CN102337249A - Method for culturing various viruses by using MHV mixed cells - Google Patents
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Abstract
The invention belongs to the technical field of biology, and relates to a method for culturing various viruses by using MHV mixed cells. According to the invention, MDCK, HEP-2, and Vero monolayer cells are prepared by using T75 cell flasks; the monolayer cells are processed through EDTA-trypsinization; obtained cell suspensions are respectively counted, and are mixed, such that a cell suspension with a cell number of 1.5*10<5>/ml is obtained; the cell suspension is added into a small culture flask, such that a mixed cell culture flask is obtained. The mixed cell culture flask can be used for separating and culturing various viruses, such as influenza B virus, influenza A virus, adenovirus, and enterovirus E71. According to the invention, three cells are prepared into a mixed cell bottle used for separating and culturing various viruses and pathogens. Compared to prior arts, the separation culture effect of the method provided by the invention is better. Traditionally, a cell line is used for separating and culturing viruses, and sensitive cell lines are selected through the conjecturing of possibly infected viruses. With the method provided by the invention, the defect is overcome, specificity and sensitivity of virus diagnosing are improved, and results can be quickly obtained.
Description
Technical field
The invention belongs to biological technical field, relate to viral isolation cultivation method, be specifically related to the cultural method of the multiple virus of a kind of MHV cell mixing separation and Culture.
Background technology
The viral infectious velocity of propagation is fast, scope wide, the social danger influence is big.Therefore, in time the pathogenic agent of diagnose infections is most important to stablizing of the social popular feelings.The research report, most viral infections are to be caused by the common virus cause of disease, cause that like influenza virus, adenovirus, respiratory syncytial virus etc. respiratory tract infection, the EV71 of enterovirus genus, CA16 are to cause the main virus causing disease of children's hand foot mouth disease.Research shows, viral proliferation has the strict host cell property of having a liking for, and must in proper host cell, grow, and therefore, the viral separation and Culture relatively cultivation of bacterium is wanted difficulty.
At present, laboratory separation and Culture virus mainly is to adopt a kind of clone, as for adopting which kind of clone then according to disease popularity characteristic and symptom, infers that the virus of PI is chosen sensitive cell line.Usually, the influenza virus separation and Culture mainly adopts mdck cell; Adenovirus, respiratory syncytial virus adopt the HEP-2 cell; EV71, CA16 adopt Vero cell and MRC-5 cell.There are two kinds of bibliographical information employings or three kinds of cellular segregation to cultivate main Respirovirus abroad.There is the special cell mixing of selling two kinds or three kinds cells of mechanism in the U.S..But so far, Shang Weijian is for the play-by-play of the cell mixing of ability separation and Culture respiratory tract and enterovirus.
Summary of the invention
The cultural method that the purpose of this invention is to provide the multiple virus of a kind of separation and Culture is specifically related to the cultural method of the multiple virus of a kind of MHV cell mixing separation and Culture.The inventive method relates generally to first, Influenza B virus, the adenovirus in the Respirovirus; EV71 in enterovirus virus separation and Culture relates in particular to MDCK, HEP-2, three kinds of cellular segregation of Vero and cultivates common Respirovirus, common enterovirus cultural method.
Present method prepares MDCK, HEP-2, Vero monolayer through T75 cell bottle, and monolayer mixes MDCK, Hep-2, Vero cell suspension counting back respectively through the EDTA-trysinization, and processing cell count is 1.5 * 10
5The cell suspension of/ml adds 3ml cultivation bottle and processes the cell mixing culturing bottle, and the cell count of wherein every strain cell is respectively 5 * 10
4/ ml.
Cell mixing culturing bottle of the present invention can be used for the multiple virus of separation and Culture, comprises first type, Influenza B virus; Adenovirus; Enterovirus EV 71, separation and Culture reach effect preferably.The present invention processes cell mixing bottle separation and Culture through three kinds of cells and identifies multiple virus causing disease, has both improved the specificity and the susceptibility of viral diagnosis, can go out the result more fast again.
The substratum that the inventive method is selected for use is a cell cultures substratum commonly used, and culture condition is at controlled temperature and CO
2Under the concentration, to first, Influenza B virus, adenovirus, EV71 virus is carried out separation and Culture, and separation and Culture result is satisfied.
Particularly, the cultural method of the multiple virus of MHV cell mixing separation and Culture of the present invention is characterized in that it comprises step:
1) selects culture reagent
Cell growth medium adds qingfengmeisu qiong (final concentration 24ug/ml), 7.5%NaHCO for the MEM mother liquor adds calf serum (final concentration is 10%)
3Transfer pH to 7.0～7.2; Virus transportation liquid adds green grass or young crops for the MEM mother liquor adds bovine serum albumin (final concentration 5ug/ml), Streptomycin sulphate (final concentration 100ug/ml) adds B fungizone (final concentration 0.5ug); Virus inoculation liquid adds green grass or young crops, Streptomycin sulphate (final concentration 50ug/ml) TPCK-pancreatin (final concentration is 2ug/ml) for the MEM mother liquor adds bovine serum albumin (final concentration 5ug/ml).
2) preparation culturing cell:
The laboratory preserve 5～15 generation mdck cell, HEP-2 cell, Vero cell, in T75 cell bottle, grow up to monolayer respectively.
The experimental cell that the present invention adopted all can obtain through commercial mode, and preserves by the ordinary method laboratory.
What said T75 Tissue Culture Flask adopted is the Corning Company products, numbering 430720
3) preparation MHV cell mixing bottle
Adopt MDCK, Hep-2, Vero cell preparation cell mixing culturing bottle to come first type, Influenza B virus, adenovirus in the separation and Culture Respirovirus; EV71 virus in the enterovirus;
Wherein, MDCK, Hep-2, Vero three strain cells in T75 cell bottle, grow up to monolayer respectively after the EDTA-trysinization mixes MDCK, Hep-2, Vero cell suspension counting back respectively, and processing cell count is 1.5 * 10
5The cell suspension of/ml, the cell count of wherein every strain cell is respectively 5 * 10
4/ ml, in above-mentioned mixed cell suspension packing culturing bottle, the 3ml/ bottle is put 37 ℃, 5%CO
2Grow up to the cell monolayer about 80% in the incubator.
Among the present invention, preferred EDTA-pancreatin is the 0.25%EDTA-pancreatin;
Among the present invention, preferred cell suspension counting adopts the white blood cell count(WBC) plate.
4) specimen inoculation:
Nasopharynx is wiped away sample disposal: the centrifugal 5min of 4 ℃ of nasopharynx samples, 2000r/min removes deposition, and negative control is set up in 0.22 micron pore size membrane filtration degerming or handle inoculation cell mixing bottle after 4 hours with green grass or young crops/Streptomycin sulphate, B fungizone simultaneously.
Above-mentioned sample adopts the simulation clinical samples, concrete preparation as following:
(1) sampling tube preparation is got 3.6ml virus transportation liquid and is added the 10ml sterilization in vitro;
(2) get first type, Influenza B virus liquid, adenopathy venom, each 10 strain of EV71 virus liquid of laboratory-86 ℃ preservation, every strain virus liquid is drawn 0.4ml and is added (dilution in 1: 10) in the above-mentioned sampling tube;
(3) each 10 volunteers of picked at random, the disinfecting silk or cotton swab is swallowed rear wall and the wiping gently of tonsil both sides the volunteer, inserts in the sampling tube, makes the clinical pharynx of simulation and wipes away sample.
Stool sample is handled: stool sample is suspended in pH7.2PBS liquid with 1: 5 ratio; The centrifugal 5min of 2000r/min removes the ight soil slag; 0.22 the degerming of micron pore size membrane filtration is inoculated the cell mixing bottle with green grass or young crops/Streptomycin sulphate, B fungizone processing again after 4 hours, set up negative control simultaneously.
5) culture is identified:
After the inoculation sample 24h, under inverted microscope, observe and observe differential protein mono-clonal immunofluorescence dyeing, RT-PCR, pcr amplification product evaluation under culturing cell cytopathic effect, the fluorescent microscope.
Among the present invention, adopt Nikon TS100 inverted microscope; Olympus BX 51 fluorescent microscopes;
Among the present invention, adopt first type, Influenza B virus mono-clonal immunofluorescence antibody I MAGEN
TMInfluenza virus Aand B (DAKO); Adenovirus mono-clonal immunofluorescence antibody D3 ultra
TMDFA Respiratory virus ID kit (DIAGNOSTIC HYBRIDS); General enterovirus mixed immunity fluorescence antibody LIGHT DIAGNOSTICS
TMPan-Enterovirus Reagent, MIILIPORE (chemicon).
5.1) cytopathic effect:
What the present invention adopted is three kinds of cytomixis culturing bottles; Therefore; If have in the culture to a certain cell sensitive virus; Under inverted microscope, can observe the pathology effect of this kind cell, following information is provided: (1) culture has cytopathogenic virus or toxin, and (2) possibly be influenza virus, adenovirus, respiratory syncytial virus or the enterovirus EV 71s of respiratory tract.
5.2) special viral protein detection:
According to the cytopathic effect of a certain cell, infer possible infective virus, adopt mono-clonal this Tobamovirus of identified by immunofluorescence or virus type.
5.3) special viral nucleic acid detection:
Adopt RT-PCR or PCR identifying virus hypotype.
5.4) the random primer amplification:
Culture has cell mixing generation cytopathic effect, and the non-detectable positive culture of special virus and special viral nucleic acid can adopt the random primer amplification method, the comparison evaluation of order-checking splicing back.
Among the present invention, Influenza A1 virus RT-PCR amplified production is 431BP; The Influenza B virus amplified production is 390bp; The adenovirus amplified production is 874bp.
The inventive method adopts the three kinds of cytomixis culturing bottles of the above; Cultivate 10 strain influenza A viruss, 10 strain Influenza B viruss, 10 strain adenovirus, 10 strain EV71 virus; Culture is after cultivating 48h; The result shows, it is all positive all to observe cytopathic effect, 10 strain influenza A viruss, 10 strain Influenza B viruss, 10 strain adenovirus, 10 strain EV71 viral monoclonal immunofluorescence fluorescent dyes; Culture all can detect special viral nucleic acid band.
The inventive method is processed cell mixing bottle separation and Culture through three kinds of cells and is identified multiple virus causing disease; Effective than the prior art separation and Culture; Overcoming traditional separation and Culture virus mainly is to use a kind of clone; Choose the defective of sensitive cell line through the virus of inferring PI, both improved the specificity and the susceptibility of viral diagnosis, can go out the result more fast again.
Description of drawings
Fig. 1, contrast MHV cell mixing bottle (24h).
Fig. 2, sample MHV cell mixing bottle (24h) is wiped away in the pharynx of Influenza B virus POLLUTION SIMULATION.
Fig. 3, photo 3. control group MHV cell mixing bottles (24h).
Fig. 4, sample MHV cell mixing bottle (24h) is wiped away in the pharynx of Influenza A1 virus POLLUTION SIMULATION.
Fig. 5, control group MHV cell mixing bottle (48h).
Fig. 6, sample MHV cell mixing bottle (48h) is wiped away in the pharynx of adenovirus POLLUTION SIMULATION.
Fig. 7, control group MHV cell mixing bottle (48h).
Fig. 8, sample MHV cell mixing bottle (48h) is wiped away in EV71 virus pollution simulation pharynx.
Fig. 9, control group MHV-resisiting influenza virus McAb*200.
Figure 10, sample MHV-resisiting influenza virus McAb*200 is wiped away in the pharynx of Influenza B virus POLLUTION SIMULATION.
Figure 11, sample MHV-resisiting influenza virus McAb*200 is wiped away in the susceptible malicious POLLUTION SIMULATION pharynx of first 1 type.
Figure 12, the anti-adenovirus McAb*200 of control group MHV-.
Figure 13, the anti-adenovirus McAb*200 of sample MHV-is wiped away in the pharynx of adenovirus POLLUTION SIMULATION.
Figure 14, the anti-adenovirus McAb*400 of sample MHV-is wiped away in the pharynx of adenovirus POLLUTION SIMULATION.
Figure 15, the anti-enterovirus McAb*200 of control group MHV-.
Figure 16, the anti-enterovirus McAb*200 of sample MHV-is wiped away in EV71 virus pollution simulation pharynx.
Figure 17, wherein: 1,2 is 390bpB type influenza; 3 contrasts; 4,5 is 210bp first 3 influenzas; 6 is 431bp first 1 influenza; 7,8,9 contrasts; 10,11,12,13,14,15 is 341bpEV71; M is that 100bp is apart from band.
Figure 18, wherein: 1,3,4,5 ducts are 874 adenovirus; M is that 200bp is apart from band.
Embodiment
1) gets the mdck cell that T75 cell bottle grows up to individual layer (7 generation～12 generation), HEP-2 cell and Vero cell; Wash once with the PH7.0PBS working fluid earlier, add the 0.25%EDTA-tryptic digestion, inhale and remove EDTA-trypsinase; The MEM growth media that adds 10 milliliter of 10% calf serum; Beat even and fine born of the same parents with curved mouth dropper, get above-mentioned enchylema 90ul and add the little plastics tubing of 200ul, add tongue then and expect blue 10ul dyeing; Get the above-mentioned staining cell of 10ul and splash into white blood cell count(WBC) plate counting, each cell viable count (dying the blue dead cell that is) is controlled to be 1.5 * 10
5/ ml.
2) be 1.5 * 10 with cell number average counting
5/ ml MDCK, HEP-2 and Vero cell equal-volume mixing, at this moment, the cell count of each cell is 5 * 10
4/ ml draws the glass cultivation bottle that 1ml cell mixing liquid is added to the 5ml amount, puts 37 ℃, 5%CO
2Incubator was cultivated 12～18 hours, and cell grows up to the individual layer about 80%.
3) get influenza A virus 10 strains of laboratory-86 ℃ preservation; Influenza B virus 10 strains; Draw every influenzae strain virus liquid 0.4ml and be added to the 10ml sterilization in vitro; Add 3.6ml virus transportation liquid (dilution in 1: 10), 10 volunteer's throat swabs of picked at random are inserted the above-mentioned influenza virus 10ml that contains respectively and are sterilized in vitro, simulate clinical throat swab sample.Pharynx is wiped away the centrifugal 5min of sample 2000r/min and is removed deposition, and supernatant adds green grass or young crops/Streptomycin sulphate (final concentration 100ug/ml), B fungizone (0.5ug/ml) was handled after 4 hours, and every part of sample is got 0.2ml and is inoculated in respectively in the cell mixing bottle.Other gets 0.2ml virus transportation liquid and is inoculated in the cell mixing bottle as negative control.Every part of parallel 5 pipes of doing of viral sample with negative control.Put 37 ℃, 5%CO
2Hatched in the incubator 1 hour, and let virus be adsorbed onto on the cell, abandon supernatant, add virus-culturing fluid 1ml, put 37 ℃, 5%CO
2Cultivate in the incubator.Begin to observe negative control group and virus inoculation group cellular form in the mixed cell bottle after 24 hours, observe CPE and produce.
4) inoculation sample group cell mixing bottle CPE++～+++after, draw out control group and sample group cell suspension, and scrape control group, sample group cell simultaneously and drip on 12 hole slides every duplicate samples and do 2 holes.Seasoning, cold acetone-methyl alcohol is fixed, after the pH7.0PBS washed with saline solution 3 times; Every hole adds first, Influenza B virus monoclonal antibody 10ul, and 37 ℃ of wet boxes are hatched 30min, PBS washed with saline solution 3 times; Dry, drip fluoroscope oil, under fluorescent microscope, observe fluorescent grain.
5) sample group and cellular control unit culture supernatant liquid adopt invitrogen company
LS Reagent reagent extracting viral nucleic acid.Use RT-polymerase chain reaction (RT-PCR) and detect influenza virus type and hypotype, amplified production detects specific band through 1.0% agarose gel electrophoresis.
The result shows; 10 parts of samples (adopting the laboratory to preserve each 10 strain Influenza A1 virus respectively) are wiped away in MDCK, Hep-2, the pharynx of Vero cell mixing bottle separation and Culture simulation influenza A virus and 10 parts of samples (adopting the laboratory to preserve each 10 strain Influenza B virus respectively) are wiped away in the pharynx of simulation Influenza B virus, and separation and Culture result is satisfied.Inverted microscope can be observed the mdck cell pathology effect that first, Influenza B virus causes the cell mixing bottle down, sees photo (like Fig. 1,2,3, shown in 4); Under fluorescent microscope, observe influenza virus specific fluorescence particle (seeing that photo is like Fig. 9,10, shown in 11); RT-PCR can detect the special product band of Influenza A1 virus of 431bp and the special product band of Influenza B virus (shown in figure 17) of 390bp
1) gets the mdck cell that T75 cell bottle grows up to individual layer (7 generation～12 generation), HEP-2 cell and Vero cell; Wash once with the PH7.0PBS working fluid earlier, add the 0.25%EDTA-tryptic digestion, inhale and remove EDTA-trypsinase; The MEM growth media that adds 10 milliliter of 10% calf serum; Beat even and fine born of the same parents with curved mouth dropper, get above-mentioned enchylema 90ul and add the little plastics tubing of 200ul, add tongue then and expect blue 10ul dyeing; Get the above-mentioned staining cell of 10ul and splash into white blood cell count(WBC) plate counting, each cell viable count (dying the blue dead cell that is) is controlled to be 1.5 * 10
5/ ml.
2) be cell number average counting 1.5 * 10
5/ ml MDCK, HEP-2 and Vero cell equal-volume mixing, at this moment, the cell count of each cell is 5 * 10
4/ ml draws the glass cultivation bottle that 1ml cell mixing liquid is added to the 5ml amount, puts 37 ℃, 5%CO
2Incubator was cultivated 12～18 hours, and cell grows up to the individual layer about 80%.
3) get laboratory-86 ℃ good adenovirus 10 strains of guarantor; Draw every influenzae strain virus liquid 0.4ml and add 10ml sterilization test tube; Add 3.6ml virus transportation liquid (dilution in 1: 10); 10 volunteer's throat swabs of picked at random are inserted the above-mentioned adenovirus 10ml that contains respectively and are sterilized in vitro, simulate clinical throat swab sample.Pharynx is wiped away the centrifugal 5min of sample 2000r/min and is removed deposition, and supernatant adds green grass or young crops/Streptomycin sulphate (final concentration 100ug/ml), B fungizone (0.5ug/ml) was handled after 4 hours, and every part of sample is got 0.2ml and is inoculated in respectively in the cell mixing bottle.Other gets 0.2ml virus transportation liquid and is inoculated in the cell mixing bottle as negative control.Every part of parallel 5 pipes of doing of viral sample with negative control.Put 37 ℃, 5%CO
2Hatched in the incubator 1 hour, and let virus be adsorbed onto on the cell, abandon supernatant, add virus-culturing fluid 1ml, put 37 ℃, 5%CO
2Cultivate in the incubator.Begin to observe negative control group and virus inoculation group cellular form in the mixed cell bottle after 24 hours, observe CPE and produce.
4) inoculation sample group cell mixing bottle CPE++～+++after, draw out control group and sample group cell suspension, and scrape control group, sample group cell simultaneously and drip on 12 hole slides every duplicate samples and do 2 holes.Seasoning, cold acetone-methyl alcohol is fixed, and after the pH7.0PBS washed with saline solution 3 times, every hole adds adenovirus monoclonal antibody 10ul, and 37 ℃ of wet boxes are hatched 30min, and PBS washed with saline solution 3 times is dried, and drips fluoroscope oil, observation fluorescent grain under fluorescent microscope.
5) sample group and cellular control unit culture supernatant liquid adopt invitrogen company
LS Reagent reagent extracting viral nucleic acid.Using polymerase chain reaction detects adenovirus, and amplified production detects specific band through 1.0% agarose gel electrophoresis.
The result shows that MDCK, Hep-2, Vero cell mixing bottle separation and Culture are simulated the adenovirus pharynx and wiped away 10 parts of samples (adopting the laboratory to preserve each 10 strain adenovirus) separation and Culture result's satisfaction.Inverted microscope can be observed the Hep-2 cytopathic effect (like Fig. 5, shown in 6) that adenovirus virus causes the cell mixing bottle down; Under fluorescent microscope, observe adenovirus specific fluorescence particle (like Figure 12,13, see photo shown in 14); PCR can detect the special product band of adenovirus (shown in figure 18) of 874bp.
1) gets mdck cell (7～12 generation), HEP-2 cell and the Vero cell that T75 cell bottle grows up to individual layer; Wash once with the PH7.0PBS working fluid earlier, add the 0.25%EDTA-tryptic digestion, inhale and remove EDTA-trypsinase; The MEM growth media that adds 10 milliliter of 10% calf serum; Beat even and fine born of the same parents with curved mouth dropper, get above-mentioned enchylema 90ul and add the little plastics tubing of 200ul, add tongue then and expect blue 10ul dyeing; Get the above-mentioned staining cell of 10ul and splash into white blood cell count(WBC) plate counting, each cell viable count (dying the blue dead cell that is) is controlled to be 1.5 * 10
5/ ml.
2) be cell number average counting 1.5 * 10
5/ ml MDCK, HEP-2 and Vero cell equal-volume mixing, at this moment, the cell count of each cell is 5 * 10
4/ ml draws the glass cultivation bottle that 1ml cell mixing liquid is added to the 5ml amount, puts 37 ℃, 5%CO
2Incubator was cultivated 12～18 hours, and cell grows up to the individual layer about 80%.
3) get EV71 virus 10 strains of laboratory-86 ℃ preservation; Draw every strain EV71 virus liquid 0.4ml and add 10ml sterilization test tube; Add 3.6ml virus transportation liquid (dilution in 1: 10); 10 volunteer's throat swabs of picked at random are inserted the above-mentioned EV71 of containing virus 10ml sterilization respectively in vitro, simulate clinical throat swab sample.Pharynx is wiped away the centrifugal 5min of sample 2000r/min and is removed deposition, and supernatant adds green grass or young crops/Streptomycin sulphate (final concentration 100ug/ml), B fungizone (0.5ug/ml) was handled after 4 hours, and every part of sample is got 0.2ml and is inoculated in respectively in the cell mixing bottle.Other gets 0.2ml virus transportation liquid and is inoculated in the cell mixing bottle as negative control.Every part of parallel 5 pipes of doing of viral sample with negative control.Put 37 ℃, 5%CO
2Hatched in the incubator 1 hour, and let virus be adsorbed onto on the cell, abandon supernatant, add virus-culturing fluid 1ml, put 37 ℃, 5%CO
2Cultivate in the incubator.Begin to observe negative control group and virus inoculation group cellular form in the mixed cell bottle after 24 hours, observe CPE and produce.
4) inoculation sample group cell mixing bottle CPE++～+++after, draw out control group and sample group cell suspension, and scrape control group, sample group cell simultaneously and drip on 12 hole slides every duplicate samples and do 2 holes.Seasoning, cold acetone-methyl alcohol is fixed, after the pH7.0PBS washed with saline solution 3 times; Every hole adds enterovirus genus specific monoclonal antibody 10ul, and 37 ℃ of wet boxes are hatched 30min, PBS washed with saline solution 3 times; Dry, drip fluoroscope oil, under fluorescent microscope, observe fluorescent grain.
5) sample group and cellular control unit culture supernatant liquid adopt invitrogen company
LS Reagent reagent extracting viral nucleic acid.Use RT-polymerase chain reaction and detect enterovirus EV 71 virus, amplified production detects specific band through 1.0% agarose gel electrophoresis.
The result shows that 10 parts of samples (for preserving each 10 strain EV71 virus in the laboratory) separation and Culture result's satisfaction is wiped away in MDCK, Hep-2, the viral pharynx of Vero cell mixing bottle separation and Culture simulation EV71.Can observe EV71 virus under the inverted microscope and cause cell mixing bottle Vero cytopathic effect (like Fig. 7, shown in 8); Under fluorescent microscope, observe EV71 virus specific fluorescence particle (like Figure 15,16); RT-PCR can detect 341 EV71 virus special product band (shown in figure 17).
Claims (7)
1. the cultural method of the multiple virus of MHV cell mixing separation and Culture is characterized in that it comprises step:
1) selects culture reagent
Cell growth medium adds calf serum for the MEM mother liquor, and final concentration is 10%, adds qingfengmeisu qiong, final concentration 24ug/ml, 7.5%NaHCO
3Transfer pH to 7.0～7.2;
Virus transportation liquid adds bovine serum albumin for the MEM mother liquor, and final concentration 5ug/ml adds green grass or young crops, Streptomycin sulphate final concentration 100ug/ml, adds B fungizone final concentration 0.5ug;
Virus inoculation liquid adds green grass or young crops, Streptomycin sulphate final concentration 50ug/ml for the MEM mother liquor adds bovine serum albumin final concentration 5ug/ml, and TPCK-pancreatin final concentration is 2ug/ml;
2) preparation culturing cell:
5～15 generation mdck cell, HEP-2 cell and Vero cell, in T75 cell bottle, grow up to monolayer respectively;
3) preparation MHV cell mixing bottle
Adopt MDCK, Hep-2, Vero cell preparation cell mixing culturing bottle, the first type in the separation and Culture Respirovirus, Influenza B virus, adenovirus; EV71 virus in the enterovirus;
4) specimen inoculation:
Nasopharynx is wiped away sample disposal: the centrifugal 5min of 4 ℃ of nasopharynx samples, 2000r/min removes deposition, and negative control is set up in 0.22 micron pore size membrane filtration degerming or handle inoculation cell mixing bottle after 4 hours with green grass or young crops/Streptomycin sulphate, B fungizone simultaneously;
Stool sample is handled: stool sample is suspended in pH7.2PBS liquid with 1: 5 ratio; The centrifugal 5min of 2000r/min removes the ight soil slag; 0.22 the degerming of micron pore size membrane filtration is inoculated the cell mixing bottle with green grass or young crops/Streptomycin sulphate, B fungizone processing again after 4 hours, set up negative control simultaneously;
5) culture is identified:
After the inoculation sample 24h, under inverted microscope, observe and observe differential protein mono-clonal immunofluorescence dyeing, RT-PCR, pcr amplification product evaluation under culturing cell cytopathic effect, the fluorescent microscope.
2. by the described method of claim 1; It is characterized in that in the step 3), MDCK, Hep-2, Vero three strain cells grow up to monolayer after the EDTA-trysinization respectively in T75 cell bottle; Respectively MDCK, Hep-2, Vero cell suspension counting back are mixed, processing cell count is 1.5 * 10
5The cell suspension of/ml, the cell count of wherein every strain cell is respectively 5 * 10
4/ ml, in above-mentioned mixed cell suspension packing culturing bottle, the 3ml/ bottle is put 37 ℃, 5%CO
2Grow up to the cell monolayer about 80% in the incubator.
3. by the described method of claim 2, it is characterized in that described EDTA-pancreatin is the 0.25%EDTA-pancreatin.
4. by the described method of claim 2, it is characterized in that described cell suspension counting adopts the white blood cell count(WBC) plate.
5. by the described method of claim 1, it is characterized in that in the step 4), nasopharynx is wiped away sample and adopted the simulation clinical samples, concrete preparation as following:
(1) sampling tube preparation is got 3.6ml virus transportation liquid and is added the 10ml sterilization in vitro;
(2) get-86 ℃ of preservations first type, Influenza B virus liquid, adenopathy venom, EV71 virus liquid each count strain, every strain virus liquid is drawn 0.4ml and is added in the above-mentioned sampling tube and to dilute at 1: 10;
(3) each picked at random volunteer, the disinfecting silk or cotton swab is swallowed rear wall and the wiping gently of tonsil both sides the volunteer, inserts in the sampling tube, makes the clinical pharynx of simulation and wipes away sample.
6. by the described method of claim 1, it is characterized in that, in the step 5), adopt first type, Influenza B virus mono-clonal immunofluorescence antibody I MAGEN
TMInfluenza virus A and B (DAKO); Adenovirus mono-clonal immunofluorescence antibody D3 ultra
TMDFA Respiratory virus ID kit (DIAGNOSTIC HYBRIDS); General enterovirus mixed immunity fluorescence antibody LIGHT DIAGNOSTICS
TMPan-Enterovirus Reagent, MIILIPORE (chemicon).
7. by claim 1 or 6 described methods, it is characterized in that described Influenza A1 virus RT-PCR amplified production is 431BP; The Influenza B virus amplified production is 390bp; The adenovirus amplified production is 874bp.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6274299A (en) * | 1985-09-26 | 1987-04-06 | Yoshio Numazaki | Plate for separating virus |
-
2010
- 2010-07-23 CN CN2010102369296A patent/CN102337249A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6274299A (en) * | 1985-09-26 | 1987-04-06 | Yoshio Numazaki | Plate for separating virus |
Non-Patent Citations (2)
| Title |
|---|
| JOSé M. NAVARRO-MARí ET AL.,: "apid detection of respiratory virus by shell vial assay using stimultaneous culture of HEp-2,LLC-MK2 and MDCK cells in a single vial", 《JOURNAL OF CLINICAL MICROBIOLOGY》, vol. 37, no. 7, 31 July 1999 (1999-07-31), pages 2346 - 2347 * |
| 蒋庆五 等: "甲3型、乙型流感病毒在MHL混合细胞中增殖的研究", 《中国卫生检验杂志》, vol. 13, no. 2, 30 April 2003 (2003-04-30), pages 150 - 151 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438092A (en) * | 2019-08-15 | 2019-11-12 | 广州金域医学检验中心有限公司 | Viral transporting culture medium and its preparation method and application |
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