CN110373484A - Serotype method, primer combination and the PCR system of actinobacillus pleuropneumoniae - Google Patents
Serotype method, primer combination and the PCR system of actinobacillus pleuropneumoniae Download PDFInfo
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Abstract
The present invention provides a kind of serotype method of actinobacillus pleuropneumoniae, primer combination and PCR system, the present invention is that the actinobacillus pleuropneumoniae of serum 1-15 type separately designs specific primer, 9 type of serum is identical with 11 type-special primer sequence of serum, totally 14 pairs of specific primers, 14 pairs of specific primers are divided into 4 groups, every group of 3 to 4 pairs of specific primers, each PCR system include one group of primer, are integrally formed a multiplex PCR system；Afterwards using the actinobacillus pleuropneumoniae strain gene group DNA of test serum type as template, PCR detection is carried out using above-mentioned four groups of primers respectively, is as a result compared with reference strain band.By carrying out multiplex PCR after being grouped primer after design primer of the present invention, the actinobacillus pleuropneumoniae bacterial strain of 13 kinds of serotypes can be identified, accuracy is high while saving identification serotype cost, and inspection range is big.
Description
Technical field
The present invention relates to molecular biology field more particularly to the serotype method of actinobacillus pleuropneumoniae, draw
Object combination and PCR system.
Background technique
Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, APP) belongs to Gram-negative
Bacterium is the pathogen of porcine contagious pleuropneumonia, can cause the contagious pleuropneumonia of pig, and in the whole world, there are hair in many countries
It is existing, massive losses are brought to pig breeding industry all over the world.Whether actinobacillus pleuropneumoniae depends on nicotinoyl according to its growth
Amine adenine-dinucleotide (NAD) can be divided into the biological I type of NAD dependent form and the biological II type of non-NAD dependent form.Due to blood
The clear difference for learning reaction, actinobacillus pleuropneumoniae can be divided into different serotype again, at present actinobacillus pleuropneumoniae
There are 15 serotypes, biological I type includes 15 type of serum 1-12 type and serum, and wherein serum 1 type, 5 types are divided into two Asias a, b again
Type；Biological II type includes serum 13,14 types, and in addition there are some bacterial strains for being difficult to be formed with existing categorizing system, such as K2:
O7.The pathogenicity of each serotype is different, and 1,5,9,11 and 12 types usually have very strong virulence, and 3 and 6 types are more warm
With, but the cross-protection between different serotypes is weaker, and there is presently no a kind of vaccines can be to actinobacillus pleuropneumoniae
All serotypes have protection, so just seeming especially for the identification of different actinobacillus pleuropneumoniae bacterial strain serotypes
It is important.
There are many serotype method of actinobacillus pleuropneumoniae at present, and main serological typing method has complement knot
It closes test (CFT), latex agglutination test (LAT), agar diffusion method (GD), enzyme linked immunosorbent assay (ELISA) (ELISA) and coagulates indirectly
Collection test (IHA), there are still many defects, although such as CFT method specificity it is stronger, but sensibility is low, complicated for operation；LAT
Method high specificity but need to obtain when detecting each serotype without pod membrane mutant strain, be technically difficult to realize；GD method behaviour
Although making simplicity, take a long time, sensitivity is not high；The range of ELISA method detection is relatively narrow；IHA method cross reaction is excessive.Simultaneously
Many scholars start the serotype method for probing into actinobacillus pleuropneumoniae on a molecular scale, existing at present many available
Report is seen in the methods of genotyping of actinobacillus pleuropneumoniae parting.Methods of genotyping is mainly PCR method, at present
Most of PCR classifying method is the difference based on exotoxin (Apx), primer is separately designed to four kinds of exotoxins, according to what is amplified
Stripe size and how much actinobacillus pleuropneumoniae is divided into different groups, can avoid the cross reaction of serological typing method
Problem, and high specificity, but there are still be only capable of differentiating the defect of part serotype.
Summary of the invention
The present invention provides the multiplex PCR serotype method of actinobacillus pleuropneumoniae, primer combination and PCR bodies
System, by being grouped after design primer, identifies the actinobacillus pleuropneumoniae bacterial strain of serotype to be measured, overcomes pig chest
The traditional serological classifying method of film Actinobacillus is at high cost, and time-consuming and existing PCR classifying method identification range is small
Defect,
The present invention provides a kind of serotype method of actinobacillus pleuropneumoniae, comprising: for serum 1-15 type
Actinobacillus pleuropneumoniae separately designs specific primer, wherein the specific primer sequence of 11 type of 9 type of serum and serum
Arrange identical, totally 14 pairs of specific primers, sequence are as follows: SEQ ID NO.1-SEQ ID NO.28；
14 pairs of specific primers are grouped:
G1 group includes the special of sequence shown in SEQ ID NO.1-2, SEQ ID NO.13-14 and SEQ ID NO.21-22
Property primer；
G2 group includes SEQ ID NO.3-4, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID NO.19-
The specific primer of sequence shown in 20；
G3 group includes SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.11-12 and SEQ ID NO.23-24
The specific primer of shown sequence；
G4 group includes the special of sequence shown in SEQ ID NO.9-10, SEQ ID NO.25-26 and SEQ ID NO.27-28
Property primer；
Using the actinobacillus pleuropneumoniae strain gene group DNA of test serum type as template, above-mentioned four groups are used respectively
Primer carries out PCR detection, is as a result compared with reference strain band.
Reference strain band is specially in advance by the actinobacillus pleuropneumoniae bacterial strain of known serotype 1-15 with above-mentioned
Four groups of primers carry out PCR, then are reference strain band obtained by gel electrophoresis.
It will be appreciated by those skilled in the art that serotype method for actinobacillus pleuropneumoniae and non-fully belonging to
In methods for the diagnosis of diseases, in biological products preparation, biological products field of quality control, the above method of the invention also has well
Application prospect.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 1 type, in G1 swimming lane
There is specific band at 1094bp and at G2 swimming lane 251bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 12 type, in G1 swimming lane
There is specific band at 350bp and at G2 swimming lane 251bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 2 type, in G2 swimming lane
There is specific band at 639bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 3 type, in G3 swimming lane
There is specific band at 664bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 4 type, in G3 swimming lane
There is specific band at 807bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 5 type, in G4 swimming lane
There is specific band at 835bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 6 type, in G3 swimming lane
Specific band at 240bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 7 type, in G1 swimming lane
There is specific band at 591bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 8 type, in G2 swimming lane
There is specific band at 370bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 9 type, in G2 swimming lane
There is specific band at 251bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 10 type, in G2 swimming lane
There is specific band at 1107bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 11 type, in G2 swimming lane
There is specific band at 251bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 13 type, in G3 swimming lane
There is specific band at 372bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 14 type, in G4 swimming lane
There is specific band at 194bp.
Further, when the actinobacillus pleuropneumoniae bacterial strain of the test serum type is 15 type, in G4 swimming lane
There is specific band at 1011bp.
Specifically, this 13 kinds of serotypes of 1-8,10 and 12-15 can be identified using serotype method of the invention, and blood
Clear 9 type is consistent with the specific band result of 11 type of serum.
Further, PCR amplification condition is as follows:
10 μ L reaction systems: 2 × Premix Taq 5 μ each 0.4 μ L of L, 10mmol/mL primer, 0.5 μ L of template DNA, so
Aseptic double-distilled water is added afterwards or ultrapure water complements to 10 μ L；
PCR response procedures: 95 DEG C of initial denaturation 5min；Then 95 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 1min,
Carry out 30 circulations；72 DEG C of extension 10min.
Electrophoretic imaging condition: with the Ago-Gel of 1% concentration under 150V voltage electrophoresis 20min, gel imaging system
Observe the purpose band size of every group of primer amplification.
Second aspect, the present invention provides a kind of primer combinations for actinobacillus pleuropneumoniae serotype, divide
Group mode is as follows:
First group includes nucleotides sequence shown in SEQ ID NO.1-2, SEQ ID NO.13-14 and SEQ ID NO.21-22
Column；
Second group includes SEQ ID NO.3-4, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID
Nucleotide sequence shown in NO.19-20；
Third group includes SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.11-12 and SEQ ID NO.23-
Nucleotide sequence shown in 24；
4th group includes nucleotides sequence shown in SEQ ID NO.9-10, SEQ ID NO.25-26 and SEQ ID NO.27-28
Column.
The third aspect, the present invention provides a kind of serotype methods and primer combination to prepare pig pleuropneumonia unwrapping wire bar
Application in bacterium detection kit or diagnostic reagent.
Fourth aspect, the present invention provides a kind of multiplex PCR system for actinobacillus pleuropneumoniae serotype,
DNA fragmentation including nucleotide sequence shown in SEQ NO.1-SEQ NO.28.
The present invention provides a kind of actinobacillus pleuropneumoniae serotype method, primer combination and PCR system, beneficial to effect
Fruit is as follows:
1, a kind of multiplex PCR serotype method that the present invention provides actinobacillus pleuropneumoniae divides specific primer
Actinobacillus pleuropneumoniae bacterial strain is detected after group, it is more simple and fast compared to serological typing method, save serum
The cost of type identification.
2, multiplex PCR serotype method of the invention can identify 13 kinds of serotypes and existing PCR serotype
Method is bigger compared to inspection range, while less using reagent, more efficient.
Detailed description of the invention
Fig. 1 is the actinobacillus pleuropneumoniae strain specificity band for the serum 1-15 type that the embodiment of the present invention 2 provides
Electrophoretogram；
Fig. 2-Figure 16 is respectively the actinobacillus pleuropneumoniae bacterial strain base for the serum 1-15 type that the embodiment of the present invention 3 provides
Because of the reference strain band electrophoretogram that group DNA profiling expands in 4 primer grouping systems, G1-G4 swimming lane is corresponding in figure
The result band that primer carries out PCR is grouped into tetra- primers of G1-G4.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art
Every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
The foundation of 1 primer grouping system of embodiment
The guarantor in pod membrane synthetic gene cluster that the present embodiment passes through the actinobacillus pleuropneumoniae for comparing serum 1-15 type
Gene and outer adipose membrane albumen (OlmA) gene are kept, specific primer is designed to each serotype；Wherein, 9 type of serum and serum 11
Type serotype uses the same characterizing gene, this makes 9 types and 11 type specificity sequences identical, totally 14 pairs of specific primers, sequence
It is classified as SEQ ID NO.1-SEQ ID NO.28.Dimer is formed to make in same PCR system primer be not easy reverse complemental, and
The specific primer is divided into 4 groups by the size of easily distinguishable amplicon, specific as follows:
1 primer grouping system of table
Each serotype primer specificity experiment of 2 actinobacillus pleuropneumoniae bacterial strain of embodiment
The present embodiment obtains each serotype specificity band by the following method:
1) the actinobacillus pleuropneumoniae bacterial strain of serum 1-15 type is crossed on TSA culture medium flat plate respectively, is cultivated
Single colonie out, picking single colonie, water-bath are boiled, centrifugation, obtain the genomic DNA template that supernatant is bacterial strain；
2) it using the genomic DNA obtained in step 1) as template, respectively using respective corresponding specific primer, carries out
PCR amplification, amplification condition are as follows:
10 μ L reaction systems: 2 × Premix Taq 5 μ each 0.4 μ L of L, 10mmol/mL primer, 0.5 μ L of template DNA, then
Aseptic double-distilled water is added or ultrapure water complements to 10 μ L；
PCR response procedures: 95 DEG C of initial denaturation 5min；Then by 95 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C extend
1min carries out 30 circulations；72 DEG C of extension 10min；
3) by pcr amplification product on 1% agar gel electrophoresis, observe what each swimming lane occurred with gel imaging system
Band records amplified band size, obtains the specific band information of each bacterial strain.
Specifically, method through this embodiment can obtain the special of 15 kinds of serological type strains of actinobacillus pleuropneumoniae
Property band, as a result as shown in Figure 1；Wherein, 9 type of serum is identical with 11 type serological type strain testing result of serum.
The multiplex PCR serotype method of 3 actinobacillus pleuropneumoniae bacterial strain of embodiment
The present embodiment carries out the serotype of actinobacillus pleuropneumoniae bacterial strain by the following method:
1) the actinobacillus pleuropneumoniae bacterial strain of serum 1-15 type is crossed on TSA culture medium flat plate respectively, is cultivated
Single colonie out, picking single colonie, water-bath are boiled, centrifugation, obtain the genomic DNA template that supernatant is bacterial strain；
2) using the genomic DNA obtained in step 1) as template, the 4 groups of primers provided respectively using embodiment 1 are carried out
PCR amplification, amplification condition are as follows:
10 μ L reaction systems: 2 × Premix Taq 5 μ each 0.4 μ L of L, 10mmol/mL primer, 0.5 μ L of template DNA, then
Aseptic double-distilled water is added or ultrapure water complements to 10 μ L；
PCR response procedures: 95 DEG C of initial denaturation 5min；Then by 95 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C extend
1min carries out 30 circulations；72 DEG C of extension 10min；
3) by pcr amplification product on 1% agar gel electrophoresis 20 minutes under 150V voltage, seen with gel imaging system
The band that each swimming lane occurs is examined, amplified band size is recorded, obtains the reference strain stripe information of 15 kinds of serotypes.
4) genomic DNA template of strain to be tested is obtained in the identical method of step 1), and repeats step 2) and step 3)
It is compared with the reference strain stripe information that step 3) obtains, judges by the specific band information for obtaining strain to be tested
Strain to be tested serotype.
Specifically, through this embodiment the reference of the actinobacillus pleuropneumoniae of the available serum 1-15 type of method
Bacterial strain stripe information, as a result as shown in Fig. 2-Figure 16, wherein serum 1-15 profile bar information is corresponding in turn to Fig. 2-Figure 16, in addition to blood
Clear 9 type and 11 type of serum are identical outer, can be able to obvious area by the quantity of band, position and size between specific band
Point.Wherein, 9 type of serum is identical with 11 type specificity band of serum；Since serum 1 type and 12 type of serum also have 9 type of serum
Characterizing gene also has serum in the specific amplification result of 12 type of serum 1 type and serum other than the specific band of itself
The specific band of 9 types.
When judging strain to be tested serotype, method through this embodiment can accurately identify 13 kinds of serotypes,
4 PCR systems are used only in the present embodiment simultaneously, simplify step, the reagent used is more efficient while less.
The experiment of 4 actinobacillus pleuropneumoniae bacterial strain serotype of embodiment
The multiplex PCR serotype method pair for the actinobacillus pleuropneumoniae bacterial strain that the present embodiment is provided with embodiment 3
67 plants of actinobacillus pleuropneumoniae bacterial strains that Wuhan Ke Qian Biological Co., Ltd. is clinically separated carry out serotype, and same
When using conventional AGP test (GD) method carry out serotype, and compare the genotyping result of two methods, it is as shown in the table:
2 multiplex PCR genotyping result of table and GD genotyping result compare
Specifically, the genotyping result of actinobacillus pleuropneumoniae multiplex PCR serotype method provided by the invention and
Agar diffusion method genotyping result is consistent, this illustrates that qualification result of the invention is accurate.Meanwhile the present invention is compared to AGP test
Method cost is lower, time-consuming shorter.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Sequence table
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<120>actinobacillus pleuropneumoniae serotype method, primer combination and PCR system
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Claims (8)
1. the serotype method of actinobacillus pleuropneumoniae characterized by comprising for the pig pleura of serum 1-15 type
Actinobacillus separately designs specific primer, and wherein 9 type of serum is identical with the specific primer sequence of 11 type of serum,
Totally 14 pairs of specific primers, sequence are as follows: SEQ ID NO.1-SEQ ID NO.28；
14 pairs of specific primers are grouped:
G1 group includes that the specificity of sequence shown in SEQ ID NO.1-2, SEQ ID NO.13-14 and SEQ ID NO.21-22 is drawn
Object；
G2 group includes SEQ ID NO.3-4, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID NO.19-20 institute
Show the specific primer of sequence；
G3 group is comprising shown in SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.11-12 and SEQ ID NO.23-24
The specific primer of sequence；
G4 group includes that the specificity of sequence shown in SEQ ID NO.9-10, SEQ ID NO.25-26 and SEQ ID NO.27-28 is drawn
Object；
Using the actinobacillus pleuropneumoniae strain gene group DNA of test serum type as template, above-mentioned four groups of primers are used respectively
PCR detection is carried out, is as a result compared with reference strain band.
2. serotype method according to claim 1, which is characterized in that when the pig pleuropneumonia of the test serum type
When Actinobacillus bacterial strain is serum 1 type, there is specific band at G1 swimming lane 1094bp and at G2 swimming lane 251bp.
3. serotype method according to claim 1, which is characterized in that when the pig pleuropneumonia of the test serum type
When Actinobacillus bacterial strain is 12 type of serum, there is specific band at G1 swimming lane 350bp and at G2 swimming lane 251bp.
4. serotype method according to claim 1-3, which is characterized in that the condition of the PCR detection are as follows:
10 μ L reaction systems: 2 × Premix Taq 5 μ each 0.4 μ L of L, 10mmol/mL primer, 0.5 μ L of template DNA, then plus
Enter aseptic double-distilled water or ultrapure water complements to 10 μ L；
PCR response procedures: 95 DEG C of initial denaturation 5min；Then 95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 1min are pressed, into
Row 30 circulations；72 DEG C of extension 10min.
5. the primer for actinobacillus pleuropneumoniae serotype combines, which is characterized in that primer is grouped as follows:
First group includes nucleotide sequence shown in SEQ ID NO.1-2, SEQ ID NO.13-14 and SEQ ID NO.21-22；
Second group includes SEQ ID NO.3-4, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID NO.19-20
Shown nucleotide sequence；
Third group includes SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.11-12 and SEQ ID NO.23-24 institute
Show nucleotide sequence；
4th group includes nucleotide sequence shown in SEQ ID NO.9-10, SEQ ID NO.25-26 and SEQ ID NO.27-28.
6. the kit containing the primer combination described in claim 5.
7. the combination of primer described in claim 5 is preparing Serotyping of Actinobacilus pleuropneumoniae detection kit or diagnosis examination
Application in agent.
8. being used for the multiplex PCR system of actinobacillus pleuropneumoniae serotype, which is characterized in that contain SEQ ID NO.1-
The DNA fragmentation of nucleotide sequence shown in 28.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925548A (en) * | 2012-08-02 | 2013-02-13 | 四川农业大学 | Actinobacillus pleuropneumoniae LAMP kit and application method thereof |
| KR20140133225A (en) * | 2013-05-10 | 2014-11-19 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Primer sets of differentiation and simultaneous detection of Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus suis and Streptococcus suis in pneumonic pigs and Multiplex primer kits thereof |
| 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 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925548A (en) * | 2012-08-02 | 2013-02-13 | 四川农业大学 | Actinobacillus pleuropneumoniae LAMP kit and application method thereof |
| KR20140133225A (en) * | 2013-05-10 | 2014-11-19 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Primer sets of differentiation and simultaneous detection of Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus suis and Streptococcus suis in pneumonic pigs and Multiplex primer kits thereof |
| 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 |
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