CN107267467B - Method for separating porcine reproductive and respiratory syndrome virus - Google Patents
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Abstract
A method for separating porcine reproductive and respiratory syndrome virus belongs to the technical field of biological products for animals. Which comprises the following steps: 1) preparing PAM primary cells; 2) collecting and processing pathological materials; 3) cell inoculation of the virus; 4) and (5) virus identification. The invention has the following beneficial effects: according to the separation method of the porcine reproductive and respiratory syndrome virus, the treatment method of the disease material does not need freeze thawing, the mechanization degree is high, and time and labor are saved; the PAM primary cells are simple and quick to prepare and low in cost, the virus separation period is greatly shortened, and the porcine reproductive and respiratory syndrome virus disease diagnosis and research can be facilitated.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a method for separating porcine reproductive and respiratory syndrome virus.
Background
Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly-contact infectious disease of pigs caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and the PRRSV can cause the immune suppression of animal bodies and further the infection of various pathogens when the PRRSV causes reproductive disorders such as early abortion, stillbirth, mummy, weak litter and the like of pregnant sows, respiratory symptoms of pigs of various ages, high death rate of piglets and the like. At present, PRRS is popular in almost all pig raising areas all over the world, which causes huge economic loss to the pig raising industry all over the world and arouses wide attention at home and abroad.
In order to better control PRRS, many scholars have conducted research from a variety of aspects, such as prevention, diagnosis, prevention and control. The PRRSV is a single-strand nonsegmented RNA virus, which belongs to arterivirus of arterivirus family of Nidovirales in biological classification, the whole length of PRRSV genome is different from 14.9-15.5kb, and comprises structural protein and non-structural protein, in the non-structural protein, ORF1b gene has unique nucleoside triphosphate binding site, and many Chinese medicines contain rich nucleoside component, and the virus can be well combined with the nucleoside component in the Chinese medicine. In view of this, we propose to use traditional Chinese medicine in the separation and extraction of PRRSV.
The traditional PRRSV virus separation method has a long period, the virus sample treatment process is relatively complicated, the pollution is easy to occur, the purity of the separated virus is not high, and the method is not beneficial to the timely diagnosis of PRRS, so that the invention provides the method for quickly separating and identifying PRRS, and has important significance for the quick diagnosis and research of the PRRS.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme of a method for rapidly separating and identifying porcine reproductive and respiratory syndrome virus, and the method provides a basis for diagnosing and researching the porcine reproductive and respiratory syndrome.
The separation method of the porcine reproductive and respiratory syndrome virus is characterized by comprising the following steps:
taking complete and fresh piglet lung, separating PAM by adopting an alveolar lavage method, injecting 100-200 mL sterile 4-DEG C precooled PBS solution into the lung from a trachea by using a funnel, massaging lung lobes, pouring out bronchoalveolar lavage fluid BALF, repeating the washing process for 3-5 times, mixing the BALF obtained each time, filtering by using 3 layers of sterile gauze, centrifuging at the temperature of 4 ℃ for 10min, collecting cells, and washing for 2 times by using PBS; adding RPMI-1640 or DMEM culture solution containing 10% newborn bovine serum, wherein the RPMI-1640 and DMEM culture solution contain L-glutamine suspension cells, counting the cells, placing the cells in an incubator at 37 ℃ and 5% CO2 for culturing for 0.5, 1, 2, 6 and 24 hours respectively, replacing the new culture solution, and finally, enabling the rest cells to be adherent cells;
2) collecting lung and lymph node from clinically suspected PRRS sick piglet, placing in 8-20 ml of physiological saline cooled at 4 ℃, controlling the temperature below 25 ℃, performing homogenization treatment by using a high-pressure homogenizer to obtain homogenate treatment liquid, adding virus treatment solution which is 1-5 times of the total volume of the physiological saline into the homogenate treatment liquid, uniformly mixing, standing for 5-15min, centrifuging at 3000-5000 r/min for 8-15 min, taking supernatant, and filtering by a 0.22um microporous filter membrane to obtain PRRSV virus stock solution;
3) culturing primary PAM cells with good adherent growth for 24 hours in the step 1), discarding cell culture solution, and adding PRRSV virus stock solution and the cell culture solution according to the volume ratio of 1:5-1:10In, the cell bottle was placed at 37 ℃ in 5% CO2Incubating in incubator for 0.5-1h, discarding virus solution, adding virus maintaining solution containing 2% fetal calf serum, culturing for 96-120 h, observing cytopathic effect every day, collecting cell sap with obvious pathological changes, and storing at-20 deg.C.
The method for separating the porcine reproductive and respiratory syndrome virus is characterized in that in the step 1), every 1000ml of PBS solution comprises the following components: 6-10 g of sodium chloride, 0.05-0.5 g of potassium chloride, 1-1.2 g of disodium hydrogen phosphate, 0.1-1g of monopotassium phosphate, 0.05-0.2 g of calcium chloride, 0.05-0.2 g of magnesium chloride containing 6 crystal water, 60-100 mg of penicillin and 60-100 mg of streptomycin; mixing the above components, dissolving in 1000ml double distilled water, adjusting pH to 6.0-6.8 with 0.5mol/L hydrochloric acid solution, filtering with 0.22um filter membrane, and storing at 4 deg.C.
The separation method of the porcine reproductive and respiratory syndrome virus is characterized in that in the step 2), each 1000ml of virus treatment solution consists of the following components: 1-5 mg of semen cuscutae extract, 0.1-2 mg of fructus schizandrae extract, 1-3 mg of mulberry polysaccharide, 0.1-0.5 mg of raspberry acid, 1-3 g of sodium citrate, 5-10 g of sodium chloride and 0.01-0.05 g of potassium chloride; mixing the above components, dissolving in 1000ml double distilled water, filtering with 0.22um filter membrane, and storing at 4 deg.C.
The chemical reagents related to the invention are all existing products and can be purchased in the market, wherein the dodder extract is produced and sold by Xian original plant engineering technology limited company, the schisandra extract is produced and sold by Chengdubesite reagent limited company, the mulberry polysaccharide is produced and sold by Dalian Meilan biotechnology limited company, and the raspberry acid is produced and sold by Shanghai source leaf biotechnology limited company.
The invention has the following beneficial effects:
1. the invention provides a method for separating porcine reproductive and respiratory syndrome virus, which adopts the technical scheme that the disease material is treated by a high-pressure homogenizer without freeze thawing, thereby not only ensuring the integrity of the virus, but also saving time and labor.
2. According to the method for separating the porcine reproductive and respiratory syndrome virus, the separated virus is directly inoculated in the PAM primary cells, so that the method is high in susceptibility and can greatly shorten the separation period of the virus.
3. The separation method of the porcine reproductive and respiratory syndrome virus provided by the invention fully utilizes the unique nucleoside triphosphate binding site of a virus molecule to be specifically combined with the traditional Chinese medicine containing the nucleoside component, so that the virus can be extracted to the maximum extent, and the propagation culture of the virus is not influenced.
Drawings
FIG. 1 shows the result of PCR identification of PPV;
FIG. 2 is a graph of an indirect immunofluorescence assay of a cell culture inoculated with a virus using rabbit anti-pig lgG;
FIG. 3 is a graph of an indirect immunofluorescence assay using rabbit anti-pig lgG on normally seeded cell cultures.
In fig. 1:1, negative control; 2: a positive control; 3: the isolated strain; m: DL1000 bp.
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 PAM Primary cells
Taking fresh lungs of healthy piglets of 10 days old, separating PAM by adopting an alveolar lavage method, using a funnel to fill 150 mL of sterile PBS solution (each 1000mL of PBS solution comprises 8g of sodium chloride, 0.2g of potassium chloride, 1.1g of disodium hydrogen phosphate, 0.8g of potassium dihydrogen phosphate, 0.1 g of calcium chloride, 0.1 g of magnesium chloride containing 6 crystal water, 80 mg of penicillin and 80 mg of streptomycin), mixing the components, dissolving the mixture in 1000mL of double distilled water, adjusting the pH value to 6.5 by using 0.5mol/L hydrochloric acid solution, filtering the mixture through a 0.22 mu m filter membrane, storing the mixture at 4 ℃ for later use, pouring out bronchoalveolar lavage fluid BALF after massaging lung lobes, repeating the washing process for 5 times, mixing the BALF obtained each time, filtering the BALF with 3 layers of sterile gauze, centrifuging the filtered mixture at 4 ℃ for 10min at 1000 r/min, and collecting cells, wash 2 times with PBS; adding suspension cells containing (L-glutamine) in RPMI-1640 or DMEM culture solution containing 10% newborn bovine serum, counting the cells, culturing in an incubator at 37 deg.C and 5% CO2 for 0.5, 1, 2, 6 and 24 hr, respectively, replacing with new culture solution, and collecting the residual cells as adherent cells.
In this example, a PBS solution containing the following components may be used: every 1000ml of PBS solution consisted of the following components: 6 g of sodium chloride, 0.05 g of potassium chloride, 1g of disodium hydrogen phosphate, 0.1 g of monopotassium phosphate, 0.05 g of calcium chloride, 0.05 g of magnesium chloride containing 6 crystal water, 60 mg of penicillin and 60 mg of streptomycin; or every 1000ml of PBS solution is composed of the following components: 10 g of sodium chloride, 0.5 g of potassium chloride, 1.2 g of disodium hydrogen phosphate, 1g of monopotassium phosphate, 0.2g of calcium chloride, 0.2g of magnesium chloride containing 6 crystal water, 100 mg of penicillin and 100 mg of streptomycin.
EXAMPLE 2 Collection and treatment of disease Agents
Collecting lung and lymph node from clinically suspected PRRS sick piglet, placing in 10 ml of physiological saline cooled at 4 ℃, controlling the temperature below 25 ℃, homogenizing with a high-pressure homogenizer to obtain homogenate treatment liquid, adding virus treatment solution (each 1000ml of virus treatment solution comprises the following components of 3 mg of semen Cuscutae extract, 0.8 mg of fructus Schisandrae extract, 2 mg of mulberry polysaccharide, 0.4 mg of raspberry acid, 2g of sodium citrate, 8g of sodium chloride and 0.03 g of potassium chloride) of which the volume is 3 times of the total volume of the physiological saline, mixing the components, dissolving in 1000ml of double distilled water, filtering with 0.22um filter membrane, storing at 4 ℃, mixing uniformly, standing for 8min, finally centrifuging at 3000 r/min and 10min, taking supernatant, filtering with 0.22um microporous filter membrane, thus obtaining PRRSV virus stock solution.
The virus treatment solution in this example may also be a solution of: each 1000ml of virus treatment solution consisted of the following components: 1 mg of dodder extract, 0.1 mg of schisandra extract, 1 mg of mulberry polysaccharide, 0.1 mg of raspberry acid, 1g of sodium citrate, 5 g of sodium chloride and 0.01 g of potassium chloride; or every 1000ml of virus treatment solution consists of the following components: 5 mg of semen cuscutae extract, 2 mg of fructus schizandrae extract, 3 mg of mulberry polysaccharide, 0.5 mg of raspberry acid, 3 g of sodium citrate, 10 g of sodium chloride and 0.05 g of potassium chloride.
Example 3: cell inoculation of viruses
And (2) discarding a cell culture solution after 24h of cultured primary PAM cells which grow well adherent to the primary PAM cells are cultured, adding a virus stock solution and the cell culture solution according to the ratio of (V: V) 1:5, setting a positive control group and a blank control group of PRRSV at the same time, placing a cell bottle in a 37 ℃ and 5% CO2 incubator for incubation for 1h, discarding the virus solution, adding a virus maintenance solution containing 2% fetal calf serum, continuing to culture for 100 h, observing cytopathic effect every day, collecting a cell solution with obvious pathological changes, and placing the cell solution at-20 ℃ for storage.
Experimental example isolation and identification of porcine reproductive and respiratory syndrome virus
1 Material
1.1 isolation of porcine reproductive and respiratory syndrome Virus
The isolation was carried out according to examples 1 to 3 of the present invention.
1.2 DMEM basal medium, fetal bovine serum was purchased from GIBCO.
1.3 porcine reproductive and respiratory syndrome virus positive serum and porcine reproductive and respiratory syndrome indirect immunofluorescence assay kit, all purchased from Chinese veterinary medicine inspection institute.
1.4 porcine reproductive and respiratory syndrome virus positive strains, which are preserved by the research and development center of Meibao-Long biotechnology, Zhejiang.
1.5 PCR-related reagents, purchased from Dalibao Biopsis.
2 method
2.1 measurement of Virus content
The PRRSV cell virus solution harvested in example 3 was diluted 10-fold in DMEM base medium, transferred to 96-well CEF cell culture plates with a full monolayer 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.
2.2 micro neutralization test
The standard positive serum of porcine reproductive and respiratory syndrome virus and the positive serum of swine fever are respectively diluted by 10 times in a gradient manner and are uniformly mixed with 200 cell virus solutions of TCID50, the mixture is placed at 37 ℃ for neutralization for 1h, the mixture is inoculated into a 96-well cell plate, each dilution is 8 wells, each dilution is 100ul, positive control without serum treatment and normal cell negative control are arranged, and the continuous observation is carried out for 5 days.
2.3 RT-PCR identification
In the test, a pair of primers is designed by applying Oligo 6.0, Primer5.0 and other software according to a PRRSV standard strain ATCC-VR 2332 gene sequence published by GenBank,
upstream primer (P1): 5'-TAAACCTTGTCAAATATGCC-3' the flow of the air in the air conditioner,
downstream primer (P2): 5'-TCGCCCTAATTGAATAGGTG-3', the amplified fragment is 490 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.
2.4 immunofluorescence assay
Marc-145 cells are inoculated into a 96-hole cell culture plate, after the cells grow into a single layer, a cell culture with CPE is inoculated, each hole is 0.1mL, the cell culture is placed at 37 ℃ for adsorption for 1h, then inoculation liquid is discarded, each hole is supplemented with maintenance liquid to 0.2mL, the cell culture is placed at 37 ℃ and is cultured in a 5% CO2 incubator for 48 h, then fixation is carried out for 20 min (the fixation liquid is acetone: methanol (V) =4: 1), the fixation liquid is discarded, 100ul PRRSV positive serum diluted by PBS1:100 times is added into each hole after PBS is used for cleaning, then the hole is placed in a 37 ℃ wet box for action for 45 min, and 1: FITC-rabbit anti-pig lgG (PBS containing 1/10000 Evans) diluted 1000 times is placed in a wet box at 37 ℃ for 30 min, washed for 3 times by PBS and observed under a fluorescence microscope with 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=10-5.75/0.1ml
TABLE 1 determination of viral titer
3.2 results of the microneutralization test
The result shows that the PRRSV standard positive serum can obviously inhibit the cell from generating CPE and can specifically neutralize the separated virus; the results show that the separated virus is the porcine reproductive and respiratory syndrome virus.
3.3 RT-PCR identification results
And (3) taking the separated porcine reproductive and respiratory syndrome 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 were in agreement with the expected results, and were all 490 bp.
3.4 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 reproductive and respiratory syndrome virus provided by the invention has the advantages of high degree of mechanization, simplicity, convenience and rapidness, greatly shortens the virus identification time, can obtain the virus with high virus content and high purification level, and finds a new susceptible cell for the culture of the porcine reproductive and respiratory syndrome virus.
Claims (2)
1. A method for separating porcine reproductive and respiratory syndrome virus is characterized by comprising the following steps:
taking complete and fresh piglet lung, separating PAM by adopting an alveolar lavage method, injecting 100-200 mL sterile 4-DEG C precooled PBS solution into the lung from a trachea by using a funnel, massaging lung lobes, pouring out bronchoalveolar lavage fluid BALF, repeating the washing process for 3-5 times, mixing the BALF obtained each time, filtering by using 3 layers of sterile gauze, centrifuging at the temperature of 4 ℃ for 10min, collecting cells, and washing for 2 times by using PBS; adding RPMI-1640 or DMEM culture solution containing 10% newborn bovine serum, wherein the RPMI-1640 and DMEM culture solution contain L-glutamine suspension cells, counting the cells, placing the cells in an incubator at 37 ℃ and 5% CO2 for culturing for 0.5, 1, 2, 6 and 24 hours respectively, replacing the new culture solution, and finally, enabling the rest cells to be adherent cells;
2) collecting lung and lymph node from clinically suspected PRRS sick piglet, placing in 8-20 ml of physiological saline cooled at 4 ℃, controlling the temperature below 25 ℃, performing homogenization treatment by using a high-pressure homogenizer to obtain homogenate treatment liquid, adding virus treatment solution which is 1-5 times of the total volume of the physiological saline into the homogenate treatment liquid, uniformly mixing, standing for 5-15min, centrifuging at 3000-5000 r/min for 8-15 min, taking supernatant, and filtering by a 0.22um microporous filter membrane to obtain PRRSV virus stock solution;
each 1000ml of virus treatment solution consisted of the following components: 1-5 mg of semen cuscutae extract, 0.1-2 mg of fructus schizandrae extract, 1-3 mg of mulberry polysaccharide, 0.1-0.5 mg of raspberry acid, 1-3 g of sodium citrate, 5-10 g of sodium chloride and 0.01-0.05 g of potassium chloride; mixing the above components, dissolving in 1000ml double distilled water, filtering with 0.22um filter membrane, and storing at 4 deg.C;
3) culturing primary PAM cells which grow well adherent after 24 hours in the step 1), discarding cell culture solution, adding PRRSV virus stock solution and the cell culture solution according to the volume ratio of 1:5-1:10, placing a cell bottle at 37 ℃ and 5% CO2Incubating in incubator for 0.5-1h, discarding virus solution, adding virus maintaining solution containing 2% fetal calf serum, culturing for 96-120 h, observing cytopathic effect every day, collecting cell sap with obvious pathological changes,storing at-20 deg.C.
2. The method for isolating porcine reproductive and respiratory syndrome virus according to claim 1, wherein the PBS solution in step 1) comprises the following components per 1000 ml: 6-10 g of sodium chloride, 0.05-0.5 g of potassium chloride, 1-1.2 g of disodium hydrogen phosphate, 0.1-1g of monopotassium phosphate, 0.05-0.2 g of calcium chloride, 0.05-0.2 g of magnesium chloride containing 6 crystal water, 60-100 mg of penicillin and 60-100 mg of streptomycin; mixing the above components, dissolving in 1000ml double distilled water, adjusting pH to 6.0-6.8 with 0.5mol/L hydrochloric acid solution, filtering with 0.22um filter membrane, and storing at 4 deg.C.
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