KR20140134398A - Primer set for detection of systemic bacterial diseases of Poultry and PCR kits thereof - Google Patents

Abstract

The present invention relates to a primer set for diagnosing systemic bacterial disease pathogens of poultry and a diagnosis kit including the primer set. More specifically, the present invention relates to: one or more primer sets selected from a primer set which consists a forward primer of sequence number 1 and a reverse primer of sequence number 2 so as to diagnose Escherichia coli (E.coli) which is a systemic bacterial disease pathogen of poultry, a primer set which consists of a forward primer of sequence number 3 and a reverse primer of sequence number 4 so as to diagnose Riemerella anatipestifer (R. anatipestifer) which is a systemic bacterial disease pathogen of poultry, and a primer set which consists of a forward primer of sequence number 5 and a reverse primer set of sequence number 6 so as to diagnose Salmonella spp. which is a systemic bacterial disease pathogen of poultry; a composition for diagnosing systemic bacterial disease pathogens of poultry including the primer set; a diagnosis kit which includes one or more of the primer sets so as to selectively and/or simultaneously diagnose systemic bacterial disease pathogens of poultry; and a method for selectively and/or simultaneously diagnosing Escherichia coli, E.coli, Riemerella anatipestifer (R. anatipestifer), and Salmonella spp. of three kinds of poultry using the diagnosis kit including the primer set.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a diagnostic kit for detecting bacterial systemic diseases of poultry and a diagnostic kit comprising the primer set,

The present invention relates to a primer set for diagnosing bacterial systemic diseases of poultry and a diagnostic kit comprising the primer set, and more particularly, to a diagnostic kit comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: A primer set capable of diagnosing Escherichia coli which is a pathogenic bacterium of poultry, a primer set comprising a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4 to diagnose a bacterial systemic disease causative microorganism of poultry, A primer set selected from the group consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6 capable of diagnosing salmonella which is a causative organism of bacterial systemic disease of poultry, a bacterium systemic disease A PCR kit for diagnosing a causative organism, a diagnostic kit capable of selectively diagnosing and / or simultaneously diagnosing bacterial systemic pathogens of poultry including at least one of the above primer sets, and a diagnostic kit including the primer set, The present invention relates to a diagnostic method capable of selectively diagnosing and / or simultaneously diagnosing Escherichia coli, E. coli, Riemerella anatipestifer, R. anatipestifer , Salmonella spp. will be.

The poultry may be any one selected from chicken, duck, goose, turkey, quail and pheasant.

Riemerella anatipestifer (R. anatipestifer ) is a non-motile, gram-negative bacillus, first reported in the United States in 1932, and 21 serotypes have so far been known. In Korea, there are four serotypes (1, 4, 7 and 16) presently known to be predominantly of type 1 and type 7, and serotype identification of Rimelera is performed by plate agglutination or agar precipitation Reaction.

Rimeleella infection caused by Rimeleella is an acute and chronic septic infectious disease characterized by fibroplastic epicarditis, hepatocellitis, airflow obstruction, and meningitis in poultry such as ducks, geese, and turkeys and wild birds. The disease causes high mortality, weight loss and quality deterioration, and is causing serious damage to poultry-related industries worldwide. The extent of the disease depends on the serotype, the age of the host, and the route of infection. It is highly susceptible to ducks of 1 to 8 weeks of age. Most ducks die within 1 to 2 days after clinical symptoms when infected with ducks under 5 weeks of age. In Korea, it occurs mainly between 3 and 5 weeks of age.

Rimeleella is normally present in the lining of the dorsal mucosa of young ducks, and infection occurs mainly through respiratory or skin wounds. The incubation period is usually 2 to 5 days. Nasal symptoms such as runny nose, cough, and poor walking, progression, etc., and the mortality rate of ducks infected with Rimeleella is 5 ~ 90%. Symptoms of dermatomyces infected with Rimella spp. Are grossly observed in the pericardium, hepatic capsule, and fibrosarcoma exudates, and necrotizing dermatitis may also be observed in spleen enlargement and chronic progression. The final diagnosis is not possible only with clinical symptoms and autopsy findings. It is done through tests such as bacterial segregation. Normally, samples such as liver are plated on a blood culture medium and cultured at 37 ° C for 48 to 72 hours and then identified .

The causes of bacterial sepsis, which must be distinguished from Rimeleella infection, include Escherichia coli infection, Pasteurella mirtosida infection, Streptococcus infection, Salmonella infection, etc. have. In the case of Rimelea infection, these diseases are very similar to clinical symptoms and visual findings. If these causative agents are overlapped, they should be identified by biochemical identification after bacterial isolation. However, biochemical identification of bacteria is disadvantageous in that it takes a lot of time and effort. In addition, Rimeleella is very difficult to diagnose because it is very similar in shape to Pasteurella.

As a result of the duck disease test results of the Korean Ministry of Agriculture, Forestry and Fisheries Quarantine Inspection in 2011, there were 9 cases of Escherichia coli infection by Escherichia coli, 7 cases of Salmonella infection by Salmonella, 6 cases of Rimelera infection by Rimella, From March to September, the disease was tested for duck farms. As a result, among the 17 farms (34 cases), 14 cases of farms (16 cases) were found to be infected with Rimelera.

In addition to the poultry such as ducks caused by the above-mentioned Rimelella, it causes economic damage to the production farmhouse and / or the production area of the poultry by infections caused by Escherichia coli and Salmonella which are bacterial systemic disease bacteria of poultry.

Accordingly, the present inventors have developed a diagnostic composition and / or a diagnostic kit capable of rapidly diagnosing E. coli, Rimella spp., And Salmonella spp., Which cause three systemic diseases in poultry, to distinguish Escherichia coli, Limerella spp. And Salmonella spp. A novel PCR primer set and a diagnostic PCR composition capable of selectively diagnosing and / or simultaneously diagnosing Escherichia coli, Limerelacia and Salmonella, which are causative microorganisms of three poultry system-causing diseases caused by the above-mentioned primer set, A diagnostic kit capable of selectively diagnosing and / or simultaneously diagnosing Escherichia coli, Limerella and Salmonella, which are causative bacteria of three poultry system disease-causing bacteria, preferably a multi-polymerase chain reaction diagnostic kit, and the diagnostic kit , Which is the causative organism of three poultry disease-causing diseases, E. coli, And salmonella were selectively finish the diagnosis and / or at the same time, diagnostic methods that can be diagnosed with.

The diagnostic PCR kit or diagnostic kit comprising a primer set capable of diagnosing Escherichia coli, Limerella and Salmonella, which are the causative microorganisms of the three poultry system disease-causing microorganisms of the present invention mentioned above, is a Multiplex polymerase chain reaction reaction, and multiplex PCR) to rapidly diagnose and / or diagnose the causative organisms of three species of poultry systemic diseases. In addition, a diagnostic PCR composition or a diagnostic kit comprising a primer set capable of diagnosing Escherichia coli, Limerelacia and Salmonella which are causative microorganisms of the poultry systemic diseases of the present invention has a specificity and sensitivity And it was confirmed that the storage period can be improved while maintaining the performance of the diagnostic PCR composition in the same state as that of the diagnostic PCR composition in the solution state by drying the polymerase chain reaction mixture necessary for the diagnostic reaction in the case of the diagnostic PCR composition, Respectively.

As a prior art related to the present invention, Korean Patent No. 10-0252383 relates to a multiplex polymerase chain reaction-based method for rapid diagnosis of pathogenic Escherichia coli causing diarrhea in livestock, more specifically, PCR primers for each of the four ciliated antigens that can be simultaneously applied to the PCR reaction of the primers and a multiplex PCR method for simultaneous diagnosis of E. coli ciliated antigen using these primers simultaneously.

Korean Patent No. 10-0984785 also discloses a kit for diagnosing Salmonella typhimurium that can accurately and rapidly quantitatively diagnose Salmonella typhimurium, a kit for diagnosis of Salmonella typhimurium containing the primer set, A method for diagnosing Salmonella typhimurium in a target sample using the primer set, an oligonucleotide probe for detecting Salmonella typhimurium, and a microarray in which the probe is immobilized.

In Korean Patent No. 10-0834026, it is possible to effectively prevent the infection of the outdoors pathogenic Rimella fungus, which occurs domestically in Korea. Thus, it is possible to prevent the death of ducks caused by the Rimereella infection, And a 7-type and a smoothing enhancer, and a method for producing the Rimeleiren vaccine.

However, the present invention differs from the prior arts described above in that the technical features of the invention are different from each other and the inventions have different configurations.

It is a first object of the present invention to provide a primer set capable of diagnosing E. coli, Rimella spp., And Salmonella spp., Which are three causative bacteria causing poultry systemic diseases.

A second object of the present invention is to provide a method for detecting pemphigus systemic diseases by including three or more kinds of bacteria that cause poultry systemic diseases by including at least one primer set capable of diagnosing the three pathogenic bacteria pneumococcal, A diagnostic kit, preferably a multiplex PCR kit, which can be selectively diagnosed and / or diagnosed at the same time, is provided. All of the reagents required for the PCR reaction are mixed, dispensed and dried in a single test volume, so that even untrained test subjects can easily diagnose three bacteria that cause poultry systemic diseases.

A third object of the present invention is to provide a diagnostic PCR composition comprising 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 of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: And a primer set selected from the group consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6, to thereby provide a PCR composition capable of diagnosing bacterial systemic disease causing bacteria in poultry.

A fourth object of the present invention is to provide a diagnostic kit comprising at least one primer set capable of diagnosing Escherichia coli, Limerelacia, Salmonella, which are the three causative bacteria causing the poultry systemic diseases, And a diagnostic method capable of selectively diagnosing and / or simultaneously diagnosing three bacterial species causing poultry systemic diseases by using a multiplex PCR kit.

The present invention relates to a primer set comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 to diagnose E. coli which is a causative agent of bacterial systemic disease of poultry, a forward primer and a sequence of SEQ ID NO: A reverse primer of SEQ. ID. NO. 5 and a reverse primer of SEQ. ID. NO. 6, which is composed of a reverse primer of No. 4, It is possible to provide a primer set which can be diagnosed.

The present invention relates to a diagnostic PCR composition comprising 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: And a primer set selected from a primer set consisting of a primer and a reverse primer set forth in SEQ ID No: 6, to thereby provide a PCR composition capable of diagnosing bacterial systemic disease causing bacteria in poultry.

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 primer set of the forward primer of SEQ ID No. 3 and the reverse primer of SEQ ID No. 4, the forward primer of SEQ ID No. 5, ( E. coli ), Riemerella anatipestifer (R. anatipestifer ), Salmonella spp ., And Salmonella spp ., Which cause at least one bacterial systemic disease of poultry by including at least one primer set consisting of a reverse primer A diagnostic kit capable of selectively diagnosing and / or simultaneously diagnosing Salmonella spp., Preferably a multiplex PCR kit can be provided.

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 primer set of the forward primer of SEQ ID No. 3 and the reverse primer of SEQ ID No. 4, the forward primer of SEQ ID No. 5, A diagnostic method capable of selectively diagnosing and / or simultaneously diagnosing Escherichia coli, Limerelacia, and Salmonella, which are bacterial systemic pathogens of three species of poultry, using a diagnostic kit comprising at least one primer set consisting of a reverse primer Can be provided.

The present invention relates to a primer set capable of diagnosing three species of poultry systemic pathogens such as Escherichia coli, Limerelacia, and Salmonella, a primer set capable of diagnosing the above three poultry systemic pathogens such as Escherichia coli, Limerelacia, Salmonella , A diagnostic kit comprising a primer set capable of diagnosing the three poultry systemic pathogens such as Escherichia coli, Limerelacia, and Salmonella, and preferably using a multiplex polymerase chain reaction kit Three kinds of poultry systemic disease bacteria can be diagnosed quickly and easily within 2 hours. In addition, it is possible to diagnose a trace amount of genes present in the culture medium due to high sensitivity, and it is possible to diagnose the cause of disease of poultry in a short time and contribute to the prevention of poultry disease.

FIG. 1 is a graph showing the results obtained by using three polymerase chain reaction (PCR) of Example 3 (liquid phase type PCR composition) of E. coli, R. melanogaster (RA), Salmonella (Sal) (E. coli), Rimeleella (RA), and Salmonella (Sal), which are causative agents of poultry systemic diseases, were identified using the polymerase chain reaction of Example 4 (dry type PCR composition) (Lane M: 100 bp DNA ladder, 1: liquid phase PCR mixture, 2: dry PCR mixture, N: negative control).
Fig. 2 shows the results of measurement of three species of poultry systemic pathogens E. coli, R. marianae (RA), and Salmonella (Sal) by using the multiple polymerase chain reaction of Example 4 (dry type PCR composition) (Lane M: 100 bp DNA ladder, 1 to 7: 20 ng to 20 fg DNA (10-step dilution for each lane) of three poultry systemic pathogens (Escherichia coli, Limerelacia, Salmonella) , N: negative control).
FIG. 3 shows the results of the sensitivity measurement of the polymerase chain reaction of Rimella bacilli according to the animal disease standard test method (Agriculture, Forestry and Fisheries Quarantine Service, 2011) (Lane M: 100 bp DNA ladder, 1 to 7: 20 ng to 20 fg DNA of dimeric Lemeria (10-step dilution for each lane), N: negative control).
FIG. 4 shows the results of the sensitivity measurement of the polymerase chain reaction of Salmonella according to the animal disease standard test method (Agriculture, Forestry and Fisheries Quarantine Service, 2011) (Lane M: 100 bp DNA ladder, 1 to 7: Salmonella 20 ng to 20 fg of DNA (10 step dilution per lane), N: negative control).

The present invention represents a primer set capable of diagnosing poultry systemic disease causative bacteria.

The present invention shows a promo set for the diagnosis of bacterial systemic disease causative bacteria of poultry comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2.

In the above, a primer set consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 can diagnose Escherichia coli, E. coli, which is a bacterial systemic disease pathogen of poultry.

The present invention shows a primer set for the diagnosis of bacterial systemic disease pathogens of poultry comprising the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO:

The primer set consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 is capable of diagnosing a bacterium, a bacterial systemic disease pathogen of the poultry, Riemerella anatipestifer, R. anatipestifer .

The present invention shows a primer set for the diagnosis of bacterial systemic pathogens of poultry comprising the forward primer of SEQ ID NO: 5 and the 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: 6 can diagnose Salmonella spp., A bacterial systemic disease causative organism of poultry.

The poultry may be any one selected from chicken, duck, goose, turkey, quail, and pheasant, and preferably chicken or duck.

A primer set capable of diagnosing the poultry systemic disease causative microorganism of the present invention is Escherichia coli gene (GenBank accession No. CP001925.1), R. anatipestifer gene ( Escherichia coli ), Escherichia coli gene (GenBank accession No. NC_014738.1), Salmonella spp. (GenBank accession No. EU 348369.1), the nucleotide sequence was selected to have a length of 22 to 23 bp and a Tm value of 60 to 65 ° C, and a primer consisting of a forward primer and a reverse primer (See Table 1). Tm values were checked using the MP primer program.

Primers of three species of poultry systemic disease causing bacteria Item primer Remarks Escherichia coli F: GTCCGAGCGCGACCAGTGAAAA SEQ ID NO: 1 R: TCGAGATCGACCGTCTCGCCAA SEQ ID NO: 2 Limerella F: CCGCACAAGCGGTGGATCATGT SEQ ID NO: 3 R: TCGCAGTGTAGCTGCCCTCTGT SEQ ID NO: 4 Salmonella F: TCTCCCCCTCTTCATGCGTTAC SEQ ID NO: 5 R: CCTTTGACGGTGCGATGAAGTT SEQ ID NO: 6

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

The primers set forth in Table 1 were prepared by individually performing a multiplex PCR and / or a multiplex PCR (multiplex PCR) using a primer set composed of a forward primer and a reverse primer for each target, ) To diagnose E. coli, Limerella and Salmonella, which are three poultry systemic disease causative bacteria, individually and / or simultaneously.

The present invention can provide a PCR composition capable of diagnosing bacterial systemic disease bacteria in poultry.

The present invention relates to a diagnostic PCR composition comprising 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: And a primer set selected from a primer set consisting of a primer and a reverse primer set forth in SEQ ID No: 6, to thereby provide a PCR composition capable of diagnosing bacterial systemic disease causing bacteria in poultry.

The present invention relates to a diagnostic PCR composition comprising 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: And a primer set selected from a primer set consisting of a primer set forth in SEQ ID No 6 and a primer set selected from the reverse primer set forth in SEQ ID No: 6, so that E. coli, Limerella and Salmonella, which are three poultry systemic disease causative bacteria, can be individually and / Diagnostic PCR compositions.

The above diagnostic PCR composition comprises 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: A primer set consisting of a primer set consisting of a reverse primer of SEQ ID NO: 6, and a component used in a conventional diagnostic PCR composition can be used. As an example of a component of such a conventional diagnostic PCR composition, a reaction buffer solution, dNTP, Mg ²⁺ Ion, and DNA polymerase.

The buffer solution for reaction may be 1 to 10 mM TrisHCl, 10 to 40 mM KCl (pH 9.0), and the dNTP may be selected from dATP, dTTP, dGTP and dCTP.

Stabilizers and / or non-reactive dyes for enhancing experimental convenience, stabilization and reactivity in the composition.

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 0.0001-0.01% by weight based on the total weight of the diagnostic PCR composition, and preferably in an amount of 0.001-0.005% by weight. If the non-reactive dye material is added in an amount of less than 0.0001% by weight based on the total weight of the diagnostic PCR composition, it is difficult to visually observe the movement of the sample due to the low concentration of the dye upon electrophoresis after the PCR reaction. When the content of the diagnostic PCR composition is more than 0.01% by weight based on the total weight of the diagnostic PCR composition, a high concentration of the water-soluble dye may act as a reaction inhibitor during the PCR. In addition, there is a problem that the movement of the sample during electrophoresis may be disturbed.

The polyhydric alcohols may be used as stabilizers for further stabilizing the composition of the present invention, and it is preferable to use at least one of glucose, glycerol, mannitol, galactitol, glucitol and sorbitol. 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 gelatin, small blue albumin, Thesit, PEG-8000 as stabilizers in addition to polyhydric alcohols.

The composition may be provided in a liquid form 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. The drying method as described above may be applied differently depending on the type and amount of enzyme used. In the present invention, preferably, a vacuum drying method using vacuum liquor or vacuum centrifugation may be used.

 Since the above composition is prepared in a stabilized state by mixing in a reaction tube and then frozen or dried, a separate mixing process is not required during the polymerase chain reaction, so that errors due to mixing during the reaction can be prevented, , The reactivity and the storage property can be improved.

In the above, the diagnostic PCR composition of the present invention comprises 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: And a primer set consisting of a reverse primer of SEQ ID NO: 6 may be used in an amount of 0.01 to 10 wt% based on the total weight of the diagnostic PCR composition.

In the above, the diagnostic PCR composition capable of individually and / or simultaneously diagnosing Escherichia coli, Limerella bacteria and Salmonella bacteria, which are three poultry systemic disease causative bacteria, is capable of diagnosing Escherichia coli, Limelele bacteria and Salmonella bacteria, The reaction buffer solution, dNTP, Mg ²⁺ ion, and DNA polymerase, which are components of the reagents, can be used, for example, ^AccuPower® Gold Multiplex PCR Premix (Cat K-2115) (Bioneer, Korea).

The present invention includes a diagnostic kit capable of diagnosing bacterial systemic disease bacteria of poultry.

The present invention includes a diagnostic kit capable of individually and / or simultaneously diagnosing Escherichia coli, Limerelacia, and Salmonella, which are three poultry bacterial systemic disease pathogens.

The above diagnostic kit can diagnose a bacterial systemic disease causative organism of any one selected from chicken, duck, goose, turkey, quail and pheasant.

The above diagnostic kit can individually and / or simultaneously diagnose Escherichia coli, Limerella and Salmonella, which are three bacterial systemic disease pathogens for poultry selected from chicken, duck, goose, turkey, quail and pheasant .

The above-described diagnostic kit may be a PCR kit using polymerase chain reaction (PCR).

The above diagnostic kit may be a multiple PCR kit using multiplex PCR.

The above-mentioned 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 of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: 3, and E. coli, Limerella and Salmonella can be diagnosed individually and / or simultaneously, including any one or more primer sets selected from the primer set consisting of the reverse primer set forth in SEQ ID NO: 6.

The above-mentioned 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 of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: 6, and a reagent such as a DNA polymerase, dNTPs, and a buffer used in a PCR reaction in addition to any one or more primer sets selected from the primer set selected from the reverse primer set forth in SEQ ID NO: Therefore, detailed description of the reagents used in the PCR reaction will be omitted below.

The present invention includes a diagnostic method capable of diagnosing bacterial systemic disease bacteria of a poultry from a target sample by using a diagnostic kit capable of diagnosing the bacterial systemic disease bacteria of the poultry mentioned above.

The present invention uses a diagnostic kit capable of diagnosing bacterial systemic disease causing bacteria of the above-mentioned poultry to individually and / or simultaneously diagnose three species of poultry bacterial systemic disease bacteria Escherichia coli, Limerella and Salmonella from the sample This includes diagnostic methods that can be performed.

Diagnostic methods for diagnosing bacterial systemic disease causing bacteria in poultry using the above diagnostic kit can diagnose bacterial systemic disease causative bacteria against any one of poultry selected from chicken, duck, goose, turkey, quail and pheasant.

Diagnostic methods for diagnosing bacterial systemic pathogens of poultry using the above-described diagnostic kit include three types of bacterial systemic disease causative microorganisms such as E. coli, RIMA, and Pseudomonas aeruginosa for poultry selected from chicken, duck, goose, turkey, quail, Respectively, can be diagnosed individually and / or simultaneously.

A diagnostic method capable of diagnosing the bacterial systemic disease causing bacteria of the above poultry is 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: , A primer set consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; And performing a reaction, preferably a polymerase chain reaction, so that the mixture can be amplified.

In order to accomplish the object of the present invention, the present invention provides a diagnostic kit comprising a primer set for the diagnosis of bacterial systemic disease bacteria of the present invention and a diagnostic kit comprising the primer set. In order to achieve the object of the present invention, It is desirable to provide a diagnostic kit comprising a primer set and the primer set.

Hereinafter, the present invention will be described in detail with reference to examples. It should be understood, however, that these examples are provided for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

≪ Example 1 > Preparation of template DNA

To perform multiple polymerase chain reaction, template DNA was extracted first. The strains used were Escherichia coli (6610, 13310) supplied by the Montreal Veterinary School of Canada, Rimeleella (ATCC 11845, ATCC 51411, ATCC 51412), Salmonella (ATCC 14028, ATCC 15479, ATCC 7001, ATCC 19945, ATCC 9184) were inoculated in a sheep blood agar plate (Asan Pharmaceutical Co., Ltd., Korea) and cultured at 37 ° C for 24 to 72 hours. Then, single colonies were collected and DNA was extracted using DNesay Blood & Tissue kit (Qiagen, USA).

The target gene sequence in the nucleotide sequence of the DNA extracted from Escherichia coli is shown in SEQ ID NO: 7, and the target gene sequence in the nucleotide sequence of DNA extracted from Limerella is shown in SEQ ID NO: 8 , And the target gene sequence among the nucleotide sequences of DNA extracted from Salmonella is shown in SEQ ID NO: 9

Each DNA extracted from Escherichia coli, Limerella and Salmonella was measured for concentration and purity using a UV spectrophotometer, and after confirming that the purity was 1.8 to 2.0, it was used as a template DNA in multiplex PCR Respectively.

The template DNA was diluted to 10 ng / μl using 1 μl TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) to facilitate the use of multiple polymerase chain reactions and stored at -70 ° C until further use.

Example 2: Primer design

Three species of E. coli gene (GenBank accession No. CP001925.1), R. anatipestifer gene (GenBank accession No. NC_014738.1), Salmonella spp. (GenBank accession No. EU 348369.1), the length is 22 to 23 bp and the Tm value is 60 to 65 ° C. The nucleotide sequence is selected so as to form a forward primer and a reverse primer consisting of a reverse primer A primer set for diagnosing the causative agent of poultry systemic diseases of the species was constructed and shown in Table 2 below.

The Tm values were checked using the MP primer program.

Three primers for poultry systemic disease causing bacteria Item primer Remarks Escherichia coli F: GTCCGAGCGCGACCAGTGAAAA SEQ ID NO: 1 R: TCGAGATCGACCGTCTCGCCAA SEQ ID NO: 2 Limerella F: CCGCACAAGCGGTGGATCATGT SEQ ID NO: 3 R: TCGCAGTGTAGCTGCCCTCTGT SEQ ID NO: 4 Salmonella F: TCTCCCCCTCTTCATGCGTTAC SEQ ID NO: 5 R: CCTTTGACGGTGCGATGAAGTT SEQ ID NO: 6

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

≪ Example 3 > Multiplex polymerase chain reaction using a liquid type PCR composition

The PCR composition to be used for the multiplex polymerase chain reaction was 3 kinds of poultry systemic disease causative bacteria listed in Table 2 of Example 2 based on ^AccuPower® Gold Multiplex PCR Premix (Cat K-2115) (Bioneer, Korea), E. coli, One pair of a forward primer and a reverse primer for diagnosis of bacteria and Salmonella were respectively combined and mixed in a total volume of 10 μl, and the mixture was dispensed into a 96-well plate.

Then, the DNAs of E. coli, Limelele and Salmonella, which are the three poultry systemic pathogens extracted in Example 1, were added as the template to the above PCR composition, and they were divided into sterilized distilled water so that the total volume was 20 쨉 l And mixed to obtain a liquid phase PCR composition.

The above-mentioned liquid type PCR composition was subjected to multiple polymerase chain reaction using MyGenie ^TM 96 Gradient Thermal Block, and the results are shown in FIG. 1 (see FIG. 1).

The multiplex PCR 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.

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 4 Multi-Polymerase Chain Reaction Using Dry-Type PCR Composition

Multiple polymerase chain reaction was performed using the dry type PCR composition in which the liquid phase type PCR composition of Example 3 was dried. It is intended to provide a diagnostic kit capable of diagnosing three kinds of poultry systemic disease causing causative bacteria which are confirmed that the PCR performance is not influenced by the freeze-drying conditions of the reagents, and the ease of use and the reproducibility are improved.

The dry PCR product to be used for the multiple polymerase chain reaction was constructed on the basis of AccuPower ^ㄾ Gold Multiplex PCR Premix (Cat K-2115) (Bioneer, Korea) as the three poultry systemic disease causative bacteria A forward primer and a reverse primer for diagnosing Escherichia coli, Limerella spp., And Salmonella spp. Were combined, and the mixture was mixed to a total volume of 10 μl. The mixture was dispensed into a 96-well plate, and 50 μl of SuperCentra 2 (Bionea, Minute drying (vacuum degree: -76 cmHg).

Three kinds of poultry systemic pathogens such as Escherichia coli, Limerella and Salmonella strains extracted in Example 1 were added as the template to the dried PCR composition, and the resulting mixture was divided into sterile distilled water to give a total volume of 20 μl The dried material was mixed so as to dissolve completely. Then, multiple polymerase chain reaction was performed using MyGenie ^TM 96 Gradient Thermal Block, and the results are shown in FIG. 1 (see FIG. 1).

The multiplex PCR 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.

As shown in FIG. 1, when the results of the multiplex PCR using the liquid phase PCR composition of Example 3 and the PCR results of the multi-PCR using the dry PCR of Example 4 were compared, (Example 1: Lane M: 100 bp DNA ladder in FIG. 1, 1: Example 3 (using a liquid type PCR composition), 2: Example 4 Type PCR composition), N: negative control,

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 existing test methods

DNAs of Escherichia coli, Limerella and Salmonella, which are the three poultry systemic disease causative microorganisms extracted in Example 1, were added as a template, respectively, and DNAs of Escherichia coli, Limerelacia and Salmonella described above were added at 20 ng to 20 fg Step dilution). The results are shown in FIG. 2. The results are shown in FIG.

The results of the measurement of Rimella bacterium using the Rimeleella (ATCC 11845, ATCC 51411, ATCC 51412) sold in the Nong Shim Fisheries Quarantine Inspection Headquarters and using the Animal Disease Standard Test (Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, 2011) The measurement results of Salmonella using Salmonella (ATCC 14028, ATCC 15479, ATCC 7001, ATCC 19945, ATCC 9184) and the Animal Disease Standard Test (Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, 2011) are shown in FIG. 4 .

As shown in the results of FIGS. 2, 3 and 4, Example 3 and Example 4 of the present invention were able to diagnose Escherichia coli template DNA to 2 pg (see FIG. 2, Lane M: 100 bp DNA ladder, N (negative control), Animal Disease Standard Test (Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, 2011), 20 ng to 20 fg DNA (10-step dilution for each lane), 3 species of poultry septicemia (Escherichia coli, Limerelacia, Salmonella) (Lane M: 100 bp DNA ladder, 1 to 7: 20 ng to 20 fg DNA of two species of poultry sepsis caused by pemphigus (Salmonella spp.)) (FIG. 3 and FIG. N: negative control) The diagnostic method for diagnosing the three species of poultry systemic disease bacteria Escherichia coli, Limerella bacteria and Salmonella bacteria according to the present invention is a standard animal disease standard test method (agriculture, forestry and fisheries quarantine inspection Headquarters, 2011), which is more than 10 times more sensitive than the conventional method.

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

60 strains of Escherichia coli, Limerella and Salmonella strains, which were kept in the Department of Disease Diagnosis and Inspection at the Agriculture, Forestry and Fisheries Inspection Headquarters, were identified as the multiprotein enzymes The diagnostic efficiency of poultry bacterial systemic disease-causing microorganisms using the chain reaction and the diagnostic efficiency of poultry bacterial systemic disease-causing microorganisms using the standard method for testing animal diseases (Agriculture, Forestry and Fisheries Inspection Headquarters, 2011) Respectively.

Comparison of diagnostic efficiency between the present invention and existing test methods Subject to diagnosis Example 4 Existing Tests positivity voice positivity voice Escherichia coli 20 40 Unable to diagnose Limerella 20 40 20 40 Salmonella 20 40 20 40

* In Table 3, the existing test method is the Standard Test for Animal Diseases (Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, 2011).

As shown in the above Table 3, the method for diagnosing the causative bacteria of three kinds of poultry bacterial system diseases according to the present invention shows the same efficiency as the conventional animal disease standard test method (Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, 2011) The results showed that the diagnostic efficiency of the three poultry bacterial systemic disease-causing bacteria was improved compared with the existing animal disease standard test method (Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, 2011) because the unmeasured E. coli can be further diagnosed.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. It will be understood that the present invention can be changed.

A diagnostic kit comprising a primer set capable of diagnosing the three poultry systemic disease pathogens of the present invention, Escherichia coli, Limerelacia, Salmonella, and preferably, a multi-polymerase chain reaction kit, The causative organism can be diagnosed quickly and easily within 2 hours. In addition, since the sensitivity of the present invention is high, it is possible to diagnose a small amount of genes existing in the culture medium. Therefore, when the present invention is applied to the diagnosis of poultry systemic disease causative bacteria, it can diagnose the cause of disease of poultry in a short time and contribute to the prevention of poultry disease. Can be used industrially in the sense that it can contribute to the productivity improvement of the poultry farmers.

<110> Animal, Plant and Fisheries Quarantine and Inspection Agency          BIONEER CORPORATION <120> Primer set for detection of systemic bacterial diseases          Poultry and PCR kits thereof <130> 9094 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> forward primer <400> 1 gtccgagcgc gaccagtgaa aa 22 <210> 2 <211> 22 <212> DNA <213> reverse primer <400> 2 tcgagatcga ccgtctcgcc aa 22 <210> 3 <211> 22 <212> DNA <213> forward primer <400> 3 ccgcacaagc ggtggatcat gt 22 <210> 4 <211> 22 <212> DNA <213> reverse primer <400> 4 tcgcagtgta gctgccctct gt 22 <210> 5 <211> 22 <212> DNA <213> forward primer <400> 5 tctccccctc ttcatgcgtt ac 22 <210> 6 <211> 22 <212> DNA <213> reverse primer <400> 6 cctttgacgg tgcgatgaag tt 22 <210> 7 <211> 1485 <212> DNA <213> E. coli gene (GenBank accession No. CP001925.1) <400> 7 ttgtcacggc cgagacttat agtcgctttg tttttatttt ttaatgtatt tgtacatgga 60 gaaaataaag tgaaacaaag cactattgca ctggcactct taccgttact gtttacccct 120 gtgacaaaag cccggacacc agaaatgcct gttctggaaa accgggctgc tcagggcgat 180 attactgcac ccggcggtgc tcgccgctta acggctgatc agaccgccgc tctgcgtgat 240 tctcttagcg ataaacctgc aaaaaatatt attttgctga ttggcgatgg gatgggggat 300 tcggaaatta ctgccgcacg taattatgcc gaaggtgcgg gcggcttttt taaaggtatc 360 gatgccttac cgcttaccgg gcaatacact cactatgcgc tgaataaaaa aaccggaaaa 420 ccggactacg tcaccgactc ggctgcatca gcaaccgcct ggtcaaccgg tgtcaaaacc 480 tataacggcg cgctgggcgt cgatattcac gaaaaagatc acccaacgat tctggaaatg 540 gcaaaagccg caggtctggc gaccggtaac gtttctaccg cagagttgca ggatgccacg 600 cccgccgcgc tggtggcgca tgtgacctcg cgcaaatgct acggtccgag cgcgaccagt 660 gaaaaatgtc cgggtaacgc tctggaaaaa ggcggaaaag gatcgattac cgaacagctg 720 cttaacgctc gtgccgacgt tacgcttggc ggcggcgcaa aaacctttgc tgaaacggca 780 accgctggtg aatggcaggg aaaaacgctg cgtgaacagg cacaggcgcg tggttatcag 840 ttggtgagcg atgctgcctc actgaattcg gtgacggaag cgaatcagca aaaacccctg 900 cttggcctgt ttgctgacgg caatatgcca gtgcgctggc taggaccgaa agcaacgtac 960 ccggtaata tcgataagcc cgcagtcacc tgtacgccta atccgcaacg taatgacagt 1020 gtaccgaccc tggcacagat gaccgacaaa gccattgaat tgttgagtaa aaatgagaaa 1080 ggctttttcc tgcaagttga aggtgcgtca atcgataaac aggatcatgc tgcgaatcct 1140 tgtgggcaaa ttggcgagac ggtcgatctc gatgaagccg tacaacgggc gctggaattc 1200 gccaaaaagg atggtaacac gctggtcata gtcaccgctg atcacgccca cgccagccag 1260 attgttgcgc cggataccaa agctccgggc ctcactcagg cgctaaatac caaagatggc 1320 gcagtgatgg tgatgagtta cgggaactcc gaagaggatt cacaagaaca taccggcagt 1380 cagttgcgta ttgcggcgta tggcccgcat gccgccaatg tcgttggact gaccgaccag 1440 accgatctct tctacaccat gaaagccgct ctggggctga aataa 1485 <210> 8 <211> 1505 <212> DNA <213> R. anatipestifer gene (GenBank accession No. NC_014738.1) <400> 8 agagtttgat cctggctcag gatgaacgct agcgggaggc ctaacacatg caagccgagc 60 ggtagagtgt cttcggatac ttgagagcgg cgtacgggtg cggaacacgt gtgcaacctg 120 cctttatctg agggatagcc tttcgaaagg aagattaata cctcataata tactgattgg 180 catcaattag tattgaaagc tccggcggat agagatgggc acgcgcaaga ttagatagtt 240 ggtgaggtaa cggctcacca agtcaatgat ctttaggggg cctgagaggg tgatccccca 300 cactggtact gagacacgga ccagactcct acgggaggca gcagtgagga atattggaca 360 atgggtgcaa gcctgatcca gccatcccgc gtgaaggacg acggccctat gggttgtaaa 420 cttcttttgt acagggataa acctactctc gtgagggtag ctgaaggtac tgtacgaata 480 agcaccggct aactccgtgc cagcagccgc ggtaatacgg agggtgcaag cgttatccgg 540 atttattggg tttaaagggt ccgcaggcgg gctagtaagt cagtggtgaa agcctacagc 600 ttaactgtag aactgccgtt gatactgcta gtcttgagta tagttgaggt agctggaatg 660 agtagtgtag cggtgaaatg catagatatt actcagaaca ccgattgcga aggcaggtta 720 ccaagttata actgacgctg agggacgaaa gcgtggggag cgaacaggat tagataccct 780 ggtagtccac gccgtaaacg atgctaactc gtttttgggc tatatggttc agagactaag 840 cgaaagtgat aagttagcca cctggggagt acgaccgcaa ggttgaaact caaaggaatt 900 gggggggcc cgcacaagcg gtggatcatg tggtttaatt cgatgatacg cgaggaacct 960 taccaaggct taaatgggaa ttgatagctg tagaaatacg gttttcttcg gacaattttc 1020 aaggtgctgc atggttgtcg tcagctcgtg ccgtgaggtg ttaggttaag tcctgcaacg 1080 agcgcaaccc ctgtcactag ttgccatcat taagttgggg actctagtga gactgcctac 1140 gcaagtagag aggaaggtgg ggatgacgtc aaatcatcac ggcccttacg ccttgggcca 1200 cacacgtgat acaatggccg gtacagaggg cagctacact gcgaagtgat gcaaatctcg 1260 aaagccggtc tcagttcgga ttggagtctg caactcgact ctatgaagct ggaatcgcta 1320 gtaatcgcgc atcagccatg gcgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380 tcaagccatg gaagctgggg gtacctgaag tcggtgaccg taaaaggagc tgcctagggt 1440 aaaactagta actagggcta agtcgtaaca aggtagccgt accggaaggt gcggctggaa 1500 catct 1505 <210> 9 <211> 1568 <212> DNA <213> Salmonella spp. gene (GenBank accession No. EU 348369.1) <400> 9 gtgctgcttt ctctacttaa cagtgctcgt ttacgacctg aattactgat tctggtacta 60 tcatttctat atcgcactga atatcgtact ggcgatattg gtgtttatgg ggtcgttcta cattgacaga 180 atcctcagtt tttcaacgtt tcctgcggta ctgttaatta ccacgctctt tcgtctggca 240 ttatcaatca gtaccagccg tcttatcttg attgaagccg atgccggtga aattatcgcc 300 acgttcgggc aattcgttat tggcgatagc ctggcggtgg gttttgttgt cttctctatt 360 gtcaccgtgg tccagtttat cgttattacc aaaggttcag aacgcgtcgc ggaagtcgcg 420 gcccgatttt ctctggatgg tatgcccggt aaacagatga gtattgatgc cgatttgaag 480 gccggtatta ttgatgcgga tgccgcgcgc gaacggcgaa gcgtactgga aagggaaagc 540 cagctttacg gttcctttga cggtgcgatg aagtttatca aaggtgacgc tattgccggc 600 atcattatta tctttgtgaa ctttattggc ggcatttcgg tggggatgac ccgccatggt 660 atggatttgt cctccgctct gtctacttat accatgctga ccattggtga tggtcttgtc 720 gcccagatcc ccgcattgtt gattgcgatt agtgccggtt ttatcgtgac tcgcgtaaat 780 ggcgatagcg ataacatggg gcggaatatc atgacgcagc tgttgaacaa cccatttgta 840 ttggttgtta cggctatttt gaccatttca atgggaactc tgccgggatt cccgctgccg 900 gtatttgtta ttttatcggt ggttttaagc gtactcttct attttaaatt ccgtgaagca 960 aaacgtagcg ccgccaaacc taaaaccagc aaaggcgagc agccgcttag tattgaggaa 1020 aaagaagggt cgtcgttagg actgattggc gatctcgata aagtctctac agagactgta 1080 ccgttgatat tacttgtgcc gaagagccgg cgtgaagatc tggaaaaagc tcaacttgcg 1140 gagcgtctac gtagtcagtt ctttattgat tatggcgtgc gcctgccgga agtattgtta 1200 cgcgatggcg agggcctgga cgataacagc atcgtattgt tgattaatga gatccgtgtt 1260 gaacaattta cggtctattt tgatttgatg cgagtggtaa attattccga tgaagtcgtg 1320 tcctttggta ttaatccaac aatccatcag caaggtagca gtcagtattt ctgggtaacg 1380 catgaagagg gggagaaact ccgggagctt ggctatgtgt tgcggaacgc gcttgatgag 1440 ctttaccact gtctggcggt gacgctggcg cgcaacgtca atgaatattt cggtattcag 1500 gaaacaaaac atatgctcga ccaactggaa gcgaaatttc ctgatttact taaagaagtg 1560 ctcagaca 1568

Claims (13)

A primer set for the diagnosis of bacterial systemic pathogens of poultry comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2. The method according to claim 1,
A primer set comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 diagnoses E. coli which is a causative organism of bacterial systemic disease of poultry. A primer set for the diagnosis of bacterial systemic pathogens of poultry comprising a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: The method of claim 3,
A primer set consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4 diagnoses a Lemereella bacterium that is a causative agent of bacterial systemic disease of poultry. A primer set for the diagnosis of bacterial systemic pathogens of poultry comprising a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6. The method of claim 5,
A primer set comprising a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6 diagnoses Salmonella, a causative organism of bacterial systemic disease in poultry. The method according to any one of claims 1 to 6,
Wherein the poultry is any one selected from chicken, duck, goose, turkey, quail, and pheasant. In a diagnostic PCR composition,
A forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6, and a reverse primer of SEQ ID NO: And a primer set selected from among primer sets selected from the group consisting of primers set in the PCR system. 9. The method of claim 8,
Wherein the poultry is any one selected from chicken, duck, goose, turkey, quail and pheasant, and the bacterial systemic disease causative microorganism of the poultry is any one or more selected from the group consisting of Escherichia coli, Limerella and Salmonella. A forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6, and a reverse primer of SEQ ID NO: Wherein the primer set comprises at least one primer set selected from the group consisting of primers set forth in SEQ ID NO: 11. The method of claim 10,
A primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 diagnoses E. coli, a primer set consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, A primer set comprising a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6 is a diagnostic kit for the diagnosis of bacterial systemic disease causing poultry of poultry. A forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6, and a reverse primer of SEQ ID NO: Wherein the primer set comprises at least one primer set selected from among primer sets selected from the group consisting of primers set in the primer set. 13. The method of claim 12,
A primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 diagnoses E. coli, a primer set consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, Wherein the primer set consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 diagnoses Salmonella.

Images