KR20130021023A - Primer for the detection of mycoplasma hyorhinis, kit for the detection of mycoplasma hyorhinis by using the primer, and kit for the diagnosis of porcine pneumonia by using the primer - Google Patents

Abstract

PURPOSE: A primer set for detecting mycoplasma hyorhinis, a mycoplasma hyorhinis detection kit using the same, and a diagnosing kit for porcine pneumonia using the same are provided to quickly and specifically detect mycoplasma hyorhinis. CONSTITUTION: A primer set for detecting mycoplasma hyorhinis contains a mycoplasma hyorhinis detection primer with a pair of primers in sequence numbers 1 and 2. The primer detects the mycoplasma hyorhinis by targeting a region between 16s rRNA and 23s rRNA, which is specific to species.

Description

Primer for the detection of Mycoplasma hyorhinis, kit for the detection of Mycoplasma hyorhinis by using the primer, and kit for the diagnosis of porcine pneumonia by using the primer}

The present invention relates to a primer having specificity and sensitivity, capable of specifically detecting only Mycoplasma hyorhinis among various Mycoplasma species.

The present invention relates to primers that target a region between 16s rRNA and 23s rRNA specific for the species of mycoplasma hyorainis pathogen.

The present invention relates to a detection kit that can apply the PCR method as a method for rapidly detecting mycoplasma hyorainis, and can detect only mycoplasma hyorainis specifically and rapidly.

The present invention relates to a diagnostic kit capable of rapidly diagnosing swine pandemic pneumonia, while applying the PCR method as a method of rapidly detecting mycoplasma hyoranisis, a pathogen, for diagnosing swine pandemic pneumonia.

Mycoplasma is a prokaryotic bacterium belonging to the Molycutes class taxonomically and resides in various organs such as the respiratory or genital organs of humans or animals and causes serious diseases. species are known.

Mycoplasmas are resistant to antibiotics that inhibit cell wall synthesis, such as penicillin or streptomycin because they do not have a cell wall, and because they are 0.15 to 0.8 μm in size, they are not removed by filter sterilization and are morphologically pleomorphic. Because of its characteristics, microorganisms are difficult to observe using optical microscopes.

Usually, in order to detect mycoplasma, the direct culture method which isolates and cultures using a medium is used.

However, in the direct culture method, the substrate or the biomaterial is easily contaminated when culturing cells or tissues, and there is a problem that it takes longer than two weeks to confirm the growth of mycoplasma with slow growth rate.

There are also problems that require different culture techniques depending on the species due to the demanding nutritional requirements of mycoplasma.

Moreover, even if contaminated with mycoplasma, it is very difficult to confirm the proliferation of mycoplasma by direct culture because it does not induce cell morphology or color change of culture medium, and no symptoms may occur during infection.

Meanwhile, swine mycoplasma pneumonia (Swine Mycoplasma Pneumonia) is one person pig influenza pneumonia (Swine Epidemic pneumonia, SEP) as a contagious respiratory disease, also known as, in the past, but considering the mycoplasma high ohnyu monitor as the main woninche (Mycoplasma hyophenamoniae), recent Mycoplasma hyorhinis has been shown to cause the same respiratory disease.

The mechanism of this disease is that mycoplasma bacteria adhere to the bronchial ciliary cells of the pig to form colonies and secrete cytotoxic to paralyze the ciliated cells, thereby inhibiting bronchial ciliary discharge and bronchial mucus production. They invade and damage epithelial cells and proliferate.

Mycoplasma bacteria then damage lymphocytes and cause immune dysfunction in pigs.

In addition, the function of Pulmonary Alveolar Macropharge (PAM) in pigs is severely impaired, resulting in secondary bacterial infections such as pleural pneumonia and Pasteurella. It is known to cause mechanisms.

Therefore, it has been found that mycoplasma hyolainis is an important pathogen for swine epidemic pneumonia as much as mycoplasma hyo pneumoniae, so the need for accurate diagnosis of mycoplasma hyolainis is emerging.

Conventionally, as a method for diagnosing a pandemic pneumonia in pigs, a bacterium identification method, a serological method, or a tissue method has been used in which a fresh specimen is collected to isolate and identify a causative pathogen.

However, the bacterium identification method is difficult to culture due to the characteristics of the pathogen, and because the growth rate is very slow, it takes 10 to 30 days or more to isolate the bacterium, and therefore it takes a long time to diagnose the pathogen. There was a problem that can not cope with the enemy.

Recently, the most preferred method of detecting mycoplasma hyoliinis is a method using a polymerase chain reaction (PCR) that amplifies a nucleic acid, a genetic material.

Here, PCR uses a small amount of DNA present in vivo as a template and amplifies only a target DNA fragment hundreds of thousands times in a short time by using oligonucleotides complementary to both ends of the target DNA sequence as primers. Is a technique.

On the other hand, when looking at the patent documents conventionally related to the detection of epidemic pneumonia and mycoplasma in pigs, first, Korean Patent Publication No. 10-1999-0073181 (Patent Document 1) a method for diagnosing swine pandemic pneumonia using an intracellular tissue hybridization method This is described.

This is characterized by diagnosing the infection of a pathogen without bacterial separation using an intracellular hybridization method in which a specific DNA probe of Mycoplasma hyopneumoniae is applied to a tissue section of a pig.

Second, Korean Patent Laid-Open Publication No. 10-2001-0032493 (Patent Document 2) discloses a protein, a DNA sequence encoding a protein, a protein-based vaccine, a DNA sequence, and a mycoplasma hyonneumoniae produced by recombinant DNA or synthetic means. A method for preventing swine pandemic pneumonia using a vaccine and the like, and a diagnostic method based on a protein or antibody generated therein for detecting the presence of an infection in Mycoplasma hyon pneumoniae.

Third, Korean Patent Laid-Open Publication No. 10-2010-0043619 (Patent Document 3) discloses a recombinant expression vector for producing ApxIIA, which is used as a recombinant antigen protein for preventing pleural pneumonia in swine, and an ApxIIA protein using the same. It is described how to produce the in water-soluble form.

Fourth, Korean Patent Publication No. 10-0615424 (Patent Document 4) discloses an oligonucleotide for genotype discrimination of mycoplasma and similar strains, a microarray including the same and a method for detecting the species using the same.

In brief, claim 1 claims a target DNA having SEQ ID NOS: 1 to 6, and claims 2 and 3 claim an oligonucleotide for differentiating mycoplasma strains having SEQ ID NOs: 7 to 21 and 28 to 127.

Here, the oligonucleotides of SEQ ID NOs: 7 to 21 and 28 to 127 mainly function as probes for the search for individual mycoplasma, and in particular, SEQ ID NOs: 41 to 48 are probe bases for the detection of mycoplasma hyorainis. The sequence is described.

However, the base sequence of the primer according to the embodiment of the present invention described below does not match.

Fifthly, Korean Patent Publication No. 10-0502765 (Patent Document 5) discloses a primer for identifying and detecting mycoplasma using PCR, and claim 4 has an identification primer having SEQ ID NOs: 28 to 31. , Claim 5 describes detection primers having SEQ ID NOs: 32-37.

Here, SEQ ID NOs: 28 to 29 and 32 to 33 are Pneumoniae groups, and SEQ ID NOs: 30 to 31 and 34 to 37 are primers for the Hominis group.

Sixth, Korean Patent Publication No. 10-0974861 (Patent Document 6) discloses a PCR primer set for detecting mycoplasma from a biological drug.

Schematically, the first and second polymerase chain reactions are carried out, but the first and second forward primers of the 1, 2 and 10 sequences and the reverse primers of the 3, 4, 5, 6 and 11 sequences are used. Forward primers of 7 and 12 sequences and reverse primers of 8, 9 and 13 sequences will be used.

However, the primers of the 1 to 13 sequences are not consistent with the primers according to the embodiments of the present invention described below.

Collectively, it is required to quickly detect mycoplasma hyorainis, a pathogen, in order to diagnose swine pandemic pneumonia. However, due to the nature of pathogens, the identification method of bacteria has been required for a long time for diagnosis. .

In addition, in the conventional patent technology, it is only known that the DNA probe method is used as a method for diagnosing swine pandemic pneumonia or based on a protein or an antibody. It has been focused on.

In addition, in the conventional patent technology, two polymerizations using a probe for probing individual mycoplasmas, a Pneumoniae or Hominis group, and 13 kinds of forward and reverse primers There have been methods for carrying out an enzyme chain reaction, but a method for detecting mycoplasma hyorainis specifically and sensitively has not been developed.

Therefore, the present inventors apply the PCR method as a method for rapidly detecting mycoplasma hyorainis, a pathogen for diagnosing the epidemic pneumonia in pigs, but by allowing the primer to retain specificity and sensitivity, mycoplasma among various mycoplasma species A technique capable of specifically detecting only hyorinis was devised.

KR 10-1999-0073181 A KR 10-2001-0032493 A KR 10-2010-0043619 A KR 10-0615424 B1 KR 10-0502765 B1 KR 10-0974861 B1

It is an object of the present invention to provide a primer having specificity and sensitivity capable of specifically detecting mycoplasma hyopneumoniae among various mycoplasma species.

It is an object of the present invention to provide a primer that targets a region between 16s rRNA and 23s rRNA that is specific for mycoplasma hyon pneumoniae species.

SUMMARY OF THE INVENTION An object of the present invention is to provide a detection kit that can apply a PCR method as a method for rapidly detecting mycoplasma hyorainis, and can detect only mycoplasma hyorainis specifically and rapidly.

An object of the present invention is to provide a diagnostic kit for rapidly detecting swine epidemic pneumonia, while applying the PCR method as a method of rapidly detecting mycoplasma hyorainis, a pathogen, for diagnosing swine pandemic pneumonia. .

The present invention aims to solve the technical problem by providing a primer for detecting Mycoplasma hyorhinis , comprising a primer pair of SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention is to solve the technical problem by detecting the mycoplasma hyorainis, by providing a primer characterized by detecting a region between the 16s rRNA and 23s rRNA specific to the species (species).

The present invention aims to solve the technical problem by providing a mycoplasma hyalurine detection kit comprising the primer as a kit for detecting mycoplasma hyalurine using a polymerase chain reaction (PCR).

The present invention, as a kit for diagnosing swine pandemic pneumonia (SEP) using a polymerase chain reaction (PCR), comprising the primer, to provide a swine pandemic pneumonia diagnostic kit, to solve the technical problem.

The primer according to the present invention has a remarkable effect of specifically reacting only mycoplasma hyorinis in 10 ² pg of DNA samples of six kinds of mycoplasma strains.

The primers according to the present invention have a remarkable effect of reacting significantly sensitively in more than 1 pg DNA samples of Mycoplasma hyorinis.

The primer according to the present invention has a remarkable effect of effectively detecting mycoplasma hyorainis from the lungs of pigs clinically due to mycoplasma hyalurinitis infection.

The primer according to the present invention targets a region between 16s rRNA and 23s rRNA specific for the species of mycoplasma hyorainis pathogen, thereby retaining specificity and sensitivity, as well as clinically rapid and remarkable. It has a remarkable effect that can detect mycoplasma hyorainis.

The present invention can provide a method that can quickly and accurately detect the mycoplasma hyorainis pathogen, a pathogen of swine pandemic pneumonia, which causes enormous damage in the field of agriculture and livestock raising, and thus can provide a method for early treatment and prevention of infection. It has a beneficial effect.

The present invention can detect mycoplasma pathogens that are frequently contaminated in cell culture or biopharmaceutical manufacture, storage, administration, and culture performed in the field of agriculture, animal husbandry, as well as in the field of agriculture and livestock with rapid and accurate sensitivity and sensitivity. It has significant effects that can be applied to the pharmaceutical and bio-biological industries.

1 is a view showing the results of specificity (specificity) experiment of the primer according to the present invention.
2 is a view showing the results of the sensitivity (sensitivity) experiment of the primer according to the present invention.
Figure 3 is a diagram showing the results of detecting mycoplasma hyalurine pathogens using clinical samples obtained from the lungs of pigs infected with pandemic pneumonia and pigs confirmed negative.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may properly define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

Therefore, the matters shown in the embodiments, reference examples, experimental examples, and drawings described herein are only the most preferred examples of the present invention, and do not represent all of the technical ideas of the present invention, and thus, these are replaced at the time of the present application. It should be understood that there may be various equivalents and variations that can be made.

First, "primer" herein means an oligonucleotide complementary to the 5 'terminal sequence and the 3' terminal sequence adjacent to the target nucleic acid used in the polymerase chain reaction.

In addition, "forward (prior) primer" and "reverse (reverse) primer" means a primer that binds to the 3 'end and 5' end of each region of the gene amplified by the polymerase chain reaction, respectively.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example 1 Preparation of a Primer Specific for Detecting Mycoplasma Hyorinis

By targeting the region between the 16s rRNA and 23s rRNA specific to the species in the mycoplasma hyorainis genome sequence, primers that can specifically detect mycoplasma hyorainis are shown below in SEQ ID NOs: 1 and 2 Prepare to include a primer pair consisting of a nucleotide sequence.

SEQ ID NO: 1. Forward primer

5'-ACT TCG GAG ACC ATT GCC TAA G-3 '

(22 mer, nucleotides 19731-19750)

SEQ ID NO: 2. Reverse primer

5'-AAA GCT CTC AAA ACT AGA CAC GAA-3 '

(24 mer, nucleotides 19889-19866)

The primer is used by Bioneer Co., Ltd. (Republic of Korea) and then purified by OPC method.

Example 2 Method for Specific Detection of Mycoplasma Hyorinis

(1) extracting DNA from a sample

Cultured cells or porcine pneumonia tissues are used as samples, and DNA is separated and extracted therefrom.

(2) preparing a primer specific for the mycoplasma hyorinis pathogen prepared in Example 1

Primers targeting the region between 16s rRNA and 23s rRNA that are specific for the species in the Mycoplasma hyorinis genome sequence are prepared.

(3) performing polymerase chain reaction (PCR) with the extracted sample DNA and primer

The composition of the reaction reagent for PCR was 1 μl forward, reverse primer (10 pmol / μl), 2.5 μl 10 × reaction buffer, 500 μM deoxynucleotide, 1.5 U Taq polymerase, and 25 μl of reaction volume. Prepare by mixing sterilized distilled water as much as possible.

The polymerase chain reaction is carried out using a thermocycler (ABI, USA).

The reaction conditions were pre-denaturation for 5 minutes at 95 ° C, followed by 1 minute at 95 ° C, 1 minute at 62 ° C, and 1 minute at 72 ° C. 30 cycles and finally post-polymerization (min) at 72 ℃ for 3 minutes (min).

The product of the polymerase chain reaction was electrophoresed on 2% agarose gel to confirm the band of 140 bp.

Reference Example 1. Experiment Preparation

Developed according to the embodiment of the present invention, the following experiments were prepared to confirm the specificity and sensitivity of the primers specific for mycoplasma hymorinis.

1-1. Mycoplasma Strain Preparation and Culture

In order to perform comparative experiments to confirm the specificity and sensitivity of the primer according to the present invention, a total of six kinds of mycoplasma strains were purchased and cultured as follows.

Mycoplasma species One Mycoplasma hyorhinis (ATCC strain 27717) 2 Mycoplasma hyopneumoniae (ATCC strain 25934) 3 Mycoplasma pneumoniae (ATCC 15531) 4 Mycoplasma pulmonis (ATCC 19612) 5 Mycoplasma hominis (ATCC 23114) 6 Mycoplasma arthritiditis (ATCC 19611)

The mycoplasma strain was cultured for 10 days at 37 rpm at 58 rpm using modified Friis medium to remove Friss medium components by centrifugation at 13,000 g for 30 minutes.

1-2. DNA Extraction Sample Preparation for Mycoplasma Strains

DNA extraction of each mycoplasma strain cultured using the bead beater-phenol extraction method (bead beater-phenol extraction method).

Pellet samples obtained by centrifuging the cultured strains at 13,000 g for 30 minutes were sterilely collected in a 2 ml tube dedicated to Mini-Bead Beater (Biospec product) containing 1 ml sterile distilled water, sliced and suspended in sterile tertiary distilled water. Add 200 μl of glass bead (0.1 mm size, Biospec product) and 200 μl of phenol-chlorform-isoamyl alcohol solution (50: 49: 1 (v / v / v)) with 5,000 rpm for 30 seconds using Mini-Bead Beater (Biospec product). Shaken.

After shaking, the mixture was centrifuged at 12,000 rpm for 15 minutes at 4 ° C, and the supernatant was transferred to a sterile 2 ml tube.

10 μl of 3 M sodium acetate and 250 μl of ice-cold ethanol were added to the mixture, and the mixture was suspended for 10 minutes at −20 ° C., followed by centrifugation at 15,000 rpm for 15 minutes.

The precipitate was washed with 70% alcohol, dried at room temperature and dissolved in 60 μl of Tris EDTA (pH 8.0) and used for the experiment.

1-3. Polymerase Chain Reaction Conditions

Primers specific for the mycoplasma hyorainis pathogen prepared in Example 1 are used, which targets a region between the 16s rRNA and the 23s rRNA that is specific for the species in the mycoplasma hyorainis genome sequence.

The composition of the reaction reagent for the polymerase chain reaction, the thermocycler for the reaction, the reaction conditions, and the confirmation of the reaction product proceed in the same manner as in Example 2.

Experimental Example 1. Confirmation of specificity of mycoplasma hyorinis of the primer according to the present invention

According to the experimental preparation of Reference Example 1, six kinds of mycoplasma strains, Mycoplasma hyorhinis , M. hyopneumoniae and Mycoplasma pneumoniae , mycoplasma pulse monitor's (M. pulmonis), mycoplasma hoe to prepare the DNA samples of 10 pg of the varnish ² (M. hominis), mycoplasma O Write ET di tooth (M. arthritiditis).

For each of the samples, a polymerase chain reaction was carried out with primers targeting the region between 16s rRNA and 23s rRNA specific to the species in the Mycoplasma hyorhinis genome sequence prepared in Example 1. Was performed.

The conditions of the polymerase chain reaction were performed in the same manner as in Example 2.

1 is a view showing the results of a specificity (specificity) experiment of the primer according to the present invention, which is shown in Table 2 below.

Mycoplasma species PCR result One M. hyorhinis +++ 2 M. hyopneumoniae - 3 M. pneumoniae - 4 M. pulmonis - 5 M. hominis - 6 M. arthritiditis -

+++: Strong reaction,-: No reaction

As shown in FIG. 1 and Table 2, the primers targeting the region between the 16s rRNA and 23s rRNA specific to the species in the mycoplasma hyorinis genome sequence developed in the present invention are Mycoplasma, which is strain # 1. It can be seen that it specifically reacted only to the hycoplasma hyorhinis DNA.

On the other hand, it can be seen that the reaction did not occur with strains 2-6.

Therefore, the result of Experiment 1 shows that the primers targeting the region between the 16s rRNA and 23s rRNA specific to the species in the mycoplasma hyorainis genome sequence developed in the present invention are specific to mycoplasma hyorainis only. It was confirmed to react.

Experimental Example 2. Confirmation of the sensitivity of the primer according to the present invention for mycoplasma hyorininess

According to the experimental preparation of Reference Example 1, the culture and DNA extraction samples of mycoplasma hyorinis strains were prepared.

The extracted Mycoplasma hyorinis Purified Mycoplasma Hyorinis DNA Samples Prepared by Quantitative DNA Quantification Using a Spectrophotometer (10)², 10, 1, 0.1 pg) for each polymerase chain reaction.

The polymerase chain reaction was carried out with primers targeting the region between the 16s rRNA and 23s rRNA specific to the species in the Mycoplasma hyorainis genome sequence prepared in Example 1.

The conditions of the polymerase chain reaction were performed in the same manner as in Example 2.

2 is a view showing the results of the sensitivity (sensitivity) experiment of the primer according to the present invention, which is shown as a table Table 3 below.

Template DNA (pg) 10 ² 10 One 0.1 Positive reaction ++ + + -

+++: Strong band, ++: moderate band, +: mild band,-: No reaction

As shown in FIG. 2 and Table 3, the primers targeting the region between the 16s rRNA and 23s rRNA specific to the species in the mycoplasma hyorinis genome sequence developed in the present invention are 1 pg or more of mycoplasma hyora It can be seen that a significant reaction occurred in the innis DNA sample.

Therefore, the result of Experiment 2 shows that the primers targeting the region between the 16s rRNA and 23s rRNA specific to the species among the mycoplasma hyorainis genome sequences developed in the present invention are significant to mycoplasma hyolineis. It was confirmed to react sensitively.

Experimental Example 3. Detection of Mycoplasma Hyorinis Pathogens Using Clinical Samples

To confirm the efficacy of the primers developed according to the present invention from the lungs of pigs clinically infected with pancreatic pneumonia due to mycoplasma hyorainis infection, a mycoplasma pathogen detection experiment using clinical samples was performed.

A slaughtered sample of pneumonia confirmed asymptomatically was collected from pigs confirmed with mycoplasma hyorainis infection at a slaughterhouse in Iksan, Jeonbuk.

In addition, a portion of the lungs was aseptically collected from a pig that was negative through pre-slaughter blood tests.

DNA samples were prepared by following the same procedure as preparing the DNA extraction sample of Reference Example 1, but three DNA samples of pigs infected with pandemic pneumonia and three DNA samples of negative pigs were prepared.

For each DNA sample, polymerase chain reaction was carried out with primers targeting the region between 16s rRNA and 23s rRNA specific to the species in the Mycoplasma hyorhinis genome sequence prepared in Example 1. Was performed.

The conditions of the polymerase chain reaction were performed in the same manner as in Example 2.

FIG. 3 is a diagram showing the results of detecting mycoplasma hyalurine pathogens using clinical samples obtained from pigs infected with pandemic pneumonia and lungs of negative pigs, and shown in Table 4 as a table.

Clinical samples (Lung) PCR result No. 1 (sero-positive) + No. 2 (sero-positive) ++ No. 3 (sero-positive) +++ No. 4 (sero-negative) - No. 5 (sero-negative) - No. 6 (sero-negative) -

++: Strong reaction,-: No reaction

As shown in FIG. 3 and Table 4, the primers targeting the region between the 16s rRNA and the 23s rRNA specific to the species in the mycoplasma hyorainis genome sequence developed in the present invention are clinically known as mycoplasma hyora. Mycoplasma hyorainis could be detected from the lungs of pigs infected with swine pandemic pneumonia caused by innis infection.

On the other hand, no. 4, 5, 6 pig lung tissue was unable to detect mycoplasma hyorainis.

Therefore, primers targeting the region between 16s rRNA and 23s rRNA specific to the species among the mycoplasma hyorainis genome sequences developed in the present invention are clinically infected with swine epidemic pneumonia caused by mycoplasma hyouranis infection. It was confirmed that mycoplasma hyorainis can be effectively detected from the lungs of pigs.

In summary, Experimental Examples 1 to 3, in 10 ² pg of DNA samples of six kinds of mycoplasma strains, the primer according to Example 1 of the present invention was specifically specific to Mycoplasma hyorainis. It was confirmed that the reaction.

In addition, it was confirmed that the primers according to Example 1 of the present invention responded significantly sensitively to DNA samples of 1 pg or more of Mycoplasma hyorainis.

In addition, it was confirmed that the primers according to Example 1 of the present invention effectively detected mycoplasma hyorainis from the lung of a pig due to mycoplasma hyalurinitis infection.

Therefore, the primer according to Example 1 of the present invention targets a region between a 16s rRNA and a 23s rRNA specific to a species in the Mycoplasma hyorhinis genome sequence, thereby significantly improving specificity and sensitivity. Of course, it can be said that clinically rapid and remarkably detectable mycoplasma hyorainis can be detected.

<110> Wonkwang University Center for Industry-Academy Cooperation <120> Primer for the detection of Mycoplasma hyorhinis, kit for the          detection of Mycoplasma hyorhinis by using the primer, and kit          for the diagnosis of porcine pneumonia by using the primer <130> 2011-03 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mhyorhinis Forward primer <400> 1 acttcggaga ccattgccta ag 22 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mhyorhinis Reverse primer <400> 2 aaagctctca aaactagaca cgaa 24

Claims (4)

A primer for detecting Mycoplasma hyorhinis , comprising primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2. The method of claim 1,
Detecting the mycoplasma hyorainis, characterized in that by detecting the target region between the 16s rRNA and 23s rRNA specific to the species (species). A kit for detecting Mycoplasma hyorhinis using polymerase chain reaction (PCR),
A mycoplasma hyorinis detection kit comprising the primers of claim 1. A kit for diagnosing swine pandemic pneumonia (SEP) using polymerase chain reaction (PCR),
Porcine epidemic pneumonia diagnostic kit comprising the primer of any one of claims 1 to 2.

Images