CN110045118A - A kind of method of quick detection Porcine epidemic diarrhea virus - Google Patents
A kind of method of quick detection Porcine epidemic diarrhea virus Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The present invention relates to a kind of methods of quickly detection Porcine epidemic diarrhea virus, the method that one kind of the invention quickly detects Porcine epidemic diarrhea virus, using the PEDV-G1 strain grown in Vero passage cell, the preparation of the Vero passage cell are as follows: after Vero cell grows up to single layer, discard nutrient solution, virus liquid is added, 1.5h is made in room temperature sense；Then maintaining liquid is added, is put into 37.5 DEG C of cultures；Virus collects venom, freeze thawing when cell growth 60h has obvious lesion, and centrifugation removes cell fragment, sets -70 DEG C or less and freeze, and the detection method is to take pig intestinal mucosa deep layer tabletting, and after acetone is fixed, fluorescent staining detects PEDV-G1 plants.Detection method provided by the invention has the characteristics of efficiently and accurately.
Description
Technical field
The present invention relates to a kind of methods of quickly detection Porcine epidemic diarrhea virus.
Background technique
Pig epidemic diarrhea is a kind of contact enteric infectious disease as caused by Porcine epidemic diarrhea virus, it is characterized in that vomitting
It spits, diarrhea, dehydration.Clinical change and symptom and pig transmissible stomach and intestine are very much like.
Pig prevalence diarrhea virus (PEDV), belongs to coronaviridae coronavirus genus.Up to the present, it has not been found that originally
Virus has different serotype.This virus is sensitive to ether, chloroform.More property are presented in virion, and tendency is round, there is capsule outside
Film.From the intestines liquid filling of illness piglet be concentrated and purify virus cannot be aggregated rabbit, mouse, pig, cavy, sheep, ox, horse,
The red blood cell of chick and people.
This disease only occurs in pig, and the pig at various ages can infection morbidity.Suckling pig, feeder pig or the disease incidence of fattening pig
Very high, especially aggrieved the most serious with suckling pig, the variation of sow disease incidence is very big, and about 15-90%.Sick pig is major source of infection.
Virus is present in intestinal villus epithelial cell and lymphonodi mesenterici, after excreting with faeces, pollutes environment, feed, drinking-water, traffic work
Tool and apparatus etc. and infect.Main infection approach is alimentary canal.If a pig farm has many nest piglets to be born or wean successively,
Virus can constantly infect the weanling pig for losing maternal antibody, make this disease in endemicity, and in this breeding farm, pig is popular
Property diarrhea can cause the weaning period intractable diarrhea of 5-8 week old piglets.This disease mostly occurs in cold season.
Pig epidemic diarrhea pathogenesis is that virus is directly entered small intestine orally and after nose infection.By immunofluorescence and
Electron microscopy, viral duplication are carried out in small intestine and colon villus epithelial cells slurry.It is not found in other organs
Virus multiplication.Since virus multiplication causes cell first, then there is small cell dysfunction in the damage of device.Intestinal villus atrophy,
The reduction of sorbent surface product is caused, small intestinal mucosa content of alkaline phosphatase substantially reduces and then absorption of nutrient ingredients is caused to hinder
Hinder, this is the main reason for causing diarrhea, to belong to osmotic diarrhea.Severe diarrhea causes to be dehydrated, and is the master for leading to sick pig death
Want reason.
Pig epidemic diarrhea incubation period is generally 5-8 days, and artificial challenge's incubation period is 8-24 hours.Main clinical condition
Shape is watery diarrhea, or in the vomiting of diarrhea.After vomiting mostly occurs in food or sucks the breast.The weight of symptom is with the age
Size and it is variant, the age is smaller, and symptom is heavier.Newborn piglet occurs after diarrhea 3-4 days in one week old, and serious dehydration is presented
And it is dead, the death rate is up to 50%, and the highest death rate is up to 100%.Sick pig body temperature is normal or slightly higher, and spirit is depressed, and appetite subtracts
It moves back or abolish.Wean pig, sow are often in down in spirits, anorexia and persistent diarrhea about one week, and are gradually recovered normal.It is a small number of
Retarded growth after pig restores.After the raising infection of same circle diarrhea all occurs for fattening pig, rehabilitation after a week, death rate 1%--
3%.
From 1973, the country is successively separated to pig prevalence with pig body partition method during studying transmissible gastroenteritis of swine
The strains such as property diarrhea China, river, Ji.1980, China successfully used these Strain of chitterlings organ cultures subculture.
1991, the pathological material of disease that the equal sick pig for being presented with epidemic diarrhea symptom from In Guangdong Province of Guangzhou animal and plant quarantine bureau Li Shugen acquires
The PED virus separated successively is adapted to Vero, Pk15 and ST passage cell growth and breeding.
It is domestic at present that there has been no the methods of more advanced reliable quickly detection Porcine epidemic diarrhea virus.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of methods of quickly detection Porcine epidemic diarrhea virus.
The method that one kind of the invention quickly detects Porcine epidemic diarrhea virus, using what is grown in Vero passage cell
PEDV-G1 plants.
The method of quickly detection Porcine epidemic diarrhea virus as described above a kind of, further explains and is, described
The preparation of Vero passage cell are as follows: after Vero cell grows up to single layer, discard nutrient solution, virus liquid is added, 1.5h is made in room temperature sense；So
After maintaining liquid is added, be put into 37.5 DEG C of cultures；Virus collects venom, freeze thawing when cell growth 60h has obvious lesion, and centrifugation is gone
Cell fragment is set -70 DEG C or less and is frozen.
A kind of method of quickly detection Porcine epidemic diarrhea virus as described above, further explaining is the inspection
Survey method is to take pig intestinal mucosa deep layer tabletting, and after acetone is fixed, fluorescent staining detects PEDV-G1 plants.
A kind of method of quickly detection Porcine epidemic diarrhea virus as described above, further explaining is that described is glimmering
Light dyes detailed process are as follows:
(1), the separation and purification of globulin
Rabbit PED hyper-immune serum 20ml is put in triangular flask, then triangular flask is put into the large beaker equipped with ice cube, is added dropwise
Isometric saturated ammonium sulfate solution stirs 5min, when there is protein precipitation, is put in 2h in refrigerator, is then centrifuged for, go
Clear liquid；It is precipitated with physiological saline solution, adds isometric 01005M, the PBS containing 011MNaCI；
(2), the desalination of globulin
Salt ion is separated with sephadex G 50 (SepHadexG50), extracts globulin；The glucan of equivalent is weighed, is passed through
After washing floating granules and sufficiently expansion with PBS, it is encased in chromatography pipe；By the globulin dropper containing sulfate ion
It is added on glucan column, is slowly added to；It is 1ml/min by column speed control, collecting 50 drops is a sample；The ball being collected into
Albumen, detects sulfate ion with 1% barium chloride, detects ammonium ion with nessler reagent, when confirming without containing sulfate ion and
When ammonium ion, it is spare that globulin is put into refrigerator；
(3), the combination of globulin and fluorescein isothiocyanate (FITC)
The ratio of FITC and globulin is 1:20~1:40；By FITC pH9.0~pH9.5,0.025M carbonate buffer
Liquid is made into 1mg/ml；Globulin is concentrated again；It is contained in the bottle for being equipped with small bar magnet, sets in refrigerator, equivalent is added dropwise while stirring
ITC；Bottleneck is stoppered with rubber stopper after adding, 4 DEG C of stirrings combine 8h；Free FITC is removed with glucan G50；It is anti-to collect fluorescence
Body, it is spare to be lyophilized when 12mg/ml to set up protein concentration；
(4), the concentration of fluorescence antibody: the setting of protein concentration is the method that absorbed water with glucan G50, is added in albumen molten night
Enter suitable glucan, after it absorbs moisture, with centrifugal process removing gel；
(5), the freeze-drying of fluorescence antibody: 25 DEG C of the temperature of freeze dryer room；Freeze-drying time 2.5h.
(6), the specific assay of fluorescence antibody, each hole that will be put into the processed coverslip of cleaning solution on 16 orifice plates
In, be added PK15 cell, add transmissible gastro-enteritis virus (TGEV), pig parvoviral (PPV), rotavirus (RV) and
Escherichia coli.
The method of quickly detection Porcine epidemic diarrhea virus as described above a kind of, further explains and is, described
TGEV is derived from United States Department of Agriculture, and PPV, RV are derived from Jiangsu veterinary institute, and Escherichia coli are derived from military medical research institute；37 DEG C of trainings
It after feeding 48h is shown in CPE, is fixed through acetone, same method carries out PED fluorescent staining, the observing response under fluorescence microscope.
The beneficial effects of the present invention are:
Detection method provided by the invention has efficiently and accurately feature.
Specific embodiment
The method that one kind of the invention quickly detects Porcine epidemic diarrhea virus, using what is grown in Vero passage cell
PEDV-G1 plants.
The method of quickly detection Porcine epidemic diarrhea virus as described above a kind of, further explains and is, described
The preparation of Vero passage cell are as follows: after Vero cell grows up to single layer, discard nutrient solution, virus liquid is added, 1.5h is made in room temperature sense；So
After maintaining liquid is added, be put into 37.5 DEG C of cultures；Virus collects venom, freeze thawing when cell growth 60h has obvious lesion, and centrifugation is gone
Cell fragment is set -70 DEG C or less and is frozen.
A kind of method of quickly detection Porcine epidemic diarrhea virus as described above, further explaining is the inspection
Survey method is to take pig intestinal mucosa deep layer tabletting, and after acetone is fixed, fluorescent staining detects PEDV-G1 plants.
A kind of method of quickly detection Porcine epidemic diarrhea virus as described above, further explaining is that described is glimmering
Light dyes detailed process are as follows:
(1), the separation and purification of globulin
Rabbit PED hyper-immune serum 20ml is put in triangular flask, then triangular flask is put into the large beaker equipped with ice cube, is added dropwise
Isometric saturated ammonium sulfate solution stirs 5min, when there is protein precipitation, is put in 2h in refrigerator, is then centrifuged for, go
Clear liquid；It is precipitated with physiological saline solution, adds isometric 01005M, the PBS containing 011MNaCI.
(2), the desalination of globulin
Salt ion is separated with sephadex G 50 (SepHadexG50), extracts globulin；The glucan of equivalent is weighed, is passed through
After washing floating granules and sufficiently expansion with PBS, it is encased in chromatography pipe；By the globulin dropper containing sulfate ion
It is added on glucan column, is slowly added to；It is 1ml/min by column speed control, collecting 50 drops is a sample；The ball being collected into
Albumen, detects sulfate ion with 1% barium chloride, detects ammonium ion with nessler reagent, when confirming without containing sulfate ion and
When ammonium ion, it is spare that globulin is put into refrigerator.
(3), the combination of globulin and fluorescein isothiocyanate (FITC).
The ratio of FITC and globulin is 1:20~1:40；By FITC pH9.0~pH9.5,0.025M carbonate buffer
Liquid is made into 1mg/ml；Globulin is concentrated again；It is contained in the bottle for being equipped with small bar magnet, sets in refrigerator, equivalent is added dropwise while stirring
ITC；Bottleneck is stoppered with rubber stopper after adding, 4 DEG C of stirrings combine 8h；Free FITC is removed with glucan G50；It is anti-to collect fluorescence
Body, it is spare to be lyophilized when 12mg/ml to set up protein concentration.
(4), the concentration of fluorescence antibody: the setting of protein concentration is the method that absorbed water with glucan G50, is added in albumen molten night
Enter suitable glucan, after it absorbs moisture, with centrifugal process removing gel.
(5), the freeze-drying of fluorescence antibody: 25 DEG C of the temperature of freeze dryer room；Freeze-drying time 2.5h.
(6), the specific assay of fluorescence antibody, each hole that will be put into the processed coverslip of cleaning solution on 16 orifice plates
In, be added PK15 cell, add transmissible gastro-enteritis virus (TGEV), pig parvoviral (PPV), rotavirus (RV) and
Escherichia coli.
The method of quickly detection Porcine epidemic diarrhea virus as described above a kind of, further explains and is, described
TGEV is derived from United States Department of Agriculture, and PPV, RV are derived from Jiangsu veterinary institute, and Escherichia coli are derived from military medical research institute；37 DEG C of trainings
It after feeding 48h is shown in CPE, is fixed through acetone, same method carries out PED fluorescent staining, the observing response under fluorescence microscope.
Field application test:
The intestinal mucosal samples of detection include 20 parts of pig farm, severe diarrhea, vomiting occur altogether, and have the pig farm of piglet death,
Take the apparent mucous membrane of small intestine deep layer tabletting of the lesions such as hyperemia, bleeding, one sample detection PEDV of a pig.
PED antibody is detected using neutralization test method.
Two weeks acquisition serum makees neutralization test after natural occurrence, artificial onset.The pig farm A is taken a blood sample 10 parts, 18 parts of the pig farm B, C
12 parts of pig farm, the pig farm D are taken a blood sample 10 parts, 20 parts of the pig farm E, 30 parts of the pig farm F.
Comparative test:
To 20 part positive reaction samples and 10 part negative reaction samples with Harbin beast grind PED fluorescence antibody make
Compare.Direct fluorescence detection operation sequence.
Glass slide after being cleaned with washing powder, then with 1N HCL impregnate, with distilled water flushing it is net after, be put into 95% alcohol
It is spare.Coverslip is rinsed well after being soaked with cleaning solution, then is dipped in spare in 95% alcohol.Take the small intestine of sick pig, especially lesion
Apparent jejunal segment makees smear, removes intestinal contents, cuts off small intestine, the tabletting on glass slide of mucous membrane of small intestine deep layer.After air-drying, drop
Add the fixed 10min of cold acetone, rinsed twice with pH7.4,0.01M PBS, if do not dyed immediately, can be placed on -20 DEG C of refrigerators and protect
It deposits spare.
Fluorescent antibody staining.
Sample is first rinsed twice with PBS, and 2/10000ths azovan blues of fluorescence antibody make 1:4 dilution, and centrifugation is removed sediment, taken
Supernatant drips on test sample, slide is put in wet box, then set 37 DEG C, 30min, takes out and is rinsed 3 times with PBS, then is steamed with double
Water rinses primary.Glycerol PBS mono- is added to drip, after covering slide, to microscopy.
It is detected with fluorescope (German OPTON).
Reaction criterion: result is determined in the case where negative control, positive control are set up.
The preservation of fluorescence antibody
The fluorescence antibody prepared, is diluted to working concentration, is distributed into every bottle of 1ml, and 4 DEG C of half a year measurement potency are put in freeze-drying.
Test result:
The titration of fluorescence antibody is that 1:16 hyperfluorescence reaction occurs, and using Liang Ge work unit, i.e. fluorescence antibody exists
Make 1:4 dilution with 2/10000ths Yi Wensilan when use.
The PED Immunofluorescent Antibody of test preparation, cannot be with transmissible gastro-enteritis virus, pig parvoviral, wheel
Shape virus and Escherichia coli etc. react, and fluorescence do not occur.
Inhibit test in, it is known that PED positive serum neutralization titer be 1:128, make 1:2,1:4,1:8 dilution, with PK15
PEDV in cell is neutralized, and can inhibit the effect of PED Immunofluorescent Antibody, fluorescence reaction does not occur.
There is serious watery diarrhea, excrement cacosmia after 16h in the piglet of artificial challenge, and anorexia dissects and sees enteron aisle spy
It is not that small intestine is hydraulically full, it is congested.4 piglets of artificial onset are slaughtered in 20h, 36h, 48h and 72h takes intestinal mucosa deep layer pressure
Piece, every takes 6 samples, fluorescence reaction can occurs.6 intestinal mucosal samples for not being inoculated with 1 control piglet of PEDV are yin
Property reaction.
Using the fluorescence antibody of this method development, 155 parts of intestinal mucosal samples that severe diarrhea pig farm occurs to 6 are examined,
Detection positive reaction 81.
100 parts of serum that the pig farm of 6 fluorescence antibody detection PEDV is detected with neutralization test method, as a result, it has been found that each pig
There is the reaction of PED positive serology in field.And the piggy of 1 artificial onset is detected, it is positive reaction.
Claims (6)
1. a kind of method of quickly detection Porcine epidemic diarrhea virus, which is characterized in that detection Porcine epidemic diarrhea virus uses
In PEDV-G1 strain of Vero passage cell growth.
2. a kind of method of quickly detection Porcine epidemic diarrhea virus as described in claim 1, which is characterized in that described
The preparation of Vero passage cell are as follows: after Vero cell grows up to single layer, discard nutrient solution, virus liquid is added, 1.5h is made in room temperature sense；So
After maintaining liquid is added, be put into 37.5 DEG C of cultures；Virus collects venom, freeze thawing when cell growth 60h has obvious lesion, and centrifugation is gone
Cell fragment is set -70 DEG C or less and is frozen.
3. a kind of method of quickly detection Porcine epidemic diarrhea virus as described in claim 1, which is characterized in that the inspection
Survey method is to take pig intestinal mucosa deep layer tabletting, and after acetone is fixed, fluorescent staining detects PEDV-G1 plants.
4. a kind of method of quickly detection Porcine epidemic diarrhea virus as claimed in claim 3, which is characterized in that described is glimmering
Light dyes detailed process are as follows:
(1), the separation and purification of globulin
Rabbit PED hyper-immune serum 20ml is put in triangular flask, then triangular flask is put into the large beaker equipped with ice cube, the bodies such as dropwise addition
Long-pending saturated ammonium sulfate solution stirs 5min, when there is protein precipitation, is put in 2h in refrigerator, is then centrifuged for, remove supernatant
Liquid；It is precipitated with physiological saline solution, adds isometric 01005M, the PBS containing 011MNaCI；
(2), the desalination of globulin
Salt ion is separated with sephadex G 50 (SepHadexG50), extracts globulin；The glucan for weighing equivalent, through with
After PBS washes floating granules and sufficiently expansion, it is encased in chromatography pipe；Globulin dropper containing sulfate ion is added
On glucan column, it is slowly added to；It is 1ml/min by column speed control, collecting 50 drops is a sample；The ball egg being collected into
It is white, sulfate ion is detected with 1% barium chloride, detects ammonium ion with nessler reagent, when confirmation does not contain sulfate ion and ammonium
When ion, it is spare that globulin is put into refrigerator；
(3), the combination of globulin and fluorescein isothiocyanate (FITC)
The ratio of FITC and globulin is 1:20~1:40；FITC pH9.0~pH9.5,0.025M carbonate buffer solution are matched
At 1mg/ml；Globulin is concentrated again；It is contained in the bottle for being equipped with small bar magnet, sets in refrigerator, the ITC of equivalent is added dropwise while stirring；
Bottleneck is stoppered with rubber stopper after adding, 4 DEG C of stirrings combine 8h；Free FITC is removed with glucan G50；Fluorescence antibody is collected, is adjusted
Determine to be lyophilized when protein concentration is 12mg/ml spare；
(4), the concentration of fluorescence antibody: the setting of protein concentration is the method that absorbed water with glucan G50, is added in albumen molten night suitable
The glucan of amount, after it absorbs moisture, with centrifugal process removing gel；
(5), the freeze-drying of fluorescence antibody: 25 DEG C of the temperature of freeze dryer room；Freeze-drying time 2.5h.
5. a kind of method of quickly detection Porcine epidemic diarrhea virus as claimed in claim 4, which is characterized in that fluorescence antibody
Specific assay, will be put into each hole on 16 orifice plates with the processed coverslip of cleaning solution, be added PK15 cell, add
Transmissible gastro-enteritis virus (TGEV), pig parvoviral (PPV), rotavirus (RV) and Escherichia coli.
6. a kind of method of quickly detection Porcine epidemic diarrhea virus as claimed in claim 4, which is characterized in that described
TGEV is derived from United States Department of Agriculture, and PPV, RV are derived from Jiangsu veterinary institute, and Escherichia coli are derived from military medical research institute；37 DEG C of trainings
It after feeding 48h is shown in CPE, is fixed through acetone, same method carries out PED fluorescent staining, the observing response under fluorescence microscope.
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| CN103756974A (en) * | 2013-09-29 | 2014-04-30 | 广东温氏食品集团股份有限公司 | Porcine epidemic diarrhea virus, and culture method and application thereof |
| CN108118032A (en) * | 2017-12-12 | 2018-06-05 | 新疆希普生物科技股份有限公司 | The culture of 2 type strain of Porcine epidemic diarrhea virus and the development of inactivated vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103756974A (en) * | 2013-09-29 | 2014-04-30 | 广东温氏食品集团股份有限公司 | Porcine epidemic diarrhea virus, and culture method and application thereof |
| CN108118032A (en) * | 2017-12-12 | 2018-06-05 | 新疆希普生物科技股份有限公司 | The culture of 2 type strain of Porcine epidemic diarrhea virus and the development of inactivated vaccine |
Non-Patent Citations (1)
| Title |
|---|
| XIAOBO WANG ET AL: "Immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes G1 and G2", 《ARCH VIROL》 * |
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Application publication date: 20190723 |