenance medium of viral plaque, all added DEAE-Dextran, in methylcellulose gum culture medium solution, also add the HEPES of 50～80mmol/L, therefore can greatly accelerate the formation of plaque, shortened the cycle of Rabies Virus Detection.
Finally, it is that sterilizing methylcellulose gum powder is directly dissolved in maintenance medium that methylcellulose gum covers substratum compound method, and preparation method is simple.
In sum, in methylcellulose gum culture medium solution, contain the HEPES of 50～80mmol/L, 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, reproducible, easy and simple to handle, economy, is easy to promote; Solve rabies virus because being difficult for without obvious cytopathy the problem of measuring, there is certain using value; Both can be used for virus clone, also can be used for viral mensuration and the detection of neutralizing antibody, and brought larger convenience to the research of rabies virus.
Accompanying drawing explanation
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 the mass ratio of 1: 99～2: 98, preferably, uses the mass ratio of 1: 99, obtains mass percent concentration and be 1% methylcellulose gum covering substratum.
The present invention has tested and in cell cultures hole, has added cell suspension and rabies virus liquid, CO 2in incubator, cultivate 6～12h, after cell attachment, form cellular layer, preferably, at cell attachment, form after monolayer cell layer, continue detecting step below.
The present invention has tested and in methylcellulose gum substratum, has used 50～80mmol/L HEPES, preferably, in the embodiment of the present invention, used the HEPES of 50mmol/L, and at Initial stage of culture, in the tectum of preparation, directly add the HEPES of 50mmol/L, 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 in cell growth medium and cell maintenance medium and has added 50～100 μ g/ml DEAE-Dextran, also can promote Plaque Formation, 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 cultivating the cell of 3～4 days, all can form clear plaque, the preferably Viola crystallina of 0.5%～1.5% (w/v).
In the embodiment of the present invention, virus strain is Flury-LEP strain (China Veterinery Drug Inspection Office, bacterial classification number: AV2012), the conventional virus in other this areas is carried out viral level mensuration as the method that Flury-HEP strain, ERA strain, CTN-1 strain and aG strain etc. also can be used for the present invention.
In the embodiment of the present invention, passage cell is BHK-21 cell (young hamster kidney passage cell), and the conventional passage cell in other this areas carries out viral level mensuration as the method that 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.
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described, in the following example, NM specific experiment method, carries out according to normal experiment method conventionally.
embodiment 1
Preparation MEM basal liquid: get 9.6g MEM and be dissolved in 1000ml water, then add 2.2g NaHCO 3, and adjust pH value to 7.2 with the HCl of 1mol/L or NaOH, and to add DEAE-Dextran to make its concentration being 80 μ g/ml, after sterile filtration, as 1 * MEM basal liquid, the MEM substratum of increase serum is not as basal liquid.The bovine serum that adds 3% volume percent in 1 * MEM basal liquid is as cell maintenance medium; The bovine serum that adds 6% volume percent in 1 * MEM basal liquid is as cell growth medium; Getting part maintenance medium, to add final concentration be that the HEPES of 50mmol/L is standby.
The method of this cell plaques Fast Measurement rabies virus, comprises the following steps:
(1) sterilized methylcellulose gum is mixed according to 1: 99 mass ratio with the maintenance medium 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 used time preheating in 37 ℃ of water-baths;
(2) digestion is in the BHK-21 of logarithmic phase cell, and with growth media dilution, in the BHK-21 of logarithmic phase cell, making cell density is 2.5 * 10 5cells/ml, obtains BHK-21 cell suspension;
(3) use maintenance medium 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 Tissue Culture Plate by 0.9ml/ hole;
(5) described gradient concentration rabies virus liquid is joined in the Tissue Culture Plate that contains cell suspension obtaining in step (4) by 0.1ml/ hole, negative control is set simultaneously, be placed in CO 2in incubator, cultivate CO 2the CO of incubator 2volume content is 5%, and temperature is 37 ℃;
After (6) 6～12h cell attachments, discarding the supernatant liquor in described Tissue Culture Plate, is that the sterilizing PBS that 0.01mol/L, pH value are 7.2 rinses one time by volumetric molar concentration, until dead cell comes off completely; Then the methylcellulose gum that the mass percent concentration that every hole adds step (1) to prepare is 1% covers substratum 1ml, as tectum, is placed in CO 2in incubator, continue to cultivate CO 2the CO of incubator 2volume content is 5%, and temperature is 35 ℃;
(7) after 96h, discard tectum, and be that the sterilizing PBS that 0.01mol/L, pH value are 7.2 rinses three times by concentration, until methylcellulose gum is rinsed well; Then every hole adds the crystal violet solution of 1ml, 1% mass percent, after the lower dyeing of room temperature condition (25 ℃) 20min, with tap water, slowly rinse 15min, until staining fluid is rinsed well, dry the plaque number of each culture hole of visual inspection Tissue Culture Plate;
(8) Tissue Culture Plate that negative control occurs without plaque, is judged to effective plate, calculates the malicious valency of rabies virus liquid to be determined 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 extension rate.
In Fig. 1 and Fig. 2, shown in arrow, be respectively the viral plaque of observing with micro-Microscopic observation, the extent of dilution that in Fig. 1, B1～B5 is corresponding 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 in 16,12,10,4 holes all within 8～30 scopes, so B3 institute corresponding 1 * 10 6for the suitableeest extension rate, as previously mentioned, v (the viral volume of every hole inoculation) is 0.1ml, according to formula, calculates:
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, according to experimental result, criticize an error and be less than log10 0.2, can realize the stdn of rabies virus poison valency and measure, and shorten half-finished round of visits in Rabies Vaccine production.
Between batch, the method for calculation of error are by same rabies virus liquid to be checked, packing in a small amount, and very low temperature is preserved, and minute 10 tests detect this malicious sample to be checked, and the result detecting for 10 times is taken the logarithm, and calculates the numerical value obtaining after the overall standard deviation of 10 samples.
Same virus liquid duplicate test 10 times, assay represents 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, calculated population sample standard deviation is 0.12, is log10 0.12.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

Claims (7)

1. for a method for the quickly measuring viral titer of rabies viruses by using cell plaques of non-diagnostic purpose, it is characterized in that, comprise the following steps:
1) methylcellulose gum is mixed according to the mass ratio of 1:99～2:98 with cell maintenance medium, methylcellulose gum is fully dissolved, obtain mass percent concentration and be 1%～2% methylcellulose gum and cover substratum;
2), after the cell of digestion in logarithmic phase, with cell growth medium dilution, obtaining cell density is 1 * 10 5cell/ml～5 * 10 5the cell suspension of cell/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) by step 2) described cell suspension and step 3) described rabies virus liquid, ratio with volume ratio 9:1 joins in the cell cultures hole of Tissue Culture Plate, get step 2 simultaneously) described cell suspension and step 1) described cell maintenance medium, the ratio of volume ratio 9:1 of usining joins in the cell cultures hole of Tissue Culture Plate as negative control, is placed in CO 2in incubator, cultivate 6～12h, after cell attachment, form cell monolayer, discard the supernatant liquor in described Tissue Culture Plate, with phosphate buffered saline buffer, rinse cell;
5) in cell cultures hole, add step 1) described methylcellulose gum covering substratum 1ml～2ml, as tectum, be placed in CO 2in incubator, continue to cultivate after 72～96h, 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 plaque number;
Wherein, step 1), 3) cell maintenance medium described in is the MEM of the bovine serum that contains 50～100 μ g/ml DEAE-Dextran and 3% volume percent; Step 2) described cell growth medium is the MEM of the bovine serum containing 50～100 μ g/ml DEAE-Dextran and 6% volume percent.
2. the method for the quickly measuring viral titer of rabies viruses by using cell plaques for non-diagnostic purpose according to claim 1, is characterized in that step 1) described in methylcellulose gum cover the HEPES that contains 50～80mmol/L in substratum.
3. the method for the quickly measuring viral titer of rabies viruses by using cell plaques for non-diagnostic purpose according to claim 1, it 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 the quickly measuring viral titer of rabies viruses by using cell plaques for non-diagnostic purpose according to claim 1, is characterized in that step 4), 5) in described CO 2cO in incubator 2volume content is 2%～10%.
5. the method for the quickly measuring viral titer of rabies viruses by using cell plaques for non-diagnostic purpose according to claim 1, is characterized in that step 4) described in culture temperature be 36 ℃～38 ℃.
6. the method for the quickly measuring viral titer of rabies viruses by using cell plaques for non-diagnostic purpose according to claim 1, is characterized in that step 5) described in culture temperature be 33 ℃～36 ℃.
7. the method for the quickly measuring viral titer of rabies viruses by using cell plaques for non-diagnostic purpose according to claim 1, it is characterized in that step 5) in staining agent be 0.5%～1.5%w/v Viola crystallina, 0.01%～0.1%w/v toluylene red or 0.5%～5%w/v iodine liquid.
CN201010273194.4A 2010-05-31 2010-09-02 Method for quickly measuring viral titer of rabies viruses by using cell plaques Active CN101921877B (en)

Priority Applications (1)

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

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
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

Publications (2)

Publication Number Publication Date
CN101921877A CN101921877A (en) 2010-12-22
CN101921877B true CN101921877B (en) 2014-04-16

Family

ID=42715727

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201010186192A Pending CN101831509A (en) 2010-05-31 2010-05-31 Method for titrating rabies viruses quickly with cell plaques
CN201010273194.4A Active CN101921877B (en) 2010-05-31 2010-09-02 Method for quickly measuring viral titer of rabies viruses by using cell plaques

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN201010186192A Pending CN101831509A (en) 2010-05-31 2010-05-31 Method for titrating rabies viruses quickly with cell plaques

Country Status (1)

Country Link
CN (2) CN101831509A (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4514497A (en) * 1983-12-30 1985-04-30 Novagene, Ltd. Modified live pseudorabies viruses

Patent Citations (2)

* Cited by examiner, † Cited by third party
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 (3)

* Cited by examiner, † Cited by third party
Title
岳军明等.甲基纤维素在狂犬病毒蚀斑中的应用.《黑龙江畜牧兽医》.1993,(第3期),29-30. *
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.《兽医大学学报》.1992,第12卷(第3期),300-301. *
邵益斌等.应用快速HEPES蚀斑法检测狂犬病毒.《中国病毒学》.1994,第9卷(第1期),70-73. *

Also Published As

Publication number Publication date
CN101831509A (en) 2010-09-15
CN101921877A (en) 2010-12-22

Similar Documents

Publication Publication Date Title
WO2021164050A1 (en) Multi-target double-dye isothermal amplification rapid detection method and kit
CN103509877A (en) Fluorescence quantitative PCR kit used for detecting PRV, and application thereof
CN112359139A (en) Method for determining heterophilic murine leukemia virus titer by tissue median infection staining
CN103616505B (en) A kind of hog cholera lapinised virus live vaccine effect detection method
CN102391996A (en) Recombination VTT and method for detecting vaccinia virus neutralizing antibody by using same
CN105759033A (en) Identification detecting method for Cox A16 and EV71 viral antigens
CN104280551A (en) Duck tembusu virus E-ELISA (E-enzyme-linked immuno sorbent assay) detection kit and preparation method thereof
CN101921877B (en) Method for quickly measuring viral titer of rabies viruses by using cell plaques
CN102094064A (en) Novel method for testing efficacy of pseudorabies live vaccine
CN109207554B (en) Method for rapidly detecting bacteriostatic effect of daily chemical product by using TTC agar culture medium
CN111154831A (en) Fluorescence detection method for total number of live bacteria
CN113049816B (en) Method for measuring titer of newcastle disease virus
CN106048090B (en) The quickly method of measurement ascovirus titre
CN102455359A (en) Kit used for detection of V-type adenovirus titer and application thereof
CN105177176A (en) Adenovirus titer detection method
CN114107558B (en) Method for determining titer of mouse parvovirus by tissue half-number infection method staining
CN101660006B (en) Aftosa virus titration method by suspended cell plaque
CN103196906B (en) Method for detecting specificity of candida albicans in clinical specimen
KR101329332B1 (en) Method for detection of neutralizaing antibody against West Nile virus
Low et al. A protocol to assess cellular bioenergetics in flavivirus-infected cells
CN114107557B (en) Method for determining reovirus type 3 titer by tissue half-number infection method staining
CN103773892B (en) Method for detecting titre of influenza virus
CN104792984B (en) A kind of canine parvovirus IgG antibody detection kit
CN103924005A (en) Detection method for influenza virus inactivation test verification
CN103743830A (en) Novel method used for quantification of baculovirus using high performance liquid chromatographic instrument

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant