KR101279396B1 - Primer set for the detection of Mycoplasma, and method and kit for the detection of Mycoplasma by using the primer set - Google Patents

Abstract

The present invention provides a consensus primer that targets a specific part of the DNA of mycoplasma genus, which quickly detects infection or contamination of mycoplasma genus bacteria in one operation. It relates to a mycoplasma detection method and kit using a primer.
The present invention relates to a primer that targets a 16s rRNA (ribosomal RNA) gene region specific for the mycoplasma genus.
The present invention relates to a method and kit for rapidly detecting mycoplasma genus bacteria by applying the PCR method using the primers.

Description

Primer set for the detection of Mycoplasma, and method and kit for the detection of Mycoplasma by using the primer set}

The present invention provides a consensus primer that targets a specific part of the DNA of mycoplasma genus, which quickly detects infection or contamination of mycoplasma genus bacteria in one operation. It relates to a mycoplasma detection method and kit using a primer.

The present invention relates to a primer that targets a 16s rRNA (ribosomal RNA) gene region specific for the mycoplasma genus.

The present invention relates to a method and kit for rapidly detecting mycoplasma genus bacteria by applying the PCR method using the primers.

Mycoplasma is a prokaryotic bacterium belonging to the Molycutes class taxonomically and hundreds of species have been reported to date.

Mycoplasma bacteria reside in various organs such as the respiratory or genital organs of humans and animals and cause serious diseases. Especially, it is a pathogen that is important for public health, such as death from pneumonia, and can cause enormous losses to the livestock industry. to be.

It is resistant to antibiotics that inhibit mycosynthesis such as penicillin or streptomycin because mycoplasmas do not have cell walls. ), Which makes it difficult to observe using an optical microscope.

In addition, mycoplasma bacteria have become a problem as a cause of frequent contamination of these cells, vaccines and drugs during laboratory cell culture, manufacture and storage of vaccines and drugs.

In particular, mycoplasma infection rate in cell culture is about 30 ~ 87%, which is mainly caused by animal tissue used in primary culture, serum used in culture, and can spread due to cross contamination.

However, it is very important to confirm whether or not mycoplasma contamination causes a serious change in cell function while reducing the rate of proliferation of cells.

In summary, it is very urgent to detect the infection of mycoplasma bacteria in humans and animals and the contamination of mycoplasma bacteria in laboratory cells.

 However, mycoplasma bacteria are not only difficult to culture and difficult to isolate, but the detection of hundreds of mycoplasma bacteria takes a long time and effort.

Therefore, there is an urgent need for a technology that can quickly detect infection or contamination of genus bacteria in a single operation.

Hereinafter, the mycoplasma detection technique used in the prior art will be examined in detail.

First, in order to detect mycoplasma, a direct culture method of separating culture using a medium has been commonly 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 may cause no symptoms during infection.

As a diagnostic method for mycoplasma, a bacterium identification method, a serological method, or a tissue-based method has been used in which a specimen is collected to isolate and identify a 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.

In recent years, the most preferred method of detecting mycoplasma bacteria 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.

However, in the case of humans, various mycoplasma species including mycoplasma pneumoniae (M. pneumoniae) is a problem causing pneumonia, and in the case of animals,M. hyopneumoniae)And mycoplasma hyorinisM. hyorhinis)Besides Other mycoplasma species are the cause of infection.

For this reason, when judging mycoplasma bacterial infection, several species-specific PCR should be performed, resulting in a long time to determine pathogen positive.

Therefore, there is a technology that can quickly detect whether infection or contamination of genus mycoplasma bacteria (genus) in a single operation, but by performing consensus PCR specific to mycoplasma genus (genus) I will be desperate.

On the other hand, when looking at the patent literature related to the detection of mycoplasma bacteria in the prior art, Korean Patent Publication No. 10-0615424 (Patent Document 1), the oligonucleotide for genotype discrimination of mycoplasma and similar species, and microarray comprising the same A fungal species detection method using the same is disclosed.

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: 7-21 and 28-127 will function primarily as probes for individual mycoplasma search.

Secondly, Korean Patent Publication No. 10-0502765 (Patent Document 2) 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, which is described below. Inconsistent with the primer according to the example.

Third, Korean Patent Publication No. 10-0974861 (Patent Document 3) 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.

In summary, it is required to quickly detect the infection and contamination of the genus mycoplasma (genus) bacteria, but due to the nature of the pathogen identification method has been a problem that takes a long time for diagnosis.

In addition, even when the PCR method is applied, several species-specific PCRs must be performed, and thus, there has been a problem that it takes a long time to determine the pathogen positive.

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 performing an enzyme chain reaction, but a method for detecting specific mycoplasma genus bacteria at once and rapidly has not been developed.

Therefore, in order to quickly detect the infection or contamination of mycoplasma genus bacteria in one operation, the present inventors have used a consensus primer that targets a specific part of the DNA of the mycoplasma genus. The present invention was developed to provide a technique for performing consensus polymerase chain reaction (Consensus PCR) using the same.

KR 10-0615424 B1 KR 10-0502765 B1 KR 10-0974861 B1

An object of the present invention is to provide a technique for quickly detecting whether infection or contamination of genus mycoplasma bacteria in one operation.

It is an object of the present invention to provide a primer that targets a 16s rRNA (ribosomal RNA) gene region specific for the mycoplasma genus.

An object of the present invention is to provide a method and kit for rapidly detecting mycoplasma genus bacteria by applying the PCR method using the primer.

The present invention is to solve the technical problem by providing a primer for detecting Mycoplasma ( Mycoplasma ) 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 targeting a 16s rRNA (ribosomal RNA) gene region specific to the genus Mycoplasma.

An object of the present invention, by applying a PCR method using the primer, to provide a method and kit for rapidly detecting mycoplasma genus (genus) bacteria, to solve the technical problem.

The primers according to the present invention have a significant effect of retaining detection sensitivity in 10 ² pg DNA samples of nine kinds of mycoplasma strains.

The primer according to the present invention has a remarkable effect of retaining detection sensitivity in 10 ² pg DNA samples of two kinds of mycoplasma and seven kinds of comparative strains.

The primer according to the present invention, by targeting the 16s rRNA (ribosomal RNA) gene region specific to the mycoplasma genus, greatly improves sensitivity and specificity, and detects mycoplasma rapidly and remarkably clinically. It has an effect that can be done.

The present invention can quickly detect mycoplasmal bacterial infections of humans and animals, and thus can easily diagnose differential diagnosis of mycoplasma infection and infection of other pathogens, thereby enabling early treatment and preventing disease spread. Has a significant effect.

The present invention can quickly and accurately detect mycoplasma pathogens contaminated frequently in the manufacture, storage, administration, and cultivation of cell cultures or biopharmaceuticals carried out in the pharmaceutical field without the direct cultivation method. It has a remarkable effect applicable to the biological industry.

The present invention has the remarkable effect of presenting a research method for discovering mycoplasma of a new species by allowing the detection of mycoplasma genus bacteria simultaneously by consensus PCR method.

1 is a view showing the nucleotide sequence structure of the primer production site 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 view showing the results of specificity (specificity) experiment of the primer according to the present invention.

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, as used herein, "primer" refers to 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, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example 1 Preparation of a Primer Specificly Detecting Mycoplasma Genus Bacteria

1 is a view showing the nucleotide sequence structure of the production site of the primer according to the present invention.

By targeting the 16s rRNA (ribosomal RNA) gene region specific to mycoplasma genus bacteria in the mycoplasma genome sequence, primers that can specifically detect bacteria belonging to the mycoplasma genus are sequenced as follows. It is prepared to include a primer pair consisting of the nucleotide sequence of the number 1 and 2.

Here, the rRNA (ribosomal RNA) gene contains information that can distinguish bacteria at the species level (species), of which 16s rRNA (ribosomal RNA) is known to have little genetic variation.

SEQ ID NO: 1. Forward primer

5'-CTC TTT GTA CCG GCC ATT GT-3 '

(20 mer, nucleotides 699283-699302)

SEQ ID NO: 2. Reverse primer

5'-GAA TGA CAG ATG GTG CAT GG-3 '

(20 mer, nucleotides 699478-699497)

The primer is used after purification by OPC method.

Example 2 Method for Specific Detection of Mycoplasma Genus Bacteria

(1) extracting DNA from a sample

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

(2) preparing a primer specific for the mycoplasma genus bacteria prepared in Example 1

In the mycoplasma genome sequence, primers targeting 16s rRNA (ribosomal RNA) gene regions specific for mycoplasma genus bacteria 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 55 ° C, and 1 minute at 72 ° C. 30 cycles and finally post-polymerization (min) at 72 ℃ for 3 minutes (min).

The product of polymerase chain reaction was electrophoresed on 2% agarose gel to identify a band of 215 bp.

Reference Example 1. Experiment Preparation

In order to confirm the sensitivity and specificity of the consensus polymerase chain reaction primer for simultaneously detecting various bacteria belonging to the genus Mycoplasma (genus) developed according to an embodiment of the present invention, the experiment was prepared as follows.

1-1. Mycoplasma Strain Preparation and Culture

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

Mycoplasma species One Mycoplasma hyopneumoniae 2 Mycoplasma hyorhinis 3 Mycoplasma pneumoniae 4 Mycoplasma hominis 5 Mycoplasma pulmonis 6 Mycoplasma arthritiditis 7 Mycoplasma bovis 8 Mycoplasma fluccurare 9 Mycoplasma mycoides

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. Preparation of Comparative Strains

In order to perform a comparative experiment for confirming the specificity of the primer according to the present invention, a total of seven kinds of comparative strains were purchased and prepared as follows.

Comparative strain One Staphylococcus aureus 2 Escherichia coli 3 Helicobacter pylori 4 Pasteurella multocida 5 Salmonella pullorum 6 Salmonella gallinarum 7 Actinobacillus pleuropneumoniae

1-3. Preparation of DNA Extraction Samples for Mycoplasma and Comparative Strains

DNA extraction of each of the cultured mycoplasma strains and the comparison strains was performed using a bead beater-phenol extraction method.

Pellet samples obtained by centrifuging the 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, chopped, and suspended in sterile tertiary distilled water. 200 μl of (0.1 mm size, Biospec product) and 200 μl of phenol-chlorform-isoamyl alcohol solution (50: 49: 1 (v / v / v)) were shaken at 5,000 rpm for 30 seconds with Mini-Bead Beater (Biospec product). It was.

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.

1-4. Polymerase Chain Reaction Conditions

A primer specific for the mycoplasma genus bacterium prepared in Example 1 is used, which targets a 16s rRNA gene region specific for the mycoplasma genus bacterium in the mycoplasma 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 sensitivity of mycoplasma genus bacteria of the primer according to the present invention

According to the experimental preparation of Reference Example 1, a total of nine kinds of mycoplasma strains, Mycoplasma hyopnenmoniae , Mycoplasma hyorhinis , and Mycoplasma pneumoniae ), mycoplasma hoe Nice (M. hominis), mycoplasma Pearl's monitor (M. Pulmonis), mycoplasma Oh bitter di itty-Tees (M. arthritiditis), mycoplasma bovis (M. bovis), mycoplasma flu curare 10 ² pg of DNA samples of M. fluccurare , Mycoplasma mycoides strains were prepared.

For each of the samples, a polymerase chain reaction was carried out with primers targeting the 16s rRNA (ribosomal RNA) gene region specific for the mycoplasma genus bacterium prepared in Example 1. The conditions were carried out 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.

Mycoplasma species PCR result One M. hyopneumoniae + 2 M. hyorhinis + 3 M. pneumoniae +++ 4 M. hominis +++ 5 M. pulmonis ++ 6 M. arthritiditis + 7 M. bovis + 8 M. fluccurare +++ 9 M. mycoides ++

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

As shown in Figure 2 and Table 3, primers targeting the 16s rRNA (ribosomal RNA) gene region specific for the mycoplasma genus bacteria developed in the present invention are strains 3, 4 and 8 ( M. pneumoniae) , M. hominis , M. fluccurare ) strongly reacted with DNA.

In addition, it was found that it reacted with DNA of the 5th and 9th strains ( M. Pulmonis , M. mycoides ) somewhat stronger, and with the DNA of the remaining strains ( M. hyopnenmoniae , M. hyorhinis, M. arthritiditis , M. bovis ). Can be.

Therefore, in view of the results of Experiment 1, primers targeting the 16s rRNA (ribosomal RNA) gene region specific to the mycoplasma genus developed in the present invention will be sensitive to the DNA of mycoplasma genus bacteria. It was confirmed to react to the extent.

Experimental Example 2. Confirmation of specificity of mycoplasma genus bacteria of the primer according to the present invention

According to the experimental preparation of Reference Example 1, DNA samples of mycoplasma and the comparative strain 10 ² pg were prepared.

Here, two kinds of mycoplasma strains were prepared: mycoplasma hyopnenmoniae and mycoplasma hyorhinis .

In addition, the comparison target strain is a total of seven types, Staphylococcus Cocos aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Helicobacter pylori (Helicobacter pylori), wave stew Pasteurella far Toshio the (Pasteurella multocida), Salmonella manipulator rurum ( Salmonella pullorum ), Salmonella gallinarum ( Salmonella gallinarum ), Actinobacillus pleuropneumoniae strains.

For each of the samples, a polymerase chain reaction was carried out with primers targeting the 16s rRNA (ribosomal RNA) gene region specific for the mycoplasma genus bacterium prepared in Example 1. The conditions were carried out in the same manner as in Example 2.

Figure 3 is a view showing the results of the specificity (specificity) experiment of the primer according to the invention, which is shown in Table 4 below.

Mycoplasma species PCR result One M. hyopneumoniae ++ 2 M. hyorhinis ++ 3 Staphylococcus aureus - 4 Escherichia coli - 5 Helicobacter pylori - 6 Pasteurella multocida - 7 Salmonella pullorum - 8 Salmonella gallinarum - 9 Actinobacillus pleuropneumoniae -

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

As shown in FIG. 3 and Table 4, the primers targeting the 16s rRNA (ribosomal RNA) gene region specific for the mycoplasma genus bacteria developed in the present invention are mycoplasma high, which is a mycoplasma genus bacterium. It can be seen that it specifically reacted to DNA of M. hyopnenmoniae and Mycoplasma hyorhinis .

On the other hand, it can be seen that there was no reaction with the DNA of a total of seven kinds of comparative strains not belonging to the genus Mycoplasma.

Therefore, when looking at the results of Experiment 2, the primer targeting the 16s rRNA (ribosomal RNA) gene region specific to the mycoplasma genus developed in the present invention is specific only to the DNA of mycoplasma genus bacteria. It was confirmed to react.

Collectively, Experimental Example 1 and Experimental Example 2, the primer according to Example 1 of the present invention by targeting the 16s rRNA (ribosomal RNA) gene region specific to the genus Mycoplasma, genus Mycoplasma ( genus) Sensitivity and specificity for detecting bacteria can be seen.

Attach an electronic file to a sequence list

Claims (4)

Including primer pair of SEQ ID NO: 1 and SEQ ID NO: 2,
Mycoplasma is detected by targeting the 16s rRNA (ribosomal RNA) gene region,
The mycoplasma may be mycoplasma hyopnenmoniae , mycoplasma hyorhinis , mycoplasma pneumoniae , mycoplasma hominis , mycoplasma Pearl's monitor (M. Pulmonis), mycoplasma Oh bitter di itty-Tees (M. arthritiditis), mycoplasma bovis (M. bovis), mycoplasma flu curare (M. fluccurare), mycoplasma Mai Koh death (M. mycoides ), comprising a primer set for detecting Mycoplasma ( Mycoplasma ).
delete Using the primer set of claim 1, the method for detecting Mycoplasma (Polymerase chain reaction (Polymerase chain reaction, PCR)).
A kit for detecting Mycoplasma using polymerase chain reaction (PCR),
Mycoplasma detection kit comprising the primer set of claim 1.

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