CN110373484B - Serotyping method, primer combination and PCR system of actinobacillus pleuropneumoniae - Google Patents
Serotyping method, primer combination and PCR system of actinobacillus pleuropneumoniae Download PDFInfo
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Abstract
The invention provides a serotyping method, primer combination and PCR system of actinobacillus pleuropneumoniae, the invention designs specific primers for actinobacillus pleuropneumoniae of type 1-15 of serum respectively, the sequences of the specific primers of type 9 and type 11 of serum are the same, 14 pairs of specific primers are divided into 4 groups, 3 to 4 pairs of specific primers are respectively arranged in each group, each PCR system comprises a group of primers, and a multiplex PCR system is integrally formed; and then, taking genome DNA of the actinobacillus pleuropneumoniae strain to be detected as a template, respectively carrying out PCR detection by using the four groups of primers, and comparing the result with a reference strain band. According to the invention, after the primers are designed, multiplex PCR is performed after the primers are grouped, so that 13 serotypes of actinobacillus pleuropneumoniae strains can be identified, the cost for identifying serotypes is saved, the accuracy is high, and the detection range is large.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a serotyping method, a primer combination and a PCR system of actinobacillus pleuropneumoniae.
Background
Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, APP) belongs to gram-negative bacteria, is a pathogenic bacterium of porcine infectious pleuropneumonia, can cause porcine infectious pleuropneumonia, is found in many countries worldwide, and brings great loss to pig industry around the world. Actinobacillus pleuropneumoniae is classified into NAD-dependent biological type I and NAD-independent biological type II according to whether its growth depends on Nicotinamide Adenine Dinucleotide (NAD). Due to the difference of serological reactions, the actinobacillus pleuropneumoniae can be divided into different serotypes, 15 serotypes exist in the actinobacillus pleuropneumoniae at present, and the biological I type comprises serotypes 1-12 and 15 types, wherein the serotypes 1 and 5 are divided into a subtype and b subtype; biological type II includes serum type 13, 14, and in addition there are some strains that are difficult to set with existing classification systems, such as K2: o7. The pathogenicity of each serotype is different, types 1, 5, 9, 11 and 12 are usually very virulent, types 3 and 6 are mild, but the cross protection between different serotypes is weak, and no vaccine can protect all serotypes of the actinobacillus pleuropneumoniae at present, so that the identification of serotypes of different actinobacillus pleuropneumoniae strains is particularly important.
The current serotyping methods of actinobacillus pleuropneumoniae are numerous, and the main serotyping methods include Complement Fixation Test (CFT), latex Agglutination Test (LAT), agar diffusion method (GD), enzyme-linked immunosorbent assay (ELISA) and indirect agglutination test (IHA), but the main serotyping methods still have many defects, such as low sensitivity and complex operation although the CFT method has strong specificity; LAT method is highly specific but requires acquisition of each serotype of capsular mutant at the time of detection, which is technically difficult to achieve; the GD method is simple and convenient to operate, but takes longer time and has low sensitivity; the ELISA method has a narrow detection range; IHA process is too cross-reactive. At the same time, many scholars begin to explore the serotyping method of the actinobacillus pleuropneumoniae at the molecular level, and a plurality of genotyping methods for the actinobacillus pleuropneumoniae typing are reported at present. The genotyping method is mainly a PCR method, most of the PCR typing methods are based on the difference of exotoxins (Apx), primers are respectively designed for the four exotoxins, and the actinobacillus pleuropneumoniae is divided into different groups according to the size of the amplified band and the number of the amplified band, so that the problem of cross reaction of the serological typing method can be avoided, the specificity is high, and the defect that only part of serotypes can be distinguished still exists.
Disclosure of Invention
The invention provides a multiplex PCR serotyping method, primer combination and PCR system of actinobacillus pleuropneumoniae, which are used for identifying the actinobacillus pleuropneumoniae strain to be detected by grouping after designing the primers, thereby overcoming the defects of high cost, long time consumption and small identification range of the traditional serologic typing method of the actinobacillus pleuropneumoniae,
the invention provides a serotyping method of actinobacillus pleuropneumoniae, which comprises the following steps: specific primers are respectively designed for the porcine actinobacillus pleuropneumoniae of the serum 1-15 type, wherein the sequences of the specific primers of the serum 9 type and the serum 11 type are the same, and 14 pairs of specific primers are formed, and the sequences are as follows: SEQ ID NO.1-SEQ ID NO.28;
grouping the 14 pairs of specific primers:
group G1 contains specific primers of sequences shown in SEQ ID NO.1-2, SEQ ID NO.13-14 and SEQ ID NO. 21-22;
group G2 contains specific primers of sequences shown in SEQ ID NO.3-4, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID NO. 19-20;
group G3 contains specific primers of sequences shown in SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.11-12 and SEQ ID NO. 23-24;
group G4 contains specific primers of sequences shown in SEQ ID NO.9-10, SEQ ID NO.25-26 and SEQ ID NO. 27-28;
and (3) taking genome DNA of the actinobacillus pleuropneumoniae strain to be detected as a template, respectively carrying out PCR detection by using the four groups of primers, and comparing the result with a reference strain band.
The reference strain band is specifically obtained by carrying out PCR on the actinobacillus pleuropneumoniae strains with the known serotypes 1-15 by using the four groups of primers and then carrying out gel electrophoresis.
Those skilled in the art will appreciate that the serotyping method for actinobacillus pleuropneumoniae is not entirely a disease diagnosis method, and the method of the invention has good application prospects in the fields of biological product preparation and biological product quality control.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 1, there is a specific band at 1094bp in lane G1 and 251bp in lane G2.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 12, there is a specific band at 350bp in lane G1 and 251bp in lane G2.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 2, there is a specific band at 639bp in lane G2.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 3, there is a specific band at 664bp in the G3 lane.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 4, there is a specific band at 807bp in lane G3.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is of type 5, there is a specific band at lane G4, 835 bp.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 6, a specific band is taken at 240bp in the G3 lane.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 7, there is a specific band at 591bp of the G1 lane.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 8, there is a specific band at 370bp in the G2 lane.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 9, there is a specific band at 251bp in the G2 lane.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is of type 10, there is a specific band at 1107bp in the G2 lane.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 11, there is a specific band at 251bp in lane G2.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 13, there is a specific band at 372bp of the G3 lane.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 14, there is a specific band at 194bp in lane G4.
Further, when the actinobacillus pleuropneumoniae strain of the serotype to be tested is type 15, there is a specific band at 1011bp of the G4 lane.
In particular, 13 serotypes 1-8, 10 and 12-15 can be identified using the serotyping methods of the invention, while the specific band results for serotype 9 and serotype 11 are consistent.
Further, the PCR amplification conditions were as follows:
10. Mu.L of reaction system: 2 XPAmix Taq 5. Mu.L, 10mmol/mL primers each 0.4. Mu.L, template DNA 0.5. Mu.L, then adding sterile double distilled water or ultrapure water to make up to 10. Mu.L;
PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; then denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s and extension at 72 ℃ for 1min, and 30 cycles are carried out; extending at 72℃for 10min.
Electrophoresis imaging conditions: electrophoresis was performed for 20min at 150V using a 1% agarose gel, and the size of the target band amplified by each set of primers was observed by a gel imaging system.
In a second aspect, the invention provides a primer combination for serotyping of actinobacillus pleuropneumoniae, the primer combination being grouped as follows:
the first group comprises the nucleotide sequences shown as SEQ ID NO.1-2, SEQ ID NO.13-14 and SEQ ID NO. 21-22;
the second group comprises the nucleotide sequences shown as SEQ ID NO.3-4, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID NO. 19-20;
the third group comprises the nucleotide sequences shown as SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.11-12 and SEQ ID NO. 23-24;
the fourth group comprises the nucleotide sequences shown as SEQ ID No.9-10, SEQ ID No.25-26 and SEQ ID No. 27-28.
In a third aspect, the invention provides a serotyping method and the use of a primer combination in the preparation of a detection kit or diagnostic reagent for actinobacillus pleuropneumoniae.
In a fourth aspect, the invention provides a multiplex PCR system for serotyping Actinobacillus pleuropneumoniae, comprising a DNA fragment comprising the nucleotide sequences shown in SEQ No.1-SEQ No.28.
The invention provides a porcine actinobacillus pleuropneumoniae serotyping method, a primer combination and a PCR system, which have the following beneficial effects:
1. the invention provides a multiplex PCR serotyping method for actinobacillus pleuropneumoniae, which is used for detecting actinobacillus pleuropneumoniae strains after specific primers are grouped, and compared with a serology typing method, the method is simpler and faster, and the cost of serotyping identification is saved.
2. The multiple PCR serotyping method can identify 13 serotypes, and has larger detection range, fewer reagents and higher efficiency compared with the existing PCR serotyping method.
Drawings
FIG. 1 is an electrophoresis chart of specific bands of actinobacillus pleuropneumoniae strains of the serum type 1-15 provided in example 2 of the present invention;
FIGS. 2 to 16 are respectively the electrophoresis patterns of the bands of the reference strains obtained by amplifying the genome DNA templates of the serum type 1-15 actinobacillus pleuropneumoniae strains provided in example 3 of the present invention in a 4 primer grouping system, wherein lanes G1 to G4 correspond to the bands of the PCR results performed by using four primer groupings G1 to G4 as primers.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 establishment of primer grouping System
This example designed specific primers for each serotype by aligning conserved genes in the capsular synthetic gene cluster of actinobacillus pleuropneumoniae from serotypes 1-15 with the exolipoprotein (OlmA) gene; wherein, the same characteristic gene is used for the serotype 9 and the serotype 11, so that the specific sequences of the type 9 and the type 11 are the same, 14 pairs of specific primers are added, and the sequences are SEQ ID NO.1-SEQ ID NO.28. In order to make the primers in the same PCR system difficult to reversely complement to form a dimer and to easily distinguish the sizes of amplicons, the specific primers are divided into 4 groups, and the specific primers are specifically as follows:
TABLE 1 primer grouping System
Example 2 Actinobacillus pleuropneumoniae Strain each serotype primer specificity experiments
The present example obtained each serotype specific band by the following method:
1) Marking the actinobacillus pleuropneumoniae strains of the serum 1-15 type on a TSA culture medium plate respectively, culturing single colonies, picking the single colonies, boiling in water bath, centrifuging, and obtaining a supernatant which is a genome DNA template of the strains;
2) Using the genomic DNA obtained in step 1) as a template, and performing PCR amplification using the corresponding specific primers, respectively, under the following amplification conditions:
10. Mu.L of reaction system: 2 XPAmix Taq 5. Mu.L, 10mmol/mL primers each 0.4. Mu.L, template DNA 0.5. Mu.L, then adding sterile double distilled water or ultrapure water to make up to 10. Mu.L;
PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; then carrying out denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s and extension at 72 ℃ for 1min, and carrying out 30 cycles; extending at 72 ℃ for 10min;
3) The PCR amplified products were electrophoresed on a 1% agarose gel, the bands appearing in each lane were observed with a gel imaging system, the amplified band sizes were recorded, and specific band information for each strain was obtained.
Specifically, specific bands of 15 serotype strains of actinobacillus pleuropneumoniae were obtained by the method of this example, and the results are shown in FIG. 1; wherein, the detection results of the serotype 9 and serotype 11 strains are the same.
Example 3 multiplex PCR serotyping method of Actinobacillus pleuropneumoniae Strain
The serotyping of the actinobacillus pleuropneumoniae strain was performed in this example by the following method:
1) Marking the actinobacillus pleuropneumoniae strains of the serum 1-15 type on a TSA culture medium plate respectively, culturing single colonies, picking the single colonies, boiling in water bath, centrifuging, and obtaining a supernatant which is a genome DNA template of the strains;
2) Using the genomic DNA obtained in step 1) as a template, PCR amplification was performed using the 4 sets of primers provided in example 1, respectively, under the following amplification conditions:
10. Mu.L of reaction system: 2 XPAmix Taq 5. Mu.L, 10mmol/mL primers each 0.4. Mu.L, template DNA 0.5. Mu.L, then adding sterile double distilled water or ultrapure water to make up to 10. Mu.L;
PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; then carrying out denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s and extension at 72 ℃ for 1min, and carrying out 30 cycles; extending at 72 ℃ for 10min;
3) The PCR amplified products were electrophoresed on a 1% agarose gel for 20 minutes at 150V, the bands appearing in each lane were observed with a gel imaging system, the amplified band sizes were recorded, and the band information of the reference strains of 15 serotypes was obtained.
4) Obtaining a genome DNA template of the strain to be tested by the same method as the step 1), repeating the step 2) and the step 3) to obtain specific band information of the strain to be tested, comparing the specific band information with the band information of the reference strain obtained in the step 3), and judging the serotype of the strain to be tested.
Specifically, the reference strain band information of the actinobacillus pleuropneumoniae of the serum 1-15 type can be obtained by the method of the embodiment, and the result is shown in fig. 2-16, wherein the band information of the serum 1-15 type sequentially corresponds to fig. 2-16, and the number, the position and the size of the specific bands can be obviously distinguished by the same type 9 and type 11 of the serum. Wherein the serotype 9 and serotype 11 specific bands are identical; since serotype 1 and serotype 12 also have genes characteristic of serotype 9, specific amplification results for serotypes 1 and 12 have specific bands for serotype 9 in addition to their own specific bands.
When the serotypes of the strain to be detected are judged, 13 serotypes can be accurately identified by the method of the embodiment, meanwhile, the embodiment only uses 4 PCR systems, the steps are simplified, the used reagents are fewer, and the efficiency is higher.
Example 4A serotyping experiment with Actinobacillus pleuropneumoniae Strain
This example provides a multiplex PCR serotyping method for actinobacillus pleuropneumoniae strains from example 3 for serotyping 67 actinobacillus pleuropneumoniae strains clinically isolated from the pre-organisms of the family Wuhanke, inc., and simultaneously serotyping using a conventional agar diffusion (GD) method, and comparing the typing results of the two methods, as shown in the following Table:
TABLE 2 comparison of multiplex PCR typing results with GD typing results
Specifically, the parting result of the actinobacillus pleuropneumoniae multiplex PCR serotyping method provided by the invention is consistent with the parting result of the agar diffusion method, which proves that the identification result of the invention is accurate. Meanwhile, compared with an agar diffusion method, the method has the advantages of lower cost and shorter time consumption.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
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Claims (8)
1. A method for serotyping a porcine actinobacillus pleuropneumoniae comprising: specific primers are respectively designed for the porcine actinobacillus pleuropneumoniae of the serum 1-15 type, wherein the sequences of the specific primers of the serum 9 type and the serum 11 type are the same, and 14 pairs of specific primers are formed, and the sequences are as follows: SEQ ID NO.1-SEQ ID NO.28;
grouping the 14 pairs of specific primers:
group G1 contains specific primers of sequences shown in SEQ ID NO.1-2, SEQ ID NO.13-14 and SEQ ID NO. 21-22;
group G2 contains specific primers of sequences shown in SEQ ID NO.3-4, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID NO. 19-20;
group G3 contains specific primers of sequences shown in SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.11-12 and SEQ ID NO. 23-24;
group G4 contains specific primers of sequences shown in SEQ ID NO.9-10, SEQ ID NO.25-26 and SEQ ID NO. 27-28;
taking genome DNA of a porcine actinobacillus pleuropneumoniae strain of a serotype to be detected as a template, respectively carrying out PCR detection by using the four groups of primers, and comparing the result with a reference strain strip;
the serotyping method is not applicable to disease diagnosis and treatment.
2. The method of claim 1, wherein when the strain of actinobacillus pleuropneumoniae to be tested is serotype 1, there is a specific band at 1094bp and 251 bp.
3. The method of claim 1, wherein when the strain of actinobacillus pleuropneumoniae to be tested is serotype 12, there is a specific band at 350bp and 251 bp.
4. A method of serotyping according to any one of claims 1 to 3, wherein the conditions for PCR detection are:
10. mu L reaction system: 2 XPAmix Taq 5. Mu.L, 10mmol/mL primers each 0.4. Mu.L, template DNA 0.5. Mu.L, then adding sterile double distilled water or ultrapure water to make up to 10. Mu.L;
PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; then carrying out denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s and extension at 72 ℃ for 1min, and carrying out 30 cycles; extending at 72℃for 10min.
5. Primer combination for serotyping of actinobacillus pleuropneumoniae, characterized in that the primers are grouped in the following manner:
a first set of primers comprising the nucleotide sequences shown as SEQ ID NOS.1-2, SEQ ID NOS.13-14 and SEQ ID NOS.21-22;
a second set of primers comprising the nucleotide sequences shown as SEQ ID No.3-4, SEQ ID No.15-16, SEQ ID No.17-18 and SEQ ID No. 19-20;
the third group comprises primers of nucleotide sequences shown as SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.11-12 and SEQ ID NO. 23-24;
the fourth set comprises primers comprising the nucleotide sequences shown as SEQ ID NOS.9-10, SEQ ID NOS.25-26 and SEQ ID NOS.27-28.
6. A kit comprising the primer set according to claim 5.
7. Use of a primer combination according to claim 5 for the preparation of a kit or diagnostic reagent for detecting a serotype of actinobacillus pleuropneumoniae.
8. A multiplex PCR system for serotyping a porcine actinobacillus pleuropneumoniae, characterized by primers comprising the nucleotide sequences shown in SEQ ID nos. 1-28, said primers being grouped in the following manner:
a first set of primers comprising the nucleotide sequences shown as SEQ ID NOS.1-2, SEQ ID NOS.13-14 and SEQ ID NOS.21-22;
a second set of primers comprising the nucleotide sequences shown as SEQ ID No.3-4, SEQ ID No.15-16, SEQ ID No.17-18 and SEQ ID No. 19-20;
the third group comprises primers of nucleotide sequences shown as SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.11-12 and SEQ ID NO. 23-24;
the fourth set comprises primers comprising the nucleotide sequences shown as SEQ ID NOS.9-10, SEQ ID NOS.25-26 and SEQ ID NOS.27-28.
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| 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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| 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 |
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