KR101911017B1 - Composition for Detecting Taylorella equigenitalis, Composition for Diagnosing Contagious Equine Metritis Caused by Taylorella equigenitalis Infection, Method of Detecting Taylorella equigenitalis, and Method of Diagnosing Contagious Equine Metritis Caused by Taylorella equigenitalis Infection - Google Patents

Abstract

The present invention relates to a composition for detecting Taylorella equigenitalis, a composition for diagnosing contagious equine metritis (CEM) caused by Taylorella equigenitalis infection, a method of detecting Taylorella equigenitalis, and a method of diagnosing contagious equine metritis caused by Taylorella equigenitalis infection and, more specifically, to a composition comprising a primer pair including a sequence complementary to a 16S rRNA gene sequence of Taylorella equigenitalis causing contagious equine metritis (CEM) as an active ingredient, a kit including the composition, a method of detecting Taylorella equigenitalis using the primer pair, and a method of diagnosing contagious equine metritis caused by Taylorella equigenitalis infection using the primer pair.

Description

A composition for the detection of tylolella equiensis, a composition for the detection of infectious hirsute enteritis caused by infection with tylolella equiensis, a method for the detection of tylolella equiognisis, and a method for the detection of infectious hirsute enteritis caused by tylolella equiensitis [0002] The present invention relates to a diagnostic method for detecting Taylorella equigenitalis, a composition for diagnosing Taylorella equigenitalis, and a method for diagnosing Taylorella equigenitalis,

The present invention relates to a composition for the detection of tylolella equiensis, a composition for the diagnosis of horse infectious uterine inflammation caused by infection with tylolella equiensitis, a method for detecting tylolella equiognitallis, The present invention relates to a method for diagnosing equine infectious endometriosis, more particularly, to a method for diagnosing equine infectious endometritis, comprising the steps of: (a) obtaining a base sequence complementary to a base sequence of 16S rRNA gene of Taylorella equigenitalis , a causative agent of contagious equine metritis A kit comprising the composition, and a method for detecting tile roullaequianetalis using the primer pair and a method for diagnosing a horse infectious hysteresis by tile roellaequiensis infection .

Tylolella < RTI ID = 0.0 > equineitalis , T. equigenitalis ) Contagious Equine Metritis (CEM), a genital disease that occurs in endemic animals such as zebras, horses (ponies, etc.), mules and donkeys by infection, ( Crowhurst RC . , Vet. Rec . , 100 (22): 476, 1977; Crowhurst , 1987), which has been reported for the first time in the world . RC . et al., Vet. Rec . , ≪ / RTI > 104 (20): 465, 1979; ter Laak EA et al ., Tijdschr Diergeneeskd , 114 (4): 189-201, 1989; Timoney PJ, J Anim Sci . , 89 (5): 1552-60, 2011; Matsuda M. et al. , Vet Microbiol . , 97 (1-2): 111-22, 2003; Bleumink-Plum NM et al. , Int . J. Syst. Bacteriol . , 43 (3): 618-21, 1993; Breuil MF et al. , Vet. Microbiol . , 148 (2-4): 260-6, 2011).

In particular, the United States succeeded in eradicating infectious endometriosis in late 1970, but it recurred in 2006 and is still occurring intermittently. In Japan, it was reported that infectious hirsenism occurred for the first time in 1980, and there was no further report from 2006 to 2010 due to ongoing management (Anzai T et al. , J. Vet. Med . Sci . , 64 (11) 999-1002, 2002).

Since the horses infected with tylolella equijetalis are infertile and the whole horse industry suffers a great deal of damage, it is designated as a disease to be managed by the World Livestock Organization (OIE), the second livestock epidemic in Korea.

In 2015, a horse infectious endometriosis disease caused by infection with tile Lilelaqueznitallis was first identified in domestic breeding horses. Since there is no effective vaccine against this disease and the nature of the disease makes it easy to relapse, Isolation and treatment through diseases are classified as essential.

On the other hand, the isolate identification method and the real-time gene diagnosis method are being used as the method for diagnosing equine infectious endometriosis. Currently, one kind of real-time gene diagnosis method is commercialized and is on sale. However, in the case of the isolate identification method of existing pathogens, it must be transported to the laboratory within 48 hours after the sampling, and a special medium should be used to isolate tile from the other strains. (Timoney PJ et al. , Vet. Rec . , 111 (5): 107-108, 1982), which has a disadvantage in that it takes a long time and has a very low separation efficiency due to the difficulty of the separation culture itself . . Atherton JG, Vet Rec, 113 (13): 299-300, 1983; Ward J. et al, Vet Rec, 114 (12):...... 298, 1984; Parlevliet JM et al, Theriogenology, 47 (6): 1169-77, 1997; Wakeley PR et al. , Vet. Microbiol . , 118 (3-4): 247-54, 2006; Matsuda M. et al. , Vet Microbiol. , 97 ): 111-22, 2003).

In addition, a kit for detecting tile roulla acuijenitallis , which is currently available for detection of tile roulla aquijenitallis, is a kit ( cador TKP PCR Reagent, cat. No. 285115, Qiagen) or tylorella aquezenitalis, Pseudomonas aeruginosa aeruginosa and Klebsiella < RTI ID = 0.0 > Although there is a multiplex kit ( cador TKP PCR Reagent, Qiagen) that can simultaneously detect pneumoniae , the supply of kit (s) is not constant (supply discontinuation in 2014) .

Under these technical backgrounds, the present inventors have found that they do not rely on the manufacturer producing the product for the detection of Tylolella axienietallis, but are inexpensive and have excellent sensitivity (sensitivity) compared to the conventional products used for detecting tylolella sequenitaltis Kit and method capable of detecting tile roullaequianetalis, which is a causative agent of horse infectious uterine typhus, with rapid and specificity, and as a result, it has been found that the composition of tile roulla aqueductis At the 16S rRNA gene site A primer pair comprising a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 as an active ingredient, a kit comprising the composition, and The detection method using the above primer pair can be carried out by using tile lorelequezenitallis (preferably tile lorelequezenitallis infected animals or tile lorella separated from a specimen (a test sample) And the present invention was completed.

It is an object of the present invention to provide a method for the preparation of tylorella < RTI ID = 0.0 > equigenitalis ) and a kit for the detection of tile roullaequianetalis comprising the composition.

It is another object of the present invention tile Pasteurella ekwi Jenny Tallis (Taylorella equigenitalis) horse infectious jagungyeom horse infectious jagungyeom by Pasteurella ekwi Jenny Tallis infected with tile comprising a diagnostic composition and the composition of (Contagious Equine Metritis, CEM) by infection And to provide a diagnostic kit of the present invention.

It is another object of the present invention to provide a method for detecting Taylorella equigenitalis .

It is another object of the present invention to Pasteurella tiled ekwi Jenny Tallis (Taylorella equigenitalis) provides a diagnostic method of the late infectious jagungyeom (Contagious Equine Metritis, CEM) according to the infection.

In order to achieve the above object, the present invention provides a primer pair comprising a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 as an active ingredient, A composition for detecting Taylorella equigenitalis , and a kit for detecting tile roullaequianetalis comprising the composition.

The present invention also relates to a method for screening a tylorella < RTI ID = 0.0 > ( TM) < / RTI > gene , comprising a primer pair consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: The present invention provides a diagnostic kit for contagious equine metritis (CEM) caused by infection with equineitalis, and a kit for the diagnosis of horse infectious uterine inflammation caused by tile roellaequianetalis infection comprising the composition.

The present invention also relates to the use of tylorella < RTI ID = 0.0 > a primer pair consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 that specifically binds to the nucleic acid of tile Lulla sequenittalis ; And amplifying the nucleic acid of the tylorella equietinis tile separated from the specimen of the sample with a template to thereby detect tylolella equiognetallis. do.

The present invention also relates to the use of tylorella < RTI ID = 0.0 > A method for diagnosing contagious equine metritis (CEM) caused by infection with equigenitalis, comprising the steps of: preparing a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 which specifically binds to the nucleic acid of tylolera sequenittalis; (Except for humans) using a primer pair composed of a reverse primer represented by the nucleotide sequence of Tailorella equiensis isolated from a specimen of a horse infectious hemorrhagic (but not human) specimen, Wherein the method comprises the step of confirming whether or not the tile is infected with Lilelaqueznitallis. The present invention also provides a method for diagnosing a horse infectious uterine disease caused by infection with tile Lilelaqueznitalis.

The 16S rRNA gene locus of the tile Lilelaquez nitalis according to the present invention A kit comprising a specific primer pair, a kit comprising the composition, and a method for detecting tile roullaequianetalis using the primer pair, can be rapidly performed because of excellent sensitivity and specificity compared to existing products and / It is possible to improve the detection efficiency of tile roulla equiensitris in a sample (a sample of a test sample), and it has a time, labor and cost saving effect compared to the conventional detection method of real-time gene diagnosis method. As a result, It is possible to make an early diagnosis of the infectious endometritis caused by the infection, which can contribute to domestic horse industry protection, livestock prevention and quarantine.

FIG. 1 is a graph showing the relationship between the 16S rRNA gene sequence of the tylorella equietinis strain (TE) and the 16S rRNA gene base of the heterologous strain in order to derive a pair of primers represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: .
2 shows the binding sequence of the primer pairs represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 according to the present invention in the 16S rRNA gene sequence (GenBank Accession No. X68645: 1 To 1499).
FIG. 3 is a graph showing the results of the detection of horse-infectious uterine bleeding in order to confirm the detection ability of the tyloreleequiensitallis in the horses diagnosed with the equine infectious uterine salt of the pair of primers represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 according to the present invention A tile of a swab sample of a horses genital was subjected to a PCR reaction with the above primer pair using a nucleic acid derived from Lilelaquezgenitalis, and the obtained PCR amplification product was subjected to electrophoresis on a gel to detect whether tylolella sequenittalis was detected .
FIG. 4 is a graph showing the sensitivity of primer pairs represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 according to the present invention. PCR was carried out using the template DNAs derived from Lelera acuijenitallis at different concentrations in PCR After the reaction, the PCR amplification product obtained was subjected to electrophoresis on a gel to confirm the detection of tile roullaequeenittalis.
FIG. 5 is a graph showing the sensitivity of a pair of primers represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 according to the present invention in the range of detectable template DNA, , PCR was carried out using the primer pair, and the PCR amplification product obtained was subjected to electrophoresis on a gel to confirm whether or not tile loreleaquezenitalis was detected.
FIG. 6 is a graph showing the specificity of the primer pair represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 according to the present invention. In order to confirm the specificity of the pair of primers shown in SEQ ID NO: 1 and SEQ ID NO: 2, , The PCR amplification product obtained was subjected to electrophoresis on a gel to confirm the detection of tylolella sequenittalis.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The term " about " used herein to describe volume, time (period), concentration, content, etc. means that there is a maximum tolerance of 10%

The present invention relates to a PCR technique using a primer pair consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2, which is specific to the 16S rRNA gene region of tylorella equiensis It has been confirmed that the tile containing the nucleic acid of Lulla equiensis is detected in the specimen (the test sample) with excellent sensitivity and specificity compared to the conventional diagnostic method.

In the present invention, a " primer " is a nucleic acid sequence having a short free 3 'end hydroxyl group, which can form a base pair with a complementary template, Lt; RTI ID = 0.0 > nucleotide < / RTI > Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates (dNTPs) at appropriate buffer solutions and temperatures. The nucleotide sequence and length of the PCR conditions, the forward (sense) primer and the reverse (antisense) primer can be modified using techniques known in the art. Such oligonucleotide primers can be easily prepared and used by those skilled in the art according to methods known in the art.

In the present invention, "forward primer" and "reverse primer" bind to 3'-terminal and 5'-terminal of a specific site of a gene amplified by gene amplification reaction, respectively, Quot; primer "

The term " Polymerase Chain Reaction (PCR) " in the present invention refers to a method of amplifying a target nucleic acid (target base sequence) from a primer pair (Primer Set) that specifically binds to a target nucleic acid ), Which is one of the representative Nucleic Acid Amplification Test (NAT).

In the present invention, the step of amplifying the target nucleic acid (target nucleotide sequence) may be performed using a PCR method, a ligase chain reaction (LCR) method, a Gap-LCR Such as Repair Chain Reaction, Transcription-mediated Amplification (TMA), Self Sustained Sequence Replication, Selective Amplification of Target Polynucleotide Sequences, PCR was performed using consensus sequence primed polymerase chain reaction (CP-PCR), arbitrary primed polymerase chain reaction (AP-PCR), Nucleic Acid Sequence Based Amplification (PCR) NASBA, Strand Displacement Amplification, Loop-mediated Isothermal Amplification (LAMP) (PCR), nested polymerase chain reaction (PCR), single tube nested polymerase chain reaction (PCR), single tube nested polymerase chain reaction (PCR) , Reverse transcription polymerase chain reaction (RT-PCR), inverse polymerase chain reaction (RTE), inverse PCR, real-time polymerase chain reaction, (RQ-PCR), and the like, but the present invention is not limited thereto.

In one embodiment, amplifying the target nucleic acid (target base sequence) comprises denaturation of the target nucleic acid; An annealing step of binding a primer pair comprising a target nucleic acid and a complementary base sequence to a complementary target nucleic acid; And an extension step.

In addition, the amplified target base sequence may be labeled with a detectable labeling substance. The labeling substance may be a fluorescent, phosphorescent or radioactive substance but is not limited thereto. For example, the labeling substance may be Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive.

In the real-time PCR reaction method of the present invention, a fluorescent substance is interchelated in a double strand DNA chain in a PCR process and amplified with a PCR product (DNA amplification product) By analyzing the melting curve showing the pattern of the melting curve, in particular the temperature (Tm) at which the DNA is melted (denatured), the amount of fluorescent substance present between the double strands of DNA is reduced by loosening the strand, Jenny Talis The presence or absence of the 16S rRNA gene site enables detection of tylolella equiGenitallis and / or diagnosis of equine infectious endometritis by tylolella equiensitis infection according to the detection.

In the present invention, the analysis of the melting curve may be performed by a FMCA (Fluorescence Melting Curve Analysis) method.

The fluorescence melting curve analysis method is a method of analyzing a melting curve using a fluorescent substance, and more specifically, a melting curve can be analyzed using a primer (s) containing a fluorescent substance. The fluorescent material may be a reporter and a quencher (for example, a scorpion PCR primer), and may be an intercalating fluorescent material.

The reporter is a group consisting of 6-carboxyfluorescein, Texas red, HEX (2 ', 4', 5 ', 7', - tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, Cy3 and Cy5 , And the quencher may be one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto.

In one embodiment of the present invention, the 16S rRNA gene (source: GenBank Accession No. X68645) of the tile Rohrlaquezittalis according to the present invention A primer pair consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 was used as a swab sample of the horses diagnosed with equine infectious endometriosis. DNA derived from Laurellaquezenitalis was specifically detected (see Test Example 1 and Figs. 1-2).

In another embodiment of the present invention, the sensitivity of the primer pairs for detection of tile roullaequianetalis was verified. As a result, the primer pair according to the present invention was able to detect up to 1 copy of the gene of tile roulla equiensis per 1 μl, The minimum detectable nucleic acid content of Laurel aquezenithalis was about 1 gag and the primer pair had a very high detection sensitivity of tylolella acijenittalis (see Test Example 2)

In a further embodiment of the present invention, the specificity of primer pairs for detection of tile roullaequianetalis was verified. As a result, the primer pairs according to the present invention were found to contain tylorella acuijenitallis (ATCC® 700933 ™, TE) In addition to Laurellaquezenitallis (a domestic isolate strain), tylorella Ashini Jenitalis ( Taylorella asinigenitalis, TA), luge Pseudomonas Labor (Pseudomonas aeruginosa, PA), keurep when Ella pneumoniae (Klebsiella pneumoniae, KP) and the tile is found to be not different from cross-reactivity Streptococcus Juba epi Demi Syracuse (Streptococcus zooepidemicus, SZ) This primer pair is suitable for differential diagnosis of Laurel aquezenitalis, and the primer pair has excellent detection specificity of tile Lullaquezenitallis (see Test Example 3).

In another embodiment of the present invention, the PCR technique using a primer pair specific to the 16S rRNA gene region of tile Lullaquezittalis according to the present invention is superior to the conventional method using a primer pair derived from a cador TKP PCR reagent It was confirmed that the tile contained in the specimen (the test sample) could be detected / differentiated and the diagnosis of the horse infectious uterine inflammation due to the infection with tile roella equijenitalis was possible (see Test Example 4).

Therefore, in one aspect, the present invention provides a primer pair comprising a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 as an active ingredient, ( Taylorella equigenitalis ).

In the present invention, the detection may be performed by a polymerase chain reaction (PCR) or a real-time polymerase chain reaction (PCR), but the present invention is not limited thereto.

The present invention relates to kits for the detection of Taylorella equigenitalis comprising the above composition from a different point of view.

In the present invention, the kit may contain reagents necessary for nucleic acid amplification, such as reaction buffer, DNA polymerase (for example, Thermus aquaticus ( Taq ), Thermus thermophilus ( Tth ), Thermus filiformis , Thermis flavus , Thermococcus a thermostable DNA polymerase from Pyrococcus furiosus ( Pfu ), a DNA polymerase joiner, and dNTPs.

In another aspect of the present invention, there is provided a primer set comprising a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer pair consisting of the reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 as an active ingredient, Taylorella equigenitalis infection (Contagious Equine Metritis, CEM).

In the present invention, the diagnosis may be performed by a polymerase chain reaction (PCR) or a real-time polymerase chain reaction (PCR), but the present invention is not limited thereto.

In another aspect, the present invention relates to a diagnostic kit for Contagious Equine Metritis (CEM) caused by infection with Taylorella equigenitalis comprising the composition.

In the present invention, the kit may contain reagents necessary for nucleic acid amplification, such as reaction buffer, DNA polymerase (for example, Thermus aquaticus ( Taq ), Thermus thermophilus ( Tth ), Thermus filiformis , Thermis flavus , Thermococcus a thermostable DNA polymerase from Pyrococcus furiosus ( Pfu ), a DNA polymerase joiner, and dNTPs.

According to another aspect of the present invention, there is provided a method for detecting tylolella equigenitalis , comprising the steps of: (a) preparing a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 which specifically binds to the nucleic acid of tylolella equijetalis ; and And amplifying the nucleic acid of the tile Lullaquezenitalis isolated from the specimen sample with a template using a primer pair composed of a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 as a template to detect tilrolella equiognetallis And a detection method.

In the present invention, the specimen sample may be derived from any particular tissue or organ selected from the group consisting of animals, preferably horses and animals, more preferably zebras, horses (such as ponies), mules and donkeys have. Representative examples of such tissues include binding, skin, muscle or nervous tissue. Representative examples of the above-mentioned organs include, but are not limited to, eyes, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testis, , Lines and inner blood vessels.

The sample of specimen includes any cell, tissue, fluid, or any other medium that can be well analyzed by the present invention from a biological source, which can be used to determine the consumption of human, animal, human or animal Samples from food prepared for this purpose are included. In addition, the sample to be analyzed includes a body fluid sample, which may be a sputum, blood, serum, plasma, lymph, milk, urine, feces, eye milk, saliva, semen, brain extract Spleen and tonsil tissue extracts.

In the present invention, the amplification is performed by polymerase chain reaction (PCR), the polymerase chain reaction is performed by heating at 95 DEG C for 2 minutes, heating the mixture at 94 DEG C for 20 seconds, heating at 55 DEG C for 30 seconds, RTI ID = 0.0 > 30 C < / RTI > for 30 seconds.

According to another aspect of the present invention, there is provided a method for diagnosing contagious equine metritis (CEM) caused by infection with Taylorella equigenitalis , comprising the steps of: ( i ) specifically binding to a nucleic acid of tylolella equiensitris And a reverse primer represented by the nucleotide sequence shown in SEQ ID NO: 2, and a nucleic acid of a tile Rohrlaquezittalis isolated from a specimen of a horse infectious hematocrit. Of the horses infected with horses infectious horsemans (except for humans) is confirmed to be infected with tile of Lulla equiensitis.

In the present invention, the horse infectious endometriosis subject may be any one selected from the group consisting of horses and animals, preferably zebras, horses (ponies, etc.), mules and donkeys.

In the present invention, the specimen sample may be derived from any specific tissue or organ selected from the group consisting of horses and animals, more preferably zebras, horses (ponies, etc.), mules and donkeys. Representative examples of such tissues include binding, skin, muscle or nervous tissue. Representative examples of the above-mentioned organs include, but are not limited to, eyes, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testis, , Lines and inner blood vessels.

The sample of specimen includes any cell, tissue, fluid, or any other medium that can be well analyzed by the present invention from a biological source, which can be used to determine the consumption of human, animal, human or animal Samples from food prepared for this purpose are included. In addition, the sample to be analyzed includes a body fluid sample, which may be a sputum, blood, serum, plasma, lymph, milk, urine, feces, eye milk, saliva, semen, brain extract Spleen and tonsil tissue extracts.

In the present invention, the amplification is performed by polymerase chain reaction (PCR), the polymerase chain reaction is performed by heating at 95 DEG C for 2 minutes, heating the mixture at 94 DEG C for 20 seconds, heating at 55 DEG C for 30 seconds, RTI ID = 0.0 > 30 C < / RTI > for 30 seconds.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

[ Test Example  One] Tile Lorella Equiznitallis  For detection primer  Design, fabrication and verification of pairs

First, bacterial species specific signature sequences, Taylorella < RTI ID = 0.0 > equigenitalis , TE) Bleumink-Pluym NM et al. , Int . J. Syst. Bacteriol . ≪ RTI ID = 0.0 > 16 , < / RTI > rRNA gene sites were designed and constructed to design and construct primer pairs (forward primer and reverse primer) , 43 (3): 618-21, 1993; Breuil MF et al. , Vet. Microbiol. , 148 (2-4): 260-6, 2011).

Specifically, a 16S rRNA gene (source: GenBank Accession No: Tile) of tile roulla equiensis obtained by searching the GenBank database of NCBI (National Center for Biotechnology Information: https://www.ncbi.nlm.nih.gov/) X68645) was analyzed with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify a heterologous bacterium, such as Taylorella asinigenitalis (TA) , Pseudomonas aeruginosa aeruginosa , PA), Klebsiella pneumoniae , KP) and Streptococcus zooepidemicus (SZ). A common conserved region of the 16S rRNA gene sequence specific to the mainstream tile Lulla equiensis strains was selected, A pair of primers capable of specifically detecting (detecting) the 16S rRNA gene region of Laurel aquezenithalis was designed and prepared (Fig. 1 shows the sequence of the 16S rRNA gene sequence of the tylorellaquezenitallis strain (TE) 1 and SEQ ID NO: 2 according to the present invention. Fig. 2 shows the binding sites of the pair of primers according to the present invention by the tile Lulla AQUIJENI (GenBank Accession No. X68645: 1 to 1499) of Talis strain (TE)), the above primer pair was used in Test Example 1 and Test Example 2 PCR was performed using conventional PCR (gel-based PCR).

The primer pair was designed such that G or C having stronger hydrogen bonding strength than A or T could be positioned at the 3 'end of the primer so that the polymerase could stably extend the DNA by increasing the binding force between the primer and the template DNA. In this specification, A (adenine), T (thymine), G (guanine) and C (cytosine) represent nucleotide symbols.

Table 1 below shows the primer pair specific to the 16S rRNA gene site of tile Roella equiensis according to the present invention.

SEQ ID NO: Name of the primer Sequence direction (5 '-> 3') Length Tm (占 폚) SEQ ID NO: 1 Forward primer
(Forward Primer, J1327F) 5'-CAGAGATGACCTTTACTATTG-3 ' 22mer 55 SEQ ID NO: 2 Reverse primer
(Reverse Primer, J1361R) 5'-AGTCCATGGTATTAACACAAAC-3 ' 22mer 55

Specifically, PCR was carried out using a primer pair according to the present invention using a DNA derived from tylolella sequenithialis as a template, and the amplified product of the PCR was amplified with EtBr (ethidium bromide, 0.5 μg / ml) in a conventional manner The presence of the target nucleic acid (DNA derived from tylolella equiensitallis) was confirmed by agarose gel electrophoresis on an added 1.5% (W / V) agarose gel (electrophoresis buffer: 1X TAE ( 40 mM Tris-acetate [pH 8.3], 1 mM EDTA).

In the PCR, 10 pmole forward primer and 10 pmole reverse primer were used per reaction sample. The PCR reaction conditions were pre-denaturation at 95 ° C for 2 minutes, followed by pre-denaturation at 94 ° C for 20 seconds, 55 ° C for 30 seconds, (DNA) in the range of about 1 ng to 100 ng or about 10 pg to 1 g at 40 cycles (cycles: 30 seconds) x 40 cycles.

In the analysis of the amplification reaction product, when a 276 bp DNA amplification product is generated, it is determined that tylolella equijetalis is detected (positive reaction). When the 276 bp DNA amplification product is not generated, It was judged that Laurellaquezenthalis was not detected (negative reaction).

As a result, as shown in FIG. 3, nucleic acids were extracted from 9 out of 139 swab samples of horse genitalia diagnosed with equine infectious endometriosis and PCR amplification was performed. Then, PCR amplification products were subjected to electrophoresis Lanes 1, 2, 3, 6 and 8 were positive and lanes 4, 5, 7 and 9 were negative. Lane 10 is a positive control (nucleic acid of T. equigenitalis ATCC® 700933 ™), lanes 11 and 12 are negative controls (template, Nucleic acid exclusion).

[ Test Example  2] Tile Lorella Aequignitis  For detection primer  Verify pair sensitivity

In order to confirm the detection sensitivity of tylolella axienietalis under the PCR conditions of Test Example 1 using a primer pair specific to the 16S rRNA gene region of the tylolera axizensital according to the present invention, the positive control group tylolella equi PCR was performed by 10-fold serial dilution of the Jenitallys DNA template, and the PCR product was subjected to gel electrophoresis, and then the sensitivity was verified.

As a result, as shown in FIG. 4, it was confirmed that the primer pair according to the present invention was able to detect up to 1 copy of the gene of tile Lilelaquez nitalis per 1 μl, which is superior in sensitivity drawing (FIG. 4: lane 1 (M: 100 bp (Copy number: 10 ⁸ ~ 10 ^-1 ), lane 12 (NC, negative control, no template DNA)) and the lane (Cat. No. D-1030, Bioneer)

Further, as shown in FIG. 5, a sample in which the tylorelequijetinis nucleic acid was diluted to 10 pg to 1ag in the content range was subjected to a PCR reaction using a primer pair according to the present invention, and the gene of tile lorelequezenithalis , It was possible to detect the gene of tylolella equiensitallis even when the nucleic acid content of the tylolella equijetalis was 1 gag, and Wakeley et al. As a result of the comparative analysis of the Ct values obtained using the analysis results based on the literature and the pair of primers derived from the cador TKP PCR Reagent, it was confirmed that the PCR result using the primer pair according to the present invention was excellent in the sensitive drawing (Fig. 5: lane 1 to 8 (tylollaequianetalis nucleic acid: 10 pg to 1ag; amplification product: 275 bp), lane 9 (M: 100 bp DNA ladder (cat.no.D-1030, Bioneer) , Lack of template DNA), lane 11 (Pos: positive control, amplified product 648 bp).

[ Test Example  3] Tile Lorella Aequignitis  For detection primer  Pair specificity validation

In order to confirm the detection specificity of tylolella equiognetallis by the PCR conditions of Test Example 1 using a primer pair specific to the 16S rRNA gene region of the tylolella equiensis according to the present invention, And the DNA template of the heterologous strain, PCR product was electrophoresed on the gel, and then the specificity was verified.

As a result, as shown in FIG. 6, the primer pair according to the present invention is composed of a tile of Lane 1 and 2, Lella Alegienitallis (ATCC® 700933 ™, TE), and tiles of Lane 11 and 12, In addition to Tallis (a domestic isolate strain), the tiles of Lanes 3 and 4, Lilera Ashini Jenitalis asinigenitalis , TA), Pseudomonas aeruginosa (PA) in lanes 5 and 6, Klebsiella pneumoniae (KP) in lanes 7 and 8, and Streptococcus epidemicus lanes 9 and 10 It was confirmed that it was not cross-reacted with Streptococcus zooepidemicus (SZ), so that it was confirmed to be applicable to differential diagnosis of tile Lullaquezenitallis , and thus it was confirmed that it is superior in the specific drawings (wherein M is 100 bp DNA ladder D-1030, Bioneer).

[ Test Example  4] Tile Lorella Equiznitallis  Detection and Horses infectious  Diagnostic method

139 swab specimens of horses diagnosed with equine infectious endometriosis were subjected to PCR using the primer pair specific to the 16S rRNA gene locus of Tylorella equiensis according to the present invention, and, as a control group Real-time PCR (Wakeley et al , Vet Microbiol, 118:.... 247-254, 2006; Bustin et al, Clinical Chemistry 55 (4): 611-622, 2009) with reference to cador PCR was performed under the PCR conditions using the primer pairs included in the TKP PCR Reagent (Cat No. 285115, Qiagen), and then PCR was performed on the tileroella aquethysinis Respectively.

Specifically, Wakeley et al. Literature and Bustin et al. As a result of the analysis based on the literature, 17 swap samples (samples 1 to 17) having a real-time PCR Cq (quantification cycle) value of 30 or less were found to be specific for the 16S rRNA gene region of tile Lulla equiensis Both primer pair and cador TKP PCR reagent- derived primer pairs were positive.

On the other hand, 35 swap samples (samples 18 to 52) with a Real-time PCR Cq value of 31 or more were positive in all cases using the primer pairs derived from the cador TKP PCR Reagent. However, 33 swap samples showed positive reaction and 2 swab samples showed negative reaction when primer pairs specific for 16S rRNA gene sites were used. In particular, among the 35 swap samples with a real-time PCR Cq value of 31 or more, two swap samples showing a negative reaction with a primer pair specific to the 16S rRNA gene region of tile roulla equiensis according to the present invention, .

In addition, 87 swap samples (samples 53-139 ) in which the real-time PCR Cq value was NA (not applicable) were all negative in the case of using a pair of primers derived from a cador TKP PCR reagent. However, In the case of primer pair specific to the 16S rRNA gene region of acizenitallis, 68 swab samples showed negative reaction and 19 swab samples showed positive reaction. In particular, among the 87 swap samples with Real-time PCR Cq value NA, 19 swab samples positive for the 16S rRNA gene locus of tile roella equiensis according to the present invention were analyzed by sequencing sequencing results showed that the nucleic acid of tylolella equijetalis was detected (see Tables 2 to 4 below).

Tables 2 to 4 below show the results of checking whether or not tile roella equiognetallis is detected from a swap sample by using a pair of primers specific to the 16S rRNA gene region of tile Roella axiensis according to the present invention.

As a result, the 16S rRNA gene region of the tile Lullaquezittalis according to the present invention The PCR technique using specific primer pairs is superior to the conventional method using the primer pairs derived from the cador TKP PCR reagent and has a sensitivity and specificity to rapidly detect and discriminate tile roellaquezetalis contained in the test sample It was confirmed that the diagnosis of horse infectious uterine inflammation by Laurel aquezenitalis infection is possible.

Taken together, the 16S rRNA gene locus of the tile Lilelaquez nitalis according to the invention The detection method of tilo lullaquezetalis using specific primer pair was superior both in sensitivity and specificity when compared with primer pair derived from cador TKP PCR reagent.

In addition, by using the above-mentioned tylolla sequenittalis detection method, the nucleic acid of the tylolella equietinis isolated from the specimen of the horse infectious hirsute saliva sample is amplified as a template, and the tylolella sequenitis virus (Diagnosis). Herein, the horse infectious endometrium may be any one selected from the group consisting of horses and animals, preferably zebras, horses (ponies, etc.), mules and donkeys.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> REPUBLIC OF KOREA (Ministry of Agriculture, Food and Rural Affairs, Animal and Plant Quarantine Agency)          MEDIAN Diagnostics Inc. <120> Composition for Detecting Taylorella equigenitalis, Composition          for Diagnosing Contagious Equine Metritis Caused by Taylorella          equigenitalis Infection, Method of Detecting Taylorella          equigenitalis, and Method of Diagnosing Contagious Equine          Metritis Caused by Taylorella equigenitalis Infection <130> YPD201706-0098 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Forward Primer for Detecting Taylorella equigenitalis: J1327F <400> 1 cagagatgac cttttactat tg 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Detecting Taylorella equigenitalis: J1361R <400> 2 agtccatggt attaacacaa ac 22

Claims (11)

A tile inducing a Contagious Equine Metritis (CEM), which comprises, as an active ingredient, a primer pair consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 A composition for the detection of Taylorella equigenitalis .
2. The method according to claim 1, wherein the detection is performed by a polymerase chain reaction (PCR) or a real-time polymerase chain reaction. Composition.
A kit for the detection of Taylorella equigenitalis , comprising the composition of claim 1.
Comprising a primer pair consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 as an active ingredient, Taylorella &lt; RTI ID = 0.0 &gt; A composition for the diagnosis of contagious equine metritis (CEM) caused by.
5. The method according to claim 4, wherein the diagnosis is by a polymerase chain reaction (PCR) or a real-time polymerase chain reaction. A composition for the diagnosis of equine infectious.
A diagnostic kit for contagious equine metritis (CEM) caused by infection with Taylorella equigenitalis , comprising the composition of claim 4.
Tylolella &lt; RTI ID = 0.0 &gt; a primer pair consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2 that specifically binds to the nucleic acid of tile Lulla sequenittalis ; And amplifying the nucleic acid of the tile Rohrlaquez nitalis isolated from the test sample with a template to thereby detect tile roellaquezenithalis.
The method according to claim 7, wherein the amplification is performed by Polymerase Chain Reaction (PCR), the polymerase chain reaction is performed by heating at 95 ° C for 2 minutes, heating the mixture at 94 ° C for 20 seconds, Heating for 30 seconds and heating at 72 DEG C for 30 seconds; and repeating the step of heating for 30 seconds and heating at 72 DEG C for 30 seconds.
Tylolella &lt; RTI ID = 0.0 &gt; A method for diagnosing contagious equine metritis (CEM) caused by infection with equigenitalis, comprising the steps of: preparing a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 which specifically binds to the nucleic acid of tylolera sequenittalis; (Except for humans) by amplifying a nucleic acid of a tile Lilelaqueznitallis isolated from a specimen of a horse infectious habitat by using a pair of primers consisting of a reverse primer represented by the nucleotide sequence of the horses infectious hirsutism, Wherein the method comprises the step of determining whether or not the tile is infected with tile roulla equianetalis.
10. The method according to claim 9, wherein said horse infectious endometriosis is a horse and an animal.
The method according to claim 9 or 10, wherein the amplification is performed by Polymerase Chain Reaction (PCR), the polymerase chain reaction is performed by heating at 95 ° C for 2 minutes, and heating the mixture at 94 ° C for 20 seconds at 40 ° C. Heating at 55 캜 for 30 seconds, and heating at 72 캜 for 30 seconds. The method for diagnosing a horse infectious uterine gland infection by infection with tile roella equenzanitales.

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