KR102353989B1 - Primers for detecting Ornithobacterium rhinotracheale infection and its use - Google Patents

Abstract

Disclosed herein are a primer set designed to specifically detect Ornithobacterium rhinotracheale, a composition comprising the primer set, a kit, and a method. The method, composition and kit according to the present application can detect the presence or absence of Ornitobacterium rhinothrachia quickly and accurately with high sensitivity with a single test.

Description

Primers for detecting Ornithobacterium rhinotracheale infection and its use

It is a technology related to the genetic diagnosis of Ornitobacterium rhinothracia, which causes respiratory diseases in birds.

Ornithobacterium rhinotracheale (ORT) is a bacterium that causes respiratory diseases in birds that occur worldwide. It causes economic damage by delaying growth, lowering the egg-laying rate, and causing death. Until recently, it has been continuously occurring in Korea and has a direct impact on the poultry industry.

The causative bacteria are Gram-negative bacteria, non-spore-forming, non-motile bacilli (rod-shaped), and taxonomically belonging to the rRNA superfamily V of the Cythophaga-Flavobacterium-Bacteroides phylum. It has been isolated from a variety of birds, including chickens, ducks, geese, quails, turkeys, pheasants, pigeons, seagulls, and ostriches, and is susceptible of all ages.

Symptoms, disease elapsed time, and mortality rate of ORT-infected chickens vary widely. Various breeding environmental factors such as high breeding density, poor hygiene or ventilation, poor breeding management, and other respiratory diseases such as coliosis, Newcastle disease, and infectious bronchitis Complex infection can be serious. On the other hand, in the case of mixed infection with various respiratory diseases, the growth rate of ORT bacteria is very slow and the proliferative power is low, making it very difficult to isolate and identify ORT bacteria.

Clinical symptoms and autopsy findings of ORT are similar to other bacterial or viral infections, so the final judgment is possible through laboratory diagnosis. Especially daejanggyunjeung of bacterial respiratory disease of birds and ORT similar lesions, such as: (Avibacterium paragallinarum woninche) (woninche: E. coli), fowl cholera (woninche:: Pasteurella multocida), rime Pasteurella infections (woninche Riemerella anatipestifer), infectious and collaboration similar to that, it is necessary to differentiate As a laboratory diagnosis of ORT infection, it is finally identified using bacterial isolation and culture, biochemical properties or genetic characteristics. Cultivation and identification of the causative agent necessary for the diagnosis of ORT infection is difficult and takes a long time compared to other bacteria. _{It requires carbon dioxide (CO 2} ) to isolate and culture the causative agent, and it is a difficult-to-cultivate slow-growing bacterium, which may interfere with diagnosis due to overgrowth of other faster-growing bacteria. In addition, the diagnostic process requires selection media, biochemical test equipment, and equipment or reagents for genetic testing, and it takes a long time for diagnosis, so it is not easy to diagnose in a general laboratory. In addition, it is necessary to differentiate it from the causative agents of other diseases, such as infectious coliza, coliosis, poultry cholera, and limerella, which have similar clinical symptoms. Therefore, it is necessary to develop an effective diagnostic method with high sensitivity to control ORT infection due to the characteristics of strains that are difficult to separate and culture.

Conventional PCR method (Avian Diseases 56, 654-658, 2012; Veterinary Research Forum, 2016, 7(4) 341 - Applied to 16S rRNA gene target primer (Avian Pathology, 28, 217-227, 1999) as an ORT causative agent diagnosis method) 346) and the real-time PCR method of the 16S rRNA gene target (Avian Diseases 57, 663-666, 2013) and the conventional PCR method of the rpoB gene target (BMC Microbiology, 19:31, 2019) have been disclosed, however, the methods presented above are There are limits to the application of some domestic isolates, and there are also problems in specificity and sensitivity.

Therefore, it is necessary to develop a highly sensitive diagnostic method due to the nature of the strains that are difficult to separate and culture. In particular, it is urgent to improve or supplement the detection efficiency of the ORT gene diagnostic method so that it can be applied to domestic isolates.

The present application intends to provide a means for detecting both Ornithobacterium rhinotracheale ( ORT) strains with high sensitivity and specificity, including standard strains and domestic isolates.

In one aspect, the present application provides a first primer set of SEQ ID NO: 1 and SEQ ID NO: 2 or a complementary sequence thereof; a second primer set of SEQ ID NO: 1 and SEQ ID NO: 3 or a complementary sequence thereof; And it provides a primer set for detecting Ornitobacterium rhinothrachia selected from the group consisting of a third primer set of SEQ ID NO: 5 and SEQ ID NO: 6 or a complementary sequence thereof.

In one embodiment, the primer set according to the present application can be used for nucleic acid amplification or real-time nucleic acid amplification, and when used for real-time nucleic acid amplification, the first and second sets and the third primer set are SEQ ID NO: 4, SEQ ID NO: 4, respectively and a probe of SEQ ID NO: 7.

The primer set according to the present application is used to detect Ornitobacterium rhinothrachia infection or infection, and because it causes respiratory symptoms such as pneumonia and stomatitis, growth retardation, decreased egg production, and death due to infection, the infection itself is prevented early. It is possible to prevent damage caused by infection in advance by detecting the

The primer set according to the present application can detect all kinds of Ornitobacterium rhinothrachia of Table 2 including standard strains and domestic isolates of Ornitobacterium rhinothrachia.

In another aspect, the present application also provides a composition or kit for detecting Ornitobacterium rhinothrachia comprising the primer set according to the present application. The compositions and kits according to the present application may be usefully used for detecting Ornitobacterium rhinothrachia infection or infection as disclosed herein.

In another aspect, the present application provides a biological sample derived from algae requiring detection of Ornitobacterium rhinothrachia; and a first primer set of SEQ ID NO: 1 and SEQ ID NO: 2 or a complementary sequence thereof in the biological sample; a second primer set of SEQ ID NO: 1 and SEQ ID NO: 3 or a complementary sequence thereof; And comprising the step of specifically amplifying Ornitobacterium rhinothrachia using a primer set selected from the group consisting of a third primer set of SEQ ID NO: 5 and SEQ ID NO: 6 or a complementary sequence thereof, A method for detecting Leum rhinothrachia is provided.

The method according to the present disclosure can be performed using a variety of biological samples, for example, avian tears, infraorbital sinuses, nasal passages, tracheal exudates, lungs, or swabs.

In one embodiment, the detection in the method according to the present application may be performed by PCR or real-time PCR.

The method according to the present application is performed by PCR in one embodiment, and the analysis result can be read by electrophoresis, and in this case, the electrophoresis result is 271 bp, 280 bp, and detection of the 169 bp product is indicative of the presence or infection of Ab. paragalinarum in the biological sample.

The method according to the present disclosure is performed by real-time PCR in another embodiment, wherein the first and second primer sets further comprise a probe of SEQ ID NO: 4, and the third primer set further comprises a probe of SEQ ID NO: 7, , the Cq value of the real-time PCR as a result of performing the real-time PCR is ≤35 indicates the presence or infection of Ornitobacterium rhinothrachia in the biological sample.

The primer set according to the present application can detect all kinds of Ornitobacterium rhinothrachia of Table 2 including standard strains and domestic isolates of Ornitobacterium rhinothrachia.

The primer set designed to specifically detect Ornitobacterium rhinothrachia according to the present application can quickly and accurately detect the presence or absence of various ORTs with high sensitivity in a single test. Accordingly, it is possible to effectively control Ornitobacterium rhinothrachia infection, which causes economic damage such as a decrease in egg production rate and growth delay.

1 is a result of agarose gel electrophoresis of various Ornitobacterium rhinothrachia detection specificities using first, second and third primer sets according to an embodiment of the present application. 1, a, b and c are the results of using the first, second and third primer sets, respectively, and the numbers on the gel are as follows: M, 100 bp DNA ladder marker; 1, ATCC51463 (Type strain); 2. ATCC51464; 3. ATCC51465; 4, 01642 ; 5, 02300; 6, 03126; 7, 14HDS; 8, 15CY; 9, 15KH; 10, 16AD002; 11, 16JH ; 12, 16NE; 13, 17AD002; 14, 17BS; 15, 17JM ; 16, 18R072; 17, 19R039; 18, 19KR; 19, 19AD065; 20, negative control (N).
2 is a result of agarose gel electrophoresis showing results of detection of bacteria and viruses other than ORT bacteria requiring differentiation using the first, second, and third primer sets according to an embodiment of the present application. In FIG. 2, ab and c are the results of using the first, second and third primer sets, respectively, and the numbers on the gel are as follows: M, 100 bp DNA ladder marker; 1, Avibacterium avium (NCTC 11297), 2. Avibacterium gallinarum (NCTC 11188); 3. Avibacterium volantium (NCTC 3438); 4. Avibacterium paragallinarum (ATCC 29545); 5, Avibacterium paragallinarum (ATCC 29975); 6. Gallibacterium anatis biovar anatis (NCTC 11413); 7, Pasteurella multocida subsp. multocida (ATCC 43137); 8, Pasteurella multocida subsp. septica (ATCC 51687); 9, Pasteurella multocida subsp. gallicida (ATCC 51689); 10, Pasteurella avium biova r 2 (CCUG 16497); 11, Pasteurella canis Muters et al. (ATCC 43326); 12, Riemerella anatipestifer (ATCC 11845); 13, Escherichia coli (ATCC 25922); 14, Enterococcus faecalis (ATCC 29212); 15, Salmonella Pullorum (ATCC 10398); 16, Salmonella Gallinarum (ATCC 98252); 17, Staphylococcus epidermidis (ATCC 12228), 18, Mycoplasma gallisepticum (ATCC 15302); 19, Mycoplasma synoviae (WVU 1853), 20, ILT virus (Nobilis ILT); 21, Fowl Pox virus (Himbaek chickenpox vaccine); N, negative control; P, ORT positive control (ATCC51463).
3 is an agarose gel electrophoresis result for measuring the detection sensitivity of ORT bacteria of three kinds of ORT novel primer set targeting the 16S rRNA gene according to an embodiment of the present application. 3, a, b, and c are results of using the first (O1-C), second (O2-C), and third (O3-C) primer sets, respectively. I. This is the experimental result for ORT DNA detection ability, and the number on the gel is as follows, M, 100 bp DNA ladder marker; 1, 10 ng/μl; 2, 1 ng/μl; 3, 100 pg/μl; 4, 10 pg/μl; 5, 1 pg/μl; 6, 100 fg/μl; 7, 10 fg/μl; 8, 1 fg/μl; N, negative control; P. positive control; Ⅱ : Experimental results for the detection sensitivity of ORT bacteria (CFU), the numbers indicate the following: M, 100 bp DNA ladder marker,; 1, 6*10 ⁶ CFU/ml: 2, 6*10 ⁵ CFU/ml: 3, 6*10 ⁴ CFU/ml: 4, 6*10 ³ CFU/ml; 5, 6*10 ² CFU/ml; 6, 6*10 ¹ CFU/ml: 7, 6*10 ⁰ CFU/ml; 8, 6*10 ^-1 CFU/ml; N, negative contrast.
4 is an agarose gel electrophoresis result for sensitivity detection of the previously published PCR method (BMC Microbiology, 19:31, 2019) using a primer set targeting the ORT rpoB gene. I : Experimental results for PCR infectious cholinergic gene (DNA) detection sensitivity, each number of the gel is as shown in Fig. 3 I ; Ⅱ : This is the experimental result for the detection sensitivity of ORT bacteria (CFU), and each number of the gel is as shown in Fig. 3 Ⅱ .
5 is an agarose gel electrophoresis result applied to a domestic isolate (17JM) using the existing ORT PCR method and the method according to the present application, and the numbers indicate the following. M, 100 bp DNA ladder marker; 1, ORT domestic isolate (17JM); 2. Voice; 3, ORT standard note (ATCC51463); A, condition I (Avian Diseases 56, 654-658, 2012); B, condition II (Veterinary Research Forum, 2016, 7(4) 341-346); C. Condition Ⅲ (BMC Microbiology, 19:31, 2019 ; D. New O1-C condition; E, New O2-C condition; F, novel O3-C condition. 17JM is an ORT domestic isolate, long time (45 cycles) A positive reaction was shown in condition I, which requires

This application is based on the discovery of a genetic diagnostic technology that can quickly and accurately detect the presence or absence of Ornithobacterium rhinotracheale (ORT) with high sensitivity with a single test.

Accordingly, in one aspect, the present application relates to a primer and a probe for detecting ORT of a sequence as shown in the table below.

[Table 1]

ORT is a bacterium that causes respiratory diseases in birds worldwide. It causes respiratory symptoms such as pneumonia and stomatitis in poultry such as chickens and turkeys, growth retardation, decreased egg production rate, and death, causing economic damage to poultry farms. Until recently, it has been continuously occurring in Korea and has a direct impact on the poultry industry.

The causative bacteria are Gram-negative bacteria, non-spore-forming, non-motile bacilli (rod-shaped), and taxonomically belonging to the rRNA superfamily V of the Cythophaga-Flavobacterium-Bacteroides phylum. It has been isolated from a variety of birds, including chickens, ducks, geese, quails, turkeys, pheasants, pigeons, seagulls, and ostriches, and is susceptible of all ages. Clinical symptoms of broilers generally appear at 3-6 weeks, with a mortality rate of 2-10%, and cause lethargy, reduced feed intake, decreased weight gain, and temporary runny nose and sneezing followed by facial edema. Infection at a young age can lead to sudden death. In broiler breeders, it is infected just before the spawning peak or tidal birth, which causes a decrease in feed intake and a slight increase in mortality along with weak respiratory symptoms. In addition, a decrease in egg laying rate, decrease in egg size, and deterioration of eggshell quality may occur. In laying hens, decreased egg production, increased number of malformed eggs, and increased mortality may be associated. The main autopsy findings show pneumonia, pleurisy, and gassing in broilers. In particular, white creamy exudates in the form of bubbles are detected in the abdominal sac, and most are accompanied by unilateral pneumonia.

Symptoms, disease elapsed time, and mortality rate of ORT-infected chickens vary widely. Various breeding environmental factors such as high breeding density, poor hygiene and ventilation, poor breeding management, and other respiratory diseases such as coliosis, Newcastle disease, and infectious bronchitis Complex infection can be serious. On the other hand, in the case of mixed infection with various respiratory diseases, the growth rate of ORT bacteria is very slow and the proliferative power is low, making it very difficult to isolate and identify ORT bacteria.

Clinical symptoms and autopsy findings of ORT are similar to other bacterial or viral infections, so the final judgment is possible through laboratory diagnosis. Escherichia coli, a bacterial respiratory disease similar to ORT in birds (caused by:E. coli),Poultry cholera (caused by:Pasteurella multocida), Limerella infection (cause:Riemerella anatipestifer), Infectious coliza (causative agent:Avibacterium paragallinarum)Such as It is similar to a lesion, so differentiation is necessary. As a laboratory diagnosis of ORT infection, it is finally identified using bacterial isolation and culture, biochemical characteristics or genetic characteristics. Cultivation and identification of the causative agent necessary for the diagnosis of ORT infection is difficult and takes a long time compared to other bacteria. Carbon dioxide (CO) for selective separation and culture of bacteria₂), which is a slow-growing, difficult-to-culture bacterium, and overgrowth of other faster-growing bacteria can interfere with diagnosis. In addition, it is difficult to diagnose in a general laboratory because the diagnostic process requires a selection medium, biochemical test equipment, equipment or reagents necessary for genetic tests, and it takes a long time for diagnosis. In addition, it is necessary to differentiate it from other disease causative agents, such as infectious coliza, poultry cholera, Escherichia coliosis, and Limerella, which have similar clinical symptoms. Therefore, it is necessary to develop an effective diagnostic method with high sensitivity for the control of ORT infection due to the nature of strains that are difficult to separate and culture.

ORT Conventional PCR diagnostics to existing woninche diagnosis is a 16S rRNA gene primers developed to target (Avian Pathology, 28, 217-227, 1999) using the PCR method (Avian Diseases 56, 654-658, 2012 and Veterinary Research Forum, 7(4) 341-346, 2016) and PCR targeting the rpoB gene (BMC Microbiology, 19:31, 2019) are applicable. However, the proposed methods require a long time of 45 cycles (Avian Diseases 56, 654-658, 2012) or have low sensitivity to domestic isolates (Veterinary Research Forum, 7(4) 341-346, 2016; BMC Microbiology, 19: 31, 2019) were shown. Meanwhile , a real-time PCR method targeting the 16S rRNA gene has been developed (Avian Diseases 57, 663-666, 2013), but this method also has low sensitivity to some domestic isolates. Therefore, it is necessary to develop a highly sensitive diagnostic method due to the nature of the strains that are difficult to separate and culture. In particular, it is urgent to improve or supplement the detection efficiency of the ORT gene diagnostic method so that it can be applied to domestic isolates.

In order to select primers applicable to domestic isolates, 16 ORT strains isolated in Korea since 2001 were collected and nucleotide sequence analysis of 16S rRNA and rpoB genes was performed. As a result, genetic mutations were observed in all of the primer regions of genes suggested by the existing diagnostic methods, confirming that there is a limit to the application of domestic isolates. Therefore, as there is a need to develop primers suitable for domestic strains, the 16S rRNA gene with excellent sensitivity was selected as the final primer candidate gene. Through analysis, a primer was selected as a common site without genetic variation.

The primer set according to the present application targets the 16S rRNA region of Ornitobacterium rhinothrachia.

In one embodiment, the primer set according to the present application comprises a first primer set of SEQ ID NO: 1 and SEQ ID NO: 2 or a complementary sequence thereof; a second primer set of SEQ ID NO: 1 and SEQ ID NO: 3 or a complementary sequence thereof; And it relates to a primer set for ORT detection selected from the group consisting of a third primer set of SEQ ID NO: 5 and SEQ ID NO: 6 or a complementary sequence thereof.

The primer set according to the present disclosure can be used in various nucleic acid amplification techniques.

In one embodiment the primer set according to the invention is used in particular for PCR.

As used herein, the term PCR (Polymerase Chain Reaction) is a method widely known in the art as a method for exponentially amplifying a starting nucleic acid material by synthesizing a nucleic acid chain using a polymerase. It is a concept that includes both qualitative analysis PCR to confirm, quantitative PCR to measure the amount of a specific nucleic acid, and real-time PCR to enable qualitative and quantitative analysis by tracking the PCR process in real time.

As used herein, the term real-time PCR is a method that can track the process in real time, and the numerical value used for qualitative or quantitative analysis is Ct or Cq. The terms are used interchangeably with each other, and the terms indicate the number of PCR cycles at the point where the amplification curve reaches a specific threshold in the amplification process, and the threshold is usually the point at which the curvature of the amplification curve becomes the maximum. It is determined and is in exponential phase. Quantification or quantitative analysis of nucleic acids using this value may be performed according to the manual of the real-time PCR device manufacturer.

In one embodiment according to the present application, in addition to the primer set in real-time PCR, a probe is further used.

Herein, the probe binds between the primer sets used together as oligonucleotides, and it is a principle that can detect and quantify the fluorescence material released from the probe according to the reaction of PCR. Accordingly, the 5' and 3' probes may be labeled with an appropriate fluorescent material, such as in the examples herein.

In one embodiment, the probe according to the present application is represented by SEQ ID NO: 4 or SEQ ID NO: 7, and in one embodiment, the SEQ ID NO: 4 is used together with the first and second primer sets, and the probe of SEQ ID NO: 7 is the first Used with 3 primer sets.

The primer or primer and probe set according to the present application is capable of detecting various Ornitobacterium rhinothrachia bacteria including standard strains and domestic strains. Since ORT causes serious damage by single or mixed infection by respiratory bacteria and viruses, it is very important to quickly detect ORT bacteria in the early stage of infection.

The primer or primer and probe set according to the present application can detect various ORTs described in Examples (Table 2) of the present application, including standard strains and domestic strains, and can be used very effectively in determining ORT infection.

The primer set or primer and probe set according to the present disclosure can be used for diagnosis of ORT infection, whether or not an infection is present or a disease associated with infection, for example pneumonia or gassenteritis.

ORT infection causes respiratory symptoms such as pneumonia and stomatitis, growth retardation, decreased egg production, and death.

Biological samples in which the primers and methods according to the present application can be used are not particularly limited as long as detection of Ornitobacterium rhinothracia is required, and bird tears, runny nose, hypoorbital sinuses, nasal sinuses, trachea, lungs, or air sac exudates or swabs including, but not limited to, water.

Birds applied to the primer or method according to the present disclosure include various birds such as chickens, ducks, geese, quails, turkeys, pheasants, pigeons, seagulls, and ostriches. In one embodiment, in particular chicken. In another aspect, the present application provides a biological sample derived from algae requiring detection of Ornitobacterium rhinothrachia; and a first primer set of SEQ ID NO: 1 and SEQ ID NO: 2 or a complementary sequence thereof; a second primer set of SEQ ID NO: 1 and SEQ ID NO: 3 or a complementary sequence thereof; And it relates to a method for detecting ORT, comprising the step of specifically amplifying the ORT using a primer set selected from the group consisting of a third primer set of SEQ ID NO: 5 and SEQ ID NO: 6 or a complementary sequence thereof.

The biological sample used in the method according to the present application and the detection method using PCR and real-time PCR may be referred to as previously described.

In one embodiment, the method according to the present application further comprises the step of determining the presence or absence of ORT in the sample based on the electrophoresis result of analyzing the nucleic acid amplification product, and the first, second and third electrophoresis results Detection of 271 bp, 280 bp, and 169 bp products, respectively, per primer set indicates the presence of ORT in the biological sample or that the subject from which the biological sample was derived was infected with the ORT.

In another embodiment, the method according to the present disclosure analyzes nucleic acid amplification by real-time PCR, in which case the first and second primer sets further include a probe of SEQ ID NO: 4, and the third primer set further include a primer set of SEQ ID NO: 7. . As a result of the analysis, the Cq value of real-time PCR was ≤35 indicates the presence of ORT in the biological sample or that the subject from which the biological sample was derived is infected with the ORT.

Hereinafter, examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Example 1. Ornitobacterium rhinothrachia ( Ornithobacterium rhinotracheale, ORT) specific primer design

ORT detection using existing diagnostic methods has reduced sensitivity for some domestic isolates, so it is necessary to develop a sensitive diagnostic method that can be applied to all domestic isolates. In addition, a differential diagnosis is necessary because the clinical symptoms and colony form are similar to those of infectious coliza, limerella, and poultry cholera, which are respiratory bacterial diseases of birds. Therefore, the development of primers or primers and probes capable of sensitively detecting all ORT isolates including standard strains was needed, and primers were designed to achieve this purpose.

In order to design primers that are specific to ORT strains and simultaneously detect various strains including domestic isolates and standard strains, here, we analyzed the base sequence of 16 ORT domestic isolates (see Table 2) since 2001. 16S ribosomal RNA ( 16S rRNA ) and β-subunit of RNA polymerase ( rpoB ) sequences were obtained through In addition, as described below, 16S rRNA and β-subunit of RNA polymerase ( rpoB ) gene sequences for various ORT bacteria registered with NCBI were obtained from NCBI.

In the case of the rpoB gene, the nucleotide sequence of 16 domestic isolates and 3 standard strains analyzed here and the nucleotide sequence of 47 strains (GenBank NOs: MH746625 ~ MH746671) obtained from NCBI were compared. No usable primer sequences were derived.

Therefore, a new primer capable of sensitively and specifically detecting ORT standard strains and domestic isolates in the 16S rRNA region was prepared. For each primer sequence, 16S rRNA gene sequencing was determined for 16 domestic isolates and 3 standard strains in Table 2, and 35 ORT strains (HF548213, HM246652, JF501953, JF501958, JF810490, JF810496, KC305846, KC305849, KC454287, KC454294, KC454301, KC454305, KC454312, KP128016, KX998670, KX998677, KX998678, KX998679, KX998685, KX998687, KX866987, KX8689, KX8984, KX8699870, KX89870, KX99870 Primers were designed through nucleotide sequence comparison analysis with KY809791, KY809793, KY809798, KY809801, MG149563), and NCBI (National Center for Biotechnology Information) Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi website) ), there was no matching nucleotide sequence except for the relevant strain. Each primer set was designed and combined to be optimized for PCR reaction, and showed excellent effects in terms of specificity and sensitivity.

Primer and probe sequences are as described in Table 1 above.

5' and 3' of the probes of SEQ ID NO: 4 and SEQ ID NO: 7 were used by labeling them with fluorescent substances HEX and BHQ (Black Hole Quencher)-1, respectively.

Example 2. Ornitobacterium rhinothrachia using PCR ( Ornithobacterium rhinotracheale, ORT) detection

(1) Securing ORT test strains and preparing samples

As shown in Table 2, standard strains were obtained from ATCC (American Type Culture Collection), and domestic isolates were used with strains stored in APQA (Agricultural, Forestry and Livestock Quarantine Headquarters).

DNA was extracted from standard strains and domestic isolates using the DNeasy ^ⓡ Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer's method.

[Table 2] ORT strains used for specificity test

(2) Securing test strains other than ORT strains and preparing samples

Other bacteria and virus strains used for specificity testing other than ORT (see Table 3) contain bacteria and viruses that can negatively affect the accurate diagnosis of ORT by coexisting in colony shape or genetically similar bacteria and sampling sites. included.

The strain used was in the form of a colony or ^{DNA was extracted using the method of the DNeasy ⓡ} Blood & Tissue Kit (Qiagen, Germany) manufacturer. ATCC (American Type Culture Collection), NCTC (National Collection of Type Cultures), CCUG (Culture Collection University of Gothenburg) and domestic commercially available vaccine strains were secured and used. For test DNA, for bacteria, ^{DNA was extracted using the DNeasy ⓡ} Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer's method, and for virus samples, Patho Gene-Spin™ DNA/RNA Extraction Kit (iNtRON Biotechnology, Korea) was used to extract DNA.

[Table 3] Ornitobacterium rhinothrachia (ORT) and other strains used for specificity test

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In the PCR of the present invention, the target sequence may be amplified by performing an amplification reaction on the DNA extracted from the sample using an ORT discrimination primer having the following nucleotide sequence. AccuPower ^® PCR PreMix (Bioneer, Korea) was used for PCR.

The PCR composition and conditions used in this example are as follows.

[Table 4-1] PCR reaction mixture composition

[Table 4-2] Primer information and PCR product size

[Table 5] PCR conditions

After PCR was completed, the gene amplification product of the test sample was electrophoresed on an agarose gel, and the size of the amplified product was confirmed using a UV transilluminator.

(2) specificity and sensitivity results

The specificity results are shown in FIGS. 1 and 2 .

As a result of PCR, when bands of 271 bp, 280 bp, and 169 bp were confirmed for each target and primer in the electrophoresis gel as shown in Table 4-2, it was determined that ORT bacteria were detected. As a result of applying the PCR method developed here to ORT bacteria including standard strains, specific detection was possible for all ORT bacteria, and it did not show cross-reaction with other bacteria and viruses other than ORT bacteria, so the specificity was very high. appeared to be

The sensitivity results are shown in Figs. 3, 4, 5 and Table 6.

[Table 6]

This sensitivity showed a sensitivity improvement of 100 times or more than that of the rpoB gene diagnosis method (BMC Microbiology 19:31, 2019) among the existing methods (see FIGS. 3 and 4 ).

In particular, as a result of comparing the ORT domestic isolate (17JM) with the conventional PCR method (see Fig. 5), a positive reaction was obtained in condition I (Avian Diseases 56, 654-658, 2012) requiring a long time (45 cycles). However, the sensitivity was low in Condition Ⅱ (Veterinary Research Forum, 7(4) 341-346, 2016) and Condition Ⅲ (BMC Microbiology, 19:31, 2019), and the sensitivity was improved as a result of applying this development method (D~F). was confirmed. As a result of gene sequencing analysis of domestic ORT isolates , gene mutations were observed in the previously announced primers of 16S rRNA and rpoB genes, which is believed to have lowered the sensitivity of the existing diagnostic method. Application is considered difficult.

Example 3. Ornitobacterium rhinothrachia detection of various serotypes using real-time PCR

(1) Test strain

For real-time PCR test, specificity tests were performed on Ornitobacterium rhinothrachia standard strains, field isolates, and Ornitobacterium rhinothrachia other strains.

(2) Specificity measurement

For real-time PCR, the probes labeled as shown in Table 6 were used, and the conditions are shown in Table 7. For Realtime PCR, Bio-rad CFX96™ Real-Time System was used.

In the real-time PCR of the present invention, a target sequence can be amplified by using a primer and a probe for ORT discrimination having the following nucleotide sequence in DNA extracted from the sample. For PCR, iQ™ Supermix (Bio-Rad Laboratories, Inc., USA) was used.

PCR compositions and conditions used in this Example are shown in Tables 6 and 7, respectively.

[Table 6-1] Realtime PCR reaction mixture composition

[Table 6-2] Primers used in Realtime PCR

[Table 7] Real-time PCR conditions

In the case of real-time PCR, when the Cycle quantification value (Cq) value was positive (Cq≤35), it was determined as ORT detection. The results are shown in Tables 8, 9 and 10. As a result of applying the PCR method developed here to standard strains and domestic isolated ORT bacteria, specific detection of all ORTs was possible, and no reaction was shown for strains other than ORT (Tables 8 and 9).

In addition, it was confirmed that the sensitivity to some domestic isolates was improved compared to the existing real-time PCR method (Avian Diseases 57, 663-666, 2013) (Table 10). Specifically, the sensitivity of the first (O1-R) set is 10 fg/μl (6*10 ⁰ CFU/ml), the second (O2-R) is 10 fg/μl (6*10 ⁰ CFU/ml), The sensitivity of the third (O3-R) set was 10 fg/μl (6*10 ⁰ CFU/ml).

[Table 8] Comparative evaluation result of ORT detection efficacy by real-time PCR method

[Table 9] Ornitobacterium rhinothrachia (ORT) and other strains used for specificity test

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[Table 10] Comparison between the existing real-time diagnosis method (Avian Diseases 57, 663-666, 2013) and the present diagnosis method

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present application as defined in the following claims are also provided. it belongs to the scope.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

<110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Primers for detecting Ornithobacterium rhinotracheale infection and its use <130> DP202001002P <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ORT16S-F1 <400> 1 gtatgcaact tgccccttatc ag 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ORT16S-R1 <400> 2 ggatcagagt tccctccatt g 21 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ORT16S-R2 <400> 3 gggatggctg gatcagag 18 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> ORT16S-P1 <400> 4 ccctacagat catcgccttg gtagtcc 27 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ORT16S-F2 <400> 5 cgtatgcaac ttgcccttat cag 23 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ORT16S-R3 <400> 6 ctacagatca tcgccttggt agtc 24 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> ORT16S-P2 <400> 7 cccggggaaa ctcggattaa tactcc 26

Claims (17)

A set of primers for detecting Ornithobacterium rhinotracheale (ORT) selected from the group consisting of:
a first primer set of SEQ ID NO: 1 and SEQ ID NO: 2 or a complementary sequence thereof;
a second primer set of SEQ ID NO: 1 and SEQ ID NO: 3 or a complementary sequence thereof; and
A third primer set of SEQ ID NO: 5 and SEQ ID NO: 6 or a complementary sequence thereof.
The method of claim 1,
The primer set is used for nucleic acid amplification or real-time nucleic acid amplification, Ornitobacterium rhinothrachia detection primer set.
3. The method of claim 2,
When used for the real-time nucleic acid amplification, the first and second sets and the third primer set further include probes of SEQ ID NO: 4, SEQ ID NO: 4 and SEQ ID NO: 7, respectively, Ornitobacterium linothrachia A set of primers for detection.
4. The method according to any one of claims 1 to 3,
The primer set is a primer set for detecting Ornitobacterium rhinothrachia, which is used to detect whether Ornitobacterium rhinothrachia is infected.
5. The method of claim 4,
The Ornitobacterium rhinothrachia is a primer set for detecting Ornitobacterium rhinothrachia, which is capable of detecting one or more of the Ornitobacterium rhinothrachia of the following table, including standard strains and domestic isolates:

A composition for detecting Ornitobacterium rhinothrachia comprising the primer set according to any one of claims 1 to 3.
7. The method of claim 6,
The Ornitobacterium rhinothrachia detection is a composition for detecting Ornitobacterium rhinothrachia that is used for diagnosing Ornitobacterium rhinothrachia infection.
A kit for detecting Ornitobacterium rhinothrachia comprising the primer set according to any one of claims 1 to 3.
9. The method of claim 8,
The Ornitobacterium rhinothrachia detection kit is used for the diagnosis of Ornitobacterium rhinothrachia infection.
providing a biological sample derived from an alga that requires detection of Ornitobacterium rhinothrachia; and
a first primer set of primers of SEQ ID NO: 1 and SEQ ID NO: 2 or a complementary sequence thereof in the biological sample; a second primer set of SEQ ID NO: 1 and SEQ ID NO: 3 or a complementary sequence thereof; And comprising the step of specifically amplifying Ornitobacterium rhinothrachia using a primer set selected from the group consisting of a third primer set of SEQ ID NO: 5 and SEQ ID NO: 6 or a complementary sequence thereof, Leeum rhinothrachia detection method.
11. The method of claim 10,
Wherein the biological sample comprises avian tears, hypoorbital sinuses, nasal passages, tracheal exudates, lungs, or air sac swabs (swab), Ornitobacterium linothracia detection method.
11. The method of claim 10,
The detection is by PCR, Ornitobacterium rhinothrachia detection method.
13. The method of claim 12,
The method comprises the steps of performing electrophoresis on the PCR product; and
Ornitobacterium rhinothrachia detection method further comprising the step of determining the presence or infection of Ornitobacterium rhinothrachia in the sample based on the electrophoresis result.
14. The method of claim 13,
As a result of the electrophoresis, detection of 271 bp, 280 bp, and 169 bp products for each of the first, second and third primer sets, respectively, indicates the presence or infection of Ornitobacterium linothracia in the biological sample, Ornitobacterium Rhinothrachia detection method.
11. The method of claim 10,
The detection is by real-time PCR,
The first and second primer sets are a probe of SEQ ID NO: 4, and the third primer set further comprises a probe of SEQ ID NO: 7, Ornitobacterium rhinothrachia detection method.
16. The method of claim 15,
As a result of the real-time PCR, the Cq value of the real-time PCR was ≤35, indicating the presence of Ornitobacterium rhinothrachia in the biological sample.
17. The method according to any one of claims 10 to 16,
The Ornitobacterium rhinothrachia is one or more strains of the strains listed below including domestic isolates, Ornitobacterium rhinothrachia detection method.

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