KR101498176B1 - Primer set for the detection of mycoplasma pulmonis, and method and kit for the detection of mycoplasma pulmonis by using the primer set - Google Patents

Abstract

The present invention relates to a primer set having specificity and sensitivity capable of specifically detecting only Mycoplasma pulmonis among various mycoplasma species bacteria.
The present invention relates to a primer set targeting a 16s ribosomal RNA sequence region of a mycoplasma pearmonis species specific.
The present invention relates to a method for detecting mycoplasma pheromone by performing PCR (Polymerase chain reaction, polymerase chain reaction) using the above primer set.
The present invention relates to a detection kit capable of specifically detecting only mycoplasma pneumoniae by applying a PCR method which is a method of rapidly detecting mycoplasma papillomonas.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer set for detecting mycoplasma pneumonias, a method for detecting mycoplasma papillomas using the primer set, and a kit and a kit for detecting the mycoplasma pumulus. PRIMER SET}

The present invention relates to a primer set having specificity and sensitivity capable of specifically detecting only Mycoplasma pulmonis among various mycoplasma species bacteria.

The present invention relates to a primer set targeting a 16s ribosomal RNA sequence region of a mycoplasma pearmonis species specific.

The present invention relates to a method for detecting mycoplasma papillomas by executing PCR using the primer set.

The present invention relates to a detection kit capable of specifically detecting only mycoplasma pneumoniae by applying a PCR method which is a method of rapidly detecting mycoplasma papillomonas.

Mycoplasma genes are taxonomic classes of the Mollicutes class of prokaryotic bacteria, and hundreds of species are known to date. It is resistant to antibiotics such as penicillin and streptomycin, which are not removed by filtration sterilization at about 0.15 ~ 0.8 ㎛ in size, and have no cell walls and inhibit cell wall synthesis. It is also a morphologically pleomorphic microorganism that is difficult to observe by optical microscopy.

Mycoplasma bacteria are well known to cause serious diseases by resident in various organs such as human or animal respiratory organs.

Among them, Maiko Plasmapulmonis Mycoplasma pulmonis species is a pathogenic agent causing MRM (Murine Respiratory Mycoplasmosis) in rodents. It has a high infection rate in rodents such as mice and rats, and has a high infection rate especially in rats.

In addition, mycoplasma pemmonis causes a respiratory mycoplasma infection of the rodent group and colonization of the membranes of the respiratory epithelium to interfere with the mucociliary clearance function. Mycoplasma infection is exacerbated by viral infections such as Sendai virus, mouse hepatitis virus, and ammonia levels in the surrounding environment.

Mycoplasma pemmonis is highly infectious when cultured or cultured when cells or tissues are manipulated in vitro, and the lungs and mucous membranes of mice are also infected.

The mouse or rat is highly preferred as an experimental animal because it has high similarity to humans in terms of genetic, nutritional, and physiological aspects and has a strong fertility. Therefore, studies have been conducted to diagnose and prevent mycoplasma infection in mice or rats.

Currently, methods for diagnosing mycoplasma infection in mice or rats have been used, including methods for isolating and identifying causative pathogens, serological methods, and tissue-based methods.

However, the method of isolating and isolating bacteria is difficult because of the nature of the pathogen, and since the growth rate is very slow, it takes more than 10 ~ 30 days to isolate the bacteria and it takes a long time to diagnose the pathogen. Therefore, there is a problem that they can not be treated immediately

In particular, the direct culture method that is commonly used for the detection of mycoplasma is easy to infect a substrate or a biomaterial during cell or tissue culture, takes more than two weeks to confirm proliferation of mycoplasma, Due to the demanding nutrient requirements, different cultivation techniques are required depending on the species.

In addition, even when infected with mycoplasma, it is extremely difficult to confirm the proliferation of mycoplasma by the direct culture method because no change occurs in the cells, the culture medium, or the like, and no symptoms may appear at the time of infection.

In recent years, various methods such as DNA fluorochrome staining, enzyme immunoassay, fluorescence staining, biochemistry, and DNA probe method have been sought as methods for detecting mycoplasma fulminans more rapidly. Recently, the most preferred method is PCR (polymerase chain reaction) which amplifies the nucleic acid as a genetic material.

PCR uses a small amount of DNA present in vivo as a template and uses an oligonucleotide complementary to both ends of the target DNA sequence as two sets of primers, that is, a primer set, It is a technique that amplifies hundreds of thousands of times in a short time. By using the gene amplification method using such PCR, it is possible to detect mycoplasma papillomasus quickly and accurately.

However, the previously developed and reported mycoplasma pemmonis primer set has a low sensitivity and specificity, so it has been difficult to accurately diagnose it.

Thus, Applicants have discovered that the target region of the 16s rRNA sequence (16s rRNA sequence) of mycoplasma fumaricum species-specific is specifically detected by detecting mycoplasma papillomas among various mycoplasma species bacteria And a detection method and kit using the primer set can be provided. Thus, the present invention has been made.

In the prior art related to the present invention, Korean Patent Registration No. 10-0502765 discloses a detection and identification method of mycoplasma.

The above prior art also devised a kit using a primer set and a primer set as the detection and identification method of mycoplasma. However, the nucleotide sequence of the primer set devised in the prior art is not limited to that of the primer set according to the embodiment of the present invention described below Does not match base sequence.

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

It is an object of the present invention to provide a primer set that targets a species specific 16s rRNA sequence region of mycoplasma pearmonis.

It is an object of the present invention to provide a method for detecting mycoplasma papillomas by performing PCR using the primer set.

It is an object of the present invention to provide a detection kit capable of specifically detecting only mycoplasma papillomas by applying a PCR method which is a method for rapidly detecting mycoplasma papillomonas.

In order to achieve the above object, the present invention aims at solving the technical problem by providing a target primer set of mycoplasma pearmonis with specificity and sensitivity.

The present invention also solves the technical problem by providing a species specific 16s rRNA sequence region of mycoplasma pemmonis.

In addition, the present invention provides a method for detecting mycoplasma papillomas by performing PCR using the primer set, thereby solving the technical problem.

The present invention also provides a detection kit capable of specifically detecting only mycoplasma papillomas by applying a PCR method which is a method of rapidly detecting mycoplasma papillomons, thereby solving the technical problem.

The primer set according to the present invention has an effect of specifically reacting 10 ⁴ pg of DNA samples of mycoplasma species strains with only mycoplasma pearmonis during PCR.

The primer set according to the present invention has the effect of stepwise diluting a DNA sample of Mycoplasma pearmonice and reacting sensitively to a DNA sample of 5 pg or more in PCR significantly.

The primer set according to the present invention targets the species-specific 16s rRNA sequence region of mycoplasma pumicornis and has a specificity and sensitivity by applying the PCR method as a rapid detection method, Can be detected.

The present invention can quickly detect mycoplasma papillomonas infected with humans and animals, so that it is possible to perform differential diagnosis of mycoplasma pneumoniae and other mycoplasma bacteria, thereby enabling early treatment, It has an effect that can be prevented.

The present invention can quickly and accurately detect mycoplasma pemmonis, which is frequently infected when cultured in the field of medicine and in the manufacture, storage and administration of biologics, without any direct culture method, It has effects that can be applied to industrial fields.

The present invention can quickly and accurately detect mycoplasma papillomonas, a pathogen of rodents such as rats and mice, and present a strategy for early treatment and prevention of infection, which can be applied to the laboratory animal industry .

[Figure 1] is a diagram showing the base sequence of species specific 16s rRNA of Mycoplasma pumulus.
[Fig. 2] is a diagram showing the results of the specificity experiment of a mycoplasma pearmonose target primer set.
[Fig. 3] is a diagram showing sensitivity test results of a mycoplasma pearmonose target primer set.

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 embodiments shown in the present specification, the reference examples, the experimental examples, and the drawings are merely the most preferred examples of the present invention, and not all of the technical ideas of the present invention are described. Therefore, It should be understood that various equivalents and modifications may be made.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other features and advantages of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG.

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

In addition, 'forward primer' and 'reverse primer' refer to a primer set which binds to 3 'and 5' ends of a certain region of a gene amplified by a polymerase chain reaction, respectively.

Example 1. Preparation of Mycoplasma pearmonis target primer set

[Figure 1] is a diagram showing the base sequence of species specific 16s rRNA of Mycoplasma pumulus.

As shown in Table 1, a set of primers comprising the nucleotide sequences of SEQ ID NOS: 1 and 2 is included, and a primer set targeting a species specific 16s rRNA sequence region in the nucleotide sequence of mycoplasma pumilon is prepared.

SEQ ID NO: Primer direction Sequence size One Omni direction 5'-CAG TAC TTG AGT TAG AAA ATG GA-3 '
(Nucleotides 256498 to 256520) 23 mer
24 mer 2 Reverse 5'-ATC TGA AAG TTT TGA AGA GTT TTG-3 '
(Nucleotides 257383 to 257405) 24 mer

After the preparation, it is purified by OPC method and used.

Experimental Example 1. Identification of specificity of mycoplasma pearmonose target primer kit

In order to confirm the specificity of the mycoplasma pearmonose target primer kit according to the present invention, experiments were conducted as follows.

(1) Preparing the experiment

1) Preparation and Culture of Mycoplasma Strain

As a comparative sample to confirm this experiment, germicoplasma species strains were purchased and cultured simultaneously with mycoplasma pearmonice as shown in [Table 2].

number Strain name ATCC registration number One Maiko Plasma High Honeymoon
( Mycoplasma hyopneumoniae ) ATCC strain 25934 2 Maiko Plasma High Orinis
( Mycoplasma hyorhinis ) ATCC strain 27717 3 Maiko Plasmapulmonis
( Mycoplasma pulmonis ) ATCC strain 19612 4 Maiko Plasma Hominis
( Mycoplasma hominis ) ATCC strain 23114 5 Maiko Plasma Athitides
( Mycoplasma arthritidis ) ATCC strain 19611

The cultivation of the Mycoplasma species strain was carried out in a modified Friis medium (Zhang et al., 1994) at 37 ° C for 10 days at 58 rpm, followed by centrifugation at 12,000 rpm for 15 minutes to remove the preexporter medium .

(2) Experimental process

1) DNA sample preparation of mycoplasma species strains

DNA extraction of each of the Mycoplasma species cultured was performed using the bead beater-phenol extraction method.

Mycoplasma species were centrifuged at 12,000 rpm for 15 minutes for 10 days and the pellet samples were aseptically collected in a 2 ml tube dedicated to a Mini-Bead Beater (Biospec product) containing 1 ml of sterilized distilled water It was cut down.

200 μl of a glass bead 0.1 mm (Biospec product) suspended in sterilized tertiary distilled water and 200 μl of phenol-chloroform-isoamyl alcohol were placed into a mini-bead beater For 30 seconds at 5,000 rpm.

The shaken pellet sample was centrifuged at 12,000 rpm for 15 minutes at 4 ° C, and then the supernatant was transferred to a sterilized 2 ml tube.

In a sterilized 2 ml tube, 10 μl of 3 M sodium acetate and 250 μl of ice-cold ethanol were added to the pellet sample, and the mixture was stood at -20 ° C. for 10 minutes and then centrifuged at 15,000 rpm for 15 minutes.

The precipitate was washed with 70% alcohol, dried at room temperature and dissolved in 60 [mu] l of Tris EDTA (pH 8.0). As a result, 10 ⁴ pg of a DNA sample of each mycoplasma strain was obtained.

2) PCR reaction condition setting and PCR Execution

Using a primer set prepared by the procedure described in Example 1, 10 ⁴ pg DNA samples of each mycoplasma strain were PCR.

The reaction reagent for PCR, that is, the polymerase chain reaction, contained 1 μl (10 pmol / μl) of the primer set prepared in the procedure described in Example 1, 2.5 μl of 10 × reaction buffer, 500 μl of dioxinucleotide, 1.5 μl of Taq And the mixture was mixed with sterilized distilled water so as to have a reaction amount of 25 μl.

PCR conditions were set as shown in [Table 3], and PCR was performed using a Thermocycler (ABI, USA).

PCR process Temperature / time Number of repetitions Denaturation (pre-denaturation) 95 ° C / 5 min -
Annealing
95 ° C / 1 minute
30 cycles
60 ° C / 1 minute 72 ° C / 1 min Post-polymerization 72 ° C / 5 min -

After PCR, the product was electrophoresed on 2% agarose gel to confirm the existence of a specific band, and a band of 911 bp was confirmed.

(3) Experimental results

[Table 4] below shows the results obtained when the Mycoplasma sp. Strain was subjected to PCR using the Mycoplasma pearmonis target primer set.

order Strain name PCR results One Maiko Plasma High Honeymoon
( Mycoplasma hyopneumoniae ) - 2 Maiko Plasmapulmonis
( Mycoplasma pulmonis ) +++ 3 Maiko Plasma High Orinis
( Mycoplasma hyorhinis ) - 4 Maiko Plasma Hominis
( Mycoplasma hominis ) - 5 Maiko Plasma Athitides
( Mycoplasma arthritidis ) -

Here, +++ represents a strong reaction and - represents a no reaction.

[Fig. 2] is a diagram showing the results of the specificity experiment of a mycoplasma pearmonose target primer set.

As a result of the experiment, as shown in [Table 4] and [Figure 2], the mycoplasma pearmonic target primer kit specifically reacted only to mycoplasma pearmonias, It was confirmed that the reaction did not occur with the mycoplasma strains Mycoplasma high hyunmonia, Mycoplasma high ornicus, Mycoplasma hominis or Mycoplasma astritis strains.

Thus, it can be concluded that the primer kit targeting the species specific 16s rRNA sequence region of mycoplasma pumilonus according to the present invention specifically detects mycoplasma pemmonis.

Experimental Example 2 Confirm sensitivity of mycoplasma pearmonis target primer kit

In order to confirm the sensitivity of the mycoplasma pearmonose target primer kit according to the present invention, experiments were conducted as follows.

(1) Preparing the experiment

1) Preparation and Culture of Mycoplasma pearmonice

The same procedure as in Experimental Example 1 was carried out. However, the experimental sample was replaced with mycoplasma pthalocyanine.

(2) Experimental process

1) Preparation of mycoplasma puromycin DNA sample

DNA extraction of the cultured Mycoplasma pearmonice was performed using the bead beater-phenol extraction method.

The pellet samples were centrifuged at 12,000 rpm for 15 minutes, and the pellet samples were aseptically collected in a 2 ml tube dedicated to a Mini-Bead Beater (Biospec product) containing 1 ml of sterilized distilled water It was cut down.

200 μl of a glass bead 0.1 mm (Biospec product) suspended in sterilized tertiary distilled water and 200 μl of phenol-chloroform-isoamyl alcohol were placed into a mini-bead beater For 30 seconds at 5,000 rpm.

The shaken pellet sample was centrifuged at 12,000 rpm for 15 minutes at 4 ° C, and then the supernatant was transferred to a sterilized 2 ml tube.

In a sterilized 2 ml tube, 10 μl of 3 M sodium acetate and 250 μl of ice-cold ethanol were added to the pellet sample, and the mixture was stood at -20 ° C. for 10 minutes and then centrifuged at 15,000 rpm for 15 minutes.

The precipitate was washed with 70% alcohol, dried at room temperature and dissolved in 60 [mu] l of Tris EDTA (pH 8.0).

The obtained Mycoplasma pearl mononuclear DNA sample was quantified using a spectrophotometer, and then some DNA samples were diluted stepwise to obtain purified mycoplasma pearmonice DNA samples 10 ⁴ , 10 ³ , 10 ² , 5, 1 pg was obtained and used in the experiment.

2) PCR reaction condition setting and PCR Execution

The same procedure as in Experimental Example 1 was carried out. However, the experimental sample was replaced with a mycoplasma pearmonice DNA sample.

(1) Experimental results

Table 5 below shows the result of PCR and electrophoresis performed by quantitatively and stepwise diluting a sample of mycoplasma pearmonice DNA.

Template
(Template)
DNA (pg) 10 ⁴ 5 x 10 ³ 10 ³ 5 × 10 ² 10 ² 5 x 10 10 5 One Positive
Reaction
(Positive reaction) +++ +++ +++ +++ ++ ++ ++ + -

Here, +++ denotes a band with a high concentration, ++ denotes a band with a medium concentration, + denotes a band with a low concentration, and - denotes a band with no band.

[Fig. 3] is a diagram showing sensitivity test results of a mycoplasma pearmonose target primer set.

As a result of the experiment, as shown in [Table 5] and [Figure 3], in the mycoplasma pearmonis target primer kit, DNA samples from 5 pg or more were detectable. In other words, it was confirmed that it was sensitive enough to the mycoplasma pearl mononuclear DNA.

Thus, it can be concluded that the primer kit targeting the species specific 16s rRNA sequence region of mycoplasma pumicornus according to the present invention accurately detects mycoplasma papillomonas.

It is to be understood that the examples and experimental examples are merely illustrative of the present invention and that the contents of the present invention are not limited by the examples and the experimental examples.

<110> Wonkwang University Center for Industry-Academy Cooperation <120> Primer set for the detection of Mycoplasma pulmonis, and method          and kit for the detection of Mycoplasma pulmonis by using the          primer set <130> 2013-01 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> forward primer of Mycoplasma pulmonis DNA <400> 1 cagtacttga gttagaaaat gga 23 <210> 2 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> reverse primer of Mycoplasma pulmonis DNA <400> 2 atctgaaagt tttgaagagt tttg 24

Claims (4)

delete delete ( Mycoplasma pulmonis ) is detected by performing PCR (Polymerase chain reaction) using a primer set,
Wherein the primer set comprises a pair of primers of SEQ ID NO: 1 and SEQ ID NO: 2,
Targeting the 16s rRNA (16s ribosomal RNA) sequence region of mycoplasma puromycin,
Wherein the sensitivity of the DNA sample is 5 pg or more and the specificity of the DNA sample is 10 ⁴ pg or more.
The PCR was carried out at 95 ° C for 5 minutes, followed by repeated 30 cycles of 95 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 1 minute, and 72 ° C for 5 minutes. A method for detecting Mycoplasma pulmonis .
A kit for detecting mycoplasma papillomas using PCR,
A mycoplasma pheromone detection kit comprising the primer set according to claim 3.

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