CN110205395A - Serotype method, primer combination and the PCR system of haemophilus parasuis - Google Patents
Serotype method, primer combination and the PCR system of haemophilus parasuis Download PDFInfo
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Abstract
The present invention provides the serotype method of haemophilus parasuis, primer combination and PCR systems, the present invention is that the haemophilus parasuis of 15 kinds of serotypes separately designs specific primer, sequence are as follows: SEQ ID NO.1-SEQ ID NO.30, and 15 pairs of specific primers are divided into six groups, every group includes 1 to 3 pair of primer, each PCR system includes one group of primer, is integrally formed a multiplex PCR system；Afterwards using the haemophilus parasuis strain gene group DNA of test serum type as template, PCR detection is carried out using above-mentioned six groups of primers respectively, is as a result compared with reference strain band.The present invention is by carrying out multiplex PCR after being grouped primer, use 6 PCR systems, 15 systems than general PCR method significantly simplify, save the cost of identification serotype, the specific band difference of different serotypes is obvious simultaneously, so that the present invention can go out 15 kinds of serotypes of haemophilus parasuis with precise Identification.
Description
Technical field
The present invention relates to molecular biology field more particularly to the serotype methods of haemophilus parasuis, primer combination
And PCR system.
Background technique
Haemophilus parasuis (Haemophilus parasuis, HPS) is the pathogen of pig Ge Laseshi disease, and a kind of leather is blue
Family name's negative bacterium, can cause the inflammation such as the scrositis, meningitis and arthritis of pig, cause heavy losses to aquaculture.HPS thallus exists
Variform can be presented under microscope, such as spherical, elongated rod shape or filiform, usually have pod membrane, Motility is more difficult, grow according to
Rely in nicotinamide adenine dinucleotide (NAD).The serotype of HPS is numerous, it is now known that and relatively common has 15 kinds of serotypes,
Every kind of serotype virulence is different, lacks cross protection between each other, it is therefore desirable to different epidemic diseases is prepared for different serotype
Seedling prevents the infection of HPS, is just particularly important to the identification of HPS bacterial strain serotype.
There are many serotype method of HPS at present, more commonly used traditional classifying method be agar diffusion method (GD) and
Connect agglutination test (IHA), but time-consuming, sensibility is low, accuracy is not high for both methods, when identification required standard positive
Serum prepares cumbersome, and the price is very expensive for purchase, and all at least 20% bacterial strain is unable to parting.With polymerase
The rise of chain reaction (PCR) method, many scholars start the serotype method for probing into HPS on a molecular scale.At present
There is the PCR method that much can be used for HPS parting to see report.Since the serotype of HPS depends primarily on pod membrane synthetic gene cluster
The antigenicity of the pod membrane of synthesis is controlled, therefore this method is generally based on pod membrane synthetic gene cluster design serum specificity and draws
Object, then bacterial strain template is expanded in PCR system from different primers, HPS is divided according to the specific band amplified
Type seldom occurs being unable to the bacterial strain of parting.Although such method has been much less workload than GD and IHA, it is needed
15 PCR systems, and parting efficiency is not still high, and since the pod membrane synthetic gene cluster of 5 types and 12 types is completely the same, it is difficult
To distinguish, therefore most of PCR classifying method cannot distinguish 5 types and 12 types, and inspection range is limited.
In conclusion traditional GD and IHA method is time-consuming and laborious in existing HPS serotype method, accuracy and sensitive
Property it is not high, be increasingly difficult to meet research and production needs, although existing PCR classifying method to a certain extent can
Overcome the defect of traditional classifying method, but its parting efficiency is still wait improve, and the secondary pig of 15 kinds of serotypes cannot be distinguished completely
Haemophilus, especially 5 types and 12 types, there are significant limitations.
Summary of the invention
For the defect for overcoming above-mentioned haemophilus parasuis tradition classifying method and existing PCR classifying method, this programme design
A kind of classifying method of multiplex PCR can reduce time and reagent cost, and can on the basis of high accuracy, high sensitivity
The serotype of a large amount of bacterial strains is identified simultaneously.
In a first aspect, the present invention provides a kind of serotype method of haemophilus parasuis, comprising: be 15 kinds of serotypes
A pair of of specific primer of every kind of design of haemophilus parasuis, sequence are as follows: SEQ ID NO.1-SEQ ID NO.30,15 kinds of blood
Clear type is haemophilus parasuis serum 1-15 type respectively.
Above-mentioned 15 pairs of specific primers are grouped:
G1 group includes the special of sequence shown in SEQ ID NO.1-2, SEQ ID NO.11-12 and SEQ ID NO.27-28
Property primer；
G2 group includes the special of sequence shown in SEQ ID NO.3-4, SEQ ID NO.15-16 and SEQ ID NO.17-18
Property primer；
G3 group includes the special of sequence shown in SEQ ID NO.5-6, SEQ ID NO.13-14 and SEQ ID NO.25-26
Property primer；
G4 group includes the specificity of sequence shown in SEQ ID NO.7-8, SEQ ID NO.9-10 and SEQ ID NO.19-20
Primer；
G5 group includes the specific primer of sequence shown in SEQ ID NO.21-22 and SEQ ID NO.29-30；
G6 group includes the specific primer of sequence shown in SEQ ID NO.23-24；
Using the haemophilus parasuis strain gene group DNA of test serum type as template, respectively using above-mentioned six groups of primers into
Row PCR detection, is as a result compared with reference strain band.
It will be appreciated by those skilled in the art that the serotype method for haemophilus parasuis not fully belongs to disease
Diagnostic method, in biological products preparation, biological products field of quality control, the above method of the invention also has good application
Prospect.
Preferably, when the haemophilus parasuis bacterial strain of test serum type is 2 type in G1 swimming lane 150bp and G2 swimming lane
There is specific band at 295bp.
Preferably, the G1 swimming lane 150bp and G5 swimming lane 468bp when the haemophilus parasuis bacterial strain of test serum type is 11 type
There is specific band at place.
Preferably, only there is specificity at G4 swimming lane 519bp when the haemophilus parasuis bacterial strain of test serum type is 5 type
Band.
Preferably, when the haemophilus parasuis bacterial strain of test serum type is 12 type in G4 swimming lane 519bp and G6 swimming lane
There is specific band at 532bp.
Preferably, there is specific item at G1 swimming lane 150bp when the haemophilus parasuis bacterial strain of test serum type is 1 type
Band.
Preferably, there is specific item at G3 swimming lane 397bp when the haemophilus parasuis bacterial strain of test serum type is 3 type
Band.
Preferably, there is specific item at G4 swimming lane 347bp when the haemophilus parasuis bacterial strain of test serum type is 4 type
Band.
Preferably, there is specific item at G1 swimming lane 550bp when the haemophilus parasuis bacterial strain of test serum type is 6 type
Band.
Preferably, there is specific item at G3 swimming lane 484bp when the haemophilus parasuis bacterial strain of test serum type is 7 type
Band.
Preferably, there is specific item at G2 swimming lane 699bp when the haemophilus parasuis bacterial strain of test serum type is 8 type
Band.
Preferably, there is specific item at G2 swimming lane 419bp when the haemophilus parasuis bacterial strain of test serum type is 9 type
Band.
Preferably, there is specificity at G4 swimming lane 942bp when the haemophilus parasuis bacterial strain of test serum type is 10 type
Band.
Preferably, there is specificity at G3 swimming lane 685bp when the haemophilus parasuis bacterial strain of test serum type is 13 type
Band.
Preferably, there is specificity at G1 swimming lane 975bp when the haemophilus parasuis bacterial strain of test serum type is 14 type
Band.
Preferably, there is specificity at G5 swimming lane 287bp when the haemophilus parasuis bacterial strain of test serum type is 15 type
Band.
Preferably, 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 combination for haemophilus parasuis serotyping, packet modes
It is as follows:
First group includes nucleotides sequence shown in SEQ ID NO.1-2, SEQ ID NO.11-12 and SEQ ID NO.27-28
Column；
Second group includes nucleotides sequence shown in SEQ ID NO.3-4, SEQ ID NO.15-16 and SEQ ID NO.17-18
Column；
Third group includes nucleotides sequence shown in SEQ ID NO.5-6, SEQ ID NO.13-14 and SEQ ID NO.25-26
Column；
4th group includes nucleotides sequence shown in SEQ ID NO.7-8, SEQ ID NO.9-10 and SEQ ID NO.19-20
Column；
5th group includes nucleotide sequence shown in SEQ ID NO.21-22 and SEQ ID NO.29-30；
6th group includes nucleotide sequence shown in SEQ ID NO.23-24.
The third aspect, the present invention provides a kind of serotype methods and primer combination to prepare haemophilus parasuis detection
Application in kit or diagnostic reagent.
Fourth aspect, the present invention provides a kind of multiplex PCR systems for haemophilus parasuis serotyping, including
The DNA fragmentation of nucleotide sequence shown in SEQ ID NO.1-30.
Compared with existing HPS classifying method, beneficial effects of the present invention are as follows:
1, the multiplex PCR classifying method of HPS provided by this programme can realize the mirror to HPS serotype in a short time
It is fixed, it is more simple and efficient compared to traditional GD and IHA method, a large amount of manpower and material resources can be saved, the cost of identification serotype is saved.
2,6 PCR systems are used to the multiplex PCR classifying method of HPS provided by this programme, compared to the existing side PCR
15 systems of method significantly simplify, while reagent used in 10 μ L PCR systems is less, more efficient.
It 3, can be special with 15 kinds of serotypes of precise Identification HPS to the multiplex PCR classifying method of HPS provided by this programme
It is not 5 types of differentiating and 12 types, recall rate is higher, and inspection range is bigger.
Detailed description of the invention
Fig. 1 is the HPS strain specificity band electrophoretogram for the serotype 1-15 that the embodiment of the present invention 2 provides；
Fig. 2-Figure 16 is respectively the HPS strain gene group DNA template for the serotype 1-15 that the embodiment of the present invention 3 provides 6
The reference strain band electrophoretogram expanded in a primer grouping system, G1-G6 swimming lane is corresponding with six primers of G1-G6 in figure
It is grouped into the result band that primer carries out PCR.
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 present embodiment designs each serotype by comparing the conservative gene in 15 serotype HPS synthetic gene clusters
Specific primer forms the big of dimer and easily distinguishable amplicon to make primer in same PCR system be not easy reverse complemental
It is small, the specific primer is divided into 6 groups, specific as follows:
Each serotype primer specificity experiment of 2 HPS bacterial strain of embodiment
The present embodiment obtains each serotype specificity band by the following method:
1) the HPS bacterial strain of 15 kinds of serotype is crossed on TSA culture medium flat plate respectively, turns out single colonie, picking list
Bacterium colony, 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, 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 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 specific band of 15 kinds of serological type strains of HPS, as a result as schemed
Shown in 1.
The multiplex PCR serotype method of 3 HPS bacterial strain of embodiment
The present embodiment carries out the serotype of HPS bacterial strain by the following method:
1) the HPS bacterial strain of 15 kinds of serotype is crossed on TSA culture medium flat plate respectively, turns out single colonie, picking list
Bacterium colony, 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 6 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, 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 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 strain stripe information of 15 kinds of serotypes of the available HPS of method, knot
Fruit is able to as shown in Fig. 2-Figure 16, between obtained 15 species specificity band by the quantity of band, position and size bright
It is aobvious to distinguish.Among these it should be noted that when only having specific band at G4 swimming lane 519bp, by being at G6 swimming lane 532bp
No have specific band to distinguish 5 types and 12 type serotypes, in the case of other G6 swimming lane not as bacterial strain serotype identification according to
According to.
When judging strain to be tested serotype, method through this embodiment can be accurate, delicately identifies 15 kinds of HPS
Serotype can distinguish 5 types and 12 type bacterial strains, while this implementation especially by the design of 12 type serotype specificity primers
6 PCR systems are used only in example, simplify step, the reagent used is less, more efficient.
The experiment of 4 HPS bacterial strain serotype of embodiment
The multiplex PCR serotype method for the HPS bacterial strain that the present embodiment is provided with embodiment 2 is to share biological before the section of Wuhan
74 HPS bacterial strains that Co., Ltd is clinically separated carry out serotype, and are carried out simultaneously using conventional AGP test (GD) method
Serotype, and the genotyping result of two methods is compared, it is as shown in the table:
Multiplex PCR genotyping result and GD genotyping result compare
Specifically, the genotyping result and agar diffusion method parting knot of HPS multiplex PCR serotype method provided by the invention
Fruit is consistent, and also identifies 10 bacterial strains (NT type) that agar diffusion method does not identify, wherein 9 are 4 types, 1 is 8
Type, this is highlighted, and qualification result of the present invention is more accurate, and identification range is wider while more simple and efficient, the spy of save the cost
Point.
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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Claims (10)
1. the serotype method of haemophilus parasuis characterized by comprising
For a pair of of specific primer of every kind of design of haemophilus parasuis HPS of 15 kinds of serotype, sequence are as follows: SEQ ID NO.1-
SEQ ID NO.30,
Above-mentioned 15 pairs of specific primers are grouped:
G1 group includes that the specificity of sequence shown in SEQ ID NO.1-2, SEQ ID NO.11-12 and SEQ ID NO.27-28 is drawn
Object；
G2 group includes that the specificity of sequence shown in SEQ ID NO.3-4, SEQ ID NO.15-16 and SEQ ID NO.17-18 is drawn
Object；
G3 group includes that the specificity of sequence shown in SEQ ID NO.5-6, SEQ ID NO.13-14 and SEQ ID NO.25-26 is drawn
Object；
G4 group includes that the specificity of sequence shown in SEQ ID NO.7-8, SEQ ID NO.9-10 and SEQ ID NO.19-20 is drawn
Object；
G5 group includes the specific primer of sequence shown in SEQ ID NO.21-22 and SEQ ID NO.29-30；
G6 group includes the specific primer of sequence shown in SEQ ID NO.23-24；
Using the haemophilus parasuis strain gene group DNA of test serum type as template, PCR is carried out using above-mentioned six groups of primers respectively
Detection, is as a result compared with reference strain band.
2. the method according to claim 1, wherein the haemophilus parasuis bacterial strain when the test serum type is
There is specific band at G1 swimming lane 150bp and G2 swimming lane 295bp when 2 type.
3. the method according to claim 1, wherein the haemophilus parasuis bacterial strain when the test serum type is
There is specific band at G1 swimming lane 150bp and G5 swimming lane 468bp when 11 type.
4. the method according to claim 1, wherein the haemophilus parasuis bacterial strain when the test serum type is
Only there is specific band at G4 swimming lane 519bp when 5 type.
5. the method according to claim 1, wherein the haemophilus parasuis bacterial strain when the test serum type is
There is specific band at G4 swimming lane 519bp and G6 swimming lane 532bp when 12 type.
6. the method according to claim 1, wherein the PCR testing conditions 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, 57 DEG C of annealing 30s, 72 DEG C of extension 1min are carried out
30 circulations；72 DEG C of extension 10min.
7. the primer for haemophilus parasuis serotyping 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.11-12 and SEQ ID NO.27-28；
Second group includes nucleotide sequence shown in SEQ ID NO.3-4, SEQ ID NO.15-16 and SEQ ID NO.17-18；
Third group includes nucleotide sequence shown in SEQ ID NO.5-6, SEQ ID NO.13-14 and SEQ ID NO.25-26；
4th group includes nucleotide sequence shown in SEQ ID NO.7-8, SEQ ID NO.9-10 and SEQ ID NO.19-20；
5th group includes nucleotide sequence shown in SEQ ID NO.21-22 and SEQ ID NO.29-30；
6th group includes nucleotide sequence shown in SEQ ID NO.23-24.
8. the kit containing primer as claimed in claim 7 combination.
9. primer combination as claimed in claim 7 is preparing 15 kinds of serotype detection kits of haemophilus parasuis or diagnostic reagent
In application.
10. being used for the multiplex PCR system of haemophilus parasuis serotyping, which is characterized in that contain SEQ ID NO.1-30 institute
Show the DNA fragmentation of nucleotide sequence.
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