CN108060139B - Isolation culture method of porcine epidemic diarrhea virus - Google Patents
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Abstract
A separation culture method of porcine epidemic diarrhea virus belongs to the technical field of biological products for animals. Collecting intestinal contents and excrement from clinically suspected PED sick piglets, respectively adding physiological saline, homogenizing the intestinal contents, uniformly oscillating and mixing excrement samples, adding a virus treatment solution, uniformly mixing, standing, centrifuging, taking supernate, filtering by using a microporous filter membrane, and filtering by using the microporous filter membrane to obtain PEDV virus stock solution; taking Vero cells which are cultured for 24 hours and grow well adherent, discarding cell culture solution, inoculating PEDV virus stock solution and maintenance solution into a cell bottle, placing the cell bottle into an incubator for culturing, observing cell lesions every day, collecting cell culture solution with obvious lesions, and placing the cell culture solution at-20 ℃ for storage. The separation culture method provided by the invention has low cost and short separation period, and is beneficial to timely diagnosis and research of porcine epidemic diarrhea virus.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a separation culture method of porcine epidemic diarrhea virus.
Background
Porcine Epidemic Diarrhea (PED) is an acute diarrhea disease caused by Porcine Epidemic Diarrhea Virus (PEDV), which has diarrhea, vomiting and dehydration of piglets as main clinical symptoms, and finally destroys electrolyte balance to cause death of sick pigs. At present, PED has a wide spread range, is reported in disease cases all over the world, has high morbidity, has the infection rate of 100 percent in young pigs and the infection mortality rate of 80 to 100 percent in piglets under 7 days old, and causes serious economic loss to the pig industry due to frequent occurrence of PED.
PEDV-induced porcine epidemic diarrhea is complex and difficult to control, and many scholars have studied from multiple aspects such as prevention, diagnosis, prevention and control. PEDV is a linear single-stranded positive-strand RNA virus, and its encoded structural proteins include S protein, E protein, M protein and N protein, where S protein is an important protein for studying the mechanism of viral infection, and can enter cells through membrane fusion after binding to a receptor (pAPN) on intestinal epithelial cells, and replication, transcription, and virion assembly are completed in cells after membrane de-encapsulation or uncoating, and pAPN plays a great role in viral propagation as viral receptor-mediated infection, and thus pAPN is applied to PEDV culture.
The traditional PEDV separation method has a long period, the virus sample treatment process is complicated, the virus sample treatment process is easy to pollute, the purity of the separated virus is not high, and the method is not beneficial to timely diagnosis of the PEDV, so that the invention of the rapid separation culture method of the PEDV has important significance for rapid diagnosis and research of the PEDV.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme of a porcine epidemic diarrhea virus isolation culture method, and provides a basis for diagnosis and research of porcine epidemic diarrhea.
The isolated culture method of the porcine epidemic diarrhea virus is characterized by comprising the following steps:
1) collecting intestinal contents and excrement from clinically suspected PED sick piglets according to the mass-volume ratio of 1: adding normal saline at a ratio of 1-1:3, homogenizing intestinal contents, uniformly mixing fecal samples by oscillation, adding a virus treatment solution according to a volume ratio of 1: 2-1: 10, uniformly mixing, standing for 5-15min, centrifuging according to a ratio of 4000 + 8000 r/min and 8-15 min, taking supernatant, filtering by a microporous filter membrane of 0.45 um, and filtering by a microporous filter membrane of 0.22um to obtain PEDV virus stock solution;
2) taking Vero cells which are cultured for 24 hours and grow well adherent, discarding cell culture solution, inoculating PEDV virus stock solution and maintenance solution into a cell bottle according to the volume ratio of 1:5-1:50, setting a positive control group and a blank control group of PEDV, culturing the cell bottle in an incubator at 37 ℃ and 5% CO2 for 96-120 hours, observing cytopathic effect every day, collecting cell culture solution with obvious pathological changes, and storing at-20 ℃.
The method for separating and culturing the porcine epidemic diarrhea virus is characterized in that the virus treatment solution in the step 1) is prepared by the following steps:
taking 5-12g of sodium chloride, 0.01-2g of potassium chloride, 0.05-1g of monopotassium phosphate, 0.01-3g of calcium chloride, 0.05-5 g of sodium citrate, 5-20 g of sodium thiosulfate, 60-100 mg of penicillin and 60-100 mg of streptomycin;
the above ingredients were mixed, dissolved in 1000ml of double distilled water and treated with 0.1M NaHCO3Adjusting pH to 7.5-9.0, filtering with 0.22um filter membrane, and storing at 4 deg.C.
The isolated culture method of the porcine epidemic diarrhea virus is characterized in that the virus treatment solution is added in the step 1) according to the volume ratio of 1: 4-1: 8.
The isolated culture method of the porcine epidemic diarrhea virus is characterized in that the maintenance liquid in the step 2) is prepared by the following steps:
taking 1-5mg of pancreatin, 1-5mg of pAPN, 6-8 g of salidroside and 3-5 g of chlorogenic acid;
mixing the above components, dissolving in 1000ml DMEM, filtering with 0.22um filter membrane, and storing at 4 deg.C.
The method for separating and culturing the porcine epidemic diarrhea virus is characterized in that in the step 2), the PEDV virus stock solution and the maintenance solution are inoculated into a cell bottle according to the volume ratio of 1:20-1: 30.
The invention has the following beneficial effects:
1. the separation method of the porcine epidemic diarrhea virus provided by the invention applies a new virus separation liquid, thereby improving the separation success rate of the virus;
2. the separation method of the porcine epidemic diarrhea virus provided by the invention fully utilizes the culture characteristic of the virus, provides a maintenance liquid for promoting virus propagation, and increases the virus content of the separated virus;
3. compared with the traditional separation method, the separation method of the porcine epidemic diarrhea virus provided by the invention has the advantages of simple operation steps, easy operation, short period and difficult pollution.
Drawings
FIG. 1 shows the result of PCR identification of PEDV;
FIG. 1-negative control; 2-the isolated strain; 3-positive control; M-DL2000 bp.
FIG. 2 is a fluorescent image of virus-inoculated cell cultures.
FIG. 3 is a fluorescence image of a normal cell culture.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following examples which are intended to illustrate the invention. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention, and that specific experimental procedures not mentioned in the following examples are generally conducted in accordance with conventional experimental procedures.
Example 1: preparation of Virus treatment solution
Sodium chloride: 8g
Potassium chloride: 1g
Potassium dihydrogen phosphate: 0.5g
Calcium chloride: 1g
Sodium citrate: 2g (purchased from Texas Huaxin laboratory instruments Co., Ltd.)
Sodium thiosulfate: 10g (purchased from national medicine group chemical reagent Co., Ltd.)
Penicillin: 80 mg of
Streptomycin: 80 mg of
The above ingredients were mixed, dissolved in 1000ml of double distilled water and treated with 0.1M NaHCO3Adjusting pH to 8.0, filtering with 0.22um filter membrane, and storing at 4 deg.C.
In this embodiment, 5g of sodium chloride, 0.01g of potassium chloride, 0.05g of potassium dihydrogen phosphate, 0.01g of calcium chloride, 0.05g of sodium citrate, 5g of sodium thiosulfate, 60mg of penicillin and 60mg of streptomycin can be used for preparation, or 12g of sodium chloride, 2g of potassium chloride, 1g of potassium dihydrogen phosphate, 3g of calcium chloride, 5g of sodium citrate, 20 g of sodium thiosulfate, 100 mg of penicillin and 100 mg of streptomycin can be used for preparation, and finally the same technical effects as those in embodiment 1 can be achieved.
Example 2: collecting and treating pathological materials
Collecting intestinal contents and excrement from clinically suspected PED sick piglets, respectively adding normal saline according to the mass-volume ratio of 1:2, homogenizing the intestinal contents, uniformly mixing excrement samples by oscillation, adding virus treatment solution according to the volume ratio of 1:5, uniformly mixing, and standing for 10 min. And finally, centrifuging at 5000 r/min for 10min, taking the supernatant, filtering by using a 0.45 um microporous filter membrane, and filtering by using a 0.22um microporous filter membrane to obtain the PEDV virus stock solution.
In the embodiment, the intestinal contents and the excrement collected by the clinically suspected PED sick piglets are respectively added with the physiological saline according to the mass-to-volume ratio of 1:1 or the intestinal contents and the excrement collected by the clinically suspected PED sick piglets are respectively added with the physiological saline according to the mass-to-volume ratio of 1: 3; the same technical effect as in example 2 can be achieved by adding the virus treatment solution in a volume ratio of 1:2, 1:6 or 1: 10.
Example 3: preparation of maintenance liquid
The method for separating the porcine epidemic diarrhea virus is characterized in that 1000ml of maintenance liquid consists of the following components:
pancreatin 3mg (purchased from Sigma)
pAPN: 2mg (purchased from Sigma)
Salidroside 7g (purchased from Shanghai Baishi Kai chemical science and technology Co., Ltd.)
Chlorogenic acid 3g (purchased from Okko chemical Co., Ltd., Shanghai)
Mixing the above components, dissolving in 1000ml DMEM, filtering with 0.22um filter membrane, and storing at 4 deg.C.
In this embodiment, pancreatin 1mg, pAPN 1mg, salidroside 6 g and chlorogenic acid 3g may be used for preparation, or pancreatin 5mg, pAPN5mg, salidroside 8g and chlorogenic acid 5g may be used for preparation, and finally the same technical effects as in embodiment 3 may be achieved.
Example 4: cell inoculation of viruses
And (2) discarding a cell culture solution after culturing the Vero cells which grow well adherent for 24 hours, inoculating a virus solution and a maintenance solution into a cell bottle according to the ratio of 1:20 (v/v), setting a positive control group and a blank control group of PEDV, culturing the cell bottle in a 37 ℃ 5% CO2 culture box for 100 hours, observing cell lesions every day, collecting the cell culture solution with obvious lesions, and storing at-20 ℃.
In this embodiment, the virus solution and the maintenance solution can be inoculated into the cell bottle at a ratio of 1:5, 1:10, 1:30 or 1:50, and finally the same technical effect as that of embodiment 4 can be achieved.
Test example: isolation and identification of porcine epidemic diarrhea virus
1 Material
1.1 isolation of porcine epidemic diarrhea Virus
Isolation according to inventive example 4;
1.2 DMEM basal medium, fetal bovine serum purchased from GIBCO company;
1.3 porcine epidemic diarrhea virus positive serum and porcine epidemic diarrhea disease indirect immunofluorescence assay kit, all purchased from Chinese veterinary medicine inspection institute;
1.4 porcine epidemic diarrhea virus positive strains, which are preserved by research and development center of Meibao Longe biotechnology Limited company in Zhejiang;
1.5 PCR-related reagents, purchased from Dalibao Biopsis.
2 method
2.1 measurement of Virus content
PEDV cell virus solution obtained in example 4 was diluted 10-fold with DMEM basal medium, transferred to 96-well monolayer-spread Vero cell culture plates at 0.1m L/well, cultured in a 5% incubator at 37 ℃ for 4 days, observed and recorded every day, and TCID50 was calculated according to the Reed-Muench method.
2.2RT-PCR identification
In the test, a pair of primers is designed by applying software such as Oligo 6.0, Primer5.0 and the like according to a PEDV gene sequence published by GenBank,
upstream primer (P1): 5'-GCATCCTTATGGCTTGCATC-3' the flow of the air in the air conditioner,
downstream primer (P2): 5'-CCTGTACGCCAGTAGCAACC-3', the amplified fragment is 243 bp.
Extracting virus RNA by using an RNA extraction kit, carrying out reverse transcription by using the virus RNA as a template, and finally adopting a 25 ul PCR reaction system: PCR Master Mix 12.5ul, upstream and downstream primers 1 ul, template DNA 1 ul, ddH2O 8.5.5 ul, the reaction program is: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1 min; and 5min at 72 ℃, performing agarose gel electrophoresis on the product, and taking a picture by using a gel imaging system to record the result, and setting a negative and positive control.
2.3 immunofluorescence assay
Inoculating Vero cells into a 96-well cell culture plate, inoculating a cell culture with CPE after the cells grow into a monolayer, placing 0.1mL of the cell culture in each well, adsorbing at 37 ℃ for 1h, then discarding an inoculation liquid, supplementing a maintenance liquid to 0.2mL of the cell culture in each well, placing the cell culture in an incubator containing 5% CO2 for 48 h, then fixing for 20 min (the fixing liquid is acetone: methanol (V) =4: 1), discarding the fixing liquid, washing with PBS, adding 100ul (diluted by PBS 1:100 times) of PRRSV positive serum in each well, then placing the mixture in a 37 ℃ wet box for 45 min, adding FITC-rabbit anti-pig lgG 100ul (the diluent is PBS containing 1/10000 Evans blue) diluted by 1: 1000 times into each hole after PBS cleaning, placing the mixture in a 37 ℃ wet box for 30 min, cleaning the mixture for 3 times by PBS, placing the mixture in a fluorescence microscope for observation, and setting negative serum and normal cells as controls.
3 results
3.1 measurement of TCID50 content
The infection amount of half cells of the separated strain is measured by a Reed-Muench method, the result of the virus CPE at each dilution is shown in the table 1, and the calculation result shows that TCID50=10-6.625/0.1ml
TABLE 1 determination of viral titer
3.2 RT-PCR identification results
And taking the separated porcine epidemic diarrhea virus as a template, carrying out agarose gel electrophoresis after PCR amplification, observing under ultraviolet light, and taking a picture by using a gel imaging system. As can be seen from FIG. 1, the amplification results of the isolated viruses are 243bp, which are consistent with the expected results.
3.3 immunofluorescence assay
The rabbit anti-pig lgG is used for carrying out indirect immunofluorescence test on a cell culture inoculated with virus and a control cell culture not inoculated with virus, and the result shows that most cells of the cell culture inoculated with virus have green flashing fluorescence on cytoplasm and cell membrane (see figure 2); no green fluorescence was observed in normal cells (see FIG. 3).
4 conclusion
In conclusion, the separation method of the porcine epidemic diarrhea virus provided by the invention is simple, convenient and rapid, greatly shortens the virus identification time, can obtain the virus with high virus content and high purification level, and provides a new virus separation solution and a new virus culture maintenance solution for the separation of the porcine epidemic diarrhea virus.
Claims (3)
1. A separation culture method of porcine epidemic diarrhea virus is characterized by comprising the following steps:
1) collecting intestinal contents and excrement from clinically suspected PED sick piglets according to the mass-volume ratio of 1: adding normal saline at a ratio of 1-1:3, homogenizing intestinal contents, uniformly mixing fecal samples by oscillation, adding a virus treatment solution according to a volume ratio of 1: 2-1: 10, uniformly mixing, standing for 5-15min, centrifuging according to a ratio of 4000 + 8000 r/min and 8-15 min, taking supernatant, filtering by a microporous filter membrane of 0.45 um, and filtering by a microporous filter membrane of 0.22um to obtain PEDV virus stock solution;
2) taking Vero cells which are cultured for 24 hours and grow well adherent, discarding cell culture solution, inoculating PEDV virus stock solution and maintenance solution into a cell bottle according to the volume ratio of 1:5-1:50, setting a positive control group and a blank control group of PEDV at the same time, placing the cell bottle at 37 ℃ and 5% CO2Culturing in incubator for 96-120 hr, observing cytopathic effect every day, collecting cell culture solution with obvious pathological changes, and storing at-20 deg.C;
the virus treatment solution is prepared by the following steps:
taking 5-12g of sodium chloride, 0.01-2g of potassium chloride, 0.05-1g of monopotassium phosphate, 0.01-3g of calcium chloride, 0.05-5 g of sodium citrate, 5-20 g of sodium thiosulfate, 60-100 mg of penicillin and 60-100 mg of streptomycin;
mixing the above components, and dissolving in 1000ml double distilled waterAnd with 0.1M NaHCO3Adjusting pH to 7.5-9.0, filtering with 0.22um filter membrane, and storing at 4 deg.C;
the maintenance liquid is prepared by the following steps:
taking 1-5mg of pancreatin, 1-5mg of pAPN, 6-8 g of salidroside and 3-5 g of chlorogenic acid;
mixing the above components, dissolving in 1000ml DMEM, filtering with 0.22um filter membrane, and storing at 4 deg.C.
2. The method for isolating and culturing the porcine epidemic diarrhea virus of claim 1, wherein the virus treatment solution is added in the step 1) in a volume ratio of 1:4 to 1: 8.
3. The method for isolating and culturing porcine epidemic diarrhea virus of claim 1, wherein the PEDV virus stock solution and the maintenance solution are inoculated into the cell flask in the step 2) at a volume ratio of 1:20 to 1: 30.
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