KR101329114B1 - Primer for the detection of Mycoplasma hyopneumoniae, and kit and method for the detection of Mycoplasma hyopneumoniae by using the primer, and kit and method for the diagnosis of porcine pneumonia by using the primer - Google Patents

Abstract

The present invention relates to primers with improved specificity and sensitivity, capable of specifically detecting mycoplasma hyopneumoniae among various mycoplasma species.
The present invention relates to a primer that targets a protein P97 region specific for the species of mycoplasma hyopneumoniae pathogen.
The present invention relates to a detection kit and a detection method capable of rapidly detecting mycoplasma high pneumoniae and specifically detecting mycoplasma high pneumoniae.
The present invention provides a diagnostic kit and diagnostic method for rapidly detecting swine epidemic pneumonia, while applying the PCR method as a method for rapidly detecting mycoplasma hyonneumoniae, a pathogen, for diagnosing swine pandemic pneumonia. will be.

Description

Primer for detecting mycoplasma hyon pneumoniae, kit and method for detecting mycoplasma hyon pneumoniae using the primer, and diagnostic kit and method for swine pandemic pneumonia using the primer (Primer for the detection of Mycoplasma hyopneumoniae, and kit and method for the detection of Mycoplasma hyopneumoniae by using the primer, and kit and method for the diagnosis of porcine pneumonia by using the primer}

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

The present invention relates to a primer that targets a protein P97 region specific for the species of mycoplasma hyopneumoniae pathogen.

The present invention relates to a detection kit and a detection method capable of rapidly detecting mycoplasma high pneumoniae and specifically detecting mycoplasma high pneumoniae.

The present invention provides a diagnostic kit and diagnostic method for rapidly detecting swine epidemic pneumonia, while applying the PCR method as a method for rapidly detecting mycoplasma hyonneumoniae, a pathogen, for diagnosing swine pandemic pneumonia. will be.

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 [(Nonpatent Literature 1), Maes et al., 2008, reference].

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 may cause no symptoms during infection.

On the other hand, swine enzootic pneumonia (SEP) is caused by Mycoplasmahyopneumoniae , which is characterized by chronic cough, stagnant growth, low mortality and high morbidity. The response to the swine industry is enormous, due to the lack of prompt and sufficient measures.

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, various methods such as DNA fluorochrome staining, enzyme immunoassay, fluorescence staining, biochemical test, and DNA probe technique have been studied as methods for more quickly identifying contamination with mycoplasma hyopneumoniae. come.

In particular, recently, the most preferred method of detection of mycoplasma hyon pneumoniae is a method using a polymerase chain reaction (PCR) to amplify 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.

 This PCR amplification method can be used to quickly and accurately detect mycoplasma hyonneumoniae infected with swine lung lesions or mycoplasma hyonneumoniae contaminated with high frequency during in vitro manipulation and culture of cells or tissues. [(Non-Patent Document 3), Calsamaglia et al., 1999, reference].

However, the sensitivity and specificity of the primers have been reduced in the mycoplasma hyon pneumoniae previously developed and reported, and thus, there has been a difficulty in accurate diagnosis [(Non-Patent Document 2), Caron et al., 2000, see].

Here, "primer" refers to oligonucleotides 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.

On the other hand, when looking at the patent documents previously associated with the swine epidemic pneumonia, first, Korean Patent Publication No. 10-1999-0073181 (Patent Document 1) describes a method for diagnosing swine epidemic pneumonia using intracellular hybridization method. .

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.

Collectively, it is required to quickly detect mycoplasma hyon pneumoniae, a pathogen, in order to diagnose swine pandemic pneumonia. However, the pathogen identification method takes a long time for diagnosis due to the nature of the pathogen. Have been.

In addition, when the PCR method is applied, the specificity and sensitivity of the primers are difficult to accurately diagnose mycoplasma hyopneumoniae.

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.

Therefore, in order to diagnose swine pandemic pneumonia, the PCR method is applied to detect mycoplasma hyon pneumoniae, a pathogen, and improves the specificity and sensitivity of primers, thereby increasing the specificity and sensitivity of mycoplasma hyon pneumoniae among various mycoplasma species. There is an urgent need for a technology that can detect only E.

KR 10-1999-0073181 A KR 10-2001-0032493 A KR 10-2010-0043619 A

Maes D, Segales J, Meyns T, Sibila M, Pieters M, Haesebrouck F. Control of Mycoplasma hyopneumoniae infections in pigs. Vet Microbiol 2008, 126, 297-309. Caron, J., M. Ouardani, and S. Dea. 2000. Detection and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis by PCR amplification of the p36 and p46 genes. J. Clin. Microbiol. 38: 1390-1396. Calsamaglia, M., C. Pijoan, and A. Trigo. 1999. Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs. J. Vet. Diagn. Investig. 11: 246-251. Zhang Q, Young TF, Ross RF. Microtiter plate adherence assay and receptor analogs for Mycoplasma hyopneumoniae. Infect Immun 1994, 62, 1616-1622.

An object of the present invention is to provide a primer having improved specificity and sensitivity, which can specifically detect mycoplasma hyopneumoniae among various mycoplasma species.

It is an object of the present invention to provide a primer that targets a protein P97 region specific for the species of mycoplasma hyopneumoniae pathogen.

SUMMARY OF THE INVENTION An object of the present invention is to provide a detection kit and a detection method capable of rapidly detecting mycoplasma high pneumoniae and specifically detecting mycoplasma high pneumoniae. .

An object of the present invention is to apply a PCR method as a method for rapidly detecting mycoplasma hyon pneumoniae, a pathogen, to diagnose swine pandemic pneumonia, and a diagnostic kit and diagnostic method for rapidly diagnosing swine pandemic pneumonia. In providing.

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

The present invention aims to solve the technical problem by providing a primer which detects the mycoplasma hyopneumoniae and targets and detects a protein P97 region specific to a species.

The present invention is to solve the technical problem by providing a method for detecting mycoplasma high pneumoniae by a polymerase chain reaction (PCR) using the primer.

The present invention, by providing a method for diagnosing swine enzootic pneumonia (SEP) by the polymerase chain reaction (PCR) using the primer, to solve the technical problem.

The present invention is to provide a detection kit for mycoplasma hyonneumoniae 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 responding to mycoplasma hyonneumoniae only in 10 ² pg of DNA samples of six kinds of mycoplasma strains.

The primers according to the present invention have a significant effect of reacting significantly sensitively in DNA samples of 10 pg or more of Mycoplasma hyopneumoniae.

The primer according to the present invention has a remarkable effect of effectively detecting mycoplasma hyopneumoniae from the lung of a pig clinically infected with mycoplasma hyopneumoniae.

The primer according to the present invention, by targeting the protein P97 region specific for the species of the pathogen to mycoplasma hyon pneumoniae, improves specificity and sensitivity as well as clinically rapid and remarkably. It has a remarkable effect of detecting mycoplasma hyopneumoniae.

The present invention can provide a strategy that can quickly and accurately detect mycoplasma hyon pneumoniae, a pathogen of swine pandemic pneumonia, which is causing enormous damage in the field of agricultural livestock raising, thereby providing a strategy for early treatment and prevention of infection. It has a very 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.
3 is a diagram showing the results of detecting pathogens in mycoplasma hyon pneumoniae using clinical samples obtained from the lungs of pigs infected with pandemic pneumonia and pigs with 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.

Hereinafter, preferred 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 Hyonneumoniae

By targeting a protein-specific protein P97 region in the mycoplasma hyopneumoniae genome sequence, primers that can specifically detect mycoplasma hyopneumoniae only are shown in SEQ ID NOs: 1 and 2 as follows. Prepare to include a primer pair consisting of the base sequence of.

SEQ ID NO: 1.Forward primer

5'-TCC AGT CAT ACC AAG GCA GAA ATT-3 '

(24 mer, nucleotides 223070-223093)

SEQ ID NO: 2. Reverse primer (Reverse primer)

5'-CGG GCA CTT TGA CTA AGA TCT GC-3 '

(23 mer, nucleotides 222827-222849)

The primer is used after purification by OPC method.

Example 2. How to specifically detect mycoplasma hyopneumoniae

(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 hyopneumoniae pathogen prepared in Example 1

Primers targeting protein P97 regions specific for species in the genome sequence are prepared in Mycoplasma hyopneumoniae.

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

Reagents for PCR, 1 μl forward, reverse primer (10 pmol / μl), 2.5 μl 10 × reaction buffer, 500 μM deoxynucleotides, 1.5 U Taq polymerase were added and sterilized to a reaction volume of 25 μl. Prepare by mixing with distilled water.

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 60 ° 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 identify a band of 240 bp.

References 1. Experimental 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 hymon pneumoniae.

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 hyopneumoniae (ATCC strain 25934) 2 Mycoplasma hyorhinis (ATCC strain 27717) 3 Mycoplasma pneumoniae (ATCC15531) 4 Mycoplasma pulmonis (ATCC19612) 5 Mycoplasma hominis (ATCC23114) 6 Mycoplasma arthritidis (ATCC19611)

Culture of the mycoplasma strain was cultured for 1 week at 58 rpm at 37 ℃ using modified Friis medium (Ref. 4, Zhang et al., 1994) and centrifuged at 13,000 g for 30 minutes to remove the Friss medium components.

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

A primer specific for the mycoplasma hyopneumoniae pathogen prepared in Example 1 is used, which targets a protein P97 region specific for the species in the mycoplasma hyopneumoniae 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 hyon pneumoniae of the primer according to the present invention

According to the experimental preparation of Reference Example 1, six types of mycoplasma strains, Mycoplasma hyopneumoniae (ATCC strain 25934), Mycoplasma hyorhinis (ATCC strain 27717), Mycoplasma pneumoniae (ATCC15531), Mycoplasma pulmonis (ATCC19612), Mycoplasma hominis (ATCC23114) 10 ² pg of DNA samples of Mycoplasma arthritidis (ATCC19611) were prepared.

For each of the samples, a polymerase chain reaction was carried out with primers targeting the Mycoplasma hyopneumoniae protein P97 region prepared in Example 1.

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. hyopneumoniae +++ 2 M. hyorhinis - 3 M. pneumoniae - 4 M. pulmonis - 5 M. hominis - 6 M. arthritidis -

+++: Strong reaction,-: No reaction

As shown in FIG. 1 and Table 2, the Mycoplasma hyopneumoniae genomic sequence protein P97 region target primer developed in the present invention is specific only to Mycoplasma hyopneumoniae DNA, which is strain No. 1. It can be seen that the reaction.

It can also be seen that the reaction did not occur with strains 2-6.

Therefore, as a result of Experiment 1, it was confirmed that the mycoplasma hyon pneumoniae protein P97 region target primer developed in the present invention specifically reacted only to mycoplasma hyon pneumoniae.

Experimental Example 2 Confirmation of the sensitivity of the primer according to the present invention to mycoplasma hyon pneumoniae

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

DNA samples (10 ³ , 10 ² , 10,1 pg) were purified and purified by using a spectrophotometer, followed by dilution of DNA using a spectrophotometer. Polymerase chain reaction was performed for each.

The polymerase chain reaction was carried out with primers targeting the Mycoplasma hyopneumoniae protein P97 region 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 ³ 10 ² 10 One Positive reaction +++ ++ + -

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

As shown in FIG. 2 and Table 3, the mycoplasma hyon pneumoniae genome sequence protein P97 region target primer developed in the present invention significantly reacted with DNA samples at 10 pg or more of mycoplasma hyon pneumoniae. It can be seen.

Therefore, as a result of Experiment 2, it was confirmed that the protein P97 region target primer of mycoplasma hyon pneumoniae developed in the present invention reacts sensitively to mycoplasma hyon pneumoniae.

Experimental Example 3. Detection of pathogens in Mycoplasma hyopneumoniae using clinical samples

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

Slaughter was collected asymptomatically from a pig that was confirmed to be infected with mycoplasma hyon pneumoniae through a pre-slaughter blood test 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 four DNA samples of pigs infected with pandemic pneumonia and two DNA samples of negative pigs were prepared.

For each DNA sample, polymerase chain reaction was carried out with primers targeting the Mycoplasma hyopneumoniae protein P97 region prepared in Example 1.

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

3 is a diagram showing the results of detecting pathogens in Mycoplasma hyopneumoniae using clinical samples obtained from pigs infected with pandemic pneumonia and pigs with negative confirmation, and Table 4 shows the results.

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

++: Strong reaction,-: No reaction

As shown in FIG. 3 and Table 4, the Mycoplasma hyopneumoniae genome sequence protein P97 region target primer developed in the present invention is clinically infected with swine epidemic pneumonia due to Mycoplasma hyopneumoniae infection. Mycoplasma high pneumoniae could be detected from the lung.

On the other hand, no. 5 and No. 6 could not detect mycoplasma hyopneumoniae.

Therefore, the mycoplasma hyon pneumoniae genome sequence protein P97 region target primer developed in the present invention is clinically applied to mycoplasma hyon pneumoniae from pig lungs clinically infected with swine epidemic pneumonia due to mycoplasma hyon pneumoniae infection. It can be confirmed that can be effectively detected.

In summary, Experimental Example 1 to Example 3, in 10 ² pg of DNA samples of six kinds of mycoplasma strains, the primer according to Example 1 of the present invention is specifically specific to mycoplasma hyonneumoniae. 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 10 pg or more of Mycoplasma hyopneumoniae.

In addition, it was confirmed that the primers according to Example 1 of the present invention effectively detected mycoplasma hyopneumoniae from pig lungs clinically infected with mycoplasma hyopneumoniae.

Therefore, the primer according to Example 1 of the present invention targets the protein P97 region specific for the species of the pathogen to mycoplasma hyon pneumoniae, thereby improving specificity and sensitivity as well as clinically rapid. In addition, it can be said that the mycoplasma hyopneumoniae can be detected remarkably.

Attach an electronic file to a sequence list

Claims (8)

Including primer pair of SEQ ID NO: 1 and SEQ ID NO: 2,
Targets only the protein P97 region specific to the species,
Mycoplasma hyopneumoniae ( M. hyopneumoniae ), Mycoplasma hyorhinis ( M. hyorhinis ), Mycoplasma pneumoniae ( M. pneumoniae ), Mycoplasma pulmonis ( M. pulmonis ), Mycoplasma hominis ( M hominis ), DNA sample sensitivity and specificity measurements of Mycoplasma aslitidis ( M. arthritidis ) is characterized by performing a polymerase chain reaction,
The sensitivity of the DNA sample is 10 pg or more, characterized in that the specificity of the DNA sample is 10 ² pg or more,
Characterized in having a detection effect in pigs naturally infected with mycoplasma hyopneumoniae,
Primer for detecting Mycoplasma hyopneumoniae .
delete delete delete A mycoplasma hyopneumoniae detection kit comprising the primer of claim 1.
delete delete delete

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