KR20160029368A - Composition and method for detecting Staphylococcus aureus causing Bacterial meningitis - Google Patents

Abstract

Disclosed is an oligonucleotide which is designed to screen a protein A (Spa) by means of a gene specific to Staphylococcus aureus and to specifically detect Staphylococcus aureus through the detection by the gene. In addition, disclosed are a composition containing the oligonucleotide, a kit, and a method. According to the method, the composition, and the kit, it is possible to detect the presence of bacteria in a quick and precise way only with a single examination.

Description

[Technical Field] The present invention relates to a composition for detecting Staphylococcus aureus causing bacterial meningitis,

The present invention relates to a technique for detecting meningitis.

Bacterial meningitis (BM) is an acute inflammation that affects the central nervous system of the brain and spinal cord. It is a high mortality and requires prompt diagnosis and treatment. Meningococcus, Haemophilus influenzae, Streptococcus pneumoniae, and streptococcus agalactiae are the main pathogens causing bacterial meningitis.

Therefore, despite the need for immediate treatment, the treatment is delayed due to the identification of pathogens and the selection of appropriate antibiotics.

First, cerebrospinal fluid (CSF) is extracted for the identification of pathogenic bacteria, and pathogenic antibiotic susceptibility tests of causative organisms are conducted for the selection of antibiotics. However, this process provides a major source of delay in treatment that lasts at least five days.

Furthermore, in the identification of causative bacteria, bacteria that have previously been exposed to antibiotic therapy or grow slowly have a problem of false negative results.

Immunostaining is used to confirm bacterial meningitis. However, this has a problem of low sensitivity and cross-reactivity with other antigens.

PCR techniques for conserved regions of existing 16S rRNA genes can distinguish a wide range of bacteria, but there are difficulties in distinguishing certain bacterial species.

Therefore, for the diagnosis and treatment of bacterial meningitis, it is necessary to develop technologies with high sensitivity and accuracy that can distinguish specific bacterial species.

Korean Patent Publication No. 2013-0097974

The present invention provides a rapid and convenient method for detecting meningococcal bacteria based on nucleic acid detection.

In one embodiment, the invention provides oligonucleotides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and complementary sequences thereof that specifically detect Staphylococcus aureus.

In one embodiment, the oligonucleotides herein may be used in, but not limited to, nucleic acid amplification, e.g., PCR, from a target organism.

Oligonucleotides according to the present invention can be used for the diagnosis of meningitis or sepsis.

In another aspect, the invention also provides a composition or kit for detecting Staphylococcus aureus comprising an oligonucleotide according to the present invention.

In another embodiment, the present invention provides a method for specifically detecting Staphylococcus aureus in a sample from a biological sample using an primer of SEQ ID NO: 1 or a primer of the complementary sequence thereof and a primer of SEQ ID NO: 2 or a complementary sequence thereof to provide.

In another aspect, the present invention relates to a method for screening a sample of a subject suspected of having meningococcal or sepsis, comprising the steps of: preparing a primer of SEQ ID NO: 1 or a complementary sequence thereof and a primer of SEQ ID NO: 2 or a complementary sequence thereof The present invention provides a method for specifically detecting Staphylococcus aureus in a biological sample using Invitro.

The oligonucleotides, compositions, methods and kits according to the present invention may be used as biological samples, for example in the detection of Staphylococcus aureus in cultured fungi, blood, or cerebrospinal fluid, and the detection may be by amplification or hybridization of nucleic acids, In one embodiment, it is detected by amplification, for example by PCR.

When PCR is used in the detection method according to the present invention, the method comprises: performing electrophoresis on the PCR product; And determining the presence and / or amount of Staphylococcus aureus in the sample based on the electrophoresis result, wherein the detection of the 317 bp product as a result of the electrophoresis indicates the presence of Staphylococcus aureus in the biological sample will be. The presence of Staphylococcus aureus in the sample is used for the diagnosis of meningitis or sepsis.

By using the composition and method of the present invention, the presence or absence of meningococcal bacteria can be detected quickly and accurately by a single test.

FIG. 1 shows the results of electrophoresis after detecting the specificity of S. aureus- specific primers according to one embodiment of the present invention by PCR method. NC was a negative control, and the same experiment was performed on six different strains other than S. aureus .
FIG. 2 shows the sensitivity of S. aureus- specific primers according to one embodiment of the present invention. PCR was performed using different concentrations of template, followed by electrophoresis.

Present in the oligonucleotide designed to identify Spa (Protein A) in specific genes of Staphylococcus aureus (Staphylococcus aureus), and specific detection of Staphylococcus aureus (Staphylococcus aureus) through the detection of the gene nucleotide, oligonucleotide wherein Compositions, kits, and methods that include nucleotides are disclosed.

Staphylococcus aureus is a typical bacterium that causes serious infiltrating diseases such as sepsis and meningitis in newborns. In addition, 10-30% of pregnant organs of the stomach or digestive organ Staphylococcus aureus ( Staphylococcus aureus ) infected through the birth canal (birth canal) can infect the newborn.

Thus, compositions, kits, or methods according to the present disclosure may be useful for diagnosing, for example, early diagnosis of severe infiltrating diseases such as sepsis and meningitis caused by Staphylococcus aureus.

It is known that Staphylococcus aureus is a causative organism of the above-mentioned diseases. For example, it is known that Pathogens associated with sepsis in newborns and young infants in developing countries, Zaidi AK, Thaver D, Ali SA, Khan TA., Pediatr Infect Dis J. 2009 Jan; 28 (1 Suppl): S10-8; And Staphylococcus aureus meningitis. A review of 104 nationwide, consecutive cases., Jensen AG, Espersen F, Skinhøj P, Rosdahl VT, Frimodt-Møller N., Arch Intern Med. 1993 Aug 23; 153 (16): 1902-8. Accordingly, the composition according to the present invention can be usefully used for diagnosis of such diseases.

In one embodiment, the present invention relates to an oligonucleotide for detecting Staphylococcus aureus of SEQ ID NO: 1 or 2 or a complementary sequence thereof.

As used herein, the term " oligonucleotide " refers to a ribonucleotide or deoxyribonucleotide of any length, including both a single strand and a double strand, including both primers and probes.

As used herein, the term " primer " refers to an oligonucleotide having a sequence complementary to at least a portion of a gene specific to a strain detected herein, and is a starting point of polymerization in a polymerization reaction using a polymerase such as a polymerase chain reaction. The primers herein also include those having high sequence similarity thereto. High sequence similarity can be determined by using at least 70% sequence similarity or similarity using GCG FastA (Genetics Computer Group, Madison, Wis. USA), MacVector 4.5 (Kodak / iBI software package) . The primers can be synthesized through known methods or synthetic companies in the art and are at least about 5 nucleotides in length.

Oligonucleotides comprising the primers herein can be labeled with suitable materials that can be visualized according to the method of detection. The labeling substance includes radioactive isotopes, polypeptide labeling substances, fluorescent substances, coloring substances and the like. Such a labeling substance may be introduced into the oligonucleotide itself when it is synthesized, and may be introduced in the polymerization process when PCR is used. Radiation agents include beta, gamma, and alpha ray emitters, including ³² P, ³⁵ S, and ¹²⁵ I. Fluorescent substances are examples of substances that are excited when they absorb energy, and substances that emit visible light while returning to the ground state in the excited state. Polypeptide labeling substances include enzymes such as biotin, digoxigenin, and enzymes such as horseradish peroxidase. The coloring material includes a chemiluminescent material that emits energy absorbed by a chemical reaction, such as oxiruminescence.

As used herein, the term " detection " means the detection of the presence or absence of nucleic acid, if any, and is not limited to, for example, based on nucleic acid-specific DNA-DNA or RNA-DNA hybridization , Northern blot analysis, Southern blot analysis, microchip analysis, PCR (including quantitative and qualitative PCR, real-time quantitative and qualitative PCR) amplification.

In one embodiment, it is used in nucleic acid amplification, especially in PCR.

As used herein, the term " PCR (Polymerase Chain Reaction) " refers to a method of amplifying an initiating nucleic acid material in a chronological manner by using a polymerase to synthesize nucleic acids, And a real-time PCR that enables qualitative and quantitative analysis by real-time tracking of quantitative PCR and PCR process for measuring the amount of a specific nucleic acid.

For PCR reactions, a pair of primers consisting of two primers is used. In one embodiment, SEQ ID NO: 1 or a complementary sequence thereof and SEQ ID NO: 2 or a complementary sequence thereof are used.

The amplicons amplified by PCR have sequences corresponding to the molecules used as templates and can be analyzed by a variety of methods known in the art. Such methods are well known in the art and include, for example, gel electrophoresis, real-time PCR analysis, single strand conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP), capillary zone electrophoresis (CZE) analyzing technology, microchips, and the like. In one embodiment herein, PCR products are analyzed by agarose gel electrophoresis along with size markers. The electrophoresis results can determine the presence of Staphylococcus aureus in the size of the product, and in one embodiment according to the invention the size of the product amplified by the primer pairs herein is 225 bp.

In this context, the present invention relates to a composition for detecting Staphylococcus aureus comprising a primer of the present invention, a kit comprising the composition, or a method.

The primers contained in the composition or kit and the detection using the primers can be referred to the above. In one embodiment according to the present application, the composition is used for nucleic acid amplification, in particular for amplification using PCR. In this case, the PCR composition comprises the buffers, Taq polymerase and MgCl ₂ necessary for the reaction of the PCR. Various buffers known in the art may be used, for example, but not limited to, Tris-HCl, pH 9.0 buffer. Taq polymerase can be obtained commercially, for example, such as AmpliTaq Gold (Applied Biosystems, USA) can be used, and suitable concentration, for example, 1.5 mM to 2.5 mM MgCl ₂ can be included.

The kit according to the present invention further comprises a positive control, a negative control, and instructions for use. As a negative control, DNA of a different kind of Gram-positive bacterium other than the detection target can be used, and as a control group, a plasmid containing the genomic DNA of the Gram-positive bacteria to be detected or the Gram-positive bacteria of the detection subject can be used.

In another embodiment, the present application also provides a method of specifically detecting Staphylococcus aureus in a virus sample from a biological sample using a primer of SEQ ID NO: 1 or a complementary sequence thereof and a primer of SEQ ID NO: 2 or a complementary sequence thereof ≪ / RTI >

The primers used in the method of the present invention and the detection method using the primers can be referred to the above.

The compositions, methods and kits according to the present invention are capable of detecting meningococcal bacteria from a variety of biological samples. A biological sample refers to an organism-derived tissue, a cell extract, and / or an inducer thereof and a culture thereof, which can detect the presence or the amount of meningococcal S. aureus using primers according to the present invention. For example, , Cerebrospinal fluid, cultured bacteria, blood, cerebrospinal fluid. The cultured bacterium can be used as a bacterium cultured using a sample such as blood or cerebrospinal fluid derived from a subject suspected of having a disease, for example, by using the method described in this embodiment, and then used for detection .

Hereinafter, the present invention will be described in detail with reference to the following examples, which are intended to be illustrative and not limit the scope of the present invention in any way.

The present invention may be practiced using conventional techniques within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombinant techniques, unless otherwise indicated. In addition, a more detailed description of common techniques can be found in Molecular Biotechnology: (Bernard et al., ASM press 1994); Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al., Eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985).

Example

Example 1 Cell Culture and Dielectric Extraction

Staphylococcus aureus , Streptococcus pneumoniae , Streptococcus agalactiae , Bacteroides fragilis , Clostridium perfringens , Clostridium difficile , and Finegoldia magna were introduced into TRYPTICASE SOY AGAR (BD Diagnostics, Germany) and incubated overnight in a 37 ° C stirred incubator. Then, each strain was centrifuged at 3000 × g, and then collected using a Higene genomic DNA prep kit (BIOFACT) according to the manufacturer's instructions. In summary, 300 μl of the R1 solution and 2 μl of lysozyme were added to the kit, followed by mixing and reacting at 37 ° C. for 1 hour. Then, the supernatant was removed by centrifugation at 13,000 rpm, and then 350 μl of the SGD1 solution was added thereto, followed by vortexing. Then, 8 μl of protease K was mixed and then cultured at 65 ° C. for 10 minutes. Then, 400 μl of the SGD2 solution was added, followed by vortexing and centrifugation at 1300 rpm for 5 minutes. The supernatant was separated, loaded on a column, centrifuged, washed twice, and then eluted with distilled water.

Example 2 Primer design and PCR reaction

The primer was designed at the site of S. aureus Spa (Staphylococcus aureus, Protein A), and the sequence is as follows: forward 5'- AATGACTCTCAAGCTCCAA-3 '(SEQ ID NO: 1), reverse 5'- TTTGTTGAAATTGTTGTCAGC- No. 2).

PCR was performed using the above primers under the following conditions. PCR was performed using 2 μl of the DNA extracted from Example 1, 2.5 μl of 10 × PCR buffer, 10 pmol forward, reverse primer, 2 μl dNTP mixture and 2.5 U Taq polymerase. The total PCR conditions were 30 minutes at 95 ° C for 30 seconds, 30 seconds at 95 ° C, 30 seconds at 57 ° C and 45 seconds at 72 ° C, and finally 10 minutes at 72 ° C. The amplified DNA was confirmed by agarose gel electrophoresis containing EtBR.

Example 3 Analysis of Staphylococcus aureus detection specificity

The specificity of S. aureus detection using the primers according to the present invention was analyzed by performing PCR as in Example 2. [ The results are shown in Fig. As shown in the figure , the amplification of the SpA gene was not observed in the six bacteria of Streptococcus pneumoniae, Streptococcus agalactiae, Bacteroides fragilis, Clostridium perfringens, Clostridium difficile, and Finegoldia magna in the absence of negative control DNA , whereas Staphylococcus specific amplification only in PCR using aureus genome.

Example 4 Sensitivity Analysis of Staphylococcus aureus Detection

The sensitivity of the detection of Staphylococcus aureus using primers according to the present invention was analyzed by performing PCR as in Example 2 and PCR was performed by diluting with 0, 10 ng, 1 ng, 100 pg, 10 pg, and 1 pg, respectively . The results are shown in Fig. Staphylococcus aureus (Staphylococcus aureus) can be detected from 1pg of DNA, except for the negative control, as shown above.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Composition and method for detecting Staphylococcus aureus <130> DP201407002P <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus aureus <400> 1 aatgactctc aagctccaa 19 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus aureus <400> 2 tttgttgaaa ttgttgtcag c 21

Claims (11)

An oligonucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and the complementary sequence thereof, which specifically detects Staphylococcus aureus.
2. The oligonucleotide of claim 1, wherein the oligonucleotide is used for nucleic acid amplification comprising PCR.
3. The oligonucleotide of claim 2, wherein the oligonucleotide is used for the diagnosis of meningitis or sepsis.
The method according to claim 1,
Wherein the oligonucleotide is labeled with a chromogenic, luminescent or fluorescent substance.
A composition for detecting S. cerevisiae Staphylococcus aureus comprising an oligonucleotide according to any one of claims 1 to 3.
A kit for detecting S. cerevisiae Staphylococcus aureus comprising an oligonucleotide according to any one of claims 1 to 3.
A method for specifically detecting Staphylococcus aureus in a virus sample from a biological sample using a primer of SEQ ID NO: 1 or a primer of a complementary sequence thereof and a primer of SEQ ID NO: 2 or a complementary sequence thereof.
8. The method of claim 7,
Wherein said biological sample is a cultured fungus, blood, or cerebrospinal fluid.
8. The method of claim 7, wherein said detection is by PCR.
10. The method of claim 9,
The method comprising: performing electrophoresis on the PCR product; And
And determining the presence or absence of Staphylococcus aureus in the sample based on the result of the electrophoresis.
11. The method of claim 10,
Wherein the detection of the 225 bp product as a result of the electrophoresis indicates the presence of Staphylococcus aureus in the biological sample.

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