CN107937498A - The Primer composition of auxiliary identification porcine contagious pleuropneumonia ApxI toxin and its application - Google Patents
The Primer composition of auxiliary identification porcine contagious pleuropneumonia ApxI toxin and its application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The present invention provides a kind of Primer composition for aiding in identification porcine contagious pleuropneumonia ApxI toxin and its application, belongs to biological technical field.Special primer pair provided by the invention, is made of the single strand dna shown in sequence table 14.Special primer pair provided by the invention, it can identify Actinobacillus pleuropneumoniae toxin, Primer composition combination double PCR provided by the invention can identify IV A toxin of Apx I A and Apx at the same time, there is high sensitivity, high specificity, the time is short, cost is low, it can be detected for clinical sample, easy to operate, easy popularization, operates and applies easy to basic unit.
Description
Technical field
The present invention relates to biological technical field, particularly relates to a kind of auxiliary identification porcine contagious pleuropneumonia ApxI toxin
Primer composition and its application.
Background technology
Porcine contagious pleuropneumonia is that the high degree in contact of a boar as caused by actinobacillus pleuropneumoniae (APP) passes
Catch an illness, and the present age it is internationally recognized endanger one of five big Important Infectious Diseases of Pig Industry industry, and the three of current large-scale pig farm
One of big respiratory infectious disease.Mainly oozed out in clinical dissect with empsyxis, necrosis and cellulosic for major lesions, it is acute
Case case fatality rate is high.This disease morbidity season is obvious, multiple in the time of the awful weathers such as temperature relatively low winter, late fall in early spring.
The pig at any age is susceptible to this disease, wherein 3 monthly ages are most susceptible.The pig of Actinobacillus pleuropneumoniae is infected, clinically
Often easily other secondary bacteriosises such as haemophilus parasuis, pasteurella multocida etc., also mycoplasma, pig are bred in breathing
Distress syndrome virus (PRRSV), pig circular ring virus (PCV) and pseudorabies virus (PRV) etc., cause the respiratory tract of complexity comprehensive
Disease is closed, aggravates the harm to infecting pig.
Actinobacillus pleuropneumoniae causes porcine contagious pleuropneumonia mainly related with more virulence factor, including fat
Polysaccharide (LPS), outer membrane protein (OMP), turn these virulence such as iron-binding protein (TBP), pod membrane (CP), hemolytic exotoxin (APX)
The factor can cause the generation of pleuropneumonia.APX therein is a kind of material that toxic action is played to pulmonary alveolar macrophage, can be pressed down
The phagocytic activity of pulmonary alveolar macrophage processed, meanwhile, APX also has cytotoxic effect, quilt to peripheral mononuclear cells and lymphocyte
It is considered to cause the main reason for infecting and making lung tissue major injury.Apx points are ApxI, Apx II, Apx III, tetra- kinds of ApxIV
The toxin of different shaped, ApxI, Apx II, Apx III are the appearance for causing Disease Clinical symptom and typical pulmonary lesion essential
Matter, is acknowledged as the Major Virulence Factors for causing porcine contagious pleuropneumonia, significant to clinical disease diagnosis.
ApxI is to cause the appearance of porcine contagious pleuropneumonia Disease Clinical symptom and typical pulmonary lesion mainly malicious
The power factor, but in diagnostic method poor specificity, it is difficult to detected all serotypes of APP, because with other Actinobacillus such as pig unwrapping wire
There are cross reaction for bacillus, Ross Actinobacillus, pig tonsil Actinobacillus etc..And all serotypes of APP all secrete ApxIV,
The inter-species specificity of ApxIV is very strong, Actinobacillus and other Gram-negative bars that can secrete ApxI toxin in other kinds
It then can't detect ApxIV in bacterium.
Patent (CN1017247209A) discloses a kind of PCR diagnostic kit for porcine infectious pleuropneumonia, it includes 400 μ
L concentration be 20mg/mL Proteinase K, 1000 μ L lysates, 1500 μ LTE buffer solutions, 250 μ LPCR enzymes, 170 μ L ultra-pure waters, 50
The concentration of the MarkerDL2000 of μ L, primer P1, P2 that 40 μ L are mixed in equal volume, primer P1, P2 are 20 μM, and 20 μ L feminine genders are right
According to and 20 μ L positive controls.Since the patent only have detected ApxIVA toxin, clinically still fubaritic this is mainly controlled
Sick virulence factor belongs to which kind in ApxI, Apx II, Apx III on earth, there is the defects of can not providing accurate diagnosis.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of precise Identification ApxIA toxin, and fast, it is sensitive, detection
Mode is simple, the Primer composition of the good auxiliary identification porcine contagious pleuropneumonia ApxI toxin of using effect and its application.
In order to solve the above technical problems, present invention offer technical solution is as follows：
On the one hand, there is provided a kind of Primer composition for aiding in identification porcine contagious pleuropneumonia ApxI toxin, includes following 4
Bar primer：
1-F 5’-TCGGTCGTAGCATTAGCG-3’
1-R5’-GACATCCCAACGCTGTTG-3’
2-F 5’-CTTTTTTGTTGTAGAAGCATCC-3’
2-R 5’-TCGTCAATAGGCGTAACAGTT-3’。
On the other hand, the Primer composition the present invention also provides above-mentioned auxiliary identification porcine contagious pleuropneumonia toxin is being made
The application of standby kit, applied to identifying porcine contagious pleuropneumonia ApxI toxin and auxiliary identify in sample to be tested whether be pig
Actinobacillus pleuropneumoniae toxin.
Another further aspect, the present invention also provides a kind of kit, including the Primer composition described in claim 1.
Preferably, which is based on dual-PCR method, after extracting gene DNA to sample to be tested, using claim 1
The Primer composition carries out the pleuropneumonia toxin in double PCR detection sample to be tested.
Preferably, the concentration of upstream and downstream primer is 1.0ng/ml in kit.
Preferably, the detection architecture of kit is 30-50 μ l.
Preferably, sample to be tested DNA concentration is 15 μ g/mL-60 μ g/mL in kit.
Preferably, the operating condition of kit is 95 DEG C of pre-degeneration 10min, and 95 DEG C are denatured 50s, 50-60 DEG C of annealing 50s,
72 DEG C extension 1min, 35 circulation, finally again 72 DEG C extension 10min.
Preferably, annealing temperature is 56 DEG C -60 DEG C.
Mentioned reagent box be applied to identification porcine contagious pleuropneumonia ApxI toxin and auxiliary identification sample to be tested in whether
For Actinobacillus pleuropneumoniae toxin.
The invention has the advantages that：
In such scheme, Primer composition combination double PCR provided by the invention identifies IV A of Apx I A and Apx poison at the same time
Element, whether the Major Virulence Factors that can accurately and rapidly detect clinical sample are I A of Apx, have high sensitivity, specificity
By force, the features such as time is short, cost is low, can be detected for clinical sample, easy to operate, easy popularization, be grasped easy to basic unit
Make and apply.
Brief description of the drawings
Fig. 1 is the site where the partial gene sequence and primer of ApxIA toxin of the present invention；
Fig. 2 is the site where the partial gene sequence and primer of IV A toxin of Apx of the present invention；
Fig. 3 is to detect ApxIA toxin primer sensitivity using PCR, and M be DNA Maker 2000, the primer of 1-10 representatives
Concentration be respectively 0.2ng/mL, 0.4ng/mL, 0.6ng/mL, 0.8ng/mL, 1.0ng/mL, 1.2ng/mL, 1.4ng/mL,
1.6ng/mL、1.8ng/mL、2.0ng/mL；
Fig. 4 is to detect IV A toxin primer sensitivity of Apx using PCR, and M is the primer of DNA Maker2000,1-10 representatives
Concentration be respectively 0.2ng/mL, 0.4ng/mL, 0.6ng/mL, 0.8ng/mL, 1.0ng/mL, 1.2ng/mL, 1.4ng/mL,
1.6ng/mL、1.8ng/mL、2.0ng/mL；
Fig. 5 is the influence that annealing temperature detects PCR ApxIA, and M is DNA Maker 2000, and 1-6 represents annealing temperature respectively
50 DEG C, 52 DEG C, 54 DEG C, 56 DEG C, 58 DEG C, 60 DEG C of degree；
Fig. 6 is the influence that annealing temperature detects PCR IV A of Apx, and M is DNA Maker 2000, and 1-6 represents annealing respectively
Temperature 50 C, 52 DEG C, 54 DEG C, 56 DEG C, 58 DEG C, 60 DEG C；
Fig. 7 is influence of the ApxIA toxin primer concentration to double PCR testing result, and M is DNAMaker 2000, and 1-10 divides
Do not represent I A gene different primers concentration 0.2ng/mL, 0.4ng/mL of APX, 0.6ng/mL, 0.8ng/mL, 1.0ng/mL,
1.2ng/mL、1.4ng/mL、1.6ng/mL、1.8ng/mL、2.0ng/mL；
Fig. 8 detects double PCR for IV A toxin primer concentrations of Apx the influence to result, and M is DNA Maker 2000,1-
10 represent respectively IV A gene different primers concentration 0.2ng/mL, 0.4ng/mL of APX, 0.6ng/mL, 0.8ng/mL, 1.0ng/mL,
1.2ng/mL、1.4ng/mL、1.6ng/mL、1.8ng/mL、2.0ng/mL；
Fig. 9 is influence of the detection annealing temperature to double PCR result, and M is DNA Maker 2000, and 1-6 is represented move back respectively
Fiery temperature 50 C, 52 DEG C, 54 DEG C, 56 DEG C, 58 DEG C, 60 DEG C；
Figure 10 is influence of the system size to double PCR result, and M is DNA Maker 2000, and 1-9 represents dual respectively
10 μ l of PCR system size, 15 μ l, 20 μ l, 25 μ l, 30 μ l, 35 μ l, 40 μ l, 45 μ l, 50 μ l；
Figure 11 be the specific detection of primer as a result, M is DNA Maker 2000,1-6 is respectively staphylococcus aureus
DNA, ox e. coli dna, enterococcus faecalis DNA, swine escherichia coli DNA, streptococcus DNA, IV A reference cultures of APX I A and APX mix
Close DNA；
Figure 12 is the Detection of Stability of primer as a result, M is DNA Maker 2000,1-7 represents 10 respectively, 20,30,60,
120th, the DNA profiling of the IV A reference cultures of ApxIA and Apx of extraction in 240,360 days；
Figure 13 be DNA profiling Concentration Testing as a result, M is DNA Maker 2000,1-5 represents the DNA of various concentrations respectively
15 μ g/mL of template, 30 μ g/mL, 60 μ g/mL, 120 μ g/mL, 240 μ g/mL, 6 (use ddH for negative control2O replaces DNA moulds
Plate).
Embodiment
To make the technical problem to be solved in the present invention, technical solution and advantage clearer, below in conjunction with attached drawing and tool
Body embodiment is described in detail.
The present invention for I A of APX in the prior art in many Actinobacillus there are cross reaction, detect the virulence factor
Poor specificity the problem of, there is provided it is a kind of aid in identification porcine contagious pleuropneumonia toxin Primer composition and its application.
The reagent and bacterial strain information used in the present invention is as follows：
1.1 strains tested
IV A reference cultures of Apx I A and Apx are bought in Chinese veterinary microorganism culture presevation administrative center.It is enterococcus faecalis, big
Enterobacteria, staphylococcus aureus and streptococcus are commercially available bacterial strain.
1.2 culture mediums, enzyme, kit and other reagents
(1) culture medium
TSB culture mediums (purchase and Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd)：The molten 100mL of 3gTSB dry powder is double
Steam in water, 120 DEG C of high pressure 30min.
LB culture mediums：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 10/L.
(2) enzyme and kit
2 × Tag Master Mix (comprising Taq DNA Polymerase, dNTP and optimization buffer system) for promise only
Praise biotech firm's product.
DNA Maker are TaRaKa Products
DNA QIAquick Gel Extraction Kits are TaRaKa Products
(3) other reagents
Nicotinamide adenine dinucleotide (NAD) is purchased from Zhengzhou Bo Yan bio tech ltd
Agar powder is purchased from Solarbio companies
TAE (50 ×) buffer solution：Weigh the Tris alkali of 121g and the Na of 18.6g2EDTA.2H2O is added in beaker
The deionized water of 400mL, is sufficiently stirred dissolving, adds the glacial acetic acid of 30mL, fully mixes, with glacial acetic acid adjustment pH value extremely
8.5, it is settled to 500mL, room temperature preservation.
TAE (1 ×) buffer solution：Measure 10mL TAE (50 ×) buffer solutions and 490mL ddH2O is sufficiently mixed in beaker
Uniformly.
1.0% Ago-Gel：The agarose pulvis of 0.2g is weighed in conical flask, the 1 × TAE for adding 20mL is molten
Liquid, after stirring shakes up, 1-2min is boiled in heating, and the EB storing liquids of 3 μ L are added after somewhat cooling down, are shaken up spare.
The preparation of 1 Primer composition of embodiment
Each bar primer shown in table 1 is synthesized, the nucleotide sequence of wherein 1-F, 1-R, 2-F, 2-R are shown in Table 1.
Table 1
| Primer | Nucleotide sequence |
| 1-F | 5’-TCGGTCGTAGCATTAGCG-3’ |
| 1-R | 5’-GACATCCCAACGCTGTTG-3’ |
| 2-F | 5’-CTTTTTTGTTGTAGAAGCATCC-3’ |
| 2-R | 5’-TCGTCAATAGGCGTAACAGTT-3 |
ApxIA partial gene sequences (Gene bank：AF 240779.1) (the sequence length as shown in the sequence 5 of sequence table
513bp), Fig. 1, IV part A gene order (GenBank of Apx are seen in site where primer：FJ 848574.1) such as the sequence of sequence table
Shown in row 6 (sequence length 1092bp), Fig. 2 is seen in site where primer.
2 primer sensitivity technique of embodiment
The culture of bacterium and the extraction of DNA
(1) I A standard bacterias of 1.5mLApx and IV A standard bacterias cultures of Apx and DNA extractions
4 test tubes are taken, are separately added into 7mL TSB, add 5 μ l NAD, 300 μ l cow's serums, wherein two 50 μ l of addition
I A standard bacterium solutions of Apx, two add 50 μ l Apx, IV A standards and are sufficiently mixed uniformly.Four test tubes are placed on shaking table at the same time, with
200r/min, 37 DEG C of culture 12h.
Taking I A standards bacterium solutions of 1.5mL Apx and IV A standards bacterium solutions of Apx respectively, 12000r/min is centrifuged in 2 EP pipes
2min, abandons supernatant.Repetitive operation is twice.The DNA of two kinds of bacterium is extracted according to DNA extraction kit specification.Measure IV A of Apx
Bacterium DNA concentration is 150 μ g/mL；I A bacterium DNA concentrations of Apx are 240 μ g/mL.
(2) culture of other bacteriums and the extraction of DNA
4 test tubes are taken, are separately added into 7mL LB, are separately added into 100 μ l Escherichia coli, enterococcus faecalis, Staphylococcus aureus
Bacterium, streptococcus, are uniformly mixed.Four test tubes are placed on shaking table at the same time, with 200r/min, 37 DEG C of culture 12h, DNA extraction steps
Ibid.
The foundation of substance PCR system
(1) IV A gene PCR systems of I A of Apx or Apx
IV A gene PCRs of Apx I A or Apx reaction total system is 20 μ l, and details are shown in Table 2.
IV A gene PCR systems of table 2Apx I A or Apx
2ng/mL primers are diluted to 0.2ng/mL, 0.4ng/mL, 0.6ng/mL, 0.8ng/mL, 1.0ng/mL, 1.2ng/
10 kinds of various concentrations of mL, 1.4ng/mL, 1.6ng/mL, 1.8ng/mL, 2.0ng/mL, using reaction system, reaction interval in table 2
Ordered pair primer sensitivity is detected.
From Fig. 3-4, IV A primer concentrations of Apx I A and Apx can detect in the range of 0.2ng/mL-2.0ng/mL
Purpose fragment.I A genes of Apx increase with primer concentration, and purpose band is brighter, and testing result is more obvious.Primer concentration is
2.0ng/mL purpose bands are the most obvious.IV A gene primer concentration of Apx is in the range of 0.2ng/mL-2.0ng/mL, except 0.2ng/
ML bands are weaker, and other bands go out without significant change, and by sequencing comparison, primer amplified provided by the invention
Target fragment be IV A gene orders of Apx I A and Apx.
Influence of 3 annealing temperature of embodiment to PCR results
In PCR reaction conditions, 6 different annealing temperatures are designed, 50 DEG C respectively, 52 DEG C, 54 DEG C, 56 DEG C, 58 DEG C, 60
℃.Utilize the reaction system in table 2, influence of the measure different annealing temperature to IV A gene PCR results of Apx I A and Apx.Never
From the point of view of the result under annealing temperature, the influence of the PCR reactions of annealing temperature contratoxin Apx I A and IV A of Apx is different.By
Fig. 5 understands that annealing temperature has a great influence I A of Apx, although all temperature of the annealing temperature between 50 DEG C -60 DEG C can obtain
To target gene fragment, but the band that 50 DEG C and 60 DEG C of band brightness is substantially not so good as under medium temperature becomes clear.Can by Fig. 6
Know, annealing temperature changes between 50 DEG C -60 DEG C influences less IV A fragments of Apx.
4 primer concentration of embodiment changes the influence to double PCR
The foundation of double PCR reaction system, total system are 50 μ l, and details are shown in Table 3.
3 double PCR system of table
From embodiment 2, IV A primer concentrations of Apx I A or Apx can detect in the range of 0.2ng/mL-2.0ng/mL
To purpose fragment.In double PCR reaction, primer is diluted to 0.2ng/mL, 0.4ng/mL, 0.6ng/mL, 0.8ng/mL,
10 kinds of various concentrations of 1.0ng/mL, 1.2ng/mL, 1.4ng/mL, 1.6ng/mL, 1.8ng/mL, 2.0ng/mL, using in table 3
Reaction system, response procedures are detected the primer sensitivity of IV A genes of Apx in double PCR I A and Apx.
In double PCR, the upstream and downstream primer (2-F and 2-R) that inventor sets IV A of Apx first is 1.0ng/mL dense
Degree, the upstream and downstream primer of I A of Apx is diluted, and as shown in Figure 7, the concentration that can expand the primer of I A genes of Apx is 0.6ng/mL, with
Primer concentration increase, the band of purpose fragment are more obvious.The upstream and downstream primer (1-F or 1-R) of I A of Apx is such as set to 1.0ng/
ML concentration, the upstream and downstream primer of IV A of Apx is diluted, as shown in Figure 8, the primer least concentration that can detect IV A genes of Apx is
0.4ng/mL.IV A primer concentrations of Apx increase purpose band becomes bright, but the band of I A purpose fragments of Apx gradually weakens directly
To disappearance, at this time, the concentration of the upstream and downstream primer of I A of Apx is preferably 0.4ng/mL-1.0ng/mL.Illustrate in double PCR, it is single
The amplification quantity of a purpose fragment and the concentration of primer are not simple linear relationship, also related with the concentration of another primer.
Inventor uses above-mentioned system, has also groped in the different DNA profiling concentration (amount of DNA of I A of Apx IV A and Apx point
Not Wei 0.1-1 μ l, 10 different volumes) under the conditions of, carry out PCR reactions, the upstream and downstream primer concentration of IV A of Apx I A and Apx is
During 1.0ng/mL, target stripe can detect that.Since length is limited, do not enumerate, kit provided by the invention, Apx I
The upstream and downstream primer concentration of IV A of A and Apx is preferably 1.0ng/mL.
Influence of the 5 system size of embodiment to result
Design 10 μ l, 15 μ l, 20 μ l, 25 μ l, 30 μ l, 35 μ l, 40 μ l, 45 μ l, 50 μ l, 9 different size of reactants
System, using the reaction system in table 3, response procedures, probes into influence of the system size to experimental result, details are shown in Table 4-12.
4 10 μ l PCR systems of table
5 15 μ l PCR systems of table
6 20 μ l reaction systems of table
7 25 μ l reaction systems of table
8 30 μ l reaction systems of table
9 35 μ l reaction systems of table
10 40 μ l reaction systems of table
11 45 μ l of table, reaction system
12 50 μ l reaction systems of table
In above-mentioned PCR system, primer concentration is 1.0ng/mL, as shown in Figure 9, when double PCR reaction system is in 10 μ l
When, two purpose fragments are without bright band；When total system is 15 μ l, only I A of APX by purpose fragment, 20 μ l-50 μ l systems it
Between two kinds of genes obtain two purpose fragments, and two purpose fragment bands are most bright during 30 μ l systems.System is in 30 μ l-50 μ l
When, IV A fragments of APX gradually brighten, and I A of APX are gradually dimmed, so the detection architecture of kit provided by the invention is preferably 30-
50μl。
Influence of 6 annealing temperature of embodiment to double PCR
Using 30 μ l PCR reaction systems in embodiment 5,6 different annealing temperatures are designed, are respectively 50 DEG C, 52 DEG C, 54
DEG C, 56 DEG C, 58 DEG C, 60 DEG C.For influence of the measure different annealing temperature to double PCR result.
As shown in Figure 10, when annealing temperature is 50 DEG C, the amplification of IV A purpose fragments of Apx is preferable, although there is I A fragments of Apx
Brightness is smaller, and at 52 DEG C, 54 DEG C, I A gene expressions of Apx enhancing, IV A gene expressions of Apx weaken.56 DEG C, 58 DEG C, 60 DEG C two kinds
Gene PCR product expression is all preferable.Test result indicates that in double PCR, two fragments influence each other, most suitable annealing temperature with
Substance PCR is simultaneously differed, so in the double PCR program of kit provided by the invention, most suitable annealing temperature is 56 DEG C -60
℃。
7 primer specificity of embodiment detects
It is right with Escherichia coli, staphylococcus aureus, enterococcus faecalis, streptococcus and pig pleura Actinobacillus
The specificity of primer is detected, and reaction system is 50 μ l, and details are shown in Table 13.
13 primer specificity of table detects
In table, DNA profiling is respectively L-form staphylococcus aureus, ox e. coli dna, enterococcus faecalis DNA, Radix Polygalae Crotalarioidis
Bacillus DNA, streptococcus DNA, IV A reference cultures hybrid dna of APX I A and APX (each 1 μ l).
As shown in Figure 11, to staphylococcus aureus, ox Escherichia coli, enterococcus faecalis, swine escherichia coli and streptococcus
DNA expanded and fail to obtain target gene fragment, only I A of Apx and IV A reference cultures of Apx can just amplify and purpose
The consistent band of clip size, illustrates that the primer specificity of this experimental design is good.
The Stability Determination of 8 double PCR of embodiment
By the reaction of the IV A reference cultures DNA tables 8 of Apx I A and Apx of extraction in 10,20,30,60,120,240,360 days
Condition (it is 1.0ng/mL that the concentration of primer therein, which is), reaction system are expanded, and detect the stability of double PCR.
As shown in Figure 12, double PCR amplification is carried out at the same time to the DNA of different time extraction, the piece of two entries can be obtained
Section.Illustrate that this experiment primer stability is good, stability can reach 100%.
9 double PCR DNA profiling Concentration Testing of embodiment
The reaction condition and response procedures (concentration of primer therein for be 1.0ng/mL) of table 8 are used, by reactant
IV ADNA templates of Apx I A and Apx replace with the DNA (1 μ l) of the pig of affected pig contagious pleuropneumonia disease ApxI types in system.Using north
Animal tissue's DNA extraction kit of Jing Suolaibao companies purchase, extracts the DNA of sample to be tested, the DNA concentration of sample is diluted
Into 15 μ g/mL, 30 μ g/mL, 60 μ g/mL, 120 μ g/mL, 240 μ g/mL5 various concentrations, double PCR detection, detection knot are carried out
Fruit is as shown in figure 13, and with the increase of DNA versions concentration to be measured, the band of I A of Apx is gradually dimmed.In double PCR, test sample is treated
This concentration is preferably 15 μ g/mL-60 μ g/mL.
Inventors tested a large the DNA profiling concentration of the pig of multiple affected pig contagious pleuropneumonia disease ApxI types, test result
It is consistent with 9 acquired results of embodiment, since length is limited, do not repeat one by one.
To sum up, the present invention optimizes by test of many times, and the DNA profiling concentration of kit is preferably 15-60 μ g/mL, reagent
The concentration of primer is preferably 1.0ng/ml in box, and the detection architecture of kit is preferably 30-50 μ l, further preferred 30 μ l；Examination
The operating condition of agent box is 95 DEG C of pre-degeneration 10min, 95 DEG C of denaturation 50s, 50-60 DEG C of annealing 50s, 72 DEG C of extension 1min, 35
Circulation, finally again 72 DEG C extension 10min, wherein, annealing temperature is preferably 56 DEG C -60 DEG C.
The above is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principles of the present invention, some improvements and modifications can also be made, these improvements and modifications
It should be regarded as protection scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of Jiangxi
<120>The Primer composition of auxiliary identification porcine contagious pleuropneumonia toxin and its application
<130> 2017
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial synthesized sequence
<400> 1
tcggtcgtag cattagcg 18
<210> 2
<211> 18
<212> DNA
<213>Artificial synthesized sequence
<400> 2
gacatcccaa cgctgttg 18
<210> 3
<211> 22
<212> DNA
<213>Artificial synthesized sequence
<400> 3
cttttttgtt gtagaagcat cc 22
<210> 4
<211> 21
<212> DNA
<213>Artificial synthesized sequence
<400> 4
tcgtcaatag gcgtaacagt t 21
<210> 5
<211> 513
<212> DNA
<213> pig
<400> 5
tcggtcgtag cattagcgat tagcccgctt tcgttcttaa atgttgcgga taagtttgaa 60
cgtgcgaaac agcttgaaca atattcggag cgctttaaaa agttcggtta tgaaggtgat 120
agtttattag cttcattcta ccgtgaaacc ggtgcgattg aagcggcatt aaccacgatt 180
aacagtgtgt taagtgcggc ttccgcaggt gttggggctg ctgcaaccgg ctcattagtc 240
ggtgcgccgg tagcagcttt agttagtgca atcaccggta ttatttcagg tattttagat 300
gcttctaaac aggcaatctt cgaacgagtt gcaacgaaat tagcgaataa gattgacgaa 360
tgggagaaaa aacacggtaa aaactatttt gaaaacggtt atgacgcccg ccattccgca 420
ttcttagaag atacctttga attgttatca caatacaata aagagtattc ggtagagcgt 480
gtcgttgcta ttacgcaaca gcgttgggat gtc 513
<210> 6
<211> 1092
<212> DNA
<213> pig
<400> 6
cttttttgtt gtagaagcat ccggcaaacg ctatattgaa aactttggta ttgaacctct 60
tggtaagcaa gaagattttg attttgtcgg cggcttttgg tctaacttag tgaatcgtgg 120
tttggaaagt attatcgacc catccggtat cggtggaacg gtaaacctta actttaccgg 180
cgaggtggaa acctacacgt tagacgaaac aaggtttaaa gcggaagcgg cgaagaaaag 240
ccattggagt ttagtgaatg cggcgaaagt atacggcggt ttagaccaaa ttattaaaaa 300
actatgggac agcggctcaa ttaagcattt atatcaagat aaagatacgg gcaaattaaa 360
accgattatt tacggcacgg ccggcaacga cagtaagatt gaaggcacta aaatcacccg 420
taggattgcg ggtaaagaag ttacgcttga tattgccaat cagaaaattg aaaaaggcgt 480
gtcagagaaa ttggggctgt ctgttagtgg ttcggatatc attaaattgt tgtttggagc 540
attgactcca actttaaata gaatgttgct atcacaactc atccagtctt tttccgatag 600
cttggctaaa cttgataatc ccttagcccc ttacactaaa aatggcgtgg tttatgtcac 660
cggcaaaggg aatgatgtgc ttaaaggaac tgaacatgag gatttgtttc tcggtggtga 720
ggggaatgat acttattatg cgagagtagg cgatacaatt gaagacgccg acggcaaagg 780
taaagtctat tttgtgagag aaaaaggggt acctaaggcg gatcctaagc gggtagagtt 840
tagcgagtac ataacgaaag aagaaataaa agaggttgaa aaggggttat taacctacgc 900
agttttagaa aattataatt gggaagagaa aacggcgact ttcgctcatg cgactatgct 960
taatgagctt tttactgatt atactaatta tcgttatgaa gttaaaggac taaaattgcc 1020
cgccgttaaa aagttaaaaa gtccgttggt ggagtttaca gctgatttat taactgttac 1080
gcctattgac ga 1092
Claims (9)
1. a kind of Primer composition for aiding in identification porcine contagious pleuropneumonia ApxI toxin, it is characterised in that include following 4
Primer：
1-F 5’-TCGGTCGTAGCATTAGCG-3’
1-R5’-GACATCCCAACGCTGTTG-3’
2-F 5’-CTTTTTTGTTGTAGAAGCATCC-3’
2-R 5’-TCGTCAATAGGCGTAACAGTT-3’。
2. the Primer composition of auxiliary identification porcine contagious pleuropneumonia ApxI toxin according to claim 1 is preparing examination
The application of agent box, it is characterised in that applied in identification porcine contagious pleuropneumonia APXI toxin and auxiliary identification sample to be tested
Whether it is Actinobacillus pleuropneumoniae toxin.
3. a kind of kit, it is characterised in that including the Primer composition described in claim 1.
4. kit according to claim 3, it is characterised in that the kit is based on dual-PCR method, to sample to be tested
After extracting gene DNA, the pleura lung in double PCR detection sample to be tested is carried out using the Primer composition described in claim 1
Scorching toxin.
5. the kit according to claim 3 or 4, it is characterised in that the concentration of the upstream and downstream primer used in kit
It is 1.0ng/ml.
6. the kit according to claim 3 or 4, it is characterised in that the detection architecture of kit is 30-50 μ l.
7. the kit according to claim 3 or 4, it is characterised in that sample to be tested DNA concentration is 15 μ g/ in kit
mL-60μg/mL。
8. the kit according to according to claim 3 or 4, it is characterised in that the operating condition of kit is 95 DEG C of pre- changes
Property 10min, 95 DEG C denaturation 50s, 50-60 DEG C annealing 50s, 72 DEG C extension 1min, 35 circulation, finally again 72 DEG C extension 10min.
9. kit according to claim 8, it is characterised in that annealing temperature is 56 DEG C -60 DEG C.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711481218.3A CN107937498A (en) | 2017-12-29 | 2017-12-29 | The Primer composition of auxiliary identification porcine contagious pleuropneumonia ApxI toxin and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711481218.3A CN107937498A (en) | 2017-12-29 | 2017-12-29 | The Primer composition of auxiliary identification porcine contagious pleuropneumonia ApxI toxin and its application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676900A (en) * | 2018-04-28 | 2018-10-19 | 湖北省农业科学院畜牧兽医研究所 | A kind of composite PCR parting kit and its application for distinguishing eight kinds of Serotyping of Actinobacilus pleuropneumoniae |
| CN112481416A (en) * | 2020-12-11 | 2021-03-12 | 山东绿都生物科技有限公司 | African swine fever virus and porcine infectious actinobacillus pleuropneumoniae dual-fluorescence PCR detection kit and use method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724709A (en) * | 2010-01-28 | 2010-06-09 | 贵州省畜牧兽医研究所 | PCR diagnostic kit for porcine infectious pleuropneumonia |
| CN103777018A (en) * | 2014-01-16 | 2014-05-07 | 江西农业大学 | Preparation method of test strip for rapidly diagnosing porcine contagious pleuropneumonia actinobacillus |
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2017
- 2017-12-29 CN CN201711481218.3A patent/CN107937498A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724709A (en) * | 2010-01-28 | 2010-06-09 | 贵州省畜牧兽医研究所 | PCR diagnostic kit for porcine infectious pleuropneumonia |
| CN103777018A (en) * | 2014-01-16 | 2014-05-07 | 江西农业大学 | Preparation method of test strip for rapidly diagnosing porcine contagious pleuropneumonia actinobacillus |
Non-Patent Citations (3)
| Title |
|---|
| A. DREYFUS等: "Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections, development and prevalidation of the ApxIV ELISA", 《VETERINARY MICROBIOLOGY》 * |
| 何培新: "《高级微生物学》", 31 August 2017, 中国轻工业出版社 * |
| 廖华媛: "育肥猪传染性胸膜肺炎的诊断及分离菌毒力基因检测", 《黑龙江畜牧兽医》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676900A (en) * | 2018-04-28 | 2018-10-19 | 湖北省农业科学院畜牧兽医研究所 | A kind of composite PCR parting kit and its application for distinguishing eight kinds of Serotyping of Actinobacilus pleuropneumoniae |
| CN108676900B (en) * | 2018-04-28 | 2020-04-21 | 湖北省农业科学院畜牧兽医研究所 | Composite PCR typing kit for distinguishing eight porcine actinobacillus pleuropneumoniae serotypes and application thereof |
| CN112481416A (en) * | 2020-12-11 | 2021-03-12 | 山东绿都生物科技有限公司 | African swine fever virus and porcine infectious actinobacillus pleuropneumoniae dual-fluorescence PCR detection kit and use method thereof |
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