KR101527847B1

KR101527847B1 - 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

Info

Publication number: KR101527847B1
Application number: KR1020130053067A
Authority: KR
Inventors: 변재원; 배유찬; 정병열; 김하영; 이명헌; 곽동미; 오태균; 김현배; 김남일; 박한오
Original assignee: 대한민국
Filing date: 2013-05-10
Publication date: 2015-06-12

Abstract

The present invention relates to a primer set for detecting and / or discriminating causative agents of bacterial respiratory disease in pigs, a composition comprising the same, and a method for detecting porcine bacterial respiratory disease causative agent capable of detecting a causative agent of porcine bacterial respiratory disease using the composition .
Four types of porcine respiratory disease causative agents ( A. pleuropneumoniae , A. suis , H. parasuis , S. suis ) can be selectively and / or simultaneously detected and / or identified using the above detection method.

Description

[0001] The present invention relates to a primer set for distinguishing bacterial respiratory diseases from pigs, and a diagnostic kit comprising the same. [0001] The present invention relates to a primer set for distinguishing bacterial respiratory diseases from pigs,

The present invention relates to a primer set capable of detecting and / or discriminating a causative agent of bacterial respiratory disease in pigs, a polymerase chain reaction (PCR) composition comprising the primer set, a diagnostic kit comprising the primer set, The present invention relates to a diagnostic method capable of detecting and / or discriminating a causative agent of a bacterial respiratory disease of pigs using a diagnostic kit comprising

The primer sets for detecting and / or distinguishing causative agents of bacterial respiratory disease in pigs include (1) a primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2, (2) a primer set consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, (3) a primer set consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6, And a primer set consisting of a primer and a reverse primer of SEQ ID NO: 8.

A primer set consisting of (1) a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 can detect and / or differentiate Actinobacillus pleuropneumoniae ( A. pleuropneumoniae ) which is a cause of swine bacterial respiratory disease.

(2) A primer set consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 can detect and / or differentiate Actinobacillus suis ( A. suis ), a causative agent of porcine bacterial respiratory disease.

(3) A primer set consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6 can detect and / or differentiate Haemophilus parasuis ( H. parasuis ), a causative agent of porcine bacterial respiratory disease.

(4) A primer set consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 can detect and / or differentiate Streptococcus suis ( S. suis ), the causative agent of porcine bacterial respiratory disease.

Bacterial respiratory diseases of pigs occur worldwide, and are causing enormous damage to the swine industry. Bacterial respiratory diseases of pigs are caused not only by stress factors such as bad ventilation and temperature change but also by secondary infections after viral infection, causing respiratory diseases and reducing animal mortality and productivity, leading to swine farms, And / or the country.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae ) is one of the causative agents of bacterial respiratory disease in pigs, and is the causative agent of pleural pneumonia. There are 15 serotypes However, in Korea, type 2 and type 5 are generally separated. Disease caused by pleural pneumonia is caused by a lot of breeding and finishing pigs, causing a serious damage to the farm.

Actinobacillus suis, A. suis is one of the causative agents of bacterial respiratory disease in pigs. It is found in healthy pigs' nodule and nasal cavity and vagina. When the immunity level of specimen and condition of specimen deteriorates, acute sepsis, Pneumonia, arthritis, miscarriage, and sole-like skin lesions. The disease is characterized by three clinical features. The first is the sepsis type. Mainly in mammals and weaned pigs. It is characterized by petechial hemorrhage on the surface of the organs of the organ, and it is important to distinguish it from sepsis cause such as Salmonella. Second, the pleuropneumoniae ( A. pleuropneumoniae ) is very similar to the respiratory type, and it is important to differentiate it from pleural pneumonia because it occurs mainly in breeding and finishing pigs. The third is characterized by skin lesions in acute septicemia in the parenchyma.

Haemophilus parasuis ( H. parasuis ) is one of the causative agents of bacterial respiratory disease in pigs. It causes respiratory pneumonia as well as systemic multiple serosa as a causative agent of P. saris. It is mainly caused by growth pigs. It grows up to 40 ~ 41 ℃ at the initial stage and has symptoms such as sneezing, anorexia, high fever, depression, weight loss, joint swelling and conjunctivitis. . Mortality rates are reported to be between 10% and 50%.

Streptococcus suis ( S. suis ) is known to cause secondary infections by stress factors such as environmental changes in pathogenic bacterium, which has been present in the tonsil such as one side, among the pathogenic agents causing the bacterial respiratory diseases of pigs. Individuals affected by this disease are found not only in the respiratory system but also in the whole body, causing meningitis and arthritis.

A. pleuropneumoniae , A. suis , H. parasuis , S. suis ), which are the causative agents of the four kinds of swine bacterial respiratory diseases mentioned above, are involved in a complex manner depending on the situation of a pig farm, However, in order to distinguish the various bacterial causative agents, it takes a considerable time to identify the bacteria after biochemical isolation and thus, it is difficult to effectively diagnose and treat them.

Currently, a diagnostic method for bacterial respiratory disease in pigs is performed by isolating respiratory pathogens from pigs affected by respiratory diseases and diagnosing pathogens by investigating biochemical and genetic characteristics. However, biochemical tests differ between laboratories, and the method of testing for each causative organism is different, making it difficult to apply it in general laboratories. Recently, genetic testing for pathogenic pathogens has been widely used.

Therefore, in order to solve the above-mentioned problem, the present invention improves the gene test method so that various bacteria isolated from the lungs affected by respiratory diseases can be diagnosed in a laboratory. The specificity and the specificity were compared with the Standard Diagnosis Method for Animal Diseases of the Ministry of Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, and the reagents required for the reaction required for the reaction were supplied in the form of dryness, .

As a prior art related to the present invention, Korean Patent Laid-Open Publication No. 1997-0074924 discloses a method for producing porcine infectious gastroenteritis, pathogenic porcine respiratory coronavirus, influenza virus, etc., which is produced by attenuating a porcine respiratory coronavirus isolate in a cell in a continuous system Respiratory Diseases There is a description of an attenuated pig respiratory coronavirus strain that can prevent damage to the body by the human body.

Korean Patent Publication No. 2009-0119176 also discloses three kinds of respiratory diseases such as Glasser's disease, Swine enzootic pneumoniae and Porcine respiratory syndrome (PRRS), WS (after-wasting systemic wasting disease) and The present invention relates to a method for measuring an inactivated mixed vaccine and a PRRS neutralizing antibody against guinea pigs for prevention of PRDC (pig compound respiratory disease) in raising finishing pigs using guinea pig. Accordingly, the present invention provides an inactivated mixed vaccine for swine respiratory diseases, which provides a practical means for preventing the progression of the respiratory diseases in advance, and a method for producing the same.

However, the present invention and the prior art are different from each other in the technical features of the invention and the inventions have different configurations.

A first object of the present invention is to provide a primer set capable of rapidly and accurately detecting and / or discriminating the four causative agents causing bacterial respiratory diseases in swine.

A second object of the present invention is to provide a method for detecting and / or discriminating four causative agents causative of bacterial respiratory disease in a swine by polymerase chain reaction (PCR) comprising the primer set of the first object and PCR And a polymerase chain reaction (Multiplex PCR).

A third object of the present invention is to provide a diagnostic kit comprising a primer set for the first object which can rapidly and accurately detect and / or discriminate four causative agents causing bacterial respiratory disease in swine, RTI ID = 0.0 > (PCR) < / RTI > diagnostic kit.

A fourth object of the present invention is to provide a diagnostic kit comprising the primer set of the first object capable of rapidly and accurately detecting and / or discriminating the four causative agents causing the bacterial respiratory disease in pigs, And to provide a diagnostic method capable of selectively and / or simultaneously detecting porcine bacterial respiratory disease causative agents using a PCR (PCR) diagnostic kit.

(1) a primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 to detect and / or differentiate Actinobacillus pleuropneumoniae ( A. pleuropneumoniae ), which is a causative agent of porcine bacterial respiratory disease; A primer set consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4 to detect and / or discriminate Actinobacillus suis ( A. suis ), a causative agent of swine bacterial respiratory disease; (3) (4) a primer set consisting of a forward primer and a reverse primer of SEQ ID NO: 6 to detect and / or differentiate Haemophilus parasuis ( H. parasuis ) causative of porcine bacterial respiratory disease; (4) 8 reverse primer to induce swine bacterial respiratory disease Streptococcus suis ( S. suis ) Can be detected and / or distinguished.

The present invention relates to a primer set comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, the forward primer of SEQ ID NO: Four types of swine bacterial respiratory disease causative organisms ( A. pleuropneumoniae ) including at least one primer set selected from the reverse primer set of SEQ ID NO: 6, a forward primer set forth in SEQ ID NO: 7 and a reverse primer set consisting of the reverse primer set forth in SEQ ID NO: , A. suis , H. parasuis , S. suis ) can be selectively and / or simultaneously detected and / or discriminated. The above-mentioned PCR composition is used for any one reaction selected from among a polymerase chain reaction (PCR) and a multiplex polymerase chain reaction (Multiplex PCR). All the reagents necessary for the PCR reaction are mixed, dispensed and dried So that even non-skilled inspectors can easily detect and / or distinguish four kinds of bacterial respiratory pathogens.

The present invention relates to a primer set comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, the forward primer of SEQ ID NO: Four types of swine bacterial respiratory disease causative organisms ( A. pleuropneumoniae ) including at least one primer set selected from the reverse primer set of SEQ ID NO: 6, a forward primer set forth in SEQ ID NO: 7 and a reverse primer set consisting of the reverse primer set forth in SEQ ID NO: , A. suis , H. parasuis , S. suis ), and preferably a polymerase chain reaction (PCR) diagnostic kit.

The present invention relates to a primer set comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, the forward primer of SEQ ID NO: A diagnostic kit comprising at least one primer set selected from a primer set consisting of a reverse primer of SEQ ID NO: 6, a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8, preferably a polymerase chain reaction ) Diagnostic kit capable of selectively and / or simultaneously detecting four kinds of porcine bacterial respiratory disease causative agents ( A. pleuropneumoniae , A. suis , H. parasuis and S. suis ).

The present invention is directed to a primer set comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2, a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: Four types of swine bacterial respiratory disease causative organisms ( A. pneumoniae) were prepared using a diagnostic kit comprising a primer set consisting of a reverse primer, a forward primer of SEQ ID NO: 7 and a primer set consisting of a reverse primer of SEQ ID NO: 8 . pleuropneumoniae , A. suis , H. parasuis , S. suis ) can be selectively and / or simultaneously detected.

In addition, the method of detecting four kinds of porcine bacterial respiratory disease agents of the present invention can quickly and easily detect four kinds of causative agents of respiratory pathogens of respiratory pathogens in porcine respiratory organism within 2 hours and can detect even a trace amount of genes because of high sensitivity, Time to diagnose the cause of bacterial respiratory disease in pigs, which can contribute to improving swine productivity in swine farms and / or pig farming areas.

Figure 1 shows the results of a multiplex polymerase chain reaction (PCR) using genomic DNA of four species of porcine respiratory tract disease ( A. pleuropneumoniae , A. suis , H. parasuis , S. suis ) using a liquid type PCR composition and a dry type PCR composition (Lane M: 100 bp DNA ladder, 1: liquid phase PCR composition, 2: dry type PCR composition, N: negative control).
FIG. 2 is a result of measuring the sensitivity of the present invention to four kinds of porcine bacterial respiratory disease causative agents ( A. pleuropneumoniae , A. suis , H. parasuis and S. suis ) through a polymerase chain reaction (Lane M: 100 bp DNA ladder, 1 to 7: 20 ng to 20 fg (10 step dilution per lane) of four respiratory pathogens ( A. pleuropneumoniae , A. suis , H. parasuis , S. suis ) and N: negative control.
FIG. 3 shows the results of the sensitivity measurement of the polymerase chain reaction of A. pleuropneumoniae according to the animal disease standard test method used by the Agriculture, Forestry and Fisheries Inspection and Inspection Headquarters (Lane M: 100 bp DNA ladder, 1 to 7: 20 ng to 20 fg (10 step dilution per lane) of three disease agents ( A. pleuropneumoniae , H. parasuis , S. suis ) and N: negative control.
FIG. 4 shows the results of the sensitivity measurement of the polymerase chain reaction of H. parasuis according to the standard animal test method used in the Agriculture, Forestry and Fisheries Inspection Headquarters (Lane M: 100 bp DNA ladder, 1 to 7: 20 ng to 20 fg (10 step dilution per lane) of three disease agents ( A. pleuropneumoniae , H. parasuis , S. suis ) and N: negative control.
FIG. 5 shows the results of the sensitivity measurement of the polymerase chain reaction of S. suis according to the animal disease standard test method used in the Agriculture, Forestry and Fisheries Inspection and Inspection Headquarters (Lane M: 100 bp DNA ladder, 1 to 7: respiratory diseases woninche three kinds (A. pleuropneumoniae, H. parasuis, S. suis) 20ng~20fg (10 binary dilutions for each lane), N: negative control) .

The present invention shows a primer set for detecting a causative agent of a bacterial respiratory disease of swine comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2.

A primer set consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 can detect Actinobacillus pleuropneumoniae ( A. pleuropneumoniae ) which is a cause of swine bacterial respiratory disease.

The present invention shows a primer set for detecting a causative agent of a bacterial respiratory disease of pigs consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4.

A primer set consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 can detect Actinobacillus suis ( A. suis ), which is a cause of swine bacterial respiratory disease.

The present invention shows a primer set for detecting a causative agent of porcine bacterial respiratory disease consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO:

A primer set consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 5 can detect Haemophilus parasuis ( H. parasuis ), which is a cause of swine bacterial respiratory disease.

The present invention shows a primer set for detecting a causative agent of a bacterial respiratory disease of swine comprising a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO:

A primer set consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 can detect Streptococcus suis ( S. suis ) which is a cause of swine bacterial respiratory disease.

The primers of SEQ ID NO: 1 to SEQ ID NO: 8 capable of detecting and / or distinguishing four species of swine bacterial respiratory disease causative genes A. pleuropneumoniae , A. suis , H. parasuis and S. suis are A. pleuropneumoniae gene ( GenBank accession No. AF021919), A. suis gene (GenBank accession No. AY838763.1), H. parasuis gene (GenBank accession No. M75065), S. suis gene (GenBank accession No. NC_012925.1) And a primer set consisting of a forward primer (reverse primer) and a reverse primer (see Table 1, below) was selected so that the length was 22 to 23 bp and the Tm value was 60 to 65 ° C. Tm values were checked using the MP primer program.

Detection and / or diagnosis of four species of bacterial respiratory diseases of pigs Causative agent Primer Remarks A. pleuropneumoniae F: GGCGTGGTTTATGTCACCGGCA SEQ ID NO: 1 R: CGTTCCCGCCCCAATCTTTGCT SEQ ID NO: 2 A. suis F: GCCTCAAGTTGCGGTGGCAGAA SEQ ID NO: 3 R: CCTTGTTTGGCTTCCGGCCCTT SEQ ID NO: 4 H. parasuis
F: TGGCGGACGGGTGAGTAATGCT SEQ ID NO: 5 R: ACTTAAGTCACCGCCTGCGTGC SEQ ID NO: 6 S. suis F: TCAAACGAGCGCGGCGTTTTTC SEQ ID NO: 7 R: TGGTGACATCGGTGTCGGTGGT SEQ ID NO: 8

* In the primer of Table 1, F represents a forward primer and R represents a reverse primer.

The primers of SEQ ID NO: 1 to SEQ ID NO: 8 include four sets of primers (see Table 1) consisting of a forward primer and a reverse primer for each target in a polymerase chain reaction (PCR) ) And multiple polymerase chain reaction (Multiplex PCR). The four primers of the present invention were mixed in one tube, and these primers were confirmed to have no cross reaction with genes of causative agents other than the pig bacterial respiratory diseases reported so far. In addition, the present invention is characterized in that a multiplex polymerase chain reaction is performed using four kinds of primers, and four kinds of pig bacterial respiratory disease genes can be selectively and / or simultaneously detected.

A primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2, a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: the primer set of the reverse primer of SEQ ID NO: 6, SEQ ID NO: cause chain four types of swine bacterial respiratory disease, including any one or more of primer set selected from the primer sets of the seven forward primers and SEQ ID NO: 8 of the reverse primer Actinobacillus pleuropneumoniae , Actinobacillus suis , Haemophilus parasuis , and Streptococcus suis , which are capable of selectively and / or simultaneously detecting the causative agent of bacterial respiratory diseases.

A composition for detecting a causative agent of a bacterial respiratory disease in pigs capable of selectively and / or simultaneously detecting Actinobacillus pleuropneumoniae , Actinobacillus suis , Haemophilus parasuis , and Streptococcus suis , which are the causative agents of the above four kinds of swine bacterial respiratory diseases, / RTI >

A PCR composition for detecting a causative agent of a bacterial respiratory disease in pigs capable of selectively and / or simultaneously detecting Actinobacillus pleuropneumoniae , Actinobacillus suis , Haemophilus parasuis , and Streptococcus suis , which are the causative agents of the above four kinds of swine bacterial respiratory diseases, 1 to SEQ ID NO: 8, a composition for use in any one of reactions selected from among a polymerase chain reaction (PCR) and a multiplex polymerase chain reaction (multiplex PCR), for example, a reaction buffer solution, dNTP, Mg ²⁺ ions, and DNA polymerase.

For the reaction buffer solution, dNTP, Mg ²⁺ ion and DNA polymerase, those used for the PCR can be used. For example, the reaction buffer solution may contain 1 to 10 mM TrisHCl, 10 to 40 mM KCl ( pH 9.0) may be used, and the dNTP may be selected from dATP, dTTP, dGTP and dCTP.

The PCR composition may include stabilizers and / or non-reactive dyes for enhancing experimental convenience, stabilization and improving reactivity.

The non-reactive dye material should be selected from a substance which does not affect the polymerase chain reaction, and is intended to be used for analysis or identification using the polymerase chain reaction product. Materials satisfying these conditions may be used as water-soluble dyes such as rhodamine, tamla, lactose, bromophenol blue, xylenecanol, bromocresol red, and cresol red. The non-reactive dye material may be contained in an amount of 0.0001 to 0.01 wt.%, Preferably 0.001 to 0.005 wt.%, Based on the weight of the entire PCR composition. If the non-reactive dye material is added in an amount of less than 0.0001% by weight, it is difficult to observe the movement of the sample visually due to the low concentration of the dye upon electrophoresis after the polymerase chain reaction, There is a problem that a high concentration of a water-soluble dye can act as a reaction inhibitor in a polymerase chain reaction. In addition, there is a problem that the movement of the sample during electrophoresis may be disturbed.

Also, a polyhydric alcohol may be used as a stabilizing substance for stabilizing the PCR composition. As an example of such a polyhydric alcohol, use of at least one substance selected from glucose, glycerol, mannitol, galactitol, glucitol and sorbitol desirable. The polyhydric alcohol may be contained in an amount of 10 to 500 mM, and preferably in an amount of 50 to 300 mM. If the polyhydric alcohol is more than 500 mM, it is difficult to dissolve in an aqueous solution due to the solubility of the water-soluble polymer itself. Due to the high viscosity, it is difficult to sufficiently mix and the volume becomes larger than necessary. For the polymerase chain reaction, It does not easily dissolve when dissolved. In addition, there is a problem that a high concentration of a water-soluble polymer can act as a reaction inhibitor in a polymerase chain reaction. When the concentration is less than 10 mM, the target enzyme and surface water molecules can not be sufficiently coated and protected. Therefore, it is difficult to expect an effective stabilization effect of the enzyme. Also, since the viscosity of the solution is too low, And there is a problem that the enzyme can not be sufficiently protected. In the present invention, it is possible to use at least one of gelatin, small blue albumin, Thesit and PEG-8000 as stabilizers in addition to polyhydric alcohols.

The above PCR composition may be used in the form of a liquid, and is preferably dried to improve stability, simplicity of storage and long-term storage. The drying can be performed by a known drying method such as general room temperature drying, warm drying, freeze drying, and vacuum drying, and any drying method can be used as long as the composition is not lost. Such a drying method may be applied differently depending on the kind and amount of enzyme used.

Since the PCR composition is mixed in one reaction tube and then frozen or dried to be used in a stabilized state, no separate mixing process is required during the polymerase chain reaction, so that errors due to mixing during the reaction can be prevented, Stability, reactivity and storability can be improved.

On the other hand, the PCR compositions capable of individually and / or simultaneously detecting Actinobacillus pleuropneumoniae , Actinobacillus suis , Haemophilus parasuis , and Streptococcus suis, which are the causative agents of the four kinds of swine bacterial respiratory diseases, are Actinobacillus pleuropneumoniae , Actinobacillus suis , Haemophilus parasuis , Streptococcus suis DNTPs, Mg ²⁺ ions, and products already containing DNA polymerases, such as ^AccuPower® Gold Multiplex PCR Premix (Cat K-2115) ( ^Bioneer® ) , Korea) can be used.

The present invention includes a diagnostic kit for pathogen detection of porcine bacterial respiratory disease.

A primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2, a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: SEQ ID NO: consisting of a reverse primer of 6 primers, SEQ ID NO: 7 of the forward primer and SEQ ID NO: 8, including any one or more of primer set selected from the primer sets of the reverse primer of pathogens four types of swine bacterial respiratory disease Actinobacillus pleuropneumoniae of , Actinobacillus suis , Haemophilus parasuis , and Streptococcus suis , which are capable of selectively and / or simultaneously detecting pathogenic bacteriological respiratory diseases.

The above-described diagnostic kit may be any one of a PCR kit or a multiplex PCR assay kit.

The present invention includes a diagnostic method capable of detecting causative agents of porcine bacterial respiratory disease.

The present invention relates to a primer set comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, the forward primer of SEQ ID NO: A diagnostic kit comprising a primer set consisting of a reverse primer of SEQ ID NO: 6, a forward primer of SEQ ID NO: 7, and a reverse primer of SEQ ID NO: 8 was used to prepare four kinds of swine bacterial respiratory diseases The present invention relates to a method for diagnosing four kinds of bacterial respiratory disease agents of pigs capable of selectively and / or simultaneously detecting Actinobacillus pleuropneumoniae , Actinobacillus suis , Haemophilus parasuis , and Streptococcus suis .

Four methods for diagnosing bacterial respiratory disease agents in pigs include mixing a sample containing template DNA obtained from four kinds of pig bacterial respiratory disease agents into the above-mentioned PCR composition; And performing a reaction so that the reaction mixture can be amplified; And analyzing the amplification product, thereby providing a diagnostic method capable of detecting a causative agent of a bacterial respiratory tract disease of swine.

As an example of this polymerase chain reaction, denaturation at 95 ° C for 5 minutes, 30 cycles at 95 ° C for 30 seconds and 65 ° C for 1 minute and 30 seconds, 72 ° C at 72 ° C It is possible to react for 5 minutes.

A method for diagnosing porcine bacterial respiratory disease disease using a primer set for detecting a pathogenic agent of a bacterial respiratory disease of pigs of the present invention, a PCR composition comprising the primer set, a diagnostic kit comprising the primer set, and a diagnostic kit comprising the primer set In order to achieve the object of the present invention, it is necessary to prepare a primer set for detection of a pathogenic agent of a bacterial respiratory disease in pigs, a PCR composition comprising the primer set, a diagnosis including the primer set It is desirable to provide a diagnostic method for a porcine bacterial respiratory disease agent using a kit and a diagnostic kit comprising the primer set.

Hereinafter, the present invention will be described in detail with reference to examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

&Lt; Example 1 > Preparation of template DNA

To perform multiple polymerase chain reaction, template DNA was extracted first. Using strain was used as a pre-sale under A. pleuropneumoniae, H. parasuis strains, A. suis strains under the pre-sale, take pre-sale for S. suis strains from the University of Veterinary Medicine, University of Montreal, Canada, from ATCC 15557 from the Republic of Korea Ministry of Agriculture, Forestry and Fisheries Quarantine and Inspection Agency. The strain was inoculated in a blood and chocolate medium (Asan Pharmaceutical Co., Ltd., Korea) and then cultured at 37 ° C for 24 to 48 hours. Single colonies were collected and cultivated in a DNesay Blood & Tissue kit (Qiagen, USA).

The DNA was measured for concentration and purity using a UV spectrophotometer, and after confirming that the purity was 1.8 to 2.0, the DNA was used as template DNA in the multiplex PCR.

The template DNA was diluted to 10 ng / ㎕ with 1 ㅧ TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) for easy use during multiplex polymerase reaction and stored at -70 캜 until next use.

The target gene sequence of the extracted DNA is shown in SEQ ID NO: 9 to SEQ ID NO: 12.

SEQ ID NO: 9 is the target gene sequence of A. pleuropneumoniae gene (GenBank accession No. AF021919), SEQ ID NO: 10 is the target gene sequence of A. suis gene (GenBank accession No. AY838763.1), SEQ ID NO: 11 is H. parasuis gene (GenBank accession No. M75065), and SEQ ID NO: 12 is the target gene sequence of S. suis gene (GenBank accession No. NC_012925.1).

Example 2: Primer design

4 kinds of swine bacterial respiratory disease-causing reasons chain of A. pleuropneumoniae gene (GenBank accession No. AF021919 ), A. suis gene (GenBank accession No. AY838763.1), H. parasuis gene (GenBank accession No. M75065), S. The nucleotide sequence was selected to be 22-23 bp in length and Tm value 60-65 ° C in the nucleotide sequence of the suis gene (GenBank accession No. NC_012925.1), and the forward primer and the reverse primer were used (See Table 2). Tm values were checked using an MP primer program

* In Table 2, F of the primer represents a forward primer and R represents a reverse primer.

&Lt; Example 3 > Standard template polymerase chain reaction

The polymerase chain reaction was carried out using the DNA template of the liquid type PCR composition and four kinds of porcine respiratory disease causative agent ( A. pleuropneumoniae , A. suis , H. parasuis , S. suis ) as follows.

^AccuPower® Gold Multiplex PCR Premix ( ^Cat®) , which is equipped with a buffer solution for reaction of 10 mM TrisHCl, 40 mM KCl (pH 9.0) and four dNTPs, dATP, dTTP, dGTP, dCTP and Mg ²⁺ ions and DNA polymerase K-2115), and the forward primer and the reverse primer for four kinds of porcine respiratory disease causative agents ( A. pleuropneumoniae , A. suis , H. parasuis , and S. suis ) described in Table 2 of Example 2 And the mixture was mixed to a total volume of 10 μl, and the mixture was dispensed into a 96-well plate. The total volume was adjusted to 20 μl by sterilized distilled water to be completely dissolved. Then, a DNA template of four kinds of swine bacterial respiratory disease causative organism ( A. pleuropneumoniae , A. suis , H. parasuis , S. suis ) extracted in Example 1 was added, and MyGenie ^TM 96 Gradient Thermal Block (available from Bioneer , Korea) was used to perform multiple polymerase chain reaction.

The polymerase chain reaction was performed at 95 ° C for 5 minutes, followed by 30 cycles at 95 ° C for 30 seconds and 1 minute 30 seconds at 65 ° C, and 72 ° C for 5 minutes.

Example 4 Polymerase Chain Reaction with Dry PCR Composition

After the PCR composition having the same composition as in Example 3 was dried, a PCR reaction was performed using the dry type PCR composition. This is to provide four types of pig bacterial respiratory disease diagnostic kits which are confirmed that the PCR performance is not affected by the drying of the reagents, and the ease of use and the reproducibility are improved.

^AccuPower® Gold Multiplex PCR Premix ( ^Cat®) , which is equipped with a buffer solution for reaction of 10 mM TrisHCl, 40 mM KCl (pH 9.0) and four dNTPs, dATP, dTTP, dGTP, dCTP and Mg ²⁺ ions and DNA polymerase K-2115), and the forward primer and the reverse primer for four kinds of porcine respiratory disease causative agents ( A. pleuropneumoniae , A. suis , H. parasuis , and S. suis ) described in Table 2 of Example 2 Were added to a total volume of 10 μl, and the mixture was dispensed into a 96-well plate and dried for 50 minutes using SuperCentra 2 (available from Bionea, Korea) : -76 cmHg).

To the above-mentioned dry PCR composition, 4 kinds of DNAs of porcine respiratory disease causative agent extracted in Example 1 were added as a template and mixed with sterilized distilled water so that the total volume was 20 쨉 l, so that the dried product was completely dissolved. Polymerase chain reaction was carried out using MyGenie ^TM 96 Gradient Thermal Block under the same conditions as in Example 3, and the results are shown in FIG. 1 (see FIG. 1).

As shown in FIG. 1, the polymerase chain reaction using the PCR kit containing the liquid type PCR composition in Example 3 and the PCR kit containing the dry type PCR composition in Example 4 were compared with each other. As a result, (Lane M: 100 bp DNA ladder, 1: liquid phase PCR composition, 2: dry type PCR composition, and N: negative control) were observed in the same reaction results for detection of respiratory disease agent )

In FIG. 1, the negative control shows the result of using distilled water (DW) instead of template DNA. The reason why DW is used as a negative control is that when DW is used instead of template DNA, no band But if the band appears, it can be judged that the positive control or the template DNA for the example is contaminated during the PCR test.

<Example 5> Sensitivity comparison between the present invention and the conventional test method

A. pleuropneumoniae, H. parasuis , A. suis , ATCC 15557 and S. suis , University of Montreal, Canada were used for the multiplex polymerisation Four types of porcine bacterial respiratory disease causative agent detection method using enzyme chain reaction, four kinds of porcine respiratory disease causative agent and pollen disease standard test method using multi-polymerase chain reaction in Example 4, A. pleuropneumoniae test method , H. parasuis test, and S. suis test (Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, 2011).

As shown in Fig. 2, four kinds of porcine bacterial respiratory disease causative agents were detected using the multiple polymerase chain reaction in Example 4 of the present invention, Lane M: 100 bp DNA ladder, 1 to 7: 20 ng to 20 fg (10-step dilution for each lane) of four respiratory disease causative agents ( A. pleuropneumoniae , A. suis , H. parasuis , S. suis ) N: negative control).

On the other hand, as a standard test for animal disease, up to 20 pg of test results (see Fig. 3) of Pleurotus pneumoniae, test results of glaze bacteria (see Fig. 4) and test results of pig streak streptococci Lane M: 100 bp DNA ladder in FIGS. 4 and 5, 1 to 7: 20 ng to 20 fg (10-step dilution for each lane) of A. pleuropneumoniae , H. parasuis , S. suis , , N: negative control). Therefore, the present invention was more than 10 times more sensitive than the conventional method.

FIG. 3 shows the results of the sensitivity measurement of the polymerase chain reaction of A. pleuropneumoniae according to the animal disease standard test method used by the Agriculture, Forestry and Fisheries Inspection and Inspection Headquarters (Lane M: 100 bp DNA ladder, 1 to 7: 20 ng to 20 fg (10 step dilution per lane) of three disease agents ( A. pleuropneumoniae , H. parasuis , S. suis ) and N: negative control.

FIG. 4 shows the results of the sensitivity measurement of the polymerase chain reaction of H. parasuis according to the standard animal test method used in the Agriculture, Forestry and Fisheries Inspection Headquarters (Lane M: 100 bp DNA ladder, 1 to 7: 20 ng to 20 fg (10 step dilution per lane) of three disease agents ( A. pleuropneumoniae , H. parasuis , S. suis ) and N: negative control.

FIG. 5 shows the results of the sensitivity measurement of the polymerase chain reaction of S. suis according to the animal disease standard test method used in the Agriculture, Forestry and Fisheries Inspection and Inspection Headquarters (Lane M: 100 bp DNA ladder, 1 to 7: respiratory diseases woninche three kinds (A. pleuropneumoniae, H. parasuis, S. suis) 20ng~20fg (10 binary dilutions for each lane), N: negative control) .

&Lt; Example 6 > Comparison of diagnostic efficiency between the invention and the conventional test method using the isolated strain

A total of 83 A. pleuropneumoniae strains , 9 A. suis strains, 94 H. parasuis strains , and 20 strains of S. suis were screened at the Department of Disease Diagnosis and Inspection at the Korean Ministry of Agriculture, Forestry and Fisheries Inspection Headquarters. The detection method of porcine bacterial respiratory disease agents of four species ( A. pleuropneumoniae , A. suis , H. parasuis , S. suis ) and the existing animal disease standard test method (Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, 2011) The diagnostic efficiencies of the four causative organisms of S. pneumoniae respiratory disease ( A. pleuropneumoniae , A. suis , H. parasuis , S. suis ) were measured and the results are shown in Table 3 below.

Comparison of the diagnostic efficiency between the method of Example 4 and the existing test method Detection target Example 4 Method Existing Tests positivity voice positivity voice A. pleuropneumoniae 83 60 83 60 A. suis 9 134 Not distinguishable H. parasuis 94 49 81 62 S. suis 20 123 20 123

As shown in Table 3 above, detection of porcine bacterial respiratory disease agents of four species ( A. pleuropneumoniae , A. suis , H. parasuis , S. suis ) using the multiple polymerase chain reaction in Example 4 of the present invention The diagnostic efficiency of H. parasuis was higher than that of conventional methods, and A. suis, which is not present in the conventional method, can be further diagnosed. Therefore, the diagnostic efficiency of pig bacterial respiratory disease is improved compared with the existing animal disease standard method.

Although the present invention has been fully described by way of example with reference to the accompanying drawings, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and similarities.

The present invention is directed to a primer set comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2, a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: ( A. pleuropneumoniae , A. suis ) including one or more primer sets selected from a primer set consisting of a reverse primer, a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: , H. parasuis , S. suis ) can be selectively and / or simultaneously diagnosed.

In addition, the diagnostic kit of the present invention can quickly and easily detect four kinds of swine bacterial respiratory diseases within two hours, and can detect even a trace amount of genes because of high sensitivity, thus causing the cause of swine bacterial respiratory disease And can contribute to productivity improvement in swine farms and / or pig farming areas, and thus, there is a possibility of industrial applicability.

<110> Animal, Plant and Fisheries Quarantine and Inspection Agency BIONEER CORPORATION <120> 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 <130> 9095 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> forward primer <400> 1 ggcgtggttt atgtcaccgg ca 22 <210> 2 <211> 22 <212> DNA <213> reverse primer <400> 2 cgttcccgcc ccaatctttg ct 22 <210> 3 <211> 22 <212> DNA <213> forward primer <400> 3 gcctcaagtt gcggtggcag aa 22 <210> 4 <211> 22 <212> DNA <213> reverse primer <400> 4 ccttgtttgg cttccggccc tt 22 <210> 5 <211> 22 <212> DNA <213> forward primer <400> 5 tggcggacgg gtgagtaatg ct 22 <210> 6 <211> 22 <212> DNA <213> reverse primer <400> 6 acttaagtca ccgcctgcgt gc 22 <210> 7 <211> 22 <212> DNA <213> forward primer <400> 7 tcaaacgagc gcggcgtttt tc 22 <210> 8 <211> 22 <212> DNA <213> reverse primer <400> 8 tggtgacatc ggtgtcggtg gt 22 <210> 9 <211> 6736 <212> DNA <213> target gene sequence of Actinobacillus pleuropneumoniae (GenBank accession No. AF021919) <400> 9 atcgatatgc cgccgggtac gggcgatatc caacttactc tttcgcaaca aattccggtt 60 accggtgcgg tggtggtaac cactccgcaa gatattgcgt tattagatgc ggtgaaaggt 120 atttcaatgt tccaaaaagt gtcggtaccg gtcttaggta tcattgaaaa tatgagcgta 180 catatctgcc aaaattgcgg tcaccacgaa gatattttcg gcaccggcgg tgcggagaaa 240 gtgggagaaga aatacggtac taaagtatta ggacaaatgc cgttgcatat tcgcttacgt 300 caagatttgg atgccggcac accgaccgtc gttgcggcac cggaacacga aaccagccga 360 gcctatattg aattagcggc aaaagtcgct tcggaattat actggcaagg ttcggttatc 420 ccgtctgaaa ttatgattcg tgaagtaaaa taagttttaa taaccacgaa aacacaaaga 480 acacaagcgg tagaatttgc agaaaaattt gcaaatccta ccgctttttt attagtacga 540 ttcgctgttg gactgctatt tgatttggtt tgtcaggata ttatgttatt gtaatgaaat 600 gttagtgaat tatttttatt aatttgaaag gaaacaaaat gaaaataaaa aaacgttaca 660 ttgcgctgtt ggtcttaggt gtcgttatca gctatgcctg gtatcaaaat tatcaatggg 720 aacagctgat gttaagcggt tattgtgaaa aggacggaag ttattttgat gataggcata 780 cgaagcaaga actgattgat agggcaatta actatatgct ggagcatcaa tctaaaaaaa 840 catacgatgc ttatactgat gaacctttag aaataaaacc atatttaaca atagaggaat 900 ttaaaaaact caatccaaat tgttgtgaaa ttacctcatg gccagcagat gcagttccac 960 aagattggga tgttcgtgtg gaaggtaagg catataggta tgtaatcgta aaatatttaa 1020 gaaccttagc aaatagagaa cctgaacgat gggaaactag tattgttttt gataattgcg 1080 gcaatcctaa aagagcaagc tacttatatt aattaaagag agaaatttat tatgacaaaa 1140 ttaactatgc aagatgtgac caatttatat ttatataaaa cgaaaactct acctaaagat 1200 agattggatg attcacttat ttctgaaata ggaaaaggag atgatgatat tgatagaaaa 1260 gaatttatgg tggggccggg acgttttgtg accgctgata actttagcgt tgtaagagat 1320 ttttttaatg ctgggaaatc acgcattatt gcgccgcaag tcccgcctat tcgttcacag 1380 caggaaaaaa tcttggtcgg tttaaaaccg ggcaaatatt ccaaagcgca gatattggaa 1440 atggagagtt atacgaaagg cggagaagtg gtaaatggca tgtttgccgg tgaagtccag 1500 acattaggct tttatgacga tggcaaaggg gatttactcg aacgcgccta tatctggaat 1560 accacaggat ttaaaatgag cgacaatgcc ttttttgtta tagaagaatc aggcaaacgc 1620 tatattgaaa actttggtat tgaacctctt ggtaagcaag aagattttga ttttgtcggc 1680 ggcttttggt ctaacttagt gaatcgtggt ttggaaagta ttatcgaccc atccggtatc 1740 ggtggaacgg taaaccttaa ctttaccggc gaggtggaaa cctacacgtt agacgaaaca 1800 aggtttaaag cggaagcggc gaagaaaagc cattggagtt tagtgaatgc ggcgaaagta 1860 tacggcggtt tagaccaaat tattaaaaaa ctatgggaca gtggctcaat taagcattta 1920 tatcaagata aagatacggg caaattaaaa ccgattattt acggcacggc cggcaacgac 1980 agtaagattg aaggcactaa aatcacccgt aggattgcgg gtaaagaagt tacgcttgat 2040 attgccaatc agaaaattga aaaaggcgtg ttagagaaat tggggctgtc tgttagtggt 2100 tcggatatca ttaaattgtt gtttggagca ttgactccaa ctttaaatag aatgttgcta 2160 tcacaactta tccagtcttt ttccgatagc ttggctaaac ttgataatcc cttagcccct 2220 tacactaaaa atggcgtggt ttatgtcacc ggcaaaggga atgatgtgct taaaggaact 2280 gaacatgagg atttgtttct cggtggtgag gggaatgata cttattatgc gagagtaggc 2340 gatacaattg aagacgccga cggcaaaggt aaagtctatt ttgtgagaga aaaaggggta 2400 cctaaggcgg atcctaagcg ggtagagttt agcgagtaca taacgaaaga agaaataaaa 2460 gaggttgaaa aggggttatt aacttacgca gttttagaaa attataattg ggaagagaaa 2520 acggcgactt tcgctcatgc gactatgctt aatgagcttt ttactgatta tactaattat 2580 cgttatgaag ttaaaggact aaaattgccc gccgttaaaa agttaaaaag tccgttggtg 2640 gagtttacag ctgatttatt aactgttacg cctattgacg aaaacggaaa agcacttagc 2700 gaaaaaagta ttacggttaa aaattttaaa aatggtgatt taggaataag gttgttggat 2760 cctaatagct attattattt ccttgaaggc caagatacgg gtttttatgg tcctgctttt 2820 tatattgaac gaaaaaacgg tggcggcgct aaaaataact cgtcgggagc aggaaatagc 2880 aaagattggg gcgggaacgg gcatggaaat caccgaaata atgcctccga cctgaataaa 2940 ccggacggaa ataatgggaa taaccaaaat aacggaagca atcaagataa tcatagcgat 3000 gtgaatgcgc caaataaccc gggacgtaac tatgatattt acgatccttt agctttagat 3060 ttagatggag atgggcttga aaccgtgtcg atgaacgggc gacaaggcgc gttattcgat 3120 cggtttttta gtgttagatc gtaaccaaga cggcattatt aatgatataa gcgagttatt tagtaataaa 3240 aatcaacttt ccgacggcag tatttctgca cacggttttg cgacattagc cgatttggat 3300 acaaaccaag atcagcgtat cgaccaaaat gataagctgt tttctaaact ccaaatttgg 3360 cgggatttaa atcaaaacgg ttttagtgaa gcgaatgagc tgtttagctt agaaagtttg 3420 aatattaaat ctttacatac cgcctatgaa gagcgtaatg attttctagc gggcaataat 3480 atccttgctc agcttgggaa gtatgaaaaa acggacggta cttttgcaca aatgggcgat 3540 ttaaatttca gttttaaccc gttttatagc cgatttaccg aagcgttaaa tttaaccgag 3600 caacaacgtc gcacaattaa tctaaccggc accggtcggg ttcgggattt gcgtgaagcc 3660 gccgcacttt ctgaggagtt ggctgcttta ttacaacagt acactaaggc ctccgatttt 3720 caggcacaac gagaattatt gcctgccatt ttagataaat gggcggcaac ggatttacag 3780 tatcaacatt atgataaaac attacttaaa acggtagaaa gtaccgatag tagtgcttct 3840 gtcgttagag tcacgccttc tcaattaagt agtatacgca atgcaaagca tgatcctacc 3900 gttatgcaaa actttgaaca gagtaaggca aaaattgcga ctttaaattc gctctacggg 3960 ttaaatatcg atcaacttta ttacacgacg gataaagaca ttcgctatat tactgataaa 4020 gtgaataata tgtatcaaac aaccgtagaa cttgcctacc gttctttact tttacaaacg 4080 cgtttgaaga aatatgttta tagcgttaat gcgaaacaat tcgaagggaa atgggtaacc 4140 gattattctc gtactgaagc cttatttaac tctactttta aacaatcgcc tgaaaatgca 4200 ttatatgatt taagcgaata cctttctttc tttaacgatc ctacggaatg gaaagaaggg 4260 ctattactgt taagccgtta tatagattat gctaaagcac aaggatttta tgaaaactgg 4320 gcggctactt ctaacttaac tattgcccgt ttaagagagg ctggagtaat ttttgcagaa 4380 tcgacggatt taaaaggcga tgaaaaaaat aatattttgt taggtagcca aaaagataat 4440 aacttatcgg gtagtgcagg tgatgatcta cttatcggcg gagagggtaa tgatacgtta 4500 aaaggcagct acggtgcaga cacctatatc tttagcaaag gacacggaca ggatatcgtt 4560 tatgaagata ccaataatga taaccgcgca agagatatcg acaccttaaa atttaccgat 4620 gtgaattatg cggaagtgaa gtttcgacga gtagataatg acttaatgtt attcggttat 4680 catgatacgg attcggtcac ggtaaaatcc ttctacagcc atgtagatta tcaatttgac 4740 aaattggagt ttgctgaccg cagtataact cgcgatgaac tgattaaagc agggcttcat 4800 ctatacggca ccgatggcaa tgatgatata aaggatcatg cggattggga cagcattttg 4860 gaaggcggca aaggcaacga tattctaaga ggtggctacg gtgcggacac ctatatcttt 4920 agcaaaggac acggacagga tatcgtttat gaagatacca ataatgataa ccgcgcaaga 4980 gatatcgaca ccttaaaatt tactgatgtg aattatgcgg aagtgaaatt ccgacgagta 5040 gataatgact taatgttatt cggttatcat gatacggatt cggtcacgat aaaatccttc 5100 tacaaccatg tagattatca atttgacaaa ttggaatttg ctgaccgcag tataactcgt 5160 gatgaactag gtaaacaagg tatggcatta tttggcactg acggtgatga taatatcaac 5220 gactggggac gtaactcggt gattgatgcc ggtgcgggta atgatacggt taatggcggt 5280 aatggcgatg acaccctcat cggcggcaaa ggtaatgata ttctaagagg tggctacggt 5340 gcggacacct atatctttag caaaggacac ggacaggata tcgtttatga agataccaat 5400 aatgataacc gcgcaagaga tatcgacacc ttaaaattta ccgatgtgaa ttatgcggaa 5460 gtgaaattcc gacgagtaga taatgactta atgttattcg gttatcatga tacggattcg 5520 gtcacggtaa aatccttcta cagccatgta gattatcaat ttgacaaatt ggagtttgct 5580 gaccgcagta taactcgcga tgaactgatt aaagcagggc ttcatctata cggcaccgat 5640 ggcaatgatg atataaagga tcatgcggat tgggacagca ttttggaagg cggcaaaggc 5700 aacgatattc taagaggtgg ctacggtgcg gacacctata tctttagcaa aggacacgga 5760 caggatatcg tttatgaaga taccaataat gataaccgag caagagatat cgacacctta 5820 aaatttactg atgtgaatta tgcggaagtg aaattccgac gagtagataa tgacttaatg 5880 ttattcggtt atcatgatac ggattcggtc acgataaaat ccttctacaa ccatgtagat 5940 tatcaatttg acaaattgga atttgctgac cgcagtataa ctcgtgatga actaggtaaa 6000 caaggtatgg cattatttgg cactgacggt gatgataata tcaacgactg gggacgtaac 6060 tcggtgattg atgccggtgc gggtaatgat acggttaatg gcggtaatgg cgatgacacc 6120 ctcatcggcg gcaaaggtaa tgatattcta agaggtggct acggtgcgga cacctatatc 6180 tttagcaaag gacacggaca ggatatcgtt tatgaagata ccaataatga taaccgcgca 6240 agagatatcg acaccttaaa atttactgat attaatttat ccgaactttg gtttagccga 6300 gaaaataacg atttgattat taaatcatta ttaagtgagg ataaagtcac ggttcaaaat 6360 tggtattcac accaagatca taaaatagaa aatattcgtt tatcgaatga gcaaacgttg 6420 gtgagcactc aggtggagaa gatggttgag tcgatggccg gctttgctca gaagcacgga 6480 ggagagatat ctcttgtgtc gcttgaagag gtaaaacaat atatcaatag cttaacagct 6540 gctttataac atacgaaaga aatcggcaca gtttttttga actgtgccga tttgatttta 6600 gtgtaagaat atagcctgat tttaagaaat ttactcttgg ctaataacta tttcccattt 6660 tataagttat tgacggatgg ttttatcaaa tatgagatca aatcttattt taaattcgct 6720 ttccattaag cgatat 6736 <210> 10 <211> 1515 <212> DNA <213> target gene sequence of Actinobacillus suis (GenBank accession No. AY838763.1) <400> 10 cggagagttt gatcctggct caggacgaac gctggcggcg tgcctaatac atgcaagtag 60 aacgctgaag agaggagcct gctcttcttg gatgagttgc gaacgggtga gtaacgcgta 120 ggtaacctgc ctggtagcgg gggataacta ttggaaacga tagctaatac cgcatgatat 180 taattattgc atgataattg attgaaagat gcaattgcat cactaccaga tggacctgcg 240 ttgtattagc tagttggtga ggtaacggcc caccaaggcg acgatacata gccgacctga 300 gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 360 agggaatctt cggcaatggg gggaaccctg accgagcaac gccgcgtgag tgaagaaggt 420 tttcggatcg taaagctctg ttgtaagaga agaacgggtg tgagagtgga aagttcacac 480 tgtgacggta tcttaccaga aagggacggc taactacgtg ccagcagccg cggtaatacg 540 taggtcccga gcgttgtccg gatttattgg gcgtaaagcg agcgcaggcg gttagataag 600 tctgaagtta aaggctgtgg cttaaccata gtatgctttg gaaactgttt aacttgagtg 660 cagaagggga gagtggaatt ccatgtgtag cggtgaaatg cgtagatata tggaggaaca 720 ccggtggcga aagcggctct ctggtctgta actgacgctg aggctcgaaa gcgtggggag 780 caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta ggtgttaggc 840 cctttccggg gcttagtgcc gcagctaacg cattaagcac tccgcctggg gagtacgacc 900 gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 960 aattcgaagc aacgcgaaga accttaccag gtcttgacat ccctctgacc gctctagaga 1020 tagagttttc cttcgggaca gaggtgacag gtggtgcatg gttgtcgtca gctcgtgtcg 1080 tgagatgttg ggttaagtcc cgcaacgagc gcaaccccta ttgttagttg ccatcattca 1140 gttgggcact ctagtgagac tgccggtaat aaaccggagg aaggtgggga tgacgtcaaa 1200 tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggctggta caacgagtcg 1260 caagccggtg acggcaagct aatctctgaa agccagtctc agttcggatt gtaggctgca 1320 actcgcctac atgaagtcgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac 1380 gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg 1440 gtgaggtaac cgtaaggagc cagccgccta aggtgggata gatgattggg gtgaagtcgt 1500 aacaaggtag ccgta 1515 <210> 11 <211> 1477 <212> DNA <213> target gene sequence of Haemophilus parasuis (GenBank accession No. M75065) <400> 11 aattgaagag tttgatcatg gctcagattg aacgctggcg gcaggcttaa cacatgcaag 60 tcgaacggta gcaggaagaa gcttncttct ttgctnacga gtggcggacg ggtgagtaat 120 gcttgggaat ctggcttatg gagggggata actacgggaa actgtagcta ataccgcgta 180 gtatcgggag atgaaagact gggaccgcaa ggccagttgc cataagatga gcccaagtgg 240 gattaggtag ttggtggggt aaaggcctac caagccgacg atctctagct ggtctgagag 300 gatgaccagc cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg 360 gaatattgcr caatgggggg aaccctgatg cagccatgcc gcgtgaatga agaaggcctt 420 cgggttgtaa agttctttcg gtgangagga agggtggtgt tttaatagaa cactacattg 480 acgttagtca cagaagaagc accggctaac tccgtgccag cagccgcggt aatacggagg 540 gtgcgagcgt taatcggaat gactgggcgt aaagggcacg caggcggtga cttaagtgag 600 atgtgaaagc cccgagctta acttgggaat tgcatttcat actgggttgc tagagtattt 660 nagggagggn tagaattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaataccg 720 aaggcgaagg cagccccttg ggaaaatact gacgctcatg tgcgaaagcg tggggagcaa 780 acaggattag ataccctggt agtccacgct gtaaacgctg tcgattnngg gattgggctn 840 agagcttggt gcccgtagct aacgtgataa atcgaccgcc tggggagtac ggccgcaagg 900 ttaaaactca aatnaattga cgggggccgc acaagcggtg gagcatgtgg tttaattcga 960 tgnaacgcga agaaccttac ctactcttga catcctaaga agaactcaga gatgagtttg 1020 tgccttcggg aacttagaga caggtgctgc atggctgtcg tcagctcgtg ttgtgaaatg 1080 ttgggttnag tcccgcaacg agcgcaaccc ttatcctttg ttgccagcga ttcggtcggg 1140 aactcaaagg agactgccag tgatnaactg gaggaaggtg gggatgacgt caagtcatca 1200 tggcccttac gagtagggct acacacgtgc tacaatggtg catacagagg gtgacgaagc 1260 cgcgaggtgg agtgaatctc agaaagtgca tctaagtccg gattggagtc tgcaactcga 1320 ctccatgaag tcggaatcgc tagtaatcgc gaatcagaat gtcgcggtga atacgttccc 1380 gggccttgta cacaccgccc gtcacaccat ggnagtgggt tgtaccagaa gtagatagct 1440 taacngaaag gggggcgttt accacggtat gattcat 1477 <210> 12 <211> 1347 <212> DNA <213> target gene sequence of Streptococcus suis (GenBank accession No. NC_012925.1) <400> 12 ttataccaaa ccttgggcaa tcatgctatc cgcaatttgt tcaaaggctg cgatgttagc 60 acctgcaagg tagtctgtac caaggtcgta tttttcagca gtttctttgg ctgtgttgaa 120 gatgttggcc atgatgtctt taagacggcc gtctacttct tcacgagtcc atgacaagcg 180 aaggctgttt tgactcattt caagggcaga tacagctaca ccaccagcgt tggcagcttt 240 tgcgagtccg tagagaacgc cattttcctt gtagactttg atggcatcaa ggtcagatgg 300 catgttggca ccttcagcca cacagtacac gccatttttt acaagggcag cagcttgttt 360 gccgttgatc tcattttgag tcgcacatgg aagggcaata tcagccttgc catcgtagtt 420 ccatacagaa cctttgaagt acttagcagt tgatttttct gcagcgtatt ctgtcaaacg 480 agcgcggcgt ttttctttga tgtccaccaa gaggtcgaag tcgataccag tttcgtcaat 540 gatgtaacca tttgagtctg aaacagaaat aacttttgca ccaagttcag tcgctttttg 600 aacagcatat tgggcaacgt taccagaacc tgagataagg acagtttggt ctttgaagga 660 tttaccgttt gctgccaaca tgttatcagt gaagtaaacc aaaccgtaac cagttgcttc 720 tgggcggatc aatgaaccac cgaagccaag aggtttacca gtcaagacac ctgcatcaaa 780 ctggcggagg cgtttgtatt gaccgtacat gtaaccgatc tcacgaccac cgacaccgat 840 gtcaccagca gggacgtcaa gtgaaggtcc gatgtgtttt tgcaattcag tcatgaagct 900 ttggcagaag cgcatgattt cagcatcagt ttttccttta ggatcaaagt ctgaaccacc 960 tttaccaccg ccgattggaa gaccagtcaa gacgtttttg aagatttgct caaaaccgag 1020 gaacttcaag atggattggt ttacagttgg gtggaagcga agaccgcctt tataaggacc 1080 tacagctgag ttgaactgaa cacggtagcc acggttgact tgaacatttc catctttatc 1140 tgtccatgga acacggaagc tgatgatacg ctcaggctca acgatacgag ccaagatgtt 1200 ttcttcgatg tattctgggt gtgcttcaaa aacaggctca agtgtagaga agagctcttc 1260 tacagcttgg aggaattctg tttcatgtgg gttgcgggct ttgactgctt caaaagaagc 1320 ttggatgtaa gctttggcat ttgacat 1347

Claims

A primer set for detecting a pathogen in a porcine bacterial respiratory disease, comprising a primer set consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO:

The method according to claim 1,
Wherein the primer set consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 detects Actinobacillus suis , a pathogen of porcine bacterial respiratory disease.

The method according to claim 1,
A primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;
A primer set consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; And
A primer set selected from the group consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8; and a set of primers selected from the group consisting of a reverse primer of SEQ ID NO:

The method of claim 3,
A primer set consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 detects Actinobacillus pleuropneumoniae , a pathogen of swine bacterial respiratory disease,
A primer set consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 detects Haemophilus parasuis , a pathogen of swine bacterial respiratory disease,
Wherein the primer set consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 detects Streptococcus suis which is a pathogen of swine bacterial respiratory disease.

A PCR composition capable of detecting a causative agent of a bacterial respiratory disease in a pig, including a primer set for detecting a pathogen of a porcine bacterial respiratory disease.

6. The method of claim 5,
A primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;
A primer set consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; And
A primer set selected from the group consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8; and a PCR primer set.

The method according to claim 6,
Wherein the cause of the bacterial respiratory disease of pigs is at least one selected from Actinobacillus pleuropneumoniae , Actinobacillus suis , Haemophilus parasuis, and Streptococcus suis .

8. The method according to any one of claims 5 to 7,
Wherein the PCR composition is prepared from a liquid composition in a liquid form or a dry composition in a dry form.

A diagnostic kit capable of detecting the pathogen of a porcine bacterial respiratory disease, comprising a primer set for detecting a pathogen of a porcine bacterial respiratory disease of Paragraph 1.

10. The method of claim 9,
A primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;
A primer set consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; And
A primer set selected from the group consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8.

A diagnostic method for detecting a causative agent of a bacterial respiratory disease of pigs using the diagnostic kit of claim 9.

12. The method of claim 11,
The diagnostic kit comprises a primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;
A primer set consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; And
A primer set selected from the group consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8.

13. The method according to claim 11 or 12,
And a step of mixing a sample containing template DNA with a primer set included in the diagnostic kit and performing a reaction selected from among a polymerase chain reaction (PCR) and a multiplex polymerase chain reaction (Multiplex PCR) A diagnostic method for detecting the causative agent of bacterial respiratory disease in pigs.

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