KR20170003855A - Method for rapidly detecting actinobacillus pleuropneumoniae from clinical samples through multiplex real-time pcr assay and the apparatus thereof - Google Patents

Abstract

The present invention relates to a method and apparatus for rapidly detecting porcine pleurisy pneumoniae from a clinical sample through multiple real-time PCR, comprising collecting a clinical sample from lung tissue of a porcine, extracting DNA from the collected clinical sample, Time multiplex PCR using specific primers that perform gene amplification by performing simultaneous species identification and serotype determination of porcine pleural pneumococcal serotypes 1, 2, 7, and 12 with the template of DNA. Speed detection method and apparatus thereof.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method and apparatus for rapid detection of swine pneumoniae from a clinical sample through multiple real-

The present invention relates to a method and apparatus for rapidly detecting swine pneumoniae from clinical samples through multiple real-time PCR, and more particularly, to a method and apparatus for collecting clinical samples from swine lung tissue and extracting DNA from the collected clinical samples And performing real-time multiplex PCR using the extracted DNA as a template by performing simultaneous species identification and serotype determination of porcine pleural fluid pneumococcal serotypes 1, 2, 7, and 12, using specific primers that perform gene amplification The present invention relates to a rapid detection method of pig pleurisy pneumoniae, and a device therefor.

In the past, bacterial pleuropneumoniae ( Actinobacillus pleuropneumoniae, App) was found in Haemophilus, Haemophilus pleuropneumoniae , which is composed of at least 12 different serotypes. Some of these are non-pathogenic, not disease-causing, but others cause very serious diseases. Strains 1, 5, 9, 11 and 12 are very fatal, and strains 3 and 6 are very mild. Determining serotype is important for understanding the spread of disease, treating and preventing disease, and genetic monitoring of serotypes in flocks and / or areas. The specificity of pleural effusions for serotypes is determined by the capsular polysaccharide on the surface of the serum.

Pleural pneumonia is transmitted from the tonsils and upper respiratory tract. Mixed respiratory disease is reported to be one of the most common clinical conditions affecting pig breeding. The causative microorganisms of swine pneumonia (or etiologic agent) affect pigs from weaning to slaughter in the weaning period, but usually affect between 8 and 16 weeks of age. The incubation period is very short, 12 hours. Toxins are highly contagious and can cause serious damage to the lungs and cause death. It spreads a short distance through droplet infection, and lives only a few days outside the pig.

PCR has become a powerful and increasingly popular tool in bacterial identification. The ability of PCR to detect genetic sequences in trace amounts of DNA is advantageous compared to serological forms of detection for many reasons. That is, strains characterized by the ability to prevent cross-reactivity between antigen and antibody and untype due to auto-junction can be classified by PCR, and DNA amplification by PCR is extremely sensitive , And PCR can be performed directly on the sample without having to wait for bacterial incubation. Multiplex PCR was developed to identify concurrently the actinobacillus pleuropneumoniae (Pasteurella multocida and Haemophilus parasuis ), the major pathogens of pigs responsible for severe economic losses in the pigs livestock industry.

The following describes briefly the prior art which exists in the technical field of the present invention, and then describes a technical matter that the present invention intends to differentiate from the prior art.

Korean Patent No. 1527847 (Apr. 21, 1995) discloses a primer set for distinguishing causative agents of bacterial respiratory disease in pigs and a diagnostic kit comprising the primer set. The primer set includes a primer set for detecting and distinguishing causative agents causing bacterial respiratory disease in pigs, A composition containing the causative agent, and a method for detecting a porcine bacterial respiratory disease agent capable of detecting a causative agent of a bacterial respiratory tract disease using the composition.

Korean Patent No. 1491793 (Feb. 23, 2015) is related to a novel 12 strains of Actinobacillus flourneumoniae serotype, a diagnostic composition and a vaccine composition containing the same, and is a novel strain having a preventive effect against swine pneumonia An actinobacillus prourunmonia serotype 12 strain KACC 91770P, a composition for diagnosing porcine pleural effusion including the strain, and a vaccine composition for preventing swine flu pneumonia comprising the strain.

The above prior arts are somewhat similar to the present invention in that they detect and prevent a causative agent against pleural pneumonia in pigs. However, the present invention relates to a method for collecting clinical samples from lung tissues of pigs and a multiplex PCR method for detecting the serotype of Pleuropneumoniae In particular, the present invention relates to the design of a specific primer that performs gene amplification from the lung tissue of a pig exposed to the aforementioned Pleural Efficiency Pneumococcus, and uses this to perform a real-time multiplex PCR analysis to determine the serotype And the method for performing the determination of serotypes is not described or suggested.

In order to solve these problems, the present invention has been applied to the identification of the most common types of serotypes 1, 2, 7, 12 and omlA, which are substantially important in the diagnostic laboratory, And the like.

It is also an object of the present invention to provide a PCR analyzer based on amplification of a specific serotype DNA region involved in biosynthesis of capsular polysaccharides (cps genes).

A method for rapid detection of swine pneumoniae from a clinical sample through multiple real-time PCR according to an embodiment of the present invention includes a step of collecting a clinical sample from lung tissue of a pig, a step of extracting DNA from the collected clinical sample Real-time multiplex PCR using specific primers to perform gene amplification by performing simultaneous identification and serotype determination of porcine pleural fluid pneumococcal serotypes 1, 2, 7 and 12 using the DNA extraction step and the extracted DNA as a template And performing multiple PCR operations to perform PCR.

Wherein the bacterial strain comprises 5 μg of NAD (BHI-NAD) per ml or a 37 ^° C brain seaweed extract in a BHI-NAD broth, the method comprising: culturing the strain of Pleuropneumoniae bacteria from the clinical sample; 0.0 > (BHI) agar plate. ≪ / RTI >

Also, the primer is an oligonucleotide primer and is characterized in that a serotype-specific primer using the cps gene of serotype 1, 2, 7 and 12 and the omlA gene is designed.

In the multiplex PCR step, a total volume of 20 μl of the PCR reaction mixture was mixed with 2 μl genomic DNA, 2 μl forward and reverse primers (5 pmol / μl each), 2 μl ultrapure water, 2 × premixes containing Prime Taq DNA Polymerase, , 4 mM MgCl2, enzyme stabilizer, sediment, and loading dye, pH 9.0, 0.5 mM dATP, dCTP, dGTP and dTTP, respectively.

Also the multiple PCR performed step is 5 minutes to 94 ^o C, for 30 seconds and 94 ^o C, for 30 seconds and 54 ^o C, 1 bun 72 ^o C for 35 cycles and 10 minutes to 72 ^o C while made the last cycle while, The amplified product is characterized by being separated by electrophoresis in 1.2% agarose gel and staining with ethidium bromide.

In the multiplex PCR step, a total volume of 20 μl of the M-PCR reaction mixture was mixed with 1 μl forward and reverse primers (5 pmol / μl each), 4 μl ultrapure water, 2 × premix with Prime Taq DNA Polymerase, 4 mM MgCl2, enzyme stabilizer, sediment, and loading dye, pH 9.0, 0.5 mM dATP, dCTP, dGTP and dTTP, respectively.

In addition, the multi-PCR The steps, and the 72 ^o C consists of the last cycle for 5 minutes and 94 ^o C, 30 cho 94 ^o C, 30 cho during Tm, 1 bun 72 ^o C for 35 cycles and 10 minutes for over 12 Each microliter reaction mixture was analyzed by electrophoresis on 1.2% agarose gel, characterized by being colored under UV light and stained with ethidium bromide (10 μg / ml).

In addition, according to one embodiment of the present invention, an apparatus for rapid detection of swine pneumoniae in swine is used for collecting clinical samples from the lung tissue of pigs and using the DNA extracted from the collected clinical samples as a template to detect swine pneumoniae serotype 1, 2, 7 And performing 12 simultaneous species identification and serotype determination to perform real-time multiplex PCR using specific primers to perform gene amplification.

The present invention relates to a method and apparatus for rapidly detecting porcine pleurisy pneumoniae from clinical samples through multiple real-time PCR, and by designing serotype-specific primers for cps gene and omlA gene using oligonucleotide primers, Simultaneous identification of serotypes 1, 2, 7, 12, and omlA genes and serotype determination are highly effective, clear, and sensitive serotyping.

FIG. 1 is a diagram showing a primer sequence and a predicted size of amplified products in a method and apparatus for rapidly detecting swine pneumoniae from a clinical sample through multi-real time PCR according to an embodiment of the present invention.
FIG. 2 is a diagram showing electrophoresis of serotype-specific primers designed using multiplex PCR in a method and apparatus for rapidly detecting swine pneumoniae from a clinical sample through multi-real time PCR according to an embodiment of the present invention .
FIG. 3 is a diagram showing primers through Gradient-PCR in a method and apparatus for rapid detection of swine pleurisy pneumoniae from a clinical sample through multi-real time PCR according to an embodiment of the present invention.
FIG. 4 is a flow chart illustrating a method and apparatus for rapidly detecting swine pneumoniae from a clinical sample through multi-real-time PCR according to an embodiment of the present invention. In the method and apparatus for detecting swine pneumoniae in a serotype of swine pneumoniae, Fig.
5 is a flow chart illustrating a method for rapidly detecting swine pneumoniae from a clinical sample through multiple real-time PCR according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings.

The DNA portion (cps regions) for a particular serotype-specific biosynthesis of the capsular polysaccharide can be identified by simultaneous species identification of serotype 1, 2, 7, and 12 of pleuropneumoniae and by multiplex PCR analysis for determination of serotype It was used to develop.

Specific primers of serotypes 1, 2, 7 and 12 were associated with primers for specific species already present in the omlA gene-based PCR assay. When multiple PCR assays were used to analyze field strains, it was possible to identify serotypes corresponding to about 94% of all strains isolated from pigs with clinical disease. More than 90% of the isolated strains cross-reacted by the latex conjugation test were serotypes 1, 2, 7 and 12. The use of specific primers in the biosynthetic part of the different Pleural Pneumoniae serotypes extends the diagnostic and genetics capabilities of this analyzer. Crystallization of serotypes by PCR is a convenient and distinct method for determining the serotype of pleuropneumoniae in diagnostic laboratories.

In addition, the present invention has recently been cloned and sequenced by Ward and Inzana, genes associated with the 5-capsule polysaccharide axeport (cpx) and polysaccharide biosynthesis (cps) of the pleural effusion, wherein the cpx genes are highly conserved, Serotype specific oligonucleotide primers specified in the cps region of Forms 1, 2, 7, and 12. The specificity and sensitivity of multiple PCR analyzers were tested and the results of multiple PCRs were compared to those obtained from classical methods of traditional serology.

Brief Description of the Drawings Fig. 1 is a diagram showing a primer sequence and a predicted size of amplified products in a method and apparatus for rapid detection of swine pleurisy pneumoniae from a clinical sample through multi-real time PCR according to the present invention.

First, materials and rooms used in the present invention will be described. First, pig lung tissue samples are collected from bronchitis-induced pigs with 5 × 10 ⁷ colony-forming units (CFU) of pleural pneumoniae serotype 5 × 10 ⁷ and stored at -20 ^° C.

5 (= 5), 5 (= 5), 6 (= 5), 7 (= 5), and 8 (= 5) isolated from the collected pig lung samples and type (N = 8) can be used to assess species characteristics in multiplex PCR assays.

On the other hand all of the bacterial strain separated comprises NAD (BHI-NAD) of 5 μg per ml or BHI-NAD liquid (broth) in a 37 ^o C brain heart leachate (BHI) agar (Difco Laboratories, Detroit, Mich. ) Lt; / RTI >

In addition, the DNA of the separately cultured bacterial strain is extracted from a colony diluted in 100 μl of ultrapure water. A loopful of the culture of the bacteria cultured for one day is also taken from the surface of the agar medium and suspended in 200 μl of sterile water.

After the loopful of the liquid corresponding to the suspended loop is obtained, DNA extraction is performed according to the AccuPrep® Genomic DNA Extraction Kit (Bioneer, South Korea) according to the protocol of the manufacturer.

The quality and concentration of the DNA is also measured using an eppendorf BioPhotometer at 260 nm and stored at -20 ^° C. The DNA thus extracted is used as a template for PCR and M-PCR.

As shown in Figure 1, the sequence of oligonucleotide primers used in the present invention consists of five different pairs depending on the type of cps-region serotype and omlA- gene.

Five other pairs of oligonucleotide primers are also used for multiplex PCR analysis and are designed as serotype-specific primers for the cps-gene (serotype 1, 2, 7, 12) and omlA- gene do.

The DNA primers designed in the present invention were selected using the Primer 3 tool program.

Polymerase chain reaction (PCR) is also performed in the eppendorf mastercycler gradient using specific primers of the target bacteria. The PCR reaction mixture in a total volume of 20 μl contained 2 μl genomic DNA, 2 μl forward and reverse primers (5 pmol / μl each), 2 μl ultrapure water, 2 × Premix with Prime Taq DNA Polymerase, 2 × reaction buffer, 4 mM MgCl 2, , sediment, loading dyes, respectively, pH 9.0, in 0.5 mM dATP, dCTP, dGTP, including dTTP following temperature and time program for 5 minutes and 94 ^o C, 30 seconds during the 94 ^o C, for 30 seconds and 54 ^o C , A 35 cycle interval of 72 ^o C for 1 minute, and a final cycle interval of 72 ^o C for 10 minutes.

In addition, the product amplified by the polymerase chain reaction can be visualized by separating the DNA product by electrophoresis in an agarose gel of 1.2% and colored with ethidium bromide to separate DNA product.

Next, the multi-polymerase chain reaction (M-PCR) will be described.

PCR is performed in an eppendorf mastercycler gradient (genetic amplifier) using 2 μl genomic DNA-specific primers of the target bacteria. The MPCR reaction mixture with a total volume of 20 μl contained 1 μl forward and reverse primers (5 pmol / μl each), 4 μl of ultrapure water, 2 × premix containing Prime Taq DNA Polymerase, 2 × reaction buffer, 4 mM MgCl 2, enzyme stabilizer, pH 9.0, comprising: a 0.5 mM dATP, dCTP, dGTP, dTTP, and then the temperature and the time the program was 5 minutes for 94 ^o C, 30 cho during the 94 ^o C, 30 cho during Tm, 1 minutes 35 of 72 ^o C for Cycle period and a final cycle period of 72 ^° C for 10 minutes. 12 [mu] l of each reaction mixture is analyzed by electrophoresis in 1.2% agarose gel. The PCR product was stained with ethidium bromide (10 μg / ml) and is shown under UV light.

As a result, serotypes of the pleural effusions are distinguished by their unique capsular polysaccharide (cps). This is because cross-reactivity often occurs in traditional serological assays and serotype determinations.

PCR also provides a practical alternative that does not use antigens and antibodies, making these capsules an ideal target for sorting by PCR.

However, cultures of pleural pneumococci succeed at 50% or less of the submitted sample volume. This is because, when pigs die before extracting the specimen, contamination by death prevents the separation of pleural pneumoniae.

However, PCR is more sensitive than bacterial cultures, and the described multiplex PCR is sufficiently clear that it will not be affected by the presence of the cause of the contamination.

FIG. 2 is a diagram showing electrophoresis of serotype-specific primers designed using multiplex PCR in a method and apparatus for rapidly detecting swine pneumoniae from a clinical sample through multi-real time PCR according to an embodiment of the present invention .

As shown in FIG. 2, the oligonucleotide primers for amplifying the primers for the omlA-gene and the parts of the cps-region are shown by electrophoresis.

As described above, four pairs of the oligonucleotide primers for identifying the Pleural Flu Pneumococcal serotypes of pigs were obtained from serotype 1, 2, 7 and 12 from the serotype-specific cps-region portion and primers for the omlA- Each pair is designed to amplify.

Ap7F and Ap7R in the oligonucleotide primers were also designed to produce a PCR fragment of approximately 1500 bp from the serotype 7 isolate while the primers for cps-region serovar (serotype variants) 1,2 and 12 were designed to produce approximately 320 bp, 720 bp And a PCR fragment of 900 bp.

In addition, the three pairs of primers are associated with primers used in conventional species-specific Pleuropneumonia PCR assays.

The HPF-HPR primer pair for the omlA- gene can also amplify the omlA- gene to approximately 1000 bp.

The primers were designed complementarily to bind to each serotype gene in order to identify the serotype of Pleuropneumoniae of pigs. The primers were designed using Gradient-PCR to find the optimal temperature for analling, Are individually optimized for serotype.

FIG. 3 is a diagram showing primers through Gradient-PCR in a method and apparatus for rapid detection of swine pleurisy pneumoniae from a clinical sample through multi-real time PCR according to an embodiment of the present invention.

As described above, the oligonucleotide primers were designed in five different pairs, and each primer was designed to amplify for the cps-region portion and the omlA gene, respectively.

As shown in FIG. 3, optimal temperatures for binding the respective primers are individually optimized by gradient-PCR.

FIG. 4 is a flow chart illustrating a method and apparatus for rapidly detecting swine pneumoniae from a clinical sample through multi-real-time PCR according to an embodiment of the present invention. In the method and apparatus for detecting swine pneumoniae in a serotype of swine pneumoniae, Fig.

As shown in FIG. 4, multiplex PCR analysis is optimized using an extract of serotype 5 bacteria. Also species-specific fragments of approximately 1000 bp and 320 bp are amplified from all the pneumococcal species analyzed.

On the other hand, isolated strains of serotype 2, 5 or 6 from one of the traditional serotyping methods are assigned to the same type of serotype by multiplex PCR analysis.

The annealing temperature is 49 ^o C to 56 ^o C.

Raising the annealing temperature to remove non-specific products also results in loss of specific PCR products, which is not helpful in optimizing PCR analysis.

Also, the cps-region of serotype 4 is successfully amplified using purified DNA, bacterial colonies, and lung samples.

Also, combining the primers does not require a change in PCR environments (e. G., A change in temperature).

The use of multiple PCR also provides advantages in using multiple primer sets in a single reaction and in simultaneous determinations.

5 is a flow chart illustrating a method for rapidly detecting swine pneumoniae from a clinical sample through multiple real-time PCR according to an embodiment of the present invention.

As shown in FIG. 5, first, a clinical sample is collected from the lung tissue of the pig (S110).

On the other hand, the clinical sample is collected from a pig infected with the Pleural Pneumoniae serotypes 1, 2, 6 and 12 as a lung tissue sample of the pig.

Next, DNA is extracted from the collected clinical sample (S120).

Next, using the extracted DNA as a template, simultaneous species identification and serotype determination of pleural pneumonia are performed, and real-time multiplex PCR is performed using a specific primer performing gene amplification (S130).

Next, the amplified product through the real-time multiplex PCR is separated by electrophoresis, and the separated product is colored with an etchant such as bromide to be visualized under UV light (S140).

On the other hand, the design of the multiplex PCR analyzer was determined by the distribution of the serotype of Pleuropneumoniae in Denmark. Also, nearly 94% of the isolated farm strains belong to serotypes 1, 2, 7 and 12. In all serotypes, the presence of nonspecific bands amplified from several serotypes, especially serotypes 7 and 12, was found to be highly conserved while maintaining the cpx band.

Also, primers selected in that region were designed in the sequence of serotype sample DNA. Successful application of multiple PCR analyzers to bacterial colonies provides an effective method for identification and serotype determination of pleural pneumoniae.

In exceptional cases of rare serotype 4, a separate 0.7-kb band was amplified from all sera. To date, most PCR analyzers reported for pleural pneumococci have been used for species identification and subclassification of isolated strains, and do not identify species and serotype simultaneously.

A variety of PCR analyzers have also been developed to identify species of pleural pneumococci. A common feature of these PCR analyzers is that the primers used for amplification are specific to pleural pneumococci other than any other pleural pneumococcal serotype. These analyzers are used only for the identification of organisms, and therefore other methods for serotyping are still needed.

Thus, the results obtained in the present invention demonstrate that multiplex PCR analyzers are a sensitive, clear, and highly effective diagnostic tool for the simultaneous identification and serotype determination of Pleural Pneumoniae serotype 1, 2, 7, 12 and omlA genes. Furthermore, the problem of serological cross-reactions can be prevented in diagnostic laboratories using multiplex PCR. The results also confirm that the genes involved in biosynthesis of the capsular polysaccharide contain serotype-specific regions of the Pleural Pneumococcal serotypes 2, 6 and 12 and omlA genes.

As described above, the present invention relates to the simultaneous identification of the pleural pneumoniae serotypes 1, 2, 7, 12 and omlA genes by designing serotype-specific primers for the cps gene and omlA gene using oligonucleotide primers There is an effect that the serotype crystallization can be carried out very clearly and sensitively and serotyped very effectively.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation. I will understand that. Accordingly, the technical scope of the present invention should be defined by the following claims.

100: oligonucleotide primer

Claims (8)

Collecting a clinical sample from the lung tissue of the pig;
A DNA extraction step of extracting DNA from the collected clinical sample; And
Using the extracted DNA as a template, simultaneous identification of species 1, 2, 7 and 12 of serotype 1, 2, 7 and 12 of porcine pleural fluid pneumococci and serotype determination are performed, The method comprising the steps of: (a) providing a rapid detection method of swine pneumoniae. The method according to claim 1,
Wherein the bacterial strain comprises 5 μg of NAD (BHI-NAD) per ml or a 37 ^o C brain heart extract in a BHI-NAD broth (BHI) agar plate. ≪ RTI ID = 0.0 > 8. < / RTI > The method according to claim 1,
The above-
Characterized in that a serotype-specific primer is designed using oligonucleotide primers and the cps genes of serotype 1, 2, 7 and 12 and the omlA gene. The method according to claim 1,
In the multiplex PCR step, a total volume of 20 μl of the PCR reaction mixture was mixed with 2 μl genomic DNA, 2 μl forward and reverse primers (5 pmol / μl each), 2 μl ultrapure water, 2 × Premix with Prime Taq DNA Polymerase, , 4 mM MgCl2, enzyme stabilizer, sediment, and loading dye, pH 9.0, 0.5 mM dATP, dCTP, dGTP and dTTP, respectively. The method of claim 4,
Wherein the multiplex PCR step comprises:
For 5 minutes and 94 ^o C, and the 94 ^o C, for 30 seconds and 54 ^o C, 1 bun 72 ^o C to 72 ^o C for 35 cycles and 10 minutes during last cycle consists of 30 seconds, an amplification product of 1.2% A method for rapid detection of porcine pneumoniae of swine, characterized in that it comprises separating by electrophoresis in agarose gel and staining with ethidium bromide. The method according to claim 1,
In this multiplex PCR step, a total volume of 20 μl of the M-PCR reaction mixture was mixed with 1 μl forward and reverse primers (5 pmol / μl each), 4 μl ultrapure water, 2 × premix with Prime Taq DNA Polymerase, 2 × reaction buffer, An enzyme stabilizer, a sediment, and a loading dye, each of which comprises pH 9.0, 0.5 mM of dATP, dCTP, dGTP, and dTTP. The method of claim 6,
Wherein the multiplex PCR step comprises:
For 5 minutes and 94 ^o C, while for 30 seconds, 94 ^o C, 30 seconds Tm, and the 72 ^o C consists of the last cycle for 1 minute for 35 cycles and 10 minutes to 72 ^o C for 12 Each reaction mixture microliters 1.2 Characterized in that it is analyzed by electrophoresis on a% agarose gel, stained with ethidium bromide (10 [mu] g / ml) and visible under UV light. By collecting clinical samples from lung tissues of pigs and using the DNA extracted from the collected clinical samples as a template, simultaneous species identification and serotype determination of porcine pleural fluid pneumococcal serotypes 1, 2, 7 and 12 were carried out, And performing a real-time multiplex PCR using a specific primer to perform the PCR.

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