KR101603284B1 - Composition and kit for detecting a laminitis in a subject, method for detecting a laminitis in a subject and method for screening a therapeutic agent for a laminitis - Google Patents

Abstract

A composition and a kit for diagnosing an individual's filamentous follicles, a method for diagnosing whether an individual has a filamentous fungus, and a method for searching for a filamentous fungi therapeutic agent.

Description

Composition and kit for detecting a laminitis in a subject, method for detecting a laminitis in a subject and method for screening a therapeutic agent for a laminitis}

The present invention relates to a composition and kit for diagnosing lobitis in an individual, a method for diagnosing whether an individual has lobitis, and a method of searching for a treatment agent for lobitis.

A leaf is a connective tissue that supports the hoof on the third phalanx of a danjeryu. It is a structure in which the epithelial layer protruding from the inside of the hoof wall and the dermal layer are interlocked with each other by forming projections with a tough basement membrane. 1 is a diagram showing the structure of a leaf blade. The dermal layer of the leaflet is a connective tissue made of type 1 collagen that is firmly connected to the bones, and blood vessels are distributed to provide nutrients necessary for growing and maintaining hoofs.

Blobitis is a disease in which a problem occurs in the basement membrane, which is a relatively weak part of the connection structure, and the solid continuity between the bone and the hooves is lost.When it becomes chronic, the third phalange penetrates the sole of the foot and causes severe pain and lameness.

Although the cause of lobitis is not clearly identified, it is known to be mainly due to gastrointestinal problems caused by carbohydrate bulimia. Short-chain carbohydrates move to the cecum in the mammal's stomach in an undigested state, and are fermented by the gram-positive bacteria of the normal microflora, acidifying the environment of the hindgut. The acidified intestinal environment causes excessive proliferation and death of Gram-positive bacteria, and endotoxins derived from killed bacteria are absorbed through the barrier with increased permeability, causing systemic toxemia. Toxemia causes the separation of the basement membrane and dermal tissue by activating the enzyme MMP (matrix metalloproteinase) present in the semi-adherent spots of the basement membrane, or it affects the blood vessels distributed in the leaflets, causing an inflammatory reaction.

Laminitis is one of the major diseases that necessitate euthanasia measures during the rearing of individuals, including horses, donkeys, and zebras. Accordingly, an accurate diagnosis is necessary to determine the treatment feasibility and prognosis, but the diagnosis is currently based only on the cardiovascular index and clinical symptoms. Therefore, there is a need to discover biomarkers and develop diagnostic methods for early diagnosis of specific leafitis.

One aspect provides a composition for diagnosing lobitis in an individual.

Another aspect provides a kit for diagnosing an individual's condyle leaf.

Another aspect provides a method of diagnosing whether an individual has leafitis.

Another aspect provides a method of screening for a therapeutic agent for lobitis in a subject.

In one aspect, an individual comprising an agent measuring the expression level of one or more miRNAs selected from the group consisting of eca-miR-199a-5p, eca-miR-27a, eca-miR-27b and eca-miR-324-5p It provides a composition for diagnosing leafitis.

The term “miRNA” may refer to microRNAs or miRs. The length of the miRNA can be about 19 to 25, or 21 to 23 nucleotides of RNA strands. Since miRNAs are transcribed from DNA but not translated into proteins, they are encoded by genes containing non-coding RNAs. miRs are processed from the known primary transcript pri-miRNA into a short stem-loop structure called pre-miRNA, and finally processed into the resulting single-stranded miRNA. The pre-miRNA can form a self-folding structure within the self-complementary region. The structure is then processed by nucleases in animals. Mature miRNA molecules are partially complementary to one or more mRNA molecules and may function to regulate the transcription of proteins. The identified sequence of miRNA can be evaluated in publicly available databases such as www.microRNA.org, www.mirbase.org, or www.mirz.unibas.ch/cgi/miRNA.cgi. miRNAs are usually assigned numbers according to naming conventions such as "mir-[number]". The number of miRNAs is assigned according to the order in which they are found for the miRNA species that have already been identified. If a miRNA is found to be similar to a known miRNA of a different organism, the name is in the form of [organism identifier]-mir-[number], and an optional organism identifier name is provided. Mature microRNAs are usually prefixed with "miR", and gene or precursor miRNAs are prefixed with "mir". In the context of the present invention, any microRNA (miRNA or miR) with the prefix mir-* or miR-* should be understood to include both precursors and/or mature species, unless expressly stated otherwise. For more information on miR naming, refer to www.mirbase.org or Ambros et al., A uniform system for microRNA annotation, RNA 9:277-279 (2003).

The eca-miR-199a-5p, eca-miR-27a, eca-miR-27b, and eca-miR-324-5p may each have a nucleotide sequence of SEQ ID NOs: 1 to 4, and are shown in Table 1. The miRNA is derived from Equus cavalus. The present inventors that the increase in the expression of one or more miRNAs selected from the group consisting of eca-miR-199a-5p, eca-miR-27a, eca-miR-27b and eca-miR-324-5 is associated with lobitis. Revealed by? Thus, the expression level of the one or more miRNAs can be used to diagnose lobitis.

The Aceession Number of eca-miR-199a-5p is. eca-miR-27a may also be referred to as MIR27A, and the Aceession Number is MIMAT0012988. The Aceession Number of eca-miR-27b is MIMAT0013114. The Aceession Number of eca-miR-324-5p is MIMAT0013033.

In the present specification, the terms "eca-miR-199a-5p", "eca-miR-27a", "eca-miR-27b" and "eca-miR-324-5p" are naturally occurring eca-miR -199a-5p, eca-miR-27a, eca-miR-27b and eca-miR-324-5p and functional variants thereof.

miRNA order eca-miR-199a-5p CCCAG UGUUC AGACU ACCUG UUC (SEQ ID NO: 1) eca-miR-27a UUCAC AGUGG CUAAG UUCCG C (SEQ ID NO: 2) eca-miR-27b UUCAC AGUGG CUAAG UUCUG C (SEQ ID NO: 3) eca-miR-324-5p CGCAU CCCCU AGGGC AUUGG UGU (SEQ ID NO: 4)

The composition for diagnosing lobitis of the individual may include an agent for measuring the amount of the one or more miRNAs. The agent may be a substance that specifically binds to the one or more miRNAs. The agent may include a primer, a probe, or an antisense sequence thereof that specifically binds to the one or more miRNAs or their complementary sequences; Or a combination thereof. The primer may be one that provides a polymerization start point in a polymerization reaction by a polymerase. The primer may be one used in a nucleic acid amplification reaction. The term “amplification” refers to increasing the copy number of a target sequence or its complementary sequence. The nucleic acid amplification reaction may be performed by any method known in the art. Amplification of nucleic acids includes methods that require multiple cycles during amplification or methods performed at a single temperature. Examples of cycling techniques include those requiring thermal cycling. Methods requiring thermal cycling include polymerase chain reaction (PCR). PCR is known in the art. PCR usually involves denaturing double-stranded DNA into single-stranded DNA by heat denaturation, and annealing a primer to the single-stranded DNA; And synthesizing a complementary strand from the primer. The isothermal amplification method is a method in which a single temperature or the main aspect of the amplification process is performed at a single temperature. In contrast to PCR, which heats the reaction product to allow additional primers to bind to separate the double strands, the isothermal method relies on a strand displacing polymerase to separate the double strands and recopy the template. do. The isothermal method can be divided into a method that relies on substitution of a primer to initiate repetitive template copying and a method that relies on continuous reuse or novel synthesis of a single primer molecule. Methods that depend on the substitution of primers include helicase dependent amplification (HDA), exonuclease dependent amplification, recombinase polymerase amplification (RPA), and loop-mediated amplification. It includes a method selected from the group consisting of (loop mediated amplification: LAMP). Methods that rely on continuous reuse or novel synthesis of single primer molecules include methods selected from the group consisting of strand displacement amplification (SDA) and nucleic acid based amplification (NASBA and TMA). The primers may be included in one, 2, 3, 4, 5, 6 or more sets according to the selected amplification method. The primer may be a primer for PCR.

The measurement of miRNA expression level is a process of confirming the presence and expression level of miRNA in an individual in order to diagnose the presence and degree of lobitis, and may be a measurement of the amount of miRNA. This can be measured by directly separating the miRNA or using a primer or probe for the miRNA. Analysis methods for this are RT-PCR, competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting. ), a nucleic acid microarray including DNA, or a combination thereof. RT-PCR is a method of analyzing RNA. It is a method of amplifying and analyzing cDNA obtained by reverse transcription of miRNA by PCR. In the amplification step of the RT-PCR, a pair of primers specifically prepared for the gene is used, and by checking the band pattern and the thickness of the band by electrophoresis after RT-PCR, it is possible to check the expression level and the presence of miRNA of the gene. And by comparing this with the normal control group, it is possible to easily determine the presence or absence of leaf salt in an individual or the degree of its onset.

In the present specification, the term "primer" is a nucleic acid sequence having a free 3-terminal hydroxyl group, which can form a base pair with a complementary template and serves as a starting point for template strand copying. Say. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. For example, PCR amplification is performed using sense and antisense primers having 7 to 50 nucleotide sequences as specific primers for the one or more miRNAs, and the presence or occurrence of leaf salt in the individual by measuring the production amount of the desired product. You can check the degree. PCR conditions, the length of the sense and antisense primers can be appropriately selected according to techniques known in the art. The primers are 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 15 to 80, 15 to 50, 15 to 30, 20 to 100, 20 to 80, 20 to 50, or 20 to It may have 30 nt.

In the present specification, the term "probe" refers to a nucleic acid fragment such as RNA or DNA capable of specifically binding to a target nucleic acid, for example, miRNA, and is labeled to confirm the presence, content, and expression level of a specific miRNA. I can. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. For example, by performing hybridization using a probe complementary to the miRNA, and measuring the expression level of miRNA through the degree of hybridization, the presence or absence of lobitis in an individual or its onset can be confirmed. Selection of an appropriate probe and conditions for hybridization can be appropriately selected according to techniques known in the art. The probe is 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 15 to 80, 15 to 50, 15 to 30, 20 to 100, 20 to 80, 20 to 50, or 20 to It may have 30 nt.

In addition, the primer or probe may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known method. In addition, such nucleic acid sequences can be modified through various methods known in the art. Examples of such modifications are methylation, encapsulation, substitution of one or more homologs of natural nucleotides, or modifications between nucleotides, such as uncharged linkers (e.g. methyl phosphonate, phosphotriester, phosphoroamidate, carbamate. Etc.) or to a charged linker (eg, phosphorothioate, phosphorodithioate, etc.). In addition, the primer or probe may be modified using a label that can directly or indirectly provide a detectable signal. Examples of the label may include radioactive isotopes, fluorescent molecules, or biotin.

The expression level may be an expression level in a sample isolated from an individual. The sample may be blood, plasma, serum, urine, feces, saliva, tears, cerebrospinal fluid, cells, tissues, or a combination thereof isolated from an individual. The tissue may be a leaf. The individual may belong to the monophyte (Perissodactypa). The subject may be of the genus Equus. The genus Equus may include horses, donkeys, or zebras. The horse may be of the species Equus ferus. The Equus ferus species may include Equus ferus caballus, Equus ferus ferus, or Equus ferus przewalskii subspecies. . The subject may include a horse, water buffalo, rhinoceros, or camel.

Another aspect comprises an agent for measuring the expression level of one or more miRNAs selected from the group consisting of eca-miR-199a-5p, eca-miR-27a, eca-miR-27b and eca-miR-324-5p, A kit for diagnosing lobitis in an individual is provided.

The "agents for measuring the expression level of one or more miRNAs", "individuals" and "for diagnosing lobitis" are as described in the description of the composition for diagnosing lobitis.

The kit may be, for example, a microarray for diagnosing lobitis, which can measure the expression level of miRNA. The microarray for diagnosing lobitis can be easily prepared by a person skilled in the art according to a method known in the art using the miRNA. The microarray may be one in which the above-described miRNA or a sequence complementary thereto, or a nucleic acid sequence corresponding thereto is attached to a substrate as a probe. The nucleic acid sequence may be selected from the group consisting of DNA, peptide nucleic acids (PNA), locked nucleic acids (LNA), combinations thereof, and analogs thereof.

When the kit is for measuring the expression level of the miRNA of the gene described above, for example, it may be a kit including essential elements necessary for performing RT-PCR. The RT-PCR kit includes test tubes or other suitable containers, reaction buffers, deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase, in addition to each primer specific for the miRNA of the marker gene. Inhibitors, DEPC-water, or sterile water. In addition, a primer pair specific to a gene used as a quantitative control may be included.

Another aspect is to measure the expression level of one or more miRNAs selected from the group consisting of eca-miR-199a-5p, eca-miR-27a, eca-miR-27b and eca-miR-324-5p in a sample isolated from an individual. It provides a method for diagnosing whether an individual has flobitis, comprising the steps of, and comparing the measured expression level of one or more miRNAs with the expression level of one or more miRNAs measured in a control group.

The individual may belong to the monophasic (Perissodactyla). Equus (Equus) can be deceiving. The genus Equus may include horses, donkeys, or zebras. The above words are Equus Perus (equus ferus ) may be a species. The EX Peru's (equus ferus) species Equus Peru's Cavalli Ruth (Equus ferus caballus ), Equus Perus Perus (Equus ferus ferus ), or Equus Perus Przwalski ( Equus ferus przewalskii ) subspecies. The subject may include a horse, water buffalo, rhinoceros, or camel.

The expression level may be the expression level of miRNA. Thus, the measurement may be the amount of miRNA. The measurement of the amount of the miRNA is as described above.

*The sample may be blood, plasma, serum, urine, feces, saliva, tears, cerebrospinal fluid, cells, tissues, or a combination thereof isolated from an individual. The tissue may be a leaf.

The method includes comparing the measured expression level of one or more miRNAs with the expression level of one or more miRNAs measured in a control. The control group may be an individual who does not suffer from lobitis.

The method may further include the step of determining that the individual suffers from lolliculitis when the measured expression level of one or more miRNAs is higher than the expression level of one or more miRNAs measured in the control group. In addition, when the measured expression level of one or more miRNAs is the same as or lower than the expression level of one or more miRNAs measured in the control, the step of determining that the individual does not suffer from lobitis may be further included.

Another aspect is the steps of administering a candidate substance to the subject; Measuring the expression level of one or more miRNAs selected from the group consisting of eca-miR-199a-5p, eca-miR-27a, eca-miR-27b and eca-miR-324-5p in the sample isolated from the individual; And comparing the measured expression level of one or more miRNAs with the expression level of one or more miRNAs measured in a control group.

The method includes administering a candidate substance to the subject. The candidate substance may be any substance that is expected to be effective in the treatment of lobitis. The administration may be appropriately selected depending on the substance to be selected. The administration is administered directly to the subject by any means, for example intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The substances can be administered systemically or locally, for example in the leaflets.

"Measuring the expression level of one or more miRNAs selected from the group consisting of eca-miR-199a-5p, eca-miR-27a, eca-miR-27b and eca-miR-324-5p in the sample isolated from the individual "And the step of comparing the expression level of one or more miRNAs measured above with the expression level of one or more miRNAs measured in a control" is as described above.

The individual may be an individual having leaf salt.

The method may further include, when the measured expression level of one or more miRNAs is lower than the expression level of one or more miRNAs measured in the control group, the candidate substance being effective for treating folliculitis.

According to the composition for diagnosing leaf salt according to an aspect, it is possible to efficiently diagnose the presence or absence of leaf salt.

According to the kit for diagnosing leaf salt according to an aspect, it is possible to efficiently diagnose the presence or absence of leaf salt.

According to the method for diagnosing whether an individual has decontamination according to an aspect, it is possible to efficiently diagnose whether an individual has decontamination.

According to the method of searching for a decontamination treatment agent according to an aspect, it is possible to effectively screen a decontamination treatment agent.

1 is a diagram showing the structure of a leaf blade.
FIG. 2 is a diagram showing a process of performing miRNAs of eca-miR-199a-5p, eca-miR-27a, eca-miR-27b, and eca-miR-324-5p as an indicator for early diagnosis of lobitis.
Figure 3 is a diagram showing the dosage and schedule of oligosaccharide administration for inducing leaf salt.
Fig. 4 is a photograph of a histological examination of a leaflet tissue caused by leafitis.
5 is a diagram showing a library production process.

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

Example 1 . miRNA Using Leaf salt Diagnosis

In this example, each of eca-miR-199a-5p, eca-miR-27a, eca-miR-27b, and eca-miR-324-5p was differentially expressed before and after artificially inducing leaf salt in horses. It was confirmed, and it was confirmed that the expression level of the miRNA can be used in the diagnosis of lobitis. FIG. 2 is a diagram showing a process of performing miRNAs of eca-miR-199a-5p, eca-miR-27a, eca-miR-27b, and eca-miR-324-5p as an index for early diagnosis of lobitis.

One. Leaf salt Experimental trigger

(1) through the administration of oligosaccharides Leaf salt cause

About 600 kg of 20-year-old Warmblood castration was used, and it was managed without exercise in a magic room for about 3 months. Fructooligosaccharide (Sun Oligo P2®, Qwon Co., Ltd., Korea) was used. Figure 3 is a diagram showing the dosage and schedule of oligosaccharide administration for inducing leaf salt. As shown in FIG. 3, after 3 days (Day 0, 1, 2), an induction dose of 1 g/kg was administered, an induction dose of 10 g/kg was injected on Day 3. The induction dose of 1 g/kg was mixed and administered in the form of powder, and the induction dose was divided into half of 6 kg, a dose of 10 g/kg, and dissolved in 4 L of tap water, as in the morning and afternoon of Day 3 It was injected twice using a nasogastric tube. On Day 4 and Day 5, clinical and blood tests were performed and then euthanized.

(1.2) Leaf salt Provoking evaluation

Physical examinations were conducted twice a day starting from Day 3. Body temperature was measured by inserting an electronic thermometer (MT 1961®, Microlife) into the rectum. From Day 3 when fructooligosaccharide was administered at 10 g/kg, changes in body temperature measured twice a day showed a continuous increase from 12 hours after administration, and a high fever of 39.5 ℃ was observed in the morning of Day 5.

The hooves' warmth was recorded as 0 at all times, i.e., when the feeling of cold was 0, when it was the same as room temperature (+), and when a feeling of heat higher than room temperature was felt (++). On Day 1, heat sensation (++) appeared on the hooves except for the left forefoot, and heat sensation (++) was observed on all four hooves from Day 1 to 2, and the temperature dropped to room temperature (+) on Day 2.

Heart rate was measured directly on the left chest using a stethoscope. The claudication rating was evaluated by the rating system proposed by the American Association of Horse Veterinarians. The changes in heart rate measured twice a day from Day 3 when oligosaccharide was administered at 10 g/kg are as follows. On Day 3, the heart rate was normal at 30 times/min. From Day 4 onwards, it continued to rise, but on Day 5 it decreased slightly, but the heart rate was still higher than normal.

In addition, we observed whether other clinical symptoms related to the gastrointestinal system appeared. He had anorexia and diarrhea on Day 4, and then continued to have anorexia. On Day 3, they showed a standing posture with their forelimbs wide open, and showed a high claudication rating between grades 4 and 5. The protein shown in Table 1 can be used as a marker for the diagnosis of lobitis.

Blood was collected from the jugular vein in the morning and afternoon of Day 5, stored in BD vacutainer EDTA-K2 10.8 mg (6 ml), and then stored in ADVIA 120 blood test equipment (Siemens Healthcare Diagnostics, Deerfield, IL, USA), and FUJI DRY-CHEM 3500i ( Fugi film, Japan, Tokyo) were used for blood and serological tests, respectively. The results of the blood test conducted on Day 5 are as follows. RBC, Hematocrit (Hct), and Hemoglobin (Hb) were observed to be higher than the reference range, and the WBC level was slightly below the reference range. Relatively severe lymphopenia was observed, and the ratio of neutrophils, basophils, monocytes and other white blood cells was measured to be slightly increased. In addition, the results of the serochemical test on Day 5 were recorded as follows. Blood glucose increased about 1.5 to 2 times of normal, and creatinine, uric acid, total bilirubin, and alkaline phosphatase (ALP) also increased about 2 times. Total protein and glutamic oxaloacetic transaminase (GOT) were slightly elevated. Excluding bilirubin, the other levels showed even higher levels just before euthanasia. The electrolyte test showed mild hyponatremia and hypomagnesemia. Creatine phosphokinase (CPK) and blood urea nitrogen (BUN) showed a slight increase in the test immediately before euthanasia.

In addition, the leaf tissues collected for histological examination were fixed in about 10% formalin, embedded in paraffin, sectioned into 4-6 μm, and stained with hematoxylin and eosin according to a conventional method. The lungs were generally red and not retracted, and bubbling fluid was present in the bronchi. The colon was generally red, and the intestinal wall was thickened with hydrophobic changes. A slight redness was observed in the cross section of the leaf tissue. Fig. 4 is a photograph of a histological examination of a leaflet tissue caused by leafitis. As shown in Fig. 4, the blood vessels of the lobe connective tissue are congested, a small number of neutrophils are infiltrated, and a hydrophobic change is shown.

2. Leaf salt In plasma before and after induction miRNA NGS Department of Analysis Leaves Specific miRNA Selection

(1) plasma sampling

Plasma before the induction of lobitis was collected from the jugular vein before administration of the induction dose on Day 0, added to 10.8 mg (6 ml) of BD vacutainer EDTA-K2, and then sampled by centrifugation at 3000 rpm for 10 minutes with a centrifuge. In addition, after induction of lobitis, plasma was collected from the jugular vein on Day 5, added to BD vacutainer EDTA-K2 10.8 mg (6 ml), and then sampled by centrifugation at 3000 rpm for 10 minutes with a centrifuge.

(2) tissue sampling

Sedation (Domosedan ^® , 10mg/kg, Orion Co./ Ketamine, 1500mg/kg, Yuhan Corporation) followed by euthanasia (Embutramide 200 mg Mebezonium iodine 50 mg Tetracaine 5 mg, T-61 ^® , 50 ml, European Union) After autopsy, 12 major organ tissues of the liver, kidneys, lungs, stomach, small intestine, colon, appendix, thyroid gland, adrenal gland, muscle, spleen, and heart of horses with lobitis-induced lobitis and from 4 hooves Was collected, quenched in liquid nitrogen, and then stored at -70°C.

(2) RNA extraction

0.75 ml of Trizol per 50 mg of leaf-leaf and 12 major organ tissues were added and homogenized. After incubating the homogenized sample at room temperature for 5 minutes, 0.2 ml of chloroform per 0.75 ml of Trizol was added and mixed by shaking for 15 seconds. The mixture was incubated at RT for 3 minutes and then centrifuged at 14000 rpm and 4° C. for 15 minutes. After centrifugation, about 500 ul of the supernatant was secured, mixed 1:1 with isopropyl alcohol, and incubated for 10 minutes at RT. Then, centrifugation was performed at 14000 rpm and 4° C. for 10 minutes, and after confirming the RNA pellet, all the supernatant was removed. After adding 1 ml of 70% EtOH as a washing process, centrifugation was performed for 5 minutes at 8000 rpm and 4°C. After removing all the supernatant, the RNA pellet was completely dried with the tube lid open at RT. When the pellet was completely dried, it was eluted by adding 20-100 ul of HPLC water. After that, DNA was completely removed by treatment with Dnase I at RT for 15 minutes to extract pure RNA.

(3) RNA concentration and quality analysis

Total RNA isolated from horse plasma was analyzed using a nano drop machine and a bioanalyzer machine for RNA concentration and quality using the following criteria.

Nucleic acid and protein have maximum absorbance at 260 nm and 280 nm, respectively. The absorbance ratio (260/280) at 260 nm and 280 nm is a criterion for evaluating the purity of DNA and RNA, and ratio 1.8 is generally pure DNA and ratio 2.0 is pure RNA. When the extracted RNA had a value of 260/280 of 1.6-2.0, it was determined that the purity was high and used for subsequent analysis.

As a result of separating total RNA from plasma of target horses, 123 ng of high purity total RNA was obtained per ml of plasma on average.

(4) NGS Within the organization using (Next Generation Sequencing) miRNA analysis

(4.1) stem loop RT- PCR Quantification using

Small RNAs of 18-32 nt size were isolated from the extracted total RNA using a size-fractionation method. After attaching the adapter to these small RNAs, a size-fractionated cDNA library was produced through a series of RT-PCR and cDNA cloning. 5 is a diagram showing a library production process.

The extracted total RNA was loaded onto a polyacrylamide gel, separated by size through electrophoresis, and then a portion corresponding to small <40-nt RNA was cut out. The <40-nt fraction was washed with a flashPAGE clean up kit (Life Technologies Corp.), dissolved in 30 ul of elution buffer, and separated. Then, it was washed with about 80% ethanol. The pellet was resuspended with 50 ul of RNase-free TE. Then, the quality of <40-nt RNA was tested.

Reverse transcriptase reaction was carried out as follows: a purified total RNA sample, 50 nM stem-loop RT primer, 1x RT buffer, 0.25 mM dNTPs, reverse transcriptase and RT reaction premix containing RNase inhibitor were mixed. 7.5 uL of reaction mixture was prepared. The prepared mixture was loaded into a 96-well plate and a sequential reaction was carried out by holding at about 16° C. for about 30 minutes, about 42° C. for about 30 minutes, about 85° C. for about 5 minutes, and about 4° C. The reverse transcription reaction was performed with the no-template control and the reverse transcriptase minus control as negative controls. In addition, the error range was reduced by performing the above reaction in duplicate or triplicate.

Real-time qPCR was carried out as follows: A PCR premix of about 18.7 uL was prepared, including forward primer, reverse primer, and hydrolysis probe. This premix was loaded into a 96-well plate, and about 1.3 ul of RT product was transferred thereto. The real-time quantitative polymerization reaction was carried out in triplicate sequentially at about 95°C for about 10 minutes at 40 cycles, about 95°C for about 15 seconds, and about 60°C for about 1 minute.

(4.2) NGS Perform (Next Generation Sequencing)

After loading the Small RNA cDNA, it was read with a 50 bp single-end using a HiSeq 2000 machine (Iluumina inc.) to perform NGS (Next Generation Sequencing). Subsequently, data cleaning was performed as follows for the NGS results obtained from the plasma of normal horses and from the plasma of horses with lobular salt, primarily according to the following criteria:

Low quality read data was removed. Then, the read data with the 5'primer contaminant was removed. Thereafter, the read data without the 3'primer was removed. Then, the read data with Poly A was removed. Thereafter, read data shorter than 18 nt were removed. After that, the read data without the insert tag was removed.

As a result, more than 95% and 94% of clean reads were confirmed, respectively. After selecting only Clean Read data from the high-quality small RNAs identified through data cleaning, the existing microRNAs, new microRNAs, rRNAs, tRNAs, snRNAs, and small RNAs of snoRNA were analyzed through standard bioinformatics analysis. The expression patterns for the were analyzed. Standard bioinformatics analysis was performed using miRBase (www.mirbase.org), which has been constructed as a database of information on miRNA nucleotide sequences and annotations published in papers.

3. Results

As a result of comparing and analyzing the expression levels of miRNAs in horse leaflets and other major organ tissues, miRNAs with high expression levels in leaflet tissues and insignificant expression levels in other major organ tissues were selected. The selected miRNA and its expression level are shown in Table 2.

miRNA Leaf tissue average ( RPM* ) Mean expression levels in major organ tissues other than the lobe ( RPM ) Multiple change eca-miR-199b-5p About 2,706.80 About 13.12 About 206.37 eca-miR-27a 2,278.84 approx. About 239.93 About 9.50 eca-miR-27b About 2,528.12 About 406.23 About 6.22 eca-miR-324-5p 9.43 approx. About 2.85 About 3.31

*RPM: Reads per million

As a result of comparing and analyzing the level of miRNA expression in the plasma before and after the induction of lobitis in horses, miRNAs with higher expression levels after lobulation induction than before induction of lobulation in horses were eca-miR-199a-5p, eca-miR-27a, eca-miR, respectively -27b and eca-miR-324-5p, which are shown in Table 3.

miRNA Expression level in plasma after induction of lobitis (RPM*) Plasma expression level (RPM) before lobitis induction Multiple change eca-miR-199a-5p About 40.99 About 5.71 7.18 approx. eca-miR-27a About 10,198.85 About 674.73 About 15.12 eca-miR-27b About 45,170.26 About 9,245.79 About 4.89 eca-miR-324-5p About 88.96 About 16.51 About 5.39

*RPM: Reads per million

<110> SNU R&DB Foundation <120> Composition and kit for detecting a laminitis in a subject, method for detecting a laminitis in a subject and method for screening a therapeutic agent for a laminitis <130> PN110440 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 23 <212> DNA <213> Equus caballus <400> 1 cccaguguuc agacuaccug uuc 23 <210> 2 <211> 21 <212> DNA <213> Equus caballus <400> 2 uucacagugg cuaaguuccg c 21 <210> 3 <211> 21 <212> DNA <213> Equus caballus <400> 3 uucacagugg cuaaguucug c 21 <210> 4 <211> 23 <212> DNA <213> Equus caballus <400> 4 cgcauccccu agggcauugg ugu 23

Claims (11)

A composition for diagnosing laminitis of an individual, comprising an agent for measuring the level of expression of the miRNA of SEQ ID NO: 2. The composition according to claim 1, wherein the agent is a substance that specifically binds to the miRNA of SEQ ID NO: 2. The composition according to claim 1, wherein the preparation is a primer, a probe, or a combination thereof that specifically binds to the miRNA of SEQ ID NO: 2 or a complementary sequence thereof. A kit for diagnosing dysplastic kidney comprising an agent for measuring the expression level of the miRNA of SEQ ID NO: 2. [Claim 4] The kit according to claim 4, wherein the agent is a substance that specifically binds to the miRNA of SEQ ID NO: 2. 5. The kit for diagnosing infectious disease of an individual according to claim 4, wherein the agent is a primer, a probe, or a combination thereof that specifically binds to the miRNA of SEQ ID NO: 2 or its complementary sequence. Determining the level of expression of the miRNA of SEQ ID NO: 2 in the sample isolated from the subject, and
Comparing the expression level of the miRNA of SEQ ID NO: 2 with the expression level of the miRNA of SEQ ID NO: 2 measured in the control. 8. The method of claim 7, further comprising the step of determining that the subject is struck by acute myelitis when the measured expression level of the miRNA of SEQ ID NO: 2 is higher than the expression level of the miRNA of SEQ ID NO: 2, Way. 9. The method of claim 7, wherein the measuring step measures the amount of miRNA of SEQ ID NO: 2. Administering the candidate substance to the individual;
Determining the level of expression of the miRNA of SEQ ID NO: 2 in a sample isolated from said subject; And
Comparing the expression level of the miRNA of SEQ ID NO: 2 with the expression level of the miRNA of SEQ ID NO: 2 measured in the control. 11. The method of claim 10, further comprising determining if the measured expression level of the miRNA of SEQ ID NO: 2 is lower than the expression level of the miRNA of SEQ ID NO: 2 as measured in a control, How it is.

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