CN106048090B - The quickly method of measurement ascovirus titre - Google Patents
The quickly method of measurement ascovirus titre Download PDFInfo
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- CN106048090B CN106048090B CN201610522012.XA CN201610522012A CN106048090B CN 106048090 B CN106048090 B CN 106048090B CN 201610522012 A CN201610522012 A CN 201610522012A CN 106048090 B CN106048090 B CN 106048090B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
Abstract
The present invention relates to field of biotechnology, specifically, disclosing a kind of rapid detection method for ascovirus titre.Logarithmic growth phase Sf9 insect cell is resuspended in this method；Set in incubator stationary culture 30 minutes it is adherent to cell, abandon supernatant culture medium, use new Sf-900IISFM culture medium instead；Cell concentration is measured using blood bead tally；96 hole cell cultures are seeded to after taking Sf9 insect cell suspension and ascovirus dilution to mix；It is placed in incubator after cultivating 24 hours, every hole adds Trypan Blue liquid, counts every hole and is colored number of cells, total cell number.Calculate every hole Sf9 insect cell death rate, the dead cell rate in every hole is lower than the dead cell rate of control group, assert the hole by vesica virus infection, it is on the contrary then think the Kong Wei by virus infection, according to the TCID of formula calculating ascovirus50.The method that the present invention measures ascovirus titre combines cell dyeing, can be used for the quick measurement of ascovirus titre, measurement result is more accurate.
Description
Technical field
The present invention relates to the methods of measurement virus titer, in particular to a kind of method of quickly measurement ascovirus titre.
Background technique
Ascovirus (ascovirus) is a kind of DNA virus for mainly infecting Lepidoptera noctuid.Ascovirus grain
Son is containing a cyclic annular supercoil double-stranded DNA, and viral genome size is according to the difference of kind from 116kbp (DpAV4) to 185kbp
(HvAV3) etc..Ascovirus genome includes two groups of repetitive sequences.First group of different size according to kind from 1.1kbp to
3.8kbp, it is currently known in physiologically not what special effect.Second group of size about 0.3~1.2kbp is encoded rod-shaped
Virus repeats to open frame gene order.Host larva shows as growth retardation, muscle after ascovirus infects host larva
The symptoms such as elasticity reduces, and food ingestion is reduced.Cell pathology feature, which is shown, infects incipient cell core hypertrophy, and nuclear membrane ruptures therewith,
Cell transition increases, and cytoplasma membrane is embedded in cell interior, and cell is divided into 20~30 folliculus.Virus is generated out of vesica,
Vesica is accumulated in hemolymph after being separated from each other.After infecting 3,4 days, viral massive duplication, the concentration in hemolymph reaches 108
A toxic particle/milliliter, hemolymph are creamy white, this is the notable feature that ascovirus infects.Therefore, from susceptible larvae collection
Hemolymph in, except also containing the vesica comprising virion containing in addition to a large amount of virion.900 due to ascovirus spy
Different cell pathology feature does not have ideal ascovirus titer determination method so far, has seriously affected ascovirus
And the flow of research of related discipline.
Now, the rapid development of the subjects such as virology and molecular biology, virus titer measuring method present simple and quick
The features such as.There are two types of the current most common titer determination methods, respectively plaque ethods and Endpoint Dilution Method,
1) plaque ethods (plaque assay) are to measure virus titer classics and the method by numerous virologists compared with approval,
Its principle are as follows: single plaque is generated by a virion, by the conversion of plaque number and dilution, obtains virus drop
Degree, is indicated with plaque forming unit (PFU/mL).This method detection time is long, vulnerable to cell quality, cell monolayer density, agar
The influence of many factors such as consistency, incubation time and the operator's qualification of sugar, and adjacent plaque may be joined to one
It rises, reduces plaque number, it is also possible to which mother's mark virus generates sub- spot, increases plaque number, causes test repeatability poor, i.e., result is not
Together.
2) Endpoint Dilution Method (end-point dilution assay) is with 50% tissue cultures infective dose (TCID50) table
Show, referring to can make half cell monolayer hole (pipe) viral dilution of lesion occur, be using a kind of wide viral level
Measuring method, this method mainly determine the titre of virus by observation cytopathy.However, since ascovirus infects insect
Cytopathy caused by cell is unobvious, and missing inspection and erroneous judgement possibility are very big, i.e., subjectivity and sense datum are to testing result
It is affected, therefore Endpoint Dilution Method measurement ascovirus titre traditionally has very big defect.
In conclusion bright due to the special cell pathology feature of ascovirus and existing viral titer determination method
Aobvious defect leads to a kind of method at present still without fast and convenient measurement ascovirus titre.
Summary of the invention
The present invention overcomes the technological deficiencies of traditional Endpoint Dilution Method and plaque ethods detection ascovirus titre, provide one
Kind quickly measures ascovirus titre method, and this method is that single layer insect cell is infected by ascovirus, causes cytopathy,
Combination cell viability stain observes whether cell is infected under the microscope, counts the insect cell death rate, and calculate viral drop
Degree.
To achieve the above object, the method for a kind of quickly measurement ascovirus titre provided by the invention, including following step
It is rapid:
1) it prepares insect cell suspension: insect cell being placed in Sf-900II SFM fluid nutrient medium and is cultivated, is obtained
To the insect cell of logarithmic growth phase, then insect cell is diluted with Sf-900II SFM fluid nutrient medium, obtains elder brother
Worm cell suspension, wherein Sf-900II SFM fluid nutrient medium is purchased from gibco company；
2) it prepares ascovirus dilution to be measured: ascovirus to be measured is used into ten times of Sf-900II SFM fluid nutrient medium
Gradient dilution is spare, obtains the ascovirus dilution to be measured of different dilutions；
3) prepare cell virus suspension: the ascovirus dilution to be measured for the different dilutions that step 2) is obtained respectively with
Step 1) obtains the mixing of insect cell suspension, obtains the cell virus suspension of different dilutions；
4) ascovirus infects: the cell virus suspension that step 3) obtains different dilutions is seeded to cell culture
It is interior, it is placed in 20~30h of culture in cell incubator, it is obvious in 20~30h inner cell apoptosis, but virus infection overlong time, quilt
The cell rupture of virus infection, releases virion and vesica, is unfavorable for subsequent dye test and dead cell number statistics；
5) Trypan Blue: taking out above-mentioned cell culture, and the dyeing of Trypan Blue liquid is added dropwise into cell culture well,
It is uniformly mixed, dyes 2~3 minutes, count the dead cell number and cell total number that every hole is colored under the microscope；
6) ascovirus titre calculates: calculating every hole cell death according to the dead cell number in every hole and cell total number
Rate, i.e., the every hole cell mortality=every hole dead cell number/hole cell total number, to count in cell culture, each
Dilution cell virus suspension infects hole count；Calculate the TCID of ascovirus50。
Further, in the step 1), insect cell is Spodopterafrugiperda Spodopterafrugiperda ovary
Cell Sf9 cell, Spodopterafrugiperda Spodoptera frugiperda gonad cell Sf21 cell, cabbage looper
Any one in Trichoplusia ni H ǜ bner egg cell system Hi5 cell.
Still further, in the step 1), in the insect cell suspension, the number of insect cell is 1.0~9.9 ×
105A/mL.
Still further, in the step 2), any one in ascovirus HvAV3, SfAV1, TnAV2 and SeAV5.
Still further, the gradient of the dilution of ascovirus dilution to be measured is 2~10 in the step 2).
Still further, the volume ratio of ascovirus dilution to be measured and insect cell suspension is 1: 8 in the step 3)
~15.
Still further, cell virus suspension is seeded to cell culture, and specific step is as follows in the step 4):
1) processing group: being inoculated with the cell virus suspension of same dilution in the cell culture well of each row or column,
2) control group: retain in the cell culture well of a row or column and be inoculated with Sf-900II SFM fluid nutrient medium and insect
The mixed liquor of cell suspension；
Wherein, the inoculum concentration in each hole is 100~200 μ L.
Still further, the mass fraction of Trypan Blue liquid is 0.3~0.6%, Trypan Blue in the step 5)
Drop dosage is 7~30 μ L.
Still further, the mean cell mortality rate of control group is calculated according to every hole cell mortality in the step 6),
That is the sum of every hole cell mortality of mean cell mortality rate=control group of control group/control group hole count；Compare the every hole of processing group
The average mortality of cell mortality and control group, judge the processing group of virus inoculation whether by virus infection,
If certain hole inner cell death rate of processing group is greater than the mean cell mortality rate of control group, determine that the hole is viral
It infects, i.e., the hole is the positive,
Or, determining the Kong Wei if certain hole inner cell death rate of processing group is less than the mean cell mortality rate of control group
By virus infection, i.e., the hole is feminine gender；
Hole count is infected by each viral dilution processing group of above-mentioned decision statistic, calculates the viral dilution processing group
Infection rate, the i.e. every group of processing infection rate=group infect hole count/total hole count of the group, to calculate capsule according to Reed-Muench method
Steep the TCID of virus50
That is, distance proportion=(the lesion rate -50% higher than 50%)/(the lesion rate-higher than 50% is lower than 50% lesion
Rate)
lgTCID50=be higher than 50% lesion viral highest dilution logarithm-distance proportion.
The beneficial effects of the present invention are:
The method of the present invention is easy to operate, result is accurate, time-consuming is short, stability is good.This method can be accurate with higher detection
Degree measurement ascovirus titre；Titre detection method measurement ascovirus titre of the present invention only needs 2 days, and traditional end
Point dilution rule needs 8-10 days, and plaque formation needs many infectious cycles, obtains final result and usually required for three weeks,
Substantially reduce detection time.Without apparent cytopathy feature, simple micro- sem observation after ascovirus infected cell
Whether cell can not be judged by virus infection, therefore traditional Endpoint Dilution Method can not accurately detect ascovirus titre.It compares
Traditional Endpoint Dilution Method and plaque ethods, the method for the invention detection cycle shorten 6-8 days, and detection efficiency is high, not only significantly
Operating time, save the cost are shortened, experimental result has more the property expected, also more precise and stable between different operation individual.This hair
The bright requirement to operator is relatively low, and uncontrollable factor is smaller, and the operational stability of experimental result is higher.
Specific embodiment
In order to better explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated, but
The contents of the present invention are not limited solely to following embodiment.
The quick measurement of 1 ascovirus HvAV-3h titre of embodiment
Specific step is as follows:
1. preparing ascovirus HvAV-3h gradient dilution liquid
By ascovirus liquid HvAV-3h to be measured Sf-900II SFM culture medium gradient dilution, mix within vortex oscillation 30 seconds.
Specific dilution is as shown in table 1.
1 ascovirus dilution scheme of table
2. preparing insect cell suspension
Routinely propagating method collects insect cell to the amount of taking fully Sf9 insect cell culture, discards supernatant after standing 30min
Culture medium uses fresh Sf-900II SFM culture medium instead, so that insect cell is suspended with rifle piping and druming, is counted with blood cell counting plate thin
Born of the same parents' number, and it is 1 × 10 that cell, which is diluted to concentration, with Sf-900IISFM culture medium5A/mL.
3. preparing cell virus suspension
The viral gradient dilution liquid of the insect cell suspension and 130 μ L that take 1300 μ L respectively is in the sterilized centrifugation of 1.5mL
Pipe, mixes gently.
4. virus infection
110 μ L cell virus suspensions are taken to be added in 96 hole cell cultures, the cell for being often classified as a gradient dilution is outstanding
Liquid, first is classified as sky, and secondary series to the 6th influenza virus dilution is respectively 10-8、10-7、10-6、10-5、10-4, the 7th is classified as not
The control group for adding viral gradient dilution liquid, carries out respective markers, sets in cell incubator and cultivates 24 hours.
The sample-adding distribution of 2 96 porose disc of table
4. insect cell dyes
96 hole culture plates are taken out, every hole adds the Trypan Blue liquid of 10 μ L0.4%, shakes gently mixing.
5. experimental result is observed
After Trypan Blue 2-3 minutes, the number of cells and cell total number that every hole is colored are counted under the microscope.
6. data processing
By counting number of cells, the cell mortality in every hole is calculated, according to the mean cell mortality rate of control group, judgement
Whether the processing group of virus inoculation is by virus infection, if the average cell that the cell mortality of processing group is greater than control group is dead
Rate, the hole by virus infection, then determine the hole for the positive, it is on the contrary then for feminine gender.
7. result calculates
The 3 insect cell death rate of table
Note: every viral dilution processing group infection rate=positive hole count/is always inoculated with hole count
4 ascovirus infection rate of table
The TCID of the virus is calculated by Reed-Muench method50Value, circular are as follows:
Distance proportion=(infection rate -50% higher than 50%)/(infection rate-higher than 50% is lower than 50% infection rate)
× lg extension rate
=(58.33%-50%)/(58.33%-33.33%)
=0.3332
lgTCID50=be higher than 50% lesion viral highest dilution logarithm-distance proportion
=-5.3332
TCID50=10lgTCID50
=10-5.3332
It follows that the titre of virus is 105.3332TCID50/10 μ L, i.e., 2.15 × 107TCID50/mL。
Above-mentioned Sf-900II SFM fluid nutrient medium is purchased from gibco company；Other materials are purchased from market.
Other unspecified parts are the prior art.Although above-described embodiment is made that the present invention and retouches in detail
State, but it is only a part of the embodiment of the present invention, rather than whole embodiments, people can also according to the present embodiment without
Other embodiments are obtained under the premise of creativeness, these embodiments belong to the scope of the present invention.
Claims (9)
1. a method of quickly measurement ascovirus titre, it is characterised in that: the following steps are included:
1) it prepares insect cell suspension: insect cell being placed in Sf-900II SFM fluid nutrient medium and is cultivated, obtain pair
The insect cell in number growth period, is then diluted insect cell with Sf-900II SFM fluid nutrient medium, it is thin to obtain insect
Born of the same parents' suspension,
2) it prepares ascovirus dilution to be measured: ascovirus to be measured is used into ten times of gradients of Sf-900II SFM fluid nutrient medium
It dilutes spare；Obtain the ascovirus dilution to be measured of different dilutions；
3) prepare cell virus suspension: the ascovirus dilution to be measured for the different dilutions that step 2) is obtained respectively with step
1) mixing of insect cell suspension is obtained, the cell virus suspension of different dilutions is obtained；
4) ascovirus infects: the cell virus suspension that step 3) obtains different dilutions being seeded in cell culture, is set
It is cultivated for 24 hours in cell incubator；
5) Trypan Blue: taking out above-mentioned cell culture, and the dyeing of Trypan Blue liquid, mixing are added dropwise into cell culture well
Uniformly, it dyes 2~3 minutes, counts the dead cell number and cell total number that every hole is colored under the microscope；
6) ascovirus titre calculates: calculating every hole cell mortality according to the dead cell number in every hole and cell total number, i.e.,
The every hole cell mortality=every hole dead cell number/hole cell total number, to count in cell culture, each dilution
Cell virus suspension infects hole count；Calculate the TCID of ascovirus50。
2. quickly measuring the method for ascovirus titre according to claim 1, it is characterised in that: in the step 1), elder brother
Worm cell is Spodopterafrugiperda Spodoptera frugiperda gonad cell Sf9 cell, Spodopterafrugiperda Spodoptera
Any one in frugiperda gonad cell Sf21 cell, cabbage looper Trichoplusia ni egg cell system Hi5 cell.
3. the method for quick measurement ascovirus titre according to claim 1 or claim 2, it is characterised in that: in the step 1),
In the insect cell suspension, the number of insect cell is 1.0~9.9 × 105A/mL.
4. the method for quick measurement ascovirus titre according to claim 1 or claim 2, it is characterised in that: in the step 2),
Ascovirus is any one in HvAV3, SfAV1, TnAV2 and SeAV5.
5. quickly measuring the method for ascovirus titre according to claim 4, it is characterised in that: in the step 2), to
The gradient for surveying the dilution of ascovirus dilution is 2~10.
6. the method for quick measurement ascovirus titre according to claim 1 or claim 2, it is characterised in that: in the step 3),
The volume ratio of ascovirus dilution to be measured and insect cell suspension is 1: 8~15.
7. the method for quick measurement ascovirus titre according to claim 1 or claim 2, it is characterised in that: in the step 4),
Cell virus suspension is seeded to cell culture, and specific step is as follows:
1) processing group: being inoculated with the cell virus suspension of same dilution in the cell culture well of each row or column,
2) control group: retain in the cell culture well of a row or column and be inoculated with Sf-900II SFM fluid nutrient medium and insect cell
The mixed liquor of suspension；
Wherein, the inoculum concentration in each hole is 100~200 μ L.
8. the method for quick measurement ascovirus titre according to claim 1 or claim 2, it is characterised in that: in the step 5),
The mass fraction of Trypan Blue liquid is 0.3~0.6%, and Trypan Blue drop dosage is 7~30 μ L.
9. the method for quick measurement ascovirus titre according to claim 1 or claim 2, it is characterised in that: in the step 6),
The mean cell mortality rate of control group, i.e. mean cell mortality rate=control group of control group are calculated according to every hole cell mortality
The sum of every hole cell mortality/control group hole count；The average mortality for comparing processing group every hole cell mortality and control group, sentences
The processing group of disconnected virus inoculation whether by virus infection,
If certain hole inner cell death rate of processing group is greater than the mean cell mortality rate of control group, determine that the hole is invaded by virus
Dye, the i.e. hole are the positive,
Or, determining that the Kong Wei is sick if certain hole inner cell death rate of processing group is less than the mean cell mortality rate of control group
Poison infects, i.e., the hole is feminine gender；
Hole count is infected by each viral dilution processing group of above-mentioned decision statistic, calculates infecting for the viral dilution processing group
Rate, the i.e. every group of processing infection rate=group infect hole count/total hole count of the group, to calculate vesica disease according to Reed-Muench method
The TCID of poison50
That is, distance proportion=(the lesion rate -50% higher than 50%)/(the lesion rate-higher than 50% is lower than 50% lesion rate),
lgTCID50=be higher than 50% lesion viral highest dilution logarithm-distance proportion.
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| CN107063976B (en) * | 2017-03-30 | 2020-04-10 | 中国农业科学院特产研究所 | System and method for calculating infection amount of half cell culture |
| CN111235309B (en) * | 2020-01-20 | 2022-09-30 | 湖南农业大学 | Absolute quantitative PCR method for rapidly determining titer of vesicular virus |
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| CN103364560A (en) * | 2013-07-23 | 2013-10-23 | 武汉中博生物股份有限公司 | Method for determining baculovirus titer |
| 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 |
| CN104531895B (en) * | 2014-11-25 | 2017-07-21 | 中国科学院苏州生物医学工程技术研究所 | A kind of method for determining virus titer |
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