KR20180110706A - Method for diagnosing subclinical Johne's Disease based on host biomarkers - Google Patents

Abstract

The present invention relates to a composition for diagnosing subclinical johne disease containing an agent, capable of amplifying or detecting a HGF or GBP6 gene, a marker gene for two kinds of subclinical johne disease, a kit for diagnosing subclinical johne disease containing the composition, and to a method for diagnosing subclinical johne disease by using the composition or the kit. When the composition for diagnosing the subclinical johne disease of the present invention is used, it is possible to effectively diagnose the subclinical johne disease, which does not show symptoms of johne even though a patient is infected with a johne disease causative organism, thereby being widely used for prevention through early diagnosis of the johne disease.

Description

[0001] The present invention relates to a method for diagnosing subclinical disease using host biomarkers,

The present invention relates to a method for diagnosing subclinical urinary incontinence, and more particularly, to a subclinical urinary incontinence diagnostic composition comprising a preparation capable of amplifying or detecting GBP6 or HGF gene, , A subclinical urinary incontinence diagnostic kit comprising the composition, and a method of diagnosing subclinical urinary incontinence using the composition or kit.

M. paratuberculosis in ruminants Infections that can occur when a strain is infected are a type of infectious chronic enteritis, and severe diarrhea and weight loss are known symptoms. Because of the high rates of infectious and infectious diseases, the Yonex disease is specifically classified as a statutory infectious disease in most countries. The special drugs used to treat the Yonezawa disease have not yet been developed and it is known that the isolation method according to the early diagnosis is the most effective way to prevent the transmission of Yonezawa disease. However, in order to use such a method, the success rate of early diagnosis of Yonsei disease should be high. Therefore, researches to develop a method for early diagnosis of YSE disease more effectively have been actively conducted.

For example, a method of diagnosing an IVD with an intradermal test based on cellular immunity is used, but in order to overcome the difficulty in diagnosing the IVD using the intradermal reaction method, The development of serological diagnostic methods based on humoral immunity has progressed and various serological diagnostic methods have been developed. However, studies on the diagnostic methods have been continuously required because of the characteristics such as slow cultivation rate and long sub - clinical type of.

Recently, a method of diagnosing Yonezawa disease using genetic markers has been developed. For example, Japanese Laid-Open Patent Application No. 2015-077098 discloses a screening test method for genetic testing of Yonezawa disease, Japanese Laid-Open Patent Application No. 2007-097490 discloses a primer for diagnosis of Yonezawa disease, Is disclosed. However, these methods are mostly used to confirm whether or not an onset disease is a urogenous disease after the occurrence of urogenous disease, and it may be applied to a subclinical period when the onset of the disease is not manifested There was no problem. If it is possible to diagnose the infection of the causative bacteria of the disease in the subclinical period of the disease, it is expected to be able to treat the disease more effectively through the early diagnosis of the disease. However, .

Under these circumstances, the inventors of the present invention have made extensive efforts to develop a method for diagnosing subclinical uremia, and as a result, when the two marker genes, GBP6 or HGF gene, are detected, And completed the present invention.

One object of the present invention is to provide a subclinical vaccine disease diagnostic composition comprising an agent capable of amplifying or detecting a marker gene of a subclinical pathogen causative pathogen.

Another object of the present invention is to provide a subclinical vaccine disease diagnostic kit comprising the composition.

Yet another object of the present invention is to provide a method for diagnosing sub-clinical infectious diseases using the composition or kit.

In order to develop a method for effectively diagnosing subclinical uremia, the present inventors have found that the incidence of the above-mentioned uremia has been confirmed, but the level of expression is significantly increased from the feces or blood of the livestock, (GBP6, HGF), which has been confirmed to be positive in most of the livestock in which the subclinical disease has been identified. Since the expression levels of the two genes were not increased in the livestock showing symptoms of Urea disease and the infection level of the Urea infection was confirmed but the expression level was significantly increased only in the livestock without symptoms of Urea disease, It was confirmed that these two genes can be used as markers capable of diagnosing subclinical diseases.

A method for diagnosing subclinical urinary incontinence in ruminants by measuring the expression levels of the above two genes has not been known in the prior art and was first developed by the present inventor.

In some embodiments for achieving the above object, the present invention, GBP6 (guanylate binding protein family member 6), HGF (hepatocyte growth factor) and subclinical yaw nebyeong disease pathogen is selected from the group consisting of a combination of (M The present invention provides a subclinical urinary incontinence diagnostic composition comprising an agent capable of amplifying or detecting a marker gene of paratuberculosis .

The term "Johne's Disease (Bovine paratuberculosis) "of the present invention refers to a disease caused by oral infections of Mycobacterium mycobacterium ( Mycobacterium avium subsp. Paratuberculosis) , Which is a disease of ruminant animals such as cattle, goats, sheep, goats, etc., which is a serious infectious disease of livestock epidemic prevention. When the causative agent is infected, the disease develops after the subclinical type for about 2 to 3 years. However, the subclinical type does not show the specific pathological symptom of the disease, but the expression of the specific gene The level is increased, and the increase in this level of expression can be confirmed through the fecal or serum of infected livestock.

The term "marker gene of a subclinical infection pathogen causative microorganism" of the present invention refers to a marker gene of a subclinical infection pathogen causative microorganism, which is expressed by a subclinical type in which the symptoms of the disease are not present, , Which is detected as an increased expression level in the feces or serum of livestock infected with the strain, and can be used as a marker for diagnosing subclinical urinary incontinence.

In the present invention, the marker gene of the subclinical infection pathogen causative microorganism is not particularly limited, but the expression level is specifically increased in the subclinical form in which the infectious agent is infected, but the symptoms of the infectious disease are not present, It can be GBP6 (guanylate binding protein family member 6) or HGF (hepatocyte growth factor), which can be detected in whole blood or serum of livestock infected with causative bacteria.

Specific base sequences of these two genes and protein information expressed from these genes are known from databases such as NCBI. For example, the base sequence of GBP6 is GenBank No. 1. NM_001192892, and the nucleotide sequence of HGF is known from GenBank No. 1. NM_001031751.

The term "agent capable of amplifying or detecting a marker gene of a subclinical infection pathogen-causing microorganism" of the present invention means amplification or detection of the gene to identify or measure the expression level of the subclinical expression vector marker gene Means, for example, a primer or a probe.

The term "primer" of the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and having a starting point for template strand copying ≪ / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

In the present invention, the nucleotide sequence of the primer is not particularly limited as long as it can amplify the marker gene of the subclinical genomic DNA by the PCR method. Preferably, the nucleotide sequence of SEQ ID NOS: 27, 28, 47, ≪ / RTI > For example, a primer capable of amplifying the GBP6 gene, which is one of the marker genes of subclinical and pathogenic bacteria, may be a primer base pair consisting of SEQ ID NOS: 47 and 48, and a primer capable of amplifying the HGF gene may be a nucleotide sequence 27, and 28, respectively.

The term "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to a gene or mRNA, including oligonucleotide probes, For example, in the form of a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection.

In the present invention, the probe may be complementary to a whole or a partial sequence of the marker gene of the subclinical infection pathogen causative microorganism like the above-mentioned primer to detect a marker gene of the subclinical infection pathogen Its base sequence is not particularly limited.

In another aspect, the present invention provides a kit for the diagnosis of subclinical urinary incontinence comprising the composition.

The term "Yoneh disease" and "subclinical Yonezawa disease ", and" marker genes of subclinical Yonezawa disease causing pathogens "

The kit of the present invention can be used for the diagnosis of subclinical diseases by measuring the expression level of the marker gene of the subclinical infection pathogen causative microorganism from the feces or serum samples of the ruminant suspected of infecting the causal agent of the pathogen. A primer or a probe for detecting the marker gene of the subclinical infection pathogen may be used as well as one or more other component compositions, solutions or devices suitable for the analysis method.

As a specific example, the subclinical vaccine diagnostic kit of the present invention may be a kit containing essential elements necessary for performing PCR analysis or multiplex-PCR analysis. The kit may further comprise an enzyme such as a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), Taq polymerase in addition to each primer pair specific for each marker gene , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like.

As another example, the kit of the present invention may contain the necessary elements necessary for performing DNA chip analysis. The kit for analyzing a DNA chip comprises a substrate to which a probe capable of complementarily binding with a marker gene or a fragment thereof of the subclinical infection pathogen is attached, and a reagent, a preparation, and an enzyme for producing the fluorescent tag probe . In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

In another aspect of the present invention, there is provided a method for diagnosing a subclinical incontinence disease using the composition or kit.

The term "Yoneh disease" and "subclinical Yonezawa disease ", and" marker genes of subclinical Yonezawa disease causing pathogens "

Specifically, the method for diagnosing the subclinical incontinence disease of the present invention comprises the steps of: (a) obtaining cDNA from a sample derived from a ruminant animal suspected of having an incidence of incontinence; (b) measuring the expression level of a marker gene of a subclinical infection-causing pathogen from the cDNA obtained using the composition or kit; And (c) if the measured expression level of the marker gene is higher than that expressed in a normal ruminant, the ruminant is infected with a pathogenic bacterium causing a pathogenic bacterium to be a subclinical type of bacteriophage do. In this case, the composition, kit, and genetic marker genes are the same as described above, and examples thereof include feces, blood, plasma, serum, lymph, cerebrospinal fluid, isolated tissues, isolated cells, saliva, And is not particularly limited.

The use of the composition for the diagnosis of subclinical urinary incontinence of the present invention enables effective diagnosis of subclinical urinary incontinence which does not cause urinary incontinence even though it is infected with the causative agent of urinary incontinence, There will be.

Figures 1 and 2 are graphs showing changes in gene expression levels in infected soldier groups 1, 2, and 3 (* P < 0.05).
FIG. 3 is a graph showing changes in the expression levels of genes between ELneg, EL45, EL100, and EL200 classified according to the ELISA level. (* P < 0.05, * P < 0.01).

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

Example  One: Subclinical type Yo Fourth  For diagnosis Marker  Selection

It is well known that Yonezawa's disease gradually develops after about two to three years of subclinical infection after the causative agent has been infected. During the subclinical period of IV disease, there is no specific onset of the disease, but due to the infected strain, it is possible to detect the gene derived from the infected strain in the feces or serum.

Thus, the individuals with Ipek disease were classified into four groups according to the infection stage through fecal PCR and serum ELISA. Specifically, Group 1 is a fecal PCR positive, serum ELISA positive population, Group 2 is fecal PCR positive, serum ELISA negative, Group 3 is fecal PCR negative, serum ELISA positive, Group 4 is fecal PCR and serum ELISA negative control. Of the 4 groups, Group 2 was subclinical infectious disease, but Group 1, which was found in both feces and serum, was infected with Yonex disease, but not in serum, Group 4, which is not identified in the serum, corresponds to a normal control group. In addition, according to the ELISA values, the infectious groups were classified into four groups. Specifically, ELneg has an ELISA value of less than 45, EL45 has a value of 45 or more and less than 100, EL100 is 100 or more and less than 200, and EL200 is 200 or more.

Total RNA was extracted from whole blood of each individual in the classified group, purified, and cDNA was synthesized using the Quantit reverse transcription kit. Then, 23 kinds of genes known to be related to Joné disease (Real-time RT-PCR) HLGF, MPL, PIGR, CD14, CHI3L1, LTF, ELANE, S100A8, S100A9, TFRC, and GBP6 The expression levels of the genes measured in Groups 1 to 3 were compared with the expression levels of the genes measured in Group 4 (control group), and genes in which expression level and expression level of the control group were distinguished among the groups were selected (Figs. 2). At this time, β-actin was used as an internal control, and primers for measuring the expression levels of the two genes were as follows.

β-Actin_F: 5'-CGCACCACTGGCATTGTCAT-3 '(SEQ ID NO: 1)

β-Actin_R: 5'-TCCAAGGCGACGTAGCAGAG-3 '(SEQ ID NO: 2)

KLRB1_F: 5'-AACCAGCATGGATGCTCAAGA-3 '(SEQ ID NO: 3)

KLRB1_R: 5'-TCTCGGATTCGTTTCCAGTGC-3 '(SEQ ID NO: 4)

KLRC1_F: 5'-ACCCAGGGATTCTGGCATTG-3 '(SEQ ID NO: 5)

KLRC1_R: 5'-ACTGGTCTCTCTGTTGCTTCC-3 '(SEQ ID NO: 6)

CCL4_F: 5'-TCCACTTGCAAACTACAGATAACA-3 '(SEQ ID NO: 7)

CCL4_R: 5'-ACCCAACAGCATCCAAACTCA-3 '(SEQ ID NO: 8)

CCL5_F: 5'-CCCAGCCAGCTGTGGTATTC-3 '(SEQ ID NO: 9)

CCL5_R: 5'-CTCGGAGCAGCTCAGTTCAA-3 '(SEQ ID NO: 10)

CXCL9_F: 5'-CTTCTGCCTCCCCATATGCC-3 '(SEQ ID NO: 11)

CXCL9_R: 5'-ACTTCTTCTCTGGGTTGGCG-3 '(SEQ ID NO: 12)

CXCL10_F: 5'-CTCTGACTGGAGTTCAAGGAGT-3 '(SEQ ID NO: 13)

CXCL10_R: 5'-TCCCTTGGCTGGTGTTGATG-3 '(SEQ ID NO: 14)

CXCR3_F: 5'-AAGCATGAGTGTGAAGGGCA-3 '(SEQ ID NO: 15)

CXCR3_R: 5'-AGGGAAACCTTGAACAATTGCAG-3 '(SEQ ID NO: 16)

IFNG_F: 5'-ACATAGCCAAGTCGGTCACG-3 '(SEQ ID NO: 17)

IFNG_R: 5'-CTCGGAACTTGACACCCACA-3 '(SEQ ID NO: 18)

MMP9_F: 5'-CCCGGATCAAGGATACAGCC-3 '(SEQ ID NO: 19)

MMP9_R: 5'-GGGCGAGGACCATACAGATG-3 '(SEQ ID NO: 20)

SERPINE1_F: 5'-CTGCGAAATTCAGGATGCGG-3 '(SEQ ID NO: 21)

SERPINE1_R: 5'-GGGTGAGAAAACCACGTTGC-3 '(SEQ ID NO: 22)

IL10_F: 5'-TCTGCCCTGCGAAAACAAGA-3 '(SEQ ID NO: 23)

IL10_R: 5'-CCTCTCTTGGAGCTCACTGAA-3 '(SEQ ID NO: 24)

HP_F: 5'-GGCCCCCGAGATTGCTAATA-3 '(SEQ ID NO: 25)

HP_R: 5'-GGGCAGCTGTCATCTTCTTCA-3 '(SEQ ID NO: 26)

HGF_F: 5'-CGACGGGCTCTTTTAGGCTC-3 '(SEQ ID NO: 27)

HGF_R: 5'-CTGTCCTTCTGCATAGGGGATG-3 '(SEQ ID NO: 28)

PIGR_F: 5'-GAGTTTGCCACCACTACCGA-3 '(SEQ ID NO: 29)

PIGR_R: 5'-TCCTTGGACGACCTCTTTGC-3 '(SEQ ID NO: 30)

MPO_F: 5'-GCCCTGGAACTTCAGAGAGATG-3 '(SEQ ID NO: 31)

MPO_R: 5'-GTCAGCACCACCGAGGTATC-3 '(SEQ ID NO: 32)

CD14_F: 5'-TGTCTGACAATCCCAGTCTCG-3 '(SEQ ID NO: 33)

CD14_R: 5'-CGCTAGATATTGGAGGGCCG-3 '(SEQ ID NO: 34)

CHI3L1_F: 5'-ATAAGAGAAGCTGGAGGCCCA-3 '(SEQ ID NO: 35)

CHI3L1_R: 5'-CCACAAAACCTGTATGAGCCG-3 '(SEQ ID NO: 36)

LTF_F: 5'-GGAAGCAGATGCCCTGAACT-3 '(SEQ ID NO: 37)

LTF_R: 5'-AGGTACCCTTCCGTTGGTCT-3 '(SEQ ID NO: 38)

ELANE_F: 5'-TTGTCTGAACGGCCTGAACT-3 '(SEQ ID NO: 39)

ELANE_R: 5'-CTCTGAACATCTGCCGGGTT-3 '(SEQ ID NO: 40)

S100A8_F: 5'-ATTTTGGGGAGACCTGGTGG-3 '(SEQ ID NO: 41)

S100A8_R: 5'-ACGGCGTGGTAATTCCCTTT-3 '(SEQ ID NO: 42)

S100A9_F: 5'-AGGCTACGGGAAGGGCAG-3 '(SEQ ID NO: 43)

S100A9_R: 5'-GCTGGCCTCCTGATTAGTGG-3 '(SEQ ID NO: 44)

TFRC_F: 5'-CAAAGTTTCTGCCAGCCCAC-3 '(SEQ ID NO: 45)

TFRC_R: 5'-AACAGAAAGAGACCGCTGGG-3 '(SEQ ID NO: 46)

GBP6_F: 5'-GAGTGAAGGACAAGCAGCCT-3 '(SEQ ID NO: 47)

GBP6_R: 5'-CAGACAGAAACCTCTGTATTCCG-3 '(SEQ ID NO: 48)

FIGS. 1 and 2 are graphs showing changes in gene expression levels in infectious army groups 1, 2, and 3, and FIG. 3 is a graph showing changes in gene expression levels among ELneg, EL45, EL100, FIG.

The results of analyzing the results of the above-mentioned FIGS. 1 to 3 are as follows:

In group 2, which belongs to the subclinical YPE, 17 genes (TFRC, S100A8, S100A9, MPO, GBP6, LTF, KLRB1, SERPINE1, PIGR, IL-10, CXCR3, CD14, MMP9, ELANE, CHI3L1, HP, (HGF, HP, TFRC, and HGF), and the expression of six genes (CCL4, CCL5, CXCL9, CXCL10, IFN-γ and KLRC1) GBP6, LTF, and CXCR3) showed statistically significant differences.

Expression of 12 genes (S100A9, TFRC, GBP6, KLRB1, SERPINE1, S100A8, HGF, PIGR, MPO, LTF, TFRC, and CXCR3) was increased by the ELISA (HGF, GBP6, and LTF) showed statistically significant differences.

From the above results, we confirmed HGF, GBP6 and LTF genes, which showed statistically significant differences when classified as PCR and ELISA positive results and genes classified by ELISA level among genes with significantly increased expression Respectively.

Therefore, by observing the expression of HGF or GBP6 gene, it is possible to effectively diagnose sub-clinical type IV disease which does not show symptoms of Yonsei disease even though it is infected with the causative agent of Yonsei disease, so that it can be widely used for prevention through early diagnosis of Yonsei disease .

<110> Seoul National University R & DB Foundation <120> Method for diagnosing subclinical Johne's Disease based on host          biomarkers <130> KPA170329-KR <160> 48 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin_F <400> 1 cgcaccactg gcattgtcat 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin_R <400> 2 tccaaggcga cgtagcagag 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KLRB1_F <400> 3 aaccagcatg gatgctcaag a 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KLRB1_R <400> 4 tctcggattc gtttccagtg c 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KLRC1_F <400> 5 acccagggat tctggcattg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KLRC1_R <400> 6 actggtctct ctgttgcttc c 21 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CCL4_F <400> 7 tccacttgca aactacagat aaca 24 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CCL4_R <400> 8 acccaacagc atccaaactc a 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL5_F <400> 9 cccagccagc tgtggtattc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL5_R <400> 10 ctcggagcag ctcagttcaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCL9_F <400> 11 cttctgcctc cccatatgcc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCL9_R <400> 12 ctcggaactt gacacccaca 20 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CXCL10_F <400> 13 ctctgactgg agttcaagga gt 22 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCL10_R <400> 14 tcccttggct ggtgttgatg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR3_F <400> 15 aagcatgagt gtgaagggca 20 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CXCR3_R <400> 16 agggaaacct tgaacaattg cag 23 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IFNG_F <400> 17 acatagccaa gtcggtcacg 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IFNG_R <400> 18 ctcggaactt gacacccaca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP9_F <400> 19 cccggatcaa ggatacagcc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP9_R <400> 20 gggcgaggac catacagatg 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SERPINE1_F <400> 21 ctgcgaaatt caggatgcgg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SERPINE1_R <400> 22 gggtgagaaa accacgttgc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL10_F <400> 23 tctgccctgc gaaaacaaga 20 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL10_R <400> 24 cctctcttgg agctcactga a 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HP_F <400> 25 ggcccccgag attgctaata 20 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HP_R <400> 26 gggcagctgt catcttcttc a 21 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HGF_F <400> 27 cgacgggctc ttttaggctc 20 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HGF_R <400> 28 ctgtccttct gcatagggga tg 22 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PIGR_F <400> 29 gagtttgcca ccactaccga 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PIGR_R <400> 30 tccttggacg acctctttgc 20 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MPO_F <400> 31 gccctggaac ttcagagaga tg 22 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MPO_R <400> 32 gtcagcacca ccgaggtatc 20 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD14_F <400> 33 tgtctgacaa tcccagtctc g 21 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD14_R <400> 34 cgctagatat tggagggccg 20 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CHI3L1_F <400> 35 ataagagaag ctggaggccc a 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CHI3L1_R <400> 36 ccacaaaacc tgtatgagcc g 21 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LTF_F <400> 37 ggaagcagat gccctgaact 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LTF_R <400> 38 aggtaccctt ccgttggtct 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ELANE_F <400> 39 ttgtctgaac ggcctgaact 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ELANE_R <400> 40 ctctgaacat ctgccgggtt 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S100A8_F <400> 41 attttgggga gacctggtgg 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S100A8_R <400> 42 acggcgtggt aattcccttt 20 <210> 43 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> S100A9_F <400> 43 aggctacggg aagggcag 18 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S100A9_R <400> 44 gctggcctcc tgattagtgg 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFRC_F <400> 45 caaagtttct gccagcccac 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFRC_R <400> 46 aacagaaaga gaccgctggg 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBP6_F <400> 47 gagtgaagga caagcagcct 20 <210> 48 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GBP6_R <400> 48 cagacagaaa cctctgtatt ccg 23

Claims (9)

A subclinical urinary incontinence diagnostic composition comprising an agent capable of amplifying or detecting a marker gene of a subclinical urinary disease selected from the group consisting of HGF (hepatocyte growth factor), GBP6 (guanylate binding protein family member 6) .
The method according to claim 1,
Wherein the agent is a primer or a probe.
3. The method of claim 2,
Wherein the primer is a base pair selected from the group consisting of SEQ ID NOS: 27, 28, 47,
The method of claim 3,
The primers include a primer set for detection of HGF gene consisting of polynucleotides comprising the nucleotide sequences of SEQ ID NOS: 27 and 28, a primer set for GBP6 gene detection consisting of polynucleotides comprising the nucleotide sequences of SEQ ID NOS: 47 and 48, &Lt; / RTI &gt;
The method according to claim 1,
Wherein said Yonex disease is caused in ruminants.
6. The method of claim 5,
Wherein the ruminant is cow, chlorine, sheep or goat.
A kit for the diagnosis of sub-clinical cancers comprising the composition of claim 1.
(a) obtaining cDNA from a tissue sample of a ruminant rheumatoid arthritis;
(b) measuring the expression level of a marker gene of a subclinical infection-causing pathogen from the cDNA obtained by using the composition of any one of claims 1 to 6 or the kit of claim 7; And
(c) if the measured expression level of the marker gene is higher than that expressed in a normal ruminant, determining that the ruminant is infected with a pathogenic bacterium and is subclinical of the disease , A subclinical method to diagnose four cases.
9. The method of claim 8,
Wherein the tissue sample is blood, plasma, serum, lymph fluid, cerebrospinal fluid, isolated tissue, isolated cells or saliva.

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