CN110468088A - The cultural method of lawsonia intracellularis and application - Google Patents
The cultural method of lawsonia intracellularis and application Download PDFInfo
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Abstract
The invention discloses a kind of cultural methods of lawsonia intracellularis, comprising the following steps: step 1: adhere-wall culture pig intestinal cell；Step 2: to cell 6~8h of adhere-wall culture, by lawsonia intracellularis, 0.01~1:1 is seeded in cell by volume with cell culture fluid, is placed in 1~96h of culture in micro- aerobic environment of improvement；Step 3: after the cell of pancreatin digestion infection, collecting infecting cell, and infection cell is resuspended with cells frozen storing liquid, it is transferred in liquid nitrogen container and saves.The application of lawsonia intracellularis by the cultural method culture is also disclosed, applied to lawsonia intracellularis be separately cultured in the research of pathogenicity feature.The present invention cultivates lawsonia intracellularis using pig intestinal cell, stringent to simulate the intracorporal vitro culture conditions of host, and cultivates lawsonia intracellularis using micro- aerobic incubator after improvement, it is easy to operate, cultivation cycle is short, at low cost, greatly increases the success rate of culture lawsonia intracellularis.
Description
Technical field
The invention belongs to field of biotechnology for animals, and in particular to a kind of cultural method of lawsonia intracellularis and application.
Background technique
Lawsonia intracellularis (Lawsonia Intracellularis, LI) belongs to Desulfovibrio, and Gram-negative bacteria is in
Arc curved shape, comma shape or S-shaped, size are 0.25~0.43 μm of 1.25~1.75 μ m, and obligatory parasitism is thin in animal intestinal tract
In born of the same parents, the porcine proliferative enteronitis characterized by enterocyte adenomatoid hyperplasia immature in ileum and colon crypt can be caused
(PPE).Though the PPE death rate is not high, which can cause the clinical symptoms such as sick pig diarrhea, slow growth, serious reduction animal
Efficiency of feed utilization in turn results in huge economic loss.It is reported that the U.S. and Britain are every year because of loss difference height caused by PPE
Up to 20,000,000 dollars, 4,000,000 pounds.In Europe, estimate that the annual disease is lost caused by every growing and fattening pigs more than 1 Euro.This
Outside, the disease is popular in world scale, and infection rate is very high.It is reported that Canada, Finland, Brazil, France, South Africa, Greece, Thailand
There are LI infection in more than 20 countries such as state, India, Japan, Korea, China, and especially in American-European countries, disease incidence is higher.Certainly
LI can be propagated through a variety of ways in right boundary, such as birds, mouse, insect animal are spread through sex intercourse, the mobility such as personnel, equipment, vehicle
It propagates.In order to preferably prevent and treat PPE, the culture and identification of LI is particularly important.
LI belongs to stringent bacterium intracellular, cannot grow, can only be grown in cell, such as IEC- in conventional culture medium
The cells such as 18, McCoys, Hep-2, henle407 and IGPC-1651；In addition, the bacterium is micro- aerobic and must be in a special gas
It can just be grown in body environment.Because of its special condition of culture, the bacterial strain that the whole world is successfully separated at present is now protected less than 25 plants
The bacterial strain deposited is less, and the country is not successfully separated the relevant report of culture so far.Nowadays, for the culture of LI, using most
Be intelligence more gas incubators and micro- aerobic production airbag.But the more gas incubators of intelligence are expensive, many laboratories are difficult to reach
To corresponding hardware requirement；And micro- aerobic production airbag is although cheap, but it is inconvenient, it is difficult to control experiment, to cell
Culture with bacterium has a certain impact.
In addition, can preferably reacting bacteria in the intracorporal invasion of host and be bred special using host cell culture bacterium
Property, increase the success rate of separation and culture LI.But cell used in LI is cultivated in report at present, is not derived from host cell,
Lawsonia intracellularis cannot be reacted completely in the infection characterization of pig stomach enteron aisle；In addition, prior art cell culture period is long, for this
The separation detection in bacterium later period is very unfavorable.
In view of the problems existing in the prior art, the present invention is by improving micro- aerobic culture condition of culture, using host cell
LI is cultivated, its culture is identified by the method for regular-PCR, quantitative fluorescent PCR and peroxidase single layer dyeing (IPMA)
As a result, and optimize bacterium infection time and dosage, and production growth curve, these study PPE for separation LI and later period
Have great importance.
Summary of the invention
It is an object of the invention to solve at least the above problems, and provide the advantages of at least will be described later.
It is a still further object of the present invention to provide a kind of cultural methods of lawsonia intracellularis, change traditional culture side LI
Method, easy to operate, cultivation cycle is short, and the bacterium is separated and identified conducive to the later period.
It is a still further object of the present invention to provide a kind of applications of lawsonia intracellularis by the cultural method culture, answer
For lawsonia intracellularis be separately cultured in the research of pathogenicity feature.
In order to realize object of the present invention and further advantage, a kind of cultural method of lawsonia intracellularis is provided, is wrapped
Include following steps:
Step 1: adhere-wall culture pig intestinal cell；
Step 2: to the pig intestinal cell 6~8h of adhere-wall culture, by volume with cell culture fluid by lawsonia intracellularis
0.01~1:1 is seeded in cell, is placed in 1~96h of culture in micro- aerobic environment of improvement；
Step 3: after the cell cultivated in pancreatin digestion infection step 2, collecting infecting cell, and be resuspended with cells frozen storing liquid
Infection cell is transferred in liquid nitrogen container and saves.
Preferably, the pig intestinal cell is pig jejunal epithelium cell IPEC-J2, chitterlings epithelial cell ZYM-
DIEC02 or chitterlings epithelial cell IPEC-1.
Preferably, adhere-wall culture pig intestinal cell in step 1, specifically includes the following steps:
S1: cell is taken out from liquid nitrogen container, is rocked in 37 DEG C of water-baths rapidly, and melt completely within 1min；
S2: the cell of thawing is added in DMEM complete medium, in 37 DEG C, 5%CO2It is cultivated in incubator；It is described thin
Concentration of the born of the same parents in complete medium is 0.5 × 106/ mL~2 × 108/mL；
S3: after cell adherent growth, slowly being cleaned cell 2~3 times with PBS, reach 80% to cell confluency degree~
When 90%, with trypsin digestion cell, cell suspension is mixed and is laid in 24 orifice plates, cell suspension and DMEM are complete in every hole
1:9 is added culture medium by volume, cultivates in incubator after mixing.
Preferably, the concentration of cell suspension is 0.5 × 10 in S36/ mL~2 × 108/mL。
Preferably, lawsonia intracellularis concentration described in step 2 is 104.9TCID50/ml。
Preferably, micro- aerobic culture environment of improvement described in step 2 is constructed by following steps:
By N2、CO2And O2After injecting air accumulator by volume for the injection of 80~95:5~10:0~10, it is then connected to micro- need
Oxygen incubator, micro- aerobic environment needed for testing the gaseous environment in incubator.
Preferably, the N2、CO2And O2Volume ratio is 80~87:8~10:5~10.
Preferably, 0.1~0.3:1 is seeded in cell lawsonia intracellularis described in step 2 by volume.
Preferably, 1~12h is cultivated in step 2 in micro- aerobic environment.
The present invention also provides a kind of applications of lawsonia intracellularis by the cultural method culture, are applied to Lawson intracellular
Bacterium be separately cultured in the research of pathogenicity feature.
The present invention is include at least the following beneficial effects:
The present invention discloses a kind of cultural method of lawsonia intracellularis, after selecting pig intestinal cell to carry out adhere-wall culture,
Lawsonia intracellularis is cultivated in its cell suspension, provides a kind of stringent simulation intracorporal body of host for lawsonia intracellularis
Outer culture environment, cultivation cycle is short, can quickly obtain a large amount of lawsonia intracellularis in a short time, increases culture lawsonia intracellularis
Success rate, conducive to the subsequent research for carrying out separation and pathological character to it.In addition, micro- aerobic after the present invention also application improvement
Incubator cultivates lawsonia intracellularis, compares the more gas incubators of existing intelligence and micro- aerobic production airbag culture lawsonia intracellularis,
Incubation and culture environment are easier to control, and simple to operation, at low cost, and more conducively laboratory carries out scientific research, also
Convenient for providing practical guidance and data support to practical aquaculture.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 is the regular-PCR qualification figure of 1LI of the embodiment of the present invention；2000 Marker of M:DL；Microbionation from left to right
Dosage is respectively 0.01mL, 0.1mL, 1mL；P: Lawson's bacterium reference culture positive control；N: negative control；
Fig. 2 is the quantitative fluorescent PCR qualification figure of 1LI of the embodiment of the present invention；Microbionation dosage is respectively from left to right
0.01mL, 0.1mL, 1mL；
Fig. 3 is the culture Fluorescence Identification figure of 1LI of the embodiment of the present invention；Microbionation dosage is respectively from left to right
0.01mL, 0.1mL, 1mL；
Fig. 4 is the regular-PCR qualification figure of 2LI of the embodiment of the present invention；2000 Marker of M:DL；Microbionation from left to right
Dosage is respectively 0.05mL, 0.15mL, 0.5mL；P: Lawson's bacterium reference culture positive control；N: negative control
Fig. 5 is the quantitative fluorescent PCR qualification figure of 2LI of the embodiment of the present invention；Microbionation dosage is respectively from left to right
0.05mL, 0.15mL, 0.5mL；
Fig. 6 is the culture Fluorescence Identification figure of 2LI of the embodiment of the present invention；Dosage of inoculation is respectively 0.05mL from left to right,
0.15mL, 0.5mL；
Fig. 7 is the regular-PCR qualification figure of 3LI of the embodiment of the present invention；2000 Marker of M:DL；Microbionation from left to right
Dosage is respectively 0.1mL, 0.2mL, 0.3mL；P: Lawson's bacterium reference culture positive control；N: negative control；
Fig. 8 is the quantitative fluorescent PCR qualification figure of 3LI of the embodiment of the present invention；Microbionation dosage is respectively from left to right
0.1mL, 0.2mL, 0.3mL；
Fig. 9 is the culture Fluorescence Identification figure of 3LI of the embodiment of the present invention；Dosage of inoculation is respectively 0.1mL from left to right,
0.2mL, 0.3mL；
Figure 10 is LI recombinant plasmid PCR qualification figure of the present invention；2000 Marker of M:DL；1: recombinant plasmid；P: positive right
According to；N: negative control；
Figure 11 is the quantitative fluorescent PCR dynamic curve diagram of LI of the present invention；From left to right corresponding plasmid template concentration according to
Secondary is 106copies/μl,107copies/μl,108Copies/ μ l, 109copies/μl,1010copies/μl,
1011copies/μl；N: negative control
Figure 12 is the quantitative fluorescent PCR canonical plotting of LI of the present invention.
Specific embodiment
The present invention is described in further detail below, to enable those skilled in the art's refer to the instruction text being capable of evidence
To implement.
Material source used in the present invention:
Fetal calf serum, DMEM basal medium are purchased from GIBCO company.
Regular-PCR and quantitative fluorescent PCR related reagent are purchased from the precious biology in Dalian.
Pig lawsonia intracellularis positive strain (less-virulent strain) is purchased from German Boehringer Ingelheim pharmaceutcal corporation, Ltd.
Pig lawsonia intracellularis monoclonal antibody, is purchased from Bio-X Diagnostcs.
Pig intestinal cell is purchased from this letter biology.
Other unlisted reagents can all be bought by regular commercial channel.
The cultural method of 1 lawsonia intracellularis of embodiment, comprising the following steps:
1) cell culture: taking out cell from liquid nitrogen container, and rock in 37 DEG C of water-baths keeps it complete within 1min rapidly
Melt.By the cell of thawing, (cell concentration is about 0.5 × 106/ mL~2 × 108/ mL) it is added in 4mL DMEM complete medium,
In 37 DEG C, 5%CO2It is cultivated in incubator.After cell adherent growth, slowly cleaned with PBS cell 2 times.To cell confluency degree
When reaching 80%, with trypsin digestion cell, cell suspension is mixed and is laid in 24 orifice plates, 0.1mL cell is added in every hole
Suspension (its concentration 0.5 × 106/ mL) and 0.9mL DMEM complete medium, it is cultivated in incubator after mixing.
2) inoculated and cultured of Lawson bacterium: when cell adhere-wall culture 6h, (concentration is pig lawsonia intracellularis positive strain
104.9TCID50/ ml) 0.01:1,0.1:1 and 1:1 are seeded in above-mentioned cell by volume respectively with cell culture fluid, it is placed in
Micro- aerobic environment (80-95%N after improvement2, 5-10%CO2, 0-10%O2) in culture.
3) infection cell freezes: after pancreatin digests infection cell, with centrifuge tube by the way that infection cell is collected by centrifugation, being used in combination
Infection cell is resuspended in cells frozen storing liquid, is transferred in liquid nitrogen container and saves for a long time.
The cultural method of 2 lawsonia intracellularis of embodiment, comprising the following steps:
1) cell culture: from liquid nitrogen container take out pig intestinal cell, rocked in 37 DEG C of water-baths rapidly make its 1min it
Interior complete thawing.By the cell of thawing, (cell concentration is about 0.5 × 106/ mL~2 × 108/ mL) be added 4mL DMEM train completely
It supports in base, in 37 DEG C, 5%CO2It is cultivated in incubator.After cell adherent growth, slowly cleaned with PBS cell 3 times.To cell
When convergence degree reaches 85%, with trypsin digestion cell, cell suspension is mixed and is laid in 24 orifice plates, every hole is added
0.1mL cell suspension (its concentration 1 × 107/ mL) and 0.9mL DMEM complete medium, it is cultivated in incubator after mixing.
2) inoculated and cultured of Lawson bacterium: when cell adhere-wall culture 7h, (concentration is pig lawsonia intracellularis positive strain
104.9TCID50/ ml) 0.05:1,0.15:1 and 0.5:1 are seeded in cell by volume respectively with cell culture fluid, and it is placed in and changes
Micro- aerobic environment (80-90%N after good2, 5-10%CO2, 5-10%O2) in culture.
3) infection cell freezes: after pancreatin digests infection cell, with centrifuge tube by the way that infection cell is collected by centrifugation, being used in combination
Infection cell is resuspended in cells frozen storing liquid, is transferred in liquid nitrogen container and saves for a long time.
Embodiment 3
The cultural method of lawsonia intracellularis, comprising the following steps:
1) cell culture: from liquid nitrogen container take out pig intestinal cell, rocked in 37 DEG C of water-baths rapidly make its 1min it
Interior complete thawing.By the cell of thawing, (cell concentration is about 0.5 × 106/ mL~2 × 108/ mL) be added 4mL DMEM train completely
It supports in base, in 37 DEG C, 5%CO2It is cultivated in incubator.After cell adherent growth, slowly cleaned with PBS cell 2 times.To cell
When convergence degree reaches 90%, with trypsin digestion cell, cell suspension is mixed and is laid in 24 orifice plates, every hole is added
0.1mL cell suspension (its concentration 2 × 108/ mL) and 0.9mL DMEM complete medium, it is cultivated in incubator after mixing.
2) inoculated and cultured of Lawson bacterium: when cell adhere-wall culture 8h, (concentration is pig lawsonia intracellularis positive strain
104.9TCID50/ ml) 0.1:1,0.2:1,0.3:1 are seeded in cell by volume respectively with cell culture fluid, after being placed in improvement
Micro- aerobic environment (80-87%N2, 8-10%CO2, 5-10%O2, preferably 83.2%N2, 8.8%CO2, 8%O2) in culture.
3) infection cell freezes: after pancreatin digests infection cell, with centrifuge tube by the way that infection cell is collected by centrifugation, being used in combination
Infection cell is resuspended in cells frozen storing liquid, is transferred in liquid nitrogen container and saves for a long time.
4 distinct methods of embodiment identify the lawsonia intracellularis of embodiment 1-3 culture
1, regular-PCR qualification test
Referring to the lawsonia intracellularis 16SrRNA sequence (accession number: L08049) that Genbank has been delivered, with Primer
It is as follows that Express 5.0 designs pair of primers:
Upstream primer (primer A): 5 ' TATGGCTGTCAAACACTCCG-3 ',
Downstream primer (primer B): 5 '-TGAAGGTATTGGTATTCTCC-3 ', amplified fragments 319bp.
DNA of bacteria is extracted using DNA extraction agent box, carries out PCR reaction by template of DNA of bacteria.It is reacted using 25 μ L
System: 2 × 12.5 μ L of PCR premix, each 9.5 μ L of 0.5 μ L, DEPC water of upstream and downstream primer (concentration is 20pmol/ μ L),
2 μ L of DNA profiling.Response procedures are as follows: 94 DEG C, 30s；56 DEG C, 30s, 72 DEG C, 10s, 30 circulations；72 DEG C, 5min.
The lawsonia intracellularis positive strain DNA that embodiment 1-3 culture is extracted with DNA of bacteria extraction agent box, utilizes DEPC
By 10 times of its serial dilution, totally 6 gradients, investigate the sensibility of this method.Large intestine is extracted with DNA of bacteria extraction agent box
The DNA of 5 kinds of bacteriums such as bacillus, salmonella, staphylococcus aureus, C.perfringens, treponema, investigates this method
Specificity.
After reaction, PCR product carries out electrophoresis with 3% gel, and 120v electrophoresis 30min is placed in gel imaging
System imaging is observed and analyzes result.
2, quantitative fluorescent PCR qualification test
The amplification of 2.1 target gene fragments
Carry out PCR reaction using specific primer to lawsonia intracellularis reference culture, target fragment needed for expanding, according to
The reaction system and response procedures of above-mentioned regular-PCR carry out.After after reaction, verified with agarose gel electrophoresis.
The recycling of 2.2 glue
Purpose band is recycled with DNA QIAquick Gel Extraction Kit.
The building of 2.3 recombinant vectors
The target fragment of recycling is reacted into 30min in 16 DEG C with pMD18-T carrier, whole process operates on ice, connects
Junctor system are as follows: 1 μ L of carrier, 2 μ L of glue recovery product, 2 μ L, Solution5 μ L of aqua sterilisa.
2.4 conversion
100 μ L competent cells, ice bath is added in the recombinant vector of connection.It takes out, is put into 45s in 42 DEG C of water-baths, quickly
It is transferred to 2min on ice.700 μ L LB liquid are added, are incubated for 1h (220rpm) in 37 DEG C of incubators.5000rpm is centrifuged 1min, discards one
Half supernatant mixes remaining bacterium solution pressure-vaccum, draws bacterium solution and applies on LB plate, be incubated overnight in 37 DEG C of insulating boxs (about 12~
14h).Second day observation bacterium colony growing state, the single colonie of growth is chosen, and is inoculated in the sterilized 10ml LB liquid that fills
In test tube, and add 10 μ L ampicillins, is incubated overnight (about 12~14h) in 37 DEG C of (180rpm) incubators.
2.5 extraction of plasmid DNA
Recombinant plasmid dna is extracted with small amount plasmid Rapid extraction kit (modified form).
2.6 recombinant plasmid concentration mensurations
With the extracted positive plasmid DNA concentration of spectrophotometric determination.
The building of 2.7 kinetic curves and standard curve
Standard items plasmid is diluted 10 times with dilution, in this, as initial concentration, 10 doubling dilutions, totally 5 gradients, and
3 repetitions are done respectively, establish reaction system, are expanded on quantitative fluorescent PCR type instrument, automatically generate mark with Computer aided analysis
Directrix curve.
2.8 sensibility and specificity
Lawsonia intracellularis less-virulent strain DNA is extracted with DNA of bacteria extraction agent box, using DEPC by its serial dilution 10
Times, totally 6 gradients, and 3 repetitions and negative control are done respectively, investigate the sensibility of this method.
With DNA of bacteria extraction agent box extract Escherichia coli, salmonella, staphylococcus aureus, C.perfringens,
The DNA of 5 kinds of bacteriums such as treponema, and 3 repetitions and negative control are done respectively, investigate the specificity of this method.
3, peroxidase single layer decoration method qualification test
(1) by primary antibody with PBS according to 1:20 volume dilution, be placed on 21 DEG C of incubation 1h, it is spare.
(2) 150 μ L paraformaldehydes are added into 12 or 24 orifice plates, 30min is made in 37 DEG C of senses.
(3) 500 μ L distilled water are added, act on 60min at 37 DEG C.
(4) orifice plate is rinsed 3 times after insulating box taking-up with PBS, each 3min；It is added dropwise 200 μ L primary antibodies into sample, 37
45min is incubated in DEG C wet box.
(5) orifice plate is taken out, PBS is rinsed 3 times, each 3min；200 μ L secondary antibodies are added dropwise into sample, are incubated in 37 DEG C of wet box
45min。
(6) orifice plate is taken out, PBS is rinsed 3 times, each 3min；100 μ L color developing agents, 37 DEG C of effect 20min are added；It uses later
PBS is rinsed 3 times, each 3min.It spontaneously dries, inverted microscope microscopy observes result.
4, result
4.1 regular-PCR results
Using lawsonia intracellularis reference culture as template, the laggard row agarose gel electrophoresis of PCR amplification is placed under ultraviolet light
Observation, and taken pictures with gel imaging system.As shown in Fig. 1, Fig. 4 and Fig. 7, separated by the lawsonia intracellularis that embodiment 1-3 is cultivated
The DNA extracted afterwards, amplification are consistent with expected results, and are 319bp.
4.2 fluorescent quantitative PCR result
PCR product is by recycling, connection, conversion, the extraction of the Zengjing Granule of positive colony, Plasmid DNA.
As shown in figure 12, by spectrophotometer, extracted plasmid concentration is 307.2ng/ μ l, standard curve y=-
3.401x+36.240 R2=0.995, amplification efficiency E=96.8%.
As shown in Fig. 2, Fig. 5 and Fig. 8, the quantitative fluorescent PCR identification of the lawsonia intracellularis of respectively embodiment 1-3 culture
Figure.As can be seen from Fig., the cultural method sensibility of lawsonia intracellularis of the present invention is good, 9.97 copies of minimum detectable/μ l；Specifically
Property is strong, with the equal no cross reaction of other enterobacteriaceaes.
4.3 peroxidase single layer decoration method test results
Using lawsonia intracellularis monoclonal antibody to the cell culture of inoculated bacteria and the control cell of non-inoculated bacteria
Culture carries out peroxidase stain test.
As shown in Fig. 3, Fig. 6, Fig. 9: the cell culture (lawsonia intracellularis cultivated by embodiment 1-3) of inoculated bacteria
There is red fluorescence on its endochylema and cell membrane；Normal cell there are no red fluorescence.
The above embodiments are only used to illustrate the present invention, rather than limitation of the present invention.Although referring to embodiment to this hair
It is bright to be described in detail, those skilled in the art should understand that, to technical solution of the present invention carry out it is various combination,
Modification or equivalent replacement should all cover and want in right of the invention without departure from the spirit and scope of technical solution of the present invention
It asks in range.
Claims (10)
1. a kind of cultural method of lawsonia intracellularis, which comprises the following steps:
Step 1: adhere-wall culture pig intestinal cell；
Step 2: to the pig intestinal cell 6~8h of adhere-wall culture, by lawsonia intracellularis and cell culture fluid by volume 0.01
~1:1 is seeded in cell, is placed in 1~96h of culture in micro- aerobic environment of improvement；
Step 3: after the cell cultivated in pancreatin digestion infection step 2, collecting infecting cell, and be resuspended and infected with cells frozen storing liquid
Cell is transferred in liquid nitrogen container and saves.
2. the cultural method of lawsonia intracellularis as described in claim 1, which is characterized in that the pig intestinal cell is pig jejunum
Epithelial cell IPEC-J2, chitterlings epithelial cell ZYM-DIEC02 or chitterlings epithelial cell IPEC-1.
3. the cultural method of lawsonia intracellularis as described in claim 1, which is characterized in that adhere-wall culture cell in step 1, tool
Body the following steps are included:
S1: cell is taken out from liquid nitrogen container, is rocked in 37 DEG C of water-baths rapidly, and melt completely within 1min；
S2: the cell of thawing is added in DMEM complete medium, in 37 DEG C, 5%CO2It is cultivated in incubator；The cell is complete
Concentration in full culture medium is 0.5 × 106/ mL~2 × 108/mL；
S3: after cell adherent growth, slowly being cleaned cell 2~3 times with PBS, when cell confluency degree reaches 80%~90%,
With trypsin digestion cell, cell suspension is mixed and is laid in 24 orifice plates, cell suspension and DMEM complete medium in every hole
1:9 is added by volume, cultivates in incubator after mixing.
4. the cultural method of lawsonia intracellularis as claimed in claim 3, which is characterized in that the concentration of cell suspension is in S3
0.5×106/ mL~2 × 108/mL。
5. the cultural method of lawsonia intracellularis as described in claim 1, which is characterized in that lawsonia intracellularis described in step 2
Concentration is 104.9TCID50/ml。
6. the cultural method of lawsonia intracellularis as described in claim 1, which is characterized in that micro- need of improvement described in step 2
Oxygen culture environment is constructed by following steps:
By N2、CO2And O2After injecting air accumulator by volume for 80~95:5~10:0~10, it is then connected to micro- aerobic incubator,
Micro- aerobic environment needed for testing the gaseous environment in incubator.
7. the cultural method of lawsonia intracellularis as claimed in claim 6, which is characterized in that the N2、CO2And O2Volume ratio is
80~87:8~10:5~10.
8. the cultural method of lawsonia intracellularis as described in claim 1, which is characterized in that lawsonia intracellularis described in step 2
0.1~0.3:1 is seeded in cell by volume with cell culture fluid.
9. the cultural method of lawsonia intracellularis as described in claim 1, which is characterized in that in step 2 in micro- aerobic environment
Cultivate 1~12h.
10. the application of the lawsonia intracellularis by the described in any item cultural method cultures of claim 1-9, which is characterized in that answer
For lawsonia intracellularis be separately cultured in the research of pathogenicity feature.
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|---|---|---|---|---|
| CN111961627A (en) * | 2020-08-27 | 2020-11-20 | 南京农业大学 | Separation and culture method of lawsonia intracellularis |
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| CN113376375A (en) * | 2021-06-07 | 2021-09-10 | 南京农业大学 | IPMA antibody detection method of Lawsonia intracellularis |
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