WO2014073858A1 - Method for detecting mrsa, and kit using same - Google Patents

Method for detecting mrsa, and kit using same Download PDF

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WO2014073858A1
WO2014073858A1 PCT/KR2013/010025 KR2013010025W WO2014073858A1 WO 2014073858 A1 WO2014073858 A1 WO 2014073858A1 KR 2013010025 W KR2013010025 W KR 2013010025W WO 2014073858 A1 WO2014073858 A1 WO 2014073858A1
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seq
mrsa
pcr
present
amplification
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French (fr)
Korean (ko)
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김정욱
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주식회사 현일바이오
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
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    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/101—Taqman
    • C—CHEMISTRY; METALLURGY
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    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/113—Real time assay
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/44—Multiple drug resistance

Definitions

  • the present invention relates to a method for detecting MRSA in a sample through simultaneous amplification and analysis of three target genes and a diagnostic kit using the same.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Huletsky et al. (4) describe a single-gene locus for a single-gene locus that amplifies a staphylococcal cassette chromosome (SCC »ec) / ⁇ 9r J (open reading frame X) junction.
  • MRSA has been endemic in Korea for many years. Among aureus isolates recovered from clinical specimens, the rate of methicillin resistance reached 67.8–74.1% in the 2000s (16). SC mec ⁇ is a mobile element that can be inserted or cleaved into the chromosome. Partial cleavage of SCCfflecr from endemic MRSA strains has been reported to result in MSSA isolates (13, 15). Thus, in highly endemic regions, assays for single-gene sites for direct detection of MRSA appear more likely to result in false-positive results due to MSSA strain J "derived from MRSA with SCOec residues.
  • Efforts have been made to develop a method that can detect or quantify 3 ⁇ 4 " mpe" fe MRSA (methici 11 in-resistant Staphylococcus aureus)%.
  • the present inventors prepared primers and probes capable of detecting mecA, SCCmec / orfX, and 16S rRNA genes of MRSA, and performed real-time quantitative PCR to prepare MRSA from a sample (preferably blood, saliva or urine).
  • the present invention has been completed by confirming that can be specifically and simply detected and quantified.
  • An object of the present invention is to provide a method for detecting or quantifying MRSA (methicillin-resistant Staphylococcus / ⁇ :).
  • Another object of the present invention is to provide a kit for detecting or diagnosing MRSA.
  • the invention comprises the following steps
  • Methods for detecting or quantifying MRSA include: (a) preparing a sample; (b) at least one forward primer selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO. Probes; Primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 and probes of SEQ ID NO: 12; And amplifying the nucleotide sequence in the sample using the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 and the probe of SEQ ID NO: 15; And (c) confirming the amplification result by fluorescence.
  • the present invention is one or more forward primers selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6 sequence, one selected from the group consisting of SEQ ID NO: 7 sequence and SEQ ID NO: 8 sequence A reverse primer and a probe of SEQ ID NO: 9; Primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 and probes of SEQ ID NO: 12; And it provides a kit for detecting or diagnosing MRSA (methiciIlin-resistant Staphylococcus aureus) comprising a primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 and a probe of SEQ ID NO: 15.
  • MRSA methiciIlin-resistant Staphylococcus aureus
  • Efforts have been made to develop methods to detect or quantify ⁇ r ⁇ lfe- MRSA (methi c 11 in-res i st ant Staphylococcus aureus) ⁇ .
  • the present inventors prepared primers and probes capable of detecting mecA, SCCmec / orfX and 16S rRNA genes of MRSA, and carried out real-time quantitative PCR to perform MRSA from a sample (preferably blood, saliva or urine). It was confirmed that can be detected and quantified specifically and simply.
  • the present invention can very effectively and simply detect the endemic MRSA.
  • the amplification of the invention is a preferred embodiment of the invention.
  • the primer of the present invention is used for amplification react ions.
  • amplification reaction means a reaction that amplifies a nucleic acid molecule.
  • Various amplification reactions are reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683, 195, 4,683,202, and 4,800, 159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al. , Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (W0 89/06700) and Davey, C. et al.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • nucleic acids Nucleic acid sequence based amplification (US Pat. Nos. 5,130, 238, 5,409 ⁇ 818, 5, 554,517, and 6,063, 603), strand displacement amplification (strand dis) lacement amplification and loop—mediated isothermal amplification; LAMP), but is not limited thereto.
  • Other amplification methods that can be used are described in US Pat. Nos. 5, 242,794, 5, 494, 810, 4,988, 617, and US Pat. No. 09 / 854,317.
  • the term "primer” refers to an oligonucleotide, wherein the conditions under which the synthesis of a primer extension product complementary to the nucleic acid chain (template) is induced, i.e., the presence of a polymerizer such as nucleotide and DNA polymerase and At conditions of suitable temperature and pH It can serve as a starting point for synthesis.
  • the primer is deoxyribonucleotide and single chain.
  • Primers used in the present invention may include naturally occurring dVP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides.
  • the primer may also include ribonucleotides.
  • the primer should be long enough to prime the synthesis of the extension product in the presence of the thickener. Suitable lengths of the primers depend on a number of factors, such as silver, utility and source of the primer.
  • annealing or “priming” refers to the placement of oligodioxynucleotides or nucleic acids in a template nucleic acid, wherein the polymerase polymerizes the nucleotides to form a nucleic acid molecule that is complementary to the template nucleic acid or portion thereof. Let's do it.
  • PCR is the most well-known method of nucleic acid amplification, and many modifications and uses have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR.
  • multiplex PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chains Inverse polymerase chain reaction (IPCR), vectorette PCR and TAIL-PC (thermal asymmetric interlaced PCR) have been developed for specific use.
  • DD-PCR differential display PCR
  • RACE rapid amplification of cDNA ends
  • IPCR inverse polymerase chains
  • vectorette PCR vectorette PCR
  • TAIL-PC thermal asymmetric interlaced PCR
  • a gene amplification reaction may be performed to simultaneously detect target genes in an analyte (eg, a sample containing a target microorganism). Therefore, the present invention performs a gene amplification reaction using a primer that binds to DNA isolated from the microorganism in the sample. Primers used in the present invention are either localized or annealed to one site of the template to form a double chain structure.
  • the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which include Ther us aquat i cusi z ⁇ ), Thermus thermophi lusi i), Thermus filiformis, Therm is flavus, Ther ococcus literal is, and Pyrococcus // contains osi / s (Pfu)
  • the reaction vessel When carrying out the polymerization reaction, it is preferable to provide the reaction vessel with an excess of components necessary for the reaction.
  • components necessary for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. It is desired to provide cofactors such as Mg ⁇ , dATP, dCTP, dGTP and dTTP to the reaction mixture such that the desired degree of amplification can be achieved.
  • All enzymes used for the amplification reaction may be active under the same reaction conditions. In fact, the supernatant ensures that all enzymes are close to the optimum reaction conditions.
  • the amplification process of the present invention can be carried out in a single reactant without changing conditions such as the addition of the counter agitate.
  • Annealing in the present invention is carried out under stringent conditions allowing specific binding between the target nucleotide sequence and the primer.
  • Stringent conditions for annealing are sequence-dependent and vary with ambient environmental variables.
  • the amplified target genes are analyzed by a suitable method to detect MRSA.
  • the target gene can be detected by performing gel electrophoresis on the amplified reaction product described above, and observing and analyzing the band formed as a result.
  • the method of the present invention is applied to the amplification reaction using the DNA of the microorganism.
  • (i) SCCy7; ecA? Primers and probes annealed to the nucleotide sequence; primers and probes annealed to the mecA nucleotide sequence; Or performing amplification reactions using primers annealed to 16S rRNA nucleotide sequences; And ( ⁇ ) analyzing the product of the amplification reaction through fluorescence, through which MRSA can be detected or quantified in the DNA extracted from the sample.
  • MRSA strains that can be detected by the methods of the invention include, but are not limited to, CCARM 3792, CCARM 3795, CCARM 3798, CCARM 3803, CCARM 3805, CCARM 3877, CCARM 3897 and CCARM 3911 It is not limited ⁇
  • hybridization means that two single stranded nucleic acids form a duplex structure by pairing complementary base sequences. Hybridization can occur when the complete complement between single stranded nucleic acid sequences (per feet match) or even when some mismatch base is present. The degree of complementarity required for the shake may vary depending on the shake reaction conditions, and in particular, may be controlled by temperature. ? The terms “annealing” and “animation” do not differ and are commonly used herein.
  • the method and kit of the present invention is a multiplex real-time PCR that simultaneously detects three genes 0 »eo4, SCOec / or / and 16S rRNA to distinguish MRSA from other microorganisms. Can be detected through.
  • the method and kit of the present invention consists of at least one forward primer selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 One reverse primer selected from the group and a probe of SEQ ID NO: 9; Primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 and probes of SEQ ID NO: 12; And a primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 and a probe of SEQ ID NO: 15.
  • the target gene used in the method and kit of the present invention is SEQ ID NO: 1 to SEQ ID NO: An SCCmec / orfX gene detected by at least one forward primer selected from the group consisting of the sixth sequence, one reverse primer selected from the group consisting of SEQ ID NO: 7 sequence, and sequence 8 sequence; A mecA gene detected by a primer pair of SEQ ID NO: 10 and SEQ ID NO: 11 and a probe of SEQ ID NO: 12; And 16S rRNA genes detected by primer pairs of SEQ ID NO: 13 and SEQ ID NO: 14 and probes of SEQ ID NO: 15.
  • Real-time PCR is a technique for monitoring and analyzing the increase of PCR amplification products in real time (Levak KJ, et a ⁇ ., PCR Methods Appl., 4 (6): 357-62 (1995)).
  • PCR reactions can be monitored by characterizing the amount of fluorescence emission in each cycle during the exponential phase, if the increase in PCR product is proportional to the initial amount of the target template. The higher the starting copy number of the nucleic acid target, the faster the fluorescence increase is observed and the lower the threshold cycle.
  • a marked increase in fluorescence above the reference value measured between 3-15 cycles means detection of accumulated PCR product.
  • real-time PCR has the following advantages: (a) Conventional PCR is measured in the plateau, whereas real-time PCR is used during the exponential growth phase. Data can be obtained; (b) the increase in the reporter fluorescence signal is directly proportional to the number of amp ⁇ cons generated; (c) the cleaved probe provides permanent record amplification of the amplicon; (d) increase the detection range; (e) requires at least 1,000 times less nucleic acid than conventional PCR methods; (f) detection of amplified DNA without separation via electrophoresis is possible; (g) small amplicon sizes can be used to obtain increased amplification effects; And (h) the risk of contamination is low.
  • a real-time PCR reaction using a step-diluted standard sample yields amplification curves arranged at equal intervals in order of increasing initial DNA content. Setting a threshold at an appropriate point here yields the point C f at which the threshold intersects the amplification curve.
  • PCR amplification products are detected by fluorescence.
  • Detection methods include interchelating methods (SYBR Green I method) and fluorescent labeling probes (TaqMan probe method).
  • the interchelating method detects both double-stranded DNA, so there is no need to prepare gene-specific probes, so the counterunggy system can be constructed at low cost. While the method using the fluorescent labeling probe is expensive, the detection specificity is high and even similar sequences can be detected.
  • the interchelating method uses a double-stranded DNA-binding die, and uses a non-sequence specific fluorescent intercalating reagent (SYBR Green I or ethidium bromide) to produce an amplicon including non-specific amplification and primer-dimer complexes.
  • SYBR Green I is a fluorescent die that binds to the minor groove of double-stranded DNA and is a reagent that shows little fluorescence in solution but shows strong fluorescence when combined with double-stranded DNA (Morrison TB, Biotechniques., 24 (6): 954-8, 960, 962 (1998).
  • SYBR Green Real-Time PCR is accompanied by optimization procedures such as melting point or dissociation curve analysis for amplicon identification. Normally, SYBR green is used for singleplex reactions, but it can be used for multiplex reactions when accompanied by melting curve analysis (Siraj AK, et al. Xlin Cancer Res., 8 (12). ): 3832-40 (2002); and Vrettou C., et al., Hum Mutat., Vol 23 (5): 513-521 (2004)).
  • the threshold cycle (C f ) value refers to the number of cycles in which the fluorescence generated in the reaction exceeds the threshold, which is inversely proportional to the logarithm of the initial copy number. Therefore, the value assigned to a particular well reflects the number of cycles in which the stratified number of amplicons in the reaction accumulates.
  • the t value is the cycle in which the increase in ⁇ was first detected.
  • Rn refers to the magnitude of the fluorescence signal generated during PCR at each time point
  • ARn refers to the fluorescence emission intensity (standardized reporter signal) of the reporter die divided by the fluorescence emission intensity of the reference die.
  • the value is also named Cp (crossing point) in LightCycler.
  • the C t value represents the point in time when the system begins to detect an increase in the fluorescence signal associated with the exponential growth of the PCR product in a log-linear phase. The period provides the most useful information about reaction.
  • the log-linear phase creeping represents the amplification efficiency (Eff) (http: // www.appl iedbiosysterns, com /).
  • TaqMan probes typically contain primers (eg, 2-30 nucleotides) comprising a 5'- uorophore and a 3'-quencher (e.g., TAMRA or non-fluorescent something (NFQ)). It is longer than l) nucleotides.
  • TaqMan probes are designed to anneal to internal sites of PCR products.
  • the TaqMan probe may be designed as an internal sequence of a 16S rRNA gene segment that is amplified by SEQ ID NO: 13 and 14.
  • TaqMan probes specifically specific for templatated DNA in the annealing step, but fluorescence is inhibited by 3 ⁇ 4 on the probe.
  • TaqMan probes that were localized in the template were degraded by the 5 'to 3' nuclease activity of TaQ DNA polymerase, and the fluorescent dye was released from the probe, thereby suppressing the inhibition by the quencher, indicating fluorescence.
  • the 5'-end of the TaqMan probe should be located downstream of the 3'-end of the extension primer.
  • the 5'-end of the TaqMan probe is cleaved by the 5 'to 3' nuclease activity of the polymerase to The fluorescent signal is generated.
  • Fluorescent reporter molecules and quencher molecules that can be used in the present invention can be any known in the art, for example: (The number in parentheses is the maximum emission wavelength in nanometers): Cy2 TM 506, Y0-PR0 TM-!
  • non-fluorescent black quencher molecules can be used which can fluoresce a wide range of wavelengths or a particular wavelength.
  • non-fluorescent black quarter molecules include, but are not limited to, BHQ and DABCYL, most preferably BHQ1 and BHQ2.
  • the reporter-quencher pair used in the probes of the invention comprises HEX, FAM, Cy5.5, BHQ1 and BHQ2—based labels.
  • the sequential sequence of the ninth sequence of the present invention uses BHQ1 as the quencher at the HEX and 3'-ends as the 5'-terminal fluorescent substance, Using FHQ and 5'-end BHQ1 as the fluorescent material at the 5'-end, the sequence 15 of the sequence Moktok of the present invention is Cy5.5 and 3'-something at the 5'-end.
  • BHQ2 Suitable reporter-something pairs are disclosed in many literatures: Pesce et al.
  • the target nucleic acid used in the present invention is not particularly limited and includes all DNA (gDNA or cDNA) or RNA molecules, more preferably gDNA. If the target nucleic acid is an RNA molecule, reverse transcription to cDNA is used.
  • Target nucleic acids include, for example, prokaryotic nucleic acids, eukaryotic cells (eg, protozoa and parasites, fungi, yeast, higher plants, lower animals and higher animals, including mammals and humans) nucleic acids, viruses (eg, herpes virus, HIV) , Influenza virus, Epstein-Barr virus, hepatitis virus, poliovirus, etc.) nucleic acid or non-loid nucleic acid.
  • the method of annealing or hybridizing the target nucleic acid to the extension primers and probes can be carried out by hybridization methods known in the art.
  • suitable isomerization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory.
  • conditions such as temperature, concentration of components, shake and reaction time, complete liquid components and their pH and ionic strength depend on various factors such as the length and GC amount of the oligonucleotide and the target nucleotide sequence.
  • Detailed conditions for the shake are described by Joseph Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer® Verlag New York Inc. NY (1999).
  • the template-dependent nucleic acid polymerase used in the present invention is an enzyme having 5 'to 3' nuclease activity.
  • the template-dependent nucleic acid polymerase used in the present invention is preferably a DNA polymerase.
  • DNA polymerases typically have 5 'to 3' nuclease activity.
  • Template-dependent nucleic acid polymerases used in the present invention include E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase.
  • the template-dependent nucleic acid polymerase is a thermostable DNA polymerase obtained from various bacterial species, which include Therms aquat i cusi ⁇ ), Thermus ther ophi lusilt), Ther us filiform is, Therm is flavus, Thermococcus literal is, Pyrococcus furiosusCPiu), Thermus antranikiani i, Thermus caldophi lus, Thermus chl iarophi lus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scmus ductus, Thermus silotoductus 17 ⁇ Contains Thermus thermophi lus, Thermotoga maritima, Thermotoga neapolitana and Thermos ipho africanus ⁇ ⁇ DNA polymerase,
  • Temporative-dependent extension reaction catalyzed by a template-dependent nucleic acid polymerase means a reaction that synthesizes a nucleotide sequence complementary to the sequence of the template.
  • the real-time PCR of the present invention is carried out by TaqMan probe method. ⁇ Effects of the Invention ⁇
  • the present invention relates to a method for detecting MRSA by amplifying and analyzing three target genes simultaneously using various primers and probes, and a diagnostic kit using the same.
  • the present invention is directed to a target gene (preferably mecA, ⁇ Xmec / orfX and
  • PCR Real-time polymerase chain reaction
  • the diagnostic kit of the present invention can easily and efficiently detect target genes in a sample through multiplex real-time PCR.
  • the methods and kits of the present invention can detect infections as well as accurate diagnosis of MRSA, and can be used to more accurately predict the prognosis of the disease and apply it to treatment based on this.
  • Results show the correlation between t values of SCOec / or / and 16S rRNA.
  • the assay method of the present invention was evaluated using 444 strains including both reference strains obtained from various international collections and clinical isolates obtained from the laboratory.
  • the reference strains include eight MRSA strains (CCARM 3792, CCARM 3795, CCARM 3798, CCARM 3803, CCARM 3805, CCARM 3877, CCARM 3897 and CCARM 3911), four MSSA strains (KCTC 1621 KCTC 1916, KCTC 1928 and ATCC). 29213), and 11 coagulase-negative Staphylococcus strains
  • KCTC 3584 Staphylococcus simulans, KCCM 41686; Staphylococcus capitis, KCCM 41466; Staphylococcus warneri, KCTC 3340; Staphylococcus haemolyticus, KCTC 3341; Staphylococcus xylosus, KCTC 3342; Staphylococcus intermedius, KCTC 3344; Staphylococcus saprophyticus, KCTC 3345; Staphylococcus cohnii, KCTC 3574; Staphylococcus caprae, KCTC 3583; and Staphylococcus auricular is, KCTC 3584), confirmed by SCOec typing (17, 18) Twenty nine MSSA isolates carrying the elements were tested with control strains.
  • Clinical isolates consisting of 209 MRSA strains, 109 MSSA strains, and 74 MRCoNS strains were mostly recovered from wound, saliva, blood and urine samples.
  • the identification and susceptibility testing of the Staphylococcus isolates described above were performed by MicroScan WalkAway 96 (Siemens Healthcare Diagnostics Inc., West Sacramento, CA, USA) and VITEK 2 (bioMerieux Inc., Durham, NC, USA) And sensitivity testing systems.
  • Reference strains and clinical isolates were incubated for 24 hours at 37 ° C in blood-agar plates (Asan Pharmaceutical, seoul, Korea). About 2-3 bacterial colonies of the reference strains and isolates were obtained in 1 ⁇ loop and suspended in 0.5 ml of distilled water. The suspension was heated in a boiling water bath for 10 minutes and then centrifuged at 13,000 X g for 5 minutes. Supernatants were used for real-time PCR.
  • the base sequences of the SCCmec / or fX junction, mecA and Staphylococcus 16S rR A genes were obtained from NCBI GenBank and aligned using Sequencher 5.0 software (Gene Codes Co., Ann Arbor, MI, USA). Based on the sequence alignment, we find regions of interest and fabricate primers and probes by the Primer 3 program (http: // f rodo.wi.mit.edu/pr imer 3 /) or by hand. It was. The position in the gene of interest of the primer sequence is as follows: (a) open reading frame (0RF) of SCCmec, staphylococcus; And (b) orfX, the attBscc site near the 3 'end. The real-time PCR primers and probes designed and used in this study are shown in Table 1. TABLE 1
  • the pair indicates the SCOec type amplified by the primer.
  • Real-time PCR reactions were performed using a Rotor-gene Q real-time PCR instrument (QIAGEN Inc., Germantown, MD, USA).
  • the PCR mixture was prepared with a total of 0.5 ⁇ l primer-probe mix, 5 ⁇ l of 2 ⁇ Rotor-Gene multiplex PCR master mix (QIAGEN Inc., Germantown, MD, USA) and 1.0 ⁇ l of template DNA. It consists of 10 ⁇ of the repellent solution.
  • PCR conditions were as follows: (a) PCR cycle, (i) pre-denaturign step, 5 min at 95 ° C .; And ( ⁇ ) 40 cycles, 15 seconds at 95 ° C and 15 seconds at 60 ° C 1 cycle; And (b) a detection step, detecting green, yellow and crimson fluorescence.
  • C t values of mecA, SCCmec / orfX and 16S rRNA genes were recorded using Rotor-Q Q software.
  • Statistical tests including correlation coefficients (r) and descriptive statistics were conducted using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) *. P values below the 5% level were considered statistically significant.
  • Assay sensitivity of real-time PCR was determined by subculture of 10-fold serial dilutions of MRSA strain CCARM 3792. The strain was incubated overnight in blood agar folate, suspended in a salt solution at the same density as McFarland turbidity number of 0.5, and serially dilute in 10-fold from 102 to 107 ⁇ DNA was QIAamp® DNA mini Kit My QIAcube (QIAGEN Inc., Germantown, MD, USA) was used to extract from bacterial dilutions (200 ⁇ ) and dissolved in 50 ⁇ . At the same time, the bacterial dilutions (200 ⁇ ) were plated on blood agar and incubated at 37 ° C for 24 hours. Thereafter, colony-forming units (CFUs) were counted.
  • CFUs colony-forming units
  • Priority real-time PCR assays were subjected to 23 strain reference strains and 29 MSSA control strains.
  • the expected PCR product was only amplified at the reference strain.
  • SCCmec / orfX was not detected in 6 of 29 control strains.
  • C tscc the C t value difference between the white mecl orfX mecA, and the 16S rRNA and mecA C t value difference (C tl6S ) were used to assess the presence of MRSA.
  • C t scc ( ⁇ 4.7; mean + 4SD) indicates simultaneous presence of MRSA and non-MRSA Staphylococcus
  • C tl6S ( ⁇ -1.72; mean-4SD) shows Staphylococcus without MRSA and mecA genes Meant that they exist together.
  • the detection limit of the assay was performed using genomic DNA purified from 1:10 4 dilution of MRSA strain CCARM 3792 stock solution, 20 CFU (colony forming unit) per PCR reaction. was measured to be
  • the simultaneous amplification method can also lead to false positive results when both MRCoNS and MSSA containing mec residues are present in the clinical sample.
  • Staphylococcus 16S rRNA gene was added to the target to reduce false positive results.
  • the number of copies of the three target genes expressed in C t values are all the same, whereas for populations with MRCoNS and MSSA containing mec residues, the number of copies of the genes is mostly Will be different.
  • MRCoNS and MSSA complexes comprising SCOec residues can be distinguished through relative quantification of the three targets.
  • a composite cocktail of Staphylococcus genomic DNA samples comprising a mixture of MRCoNS and MSSA, including a mixture of genomic DNA derived from MRSA and non-MRSA and Staphylococcus and a SCOec residue, was used to amplify the three targets.
  • the amplified samples were then analyzed (no results shown).
  • MSSA containing SCC / sec residues is estimated to be about 3% of aureus ⁇ recovered from clinical samples, with approximately 30% of 5. aureus isolates containing MSSA and 10% SCOec residues. This is because MSSA, according to Becker's study, showed that nasal colonization by MSSA and MRCoNS was observed in about 3 patients (8).
  • the MSSA containing mec residues was at least 90% of the mixed populations with C tscc values of 15 or more.
  • the three targets are simultaneously available from mixed populations of MRCoNS and MSSA containing mec residues. It is very unlikely to be amplified.

Abstract

The present invention relates to a method for detecting MRSA by simultaneously amplifying and analyzing three target genes using various primers and probes, and a diagnostic kit using the same. The present invention can effectively detect MRSA and easily distinguish the same from other strains through a multiplex real-time polymerase chain reaction (PCR) using target genes (preferably, mecA, SCCmec/orfX and 16S rRNA)-specific primers and probes. In addition, the diagnostic kit of the present invention can conveniently and effectively detect target genes in a sample through the multiplex real-time PCR. Therefore, the method and the kit of the present invention can detect MRSA infection and precisely diagnose MRSA, and more precisely predict the prognosis of diseases and treat the diseases on the basis of the result.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
MRSA 검출 방법 및 이를 이용한 키트  MRSA detection method and kit using same
[기술분야】 [Technical Field]
본 특허출원은 2012년 11월 09일에 대한민국 특허청에 제출된 대한민국 특허출원 제 10-2012-0126614호에 대하여 우선권을 주장하며， 상기 특허출원의 개시 사항은 본 명세서에 참조로서 삽입된다.  This patent application claims priority to Korean Patent Application No. 10-2012-0126614 filed with the Korean Patent Office on November 09, 2012, the disclosure of which is incorporated herein by reference.
본 발명은 3개의 타겟 유전자의 동시 증폭 및 분석을 통해 시료 내 MRSA를 검출하는 방법 및 이를 이용한 진단키트에 관한 것이다.  The present invention relates to a method for detecting MRSA in a sample through simultaneous amplification and analysis of three target genes and a diagnostic kit using the same.
【배경기술】 Background Art
병원 및 지역 공동체 (co讓 unity settings)에서 빈번하게 나타나는 메티실린-저항성 스타필로코커스 아우레우스 (methicillin-resistant Staphylococcus aureus, MRSA)의 만연은 전세계적으로 공공 건강에 대한 큰 위험이다. 따라서, 신속하고 정확한 검출 및 적절한 중재는 MRSA의 유병률 (prevalence)을 감소시킨다 (1-3). 최근에， MRSA을 분자적으로 신속하게 검출하는 방법이 개발되었다. Huletsky 등 (4)은 스타필로코커스 카세트 염색체 (staphylococcal cassette chromosome, SCC»ec)/<9r J(open reading frame X) 정션 (junction)을 증폭하는 단일-유전자 위치 (single- locus)에 대한 실 -시간 (real time) PCR 어세이를 최초로 제안하였으며， 현재까지 5 ¾?ec/o / 정션의 검출에 기반하여 MRSA를 동정하는 상업적으로 유용한 많은 어세이 방법들이 있다 (5-7). 상술한 어세이 방법들은 스크리닝 표본들로부터 MRSA의 직접적 검출을 위해 mecA 유전자와 스타필로코커스 아우레우스 ( aureus) 특이적 유전자의 동시 검출에 기반 한 이중 유전자 위치에 대한 어세이 (double- locus assays)보다 우수한 장점을 가진다. MSS methici 11 in-sensitive S. aureus)와 The prevalence of methicillin-resistant Staphylococcus aureus (MRSA), a frequent occurrence in hospitals and community settings, is a great risk to public health worldwide. Thus, rapid and accurate detection and appropriate interventions reduce the prevalence of MRSA (1-3). Recently, a method for molecularly rapid detection of MRSA has been developed. Huletsky et al. (4) describe a single-gene locus for a single-gene locus that amplifies a staphylococcal cassette chromosome (SCC »ec) / <9r J (open reading frame X) junction. We first proposed a real time PCR assay, and to date there are many commercially available assay methods for identifying MRSA based on the detection of 5 ¾ ec / o / junction (5-7). The assay methods described above are based on the simultaneous detection of mecA and Staphylococcus aureus specific genes for the direct detection of MRSA from screening samples. Has a better advantage. MSS methici 11 in-sensitive S. aureus)
MRCoNSCmethici 11 in-resistant coagulase— negative staphylococci) 7} 섞여 있는 비강 면봉 (nasal swabs)검체 등 임상 시료에서 상기 이증 유전자 위치에 대한 어세이들은 거짓 -양성 (false positive) MRSA 검출과 관련되기 때문이다 (8). 그러나 거짓 -양성 MRSA 검출은 SCC»ec 잔여물 (remnants)을 가진 MSSA 균주들이 MRSA로 오인됨으로써 단일 유전자 위치 어세이에서도 보고되고 있다 (6， 7， 9-15) . Assays for the location of these genes in clinical samples, such as mixed nasal swabs, are associated with the detection of false-positive MRSA (8). . However, false-positive MRSA detection results in SCC »ec remnants MSSA strains have been reported in single gene location assays as being misdiagnosed as MRSA (6, 7, 9-15).
MRSA는 수년 동안 대한민국에서 만연된 풍토병이었다. 임상 표본들로부터 회수된 5. aureus 분리체들 중 쩨티실린 저항성을 나타내는 비율은 2000년대에 67.8— 74.1%에 달했다 (16). SC mec^ 염색체에 삽입되거나 또는 절단될 수 있는 모바일 엘리먼트 (mobile element)이다. 풍토성 MRSA 스트레인들로부터 SCCfflecr의 부분적 절단은 MSSA 분리체들에서 초래된다고 보고되었다 (13， 15). 따라서, 고풍토성 지역에서, MRSA의 직접 검출을 위한 단일-유전자 위치에 대한 어세이는 SCOec 잔여물을 가지는 MRSA에서 유래된 MSSA 균주 J "로 인해 거짓 -양성 결과들올 초래할 가능성이 더 높은 듯 하다. 따라서 , MRSA를 신속하고 정확하게 분석할 수 있는 기술의 개발 필요성이 당업계에서 시급히 요구되고 있는 실정이다. 본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.  MRSA has been endemic in Korea for many years. Among aureus isolates recovered from clinical specimens, the rate of methicillin resistance reached 67.8–74.1% in the 2000s (16). SC mec ^ is a mobile element that can be inserted or cleaved into the chromosome. Partial cleavage of SCCfflecr from endemic MRSA strains has been reported to result in MSSA isolates (13, 15). Thus, in highly endemic regions, assays for single-gene sites for direct detection of MRSA appear more likely to result in false-positive results due to MSSA strain J "derived from MRSA with SCOec residues. Therefore, there is an urgent need in the art for the development of a technique capable of analyzing MRSA quickly and accurately, and numerous papers and patent documents are referred to and cited throughout the present specification. The disclosures of the papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.
【발명의 내용】 [Content of invention]
【해결하려는 과제】  [Problem to solve]
¾ "명지 "fe MRSA(methici 11 in-resistant Staphylococcus aureus) % 검출 또는 정량할 수 있는 방법을 개발하고자 노력하였다. 그 결과， 본 발명자는 MRSA의 mecA, SCCmec/orfX 및 16S rRNA 유전자를 검출할 수 있는 프라이머 및 프로브를 제작하여 실 -시간 정량적 PCR을 실시함으로써 시료 (바람직하게는， 혈액, 타액 또는 소변)로부터 MRSA를 특이적이고 간단하게 검출 및 정량할 수 있음을 확인함으로써, 본 발명을 완성하게 되었다. Efforts have been made to develop a method that can detect or quantify ¾ " mpe" fe MRSA (methici 11 in-resistant Staphylococcus aureus)%. As a result, the present inventors prepared primers and probes capable of detecting mecA, SCCmec / orfX, and 16S rRNA genes of MRSA, and performed real-time quantitative PCR to prepare MRSA from a sample (preferably blood, saliva or urine). The present invention has been completed by confirming that can be specifically and simply detected and quantified.
본 발명의 목적은 MRSA(methicillin-resistant Staphylococcus /謂：)으) 검출 또는 정량방법을 제공한다.  An object of the present invention is to provide a method for detecting or quantifying MRSA (methicillin-resistant Staphylococcus / 謂 :).
본 발명의 다른 목적은 MRSA 검출 또는 진단용 키트를 제공하는 데 있다. 본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명， 청구범위 및 도면에 의해 보다 명확하게 된다. Another object of the present invention is to provide a kit for detecting or diagnosing MRSA. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
【과제의 해결 수단】 [Measures of problem]
본 발명의 일 양태에 따르면, 본 발명은 다음의 단계를 포함하는 According to one aspect of the invention, the invention comprises the following steps
MRSA(methici 11 in-resistant Staphylococcus aureus)^] 검출 또는 정량방법을 제공한다: (a) 시료를 준비하는 단계; (b) 서열목록 제 1서열 내지 서열목록 제 6서열로 구성된 군으로부터 선택된 하나 이상의 정방향 프라이머， 서열목록 제 7서열 및 서열목톡 제 8서열로 구성된 군으로부터 선택된 하나의 역방향 프라이머 및 서열목톡 제 9서열의 프로브; 서열목톡 제 10서열 및 서열목록 제 11서열의 프라이머쌍 및 서열목록 제 12서열의 프로브; 및 서열목록 제 13서열 및 서열목록 제 14서열의 프라이머쌍 및 서열목톡 제 15서열의 프로브를 이용하여 상기 시료 내의 뉴클레오타이드 서열을 증폭하는 단계; 및 (c) 상기 증폭 결과를 형광으로 확인하는 단계 . 본 발명의 다른 양태에 따르면， 본 발명은 서열목톡 제 1서열 내지 서열목톡 제 6서열로 구성된 군으로부터 선택된 하나 이상의 정방향 프라이머， 서열목록 제 7서열 및 서열목톡 제 8서열로 구성된 군으로부터 선택된 하나의 역방향 프라이머 및 서열목톡 제 9서열의 프로브; 서열목톡 제 10서열 및 서열목록 제 11서열의 프라이머쌍 및 서열목록 제 12서열의 프로브; 및 서열목톡 제 13서열 및 서열목록 제 14서열의 프라이머쌍 및 서열목록 제 15서열의 프로브를 포함하는 MRSA(methiciIlin-resistant Staphylococcus aureus) 검출 또는 진단용 키트를 제공한다. Methods for detecting or quantifying MRSA (methici 11 in-resistant Staphylococcus aureus) ^] include: (a) preparing a sample; (b) at least one forward primer selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO. Probes; Primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 and probes of SEQ ID NO: 12; And amplifying the nucleotide sequence in the sample using the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 and the probe of SEQ ID NO: 15; And (c) confirming the amplification result by fluorescence. According to another aspect of the invention, the present invention is one or more forward primers selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6 sequence, one selected from the group consisting of SEQ ID NO: 7 sequence and SEQ ID NO: 8 sequence A reverse primer and a probe of SEQ ID NO: 9; Primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 and probes of SEQ ID NO: 12; And it provides a kit for detecting or diagnosing MRSA (methiciIlin-resistant Staphylococcus aureus) comprising a primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 and a probe of SEQ ID NO: 15.
^r^^lfe- MRSA(methi c i 11 in-res i st ant Staphylococcus aureus)^ 검출 또는 정량할 수 있는 방법을 개발하고자 노력하였다. 그 결과, 본 발명자는 MRSA의 mecA, SCCmec/orfX 및 16S rRNA 유전자를 검출할 수 있는 프라이머 및 프로브를 제작하여 실 -시간 정량적 PCR을 실시함으로써 시료 (바람직하게는， 혈액, 타액 또는 소변)로부터 MRSA를 특이적이고 간단하게 검출 및 정량할 수 있음을 확인하였다. Efforts have been made to develop methods to detect or quantify ^ r ^^ lfe- MRSA (methi c 11 in-res i st ant Staphylococcus aureus) ^. As a result, the present inventors prepared primers and probes capable of detecting mecA, SCCmec / orfX and 16S rRNA genes of MRSA, and carried out real-time quantitative PCR to perform MRSA from a sample (preferably blood, saliva or urine). It was confirmed that can be detected and quantified specifically and simply.
본 발명에 따르면, 본 발명은 풍토병인 MRSA를 매우 효과적이고 간편하게 검출할 수 있다. 본 발명의 바람직한 구현예에 따르면， 본 발명의 증폭은According to the present invention, the present invention can very effectively and simply detect the endemic MRSA. According to a preferred embodiment of the invention, the amplification of the invention
PCR(polymerase chain react ion)에 따라 실시된다. 본 발명의 바람직한 구현예에 따르면, 본 발명의 프라이머는 유전자 증폭 반웅 (amplification react ions)에 이용된다. It is carried out according to polymerase chain react ion (PCR). According to a preferred embodiment of the present invention, the primer of the present invention is used for amplification react ions.
본 명세서에 기재된 용어"증폭 반웅" 은 핵산 분자를 증폭하는 반웅을 의미한다. 다양한 증폭 반웅들이 당업계에 보고 되어 있으며, 이는 중합효소 연쇄반웅 (PCR) (미국 특허 제 4,683, 195, 4,683,202, 및 4,800， 159호)， 역전사-중합효소 연쇄반웅 (RT-PCR)(Sambrook 등， Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spring Harbor Press(2001)) , Miller, H. I. (W0 89/06700) 및 Davey, C. 등 (EP 329, 822)의 방법， 멀티플렉스 PCR(McPherson and Moller, 2000)， 리가아제 연쇄 반응 (ligase chain reaction; LCR), Gap-LCR(W0 90/01069), 복구 연쇄 반웅 (repair chain reaction; EP 439, 182) , 전사一중재 증폭 (transcription-mediated amplification; TMA) (WO 88/10315), 자가 유지 염기서열 복제 (self sustained sequence replication) (W0 90/06995) , 타깃 폴리뉴클레오티드 염기서열의 선택적 증폭 (selective amplification of target polynucleotide sequences) (미국 특허 제 6，410，276호), 컨센서스 서열 프라이밍 중합효소 연쇄 반웅 (consensus sequence primed polymerase chain reaction; CP-PCR) (미국 특허 제 4,437,975호), 임의적 프라이밍 중합효소 연쇄 .반웅 (arbitrari ly primed polymerase chain reaction; AP— PCR) (미국 특허 제 5,413,909호 및 제 5,861, 245호), 핵산 염기서열 기반 증폭 (nucleic acid sequence based amplification; NASBA) (미국 특허 제 5,130, 238호, 제 5，409ᅳ 818호, 제 5, 554,517호, 및 제 6,063, 603호)， 가닥 치환 증폭 (strand dis lacement amplification) 및 고리—증재 항온성 증폭 (loop— mediated isothermal amplification; LAMP)를 포함하나， 이에 한정되지는 않는다. 사용 가능한 다른 증폭 방법들은 미국특허 제 5, 242,794호, 계 5, 494, 810호， 제 4 ,988， 617호 및 미국 특허 제 09/854 ,317호에 기술되어 있다.  The term "amplification reaction" as described herein means a reaction that amplifies a nucleic acid molecule. Various amplification reactions are reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683, 195, 4,683,202, and 4,800, 159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al. , Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (W0 89/06700) and Davey, C. et al. (EP 329, 822), multiplex PCR (McPherson and Moller, 2000), ligase chain reaction (LCR), Gap-LCR (W0 90/01069), repair chain reaction (EP 439, 182), transcription-mediated amplification TMA (WO 88/10315), self sustained sequence replication (W0 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6, 410，276), consensus sequence primed polymerase chain reaction (CP-PCR) (US Pat. No. 4,437,975), arbitrarily ly primed polymerase chain reaction (AP—PCR) (US Pat. Nos. 5,413,909 and 5,861,245), nucleic acids Nucleic acid sequence based amplification (NASBA) (US Pat. Nos. 5,130, 238, 5,409 ᅳ 818, 5, 554,517, and 6,063, 603), strand displacement amplification (strand dis) lacement amplification and loop—mediated isothermal amplification; LAMP), but is not limited thereto. Other amplification methods that can be used are described in US Pat. Nos. 5, 242,794, 5, 494, 810, 4,988, 617, and US Pat. No. 09 / 854,317.
본 명세서에서 사용되는 용어 "프라이머" 는 올리고뉴클레오타이드를 의미하는 것으로， 핵산쇄 (주형)에 상보적인 프라이머 연장 산물의 합성이 유도되는 조건， 즉， 뉴클레오타이드와 DNA 중합효소와 같은 중합제의 존재ᅳ 그리고 적합한 온도와 pH의 조건에서 합성의 개시점으로 작용할 수 있다. 바람직하게는， 프라이머는 디옥시리보뉴클레오타이드이며 단일쇄이다. 본 발명에서 이용되는 프라이머는 자연 (naturally occurring) d應 P (즉, dAMP, dGMP, dCMP 및 dTMP), 변형 뉴클레오타이드 또는 비 -자연 뉴클레오타이드를 포함할 수 있다. 또한， 프라이머는 리보뉴클레오타이드도 포함할 수 있다. As used herein, the term "primer" refers to an oligonucleotide, wherein the conditions under which the synthesis of a primer extension product complementary to the nucleic acid chain (template) is induced, i.e., the presence of a polymerizer such as nucleotide and DNA polymerase and At conditions of suitable temperature and pH It can serve as a starting point for synthesis. Preferably, the primer is deoxyribonucleotide and single chain. Primers used in the present invention may include naturally occurring dVP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also include ribonucleotides.
프라이머는, 증합제의 존재 하에서 연장 산물의 합성을 프라이밍시킬 수 있을 정도로 층분히 길어야 한다. 프라이머의 적합한 길이는 다수의 요소, 예컨대, 은도, 웅용분야 및 프라이머의 소스 (source)에 따라 결정된다. 용어 "어닐링" 또는 "프라이밍" 은 주형 핵산에 올리고디옥시뉴클레오타이드 또는 핵산이 병치 (apposition)되는 것을 의미하며， 상기 병치는 중합효소가 뉴클레오타이드를 중합시켜 주형 핵산 또는 그의 일부분에 상보적인 핵산 분자를 형성하게 한다.  The primer should be long enough to prime the synthesis of the extension product in the presence of the thickener. Suitable lengths of the primers depend on a number of factors, such as silver, utility and source of the primer. The term "annealing" or "priming" refers to the placement of oligodioxynucleotides or nucleic acids in a template nucleic acid, wherein the polymerase polymerizes the nucleotides to form a nucleic acid molecule that is complementary to the template nucleic acid or portion thereof. Let's do it.
PCR은 가장 잘 알려진 핵산 증폭 방법으로, 그의 많은 변형과 웅용들이 개발되어 있다. 예를 들어， PCR의 특이성 또는 민감성을 증진시키기 위해 전통적인 PCR 절차를 변형시켜 터치다운 (touchdown) PCR, 핫 스타트 (hot start) PCR, 네스티드 (nested) PCR 및 부스터 (booster) PCR이 개발되었다. 또한， 멀티플렉스 PCR, 실 -시간 (real-time) PCR, 분별 디스풀레이 PCR(differential display PCR: DD-PCR) , cDNA 말단의 신속 증폭 (rapid amplification of cDNA ends: RACE), 인버스 중합효소 연쇄반웅 (inverse polymerase chain reaction:. IPCR) , 백토레트 (vectorette) PCR 및 TAIL-PC (thermal asymmetric interlaced PCR)이 특정한 웅용을 위해 개발되었다. PCR에 대한 자세한 내용은 McPherson, M.J. , 및 Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000)에 기재되어 있으몌 그의 교시사항은 본 명세서에 참조로 삽입된다.  PCR is the most well-known method of nucleic acid amplification, and many modifications and uses have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, multiplex PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chains Inverse polymerase chain reaction (IPCR), vectorette PCR and TAIL-PC (thermal asymmetric interlaced PCR) have been developed for specific use. For more information on PCR, see McPherson, M.J. , And Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. The teachings of which are described in (2000) are incorporated herein by reference.
본 발명의 방법을 프라이머를 이용하여 실시하는 경우에는, 유전자 증폭 반응을 실시하여 분석 대상 (예컨대, 타겟 미생물을 포함하는 시료)에서 타겟 유전자들을 동시에 검출할 수 있다. 따라서， 본 발명은 시료 내의 미생물에서 분리된 DNA에 결합하는 프라이머를 이용하여 유전자 증폭 반응을 실시한다. 본 발명에 이용되는 프라미머는 주형의 한 부위에 흔성화 또는 어닐링되어， 이중쇄 구조를 형성한다. 이러한 이중쇄 구조를 형성하는 데 적합한 핵산 흔성화의 조건은 Joseph Sambrook, 등， Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N.Y.(2001) 및 Haymes, B. D.， 등, Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985)에 개시되어 있다. 다양한 DNA 중합효소가 본 발명의 증폭에 이용될 수 있으며, E. coli DNA 중합효소 I의 "클레나우" 단편， 열안정성 DNA 중합효소 및 박테리오파아지 T7 DNA 중합효소를 포함한다. 바람직하게는， 중합효소는 다양한 박테리아 종으로부터 얻을 수 있는 열안정성 DNA 중합효소이고， 이는 Ther us aquat i cusi z^) , Thermus thermophi lusi i ) , Thermus filiformis, Therm is flavus, Ther ococcus literal is, 및 Pyrococcus //osi/s(Pfu)를 포함한다. When the method of the present invention is carried out using a primer, a gene amplification reaction may be performed to simultaneously detect target genes in an analyte (eg, a sample containing a target microorganism). Therefore, the present invention performs a gene amplification reaction using a primer that binds to DNA isolated from the microorganism in the sample. Primers used in the present invention are either localized or annealed to one site of the template to form a double chain structure. Conditions for nucleic acid localization suitable for forming such double-stranded structures include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Various DNA polymerases can be used for amplification of the present invention and include "Clenau" fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which include Ther us aquat i cusi z ^), Thermus thermophi lusi i), Thermus filiformis, Therm is flavus, Ther ococcus literal is, and Pyrococcus // contains osi / s (Pfu)
중합 반웅을 실시할 때, 반응 용기에 반응에 필요한 성분들을 과량으로 제공하는 것이 바람직하다. 증폭 반응에 필요한 성분들의 과량은， 증폭반웅이 성분의 농도에 실질적으로 제한되지 않는 정도의 양을 의미한다. Mg^와 같은 조인자, dATP, dCTP, dGTP 및 dTTP를 소망하는 증폭 정도가 달성될 수 있을 정도로 반웅 흔합물에 제공하는 것이 소망된다. 증폭 반웅에 이용되는 모든 효소들은 동일한반웅 조건에서 활성 상태일 수 있다. 사실, 완층액은 모든 효소들이 최적의 반웅 조건에 근접하도록 한다. 따라서， 본 발명의 증폭 과정은 반웅물의 첨가와 같은 조건의 변화 없이 단일 반응물에서 실시될 수 있다.  When carrying out the polymerization reaction, it is preferable to provide the reaction vessel with an excess of components necessary for the reaction. Excess of components necessary for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. It is desired to provide cofactors such as Mg ^, dATP, dCTP, dGTP and dTTP to the reaction mixture such that the desired degree of amplification can be achieved. All enzymes used for the amplification reaction may be active under the same reaction conditions. In fact, the supernatant ensures that all enzymes are close to the optimum reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as the addition of the counter agitate.
본 발명에 있어서 어닐링은 타겟 뉴클레오타이드 서열과 프라이머 사이에 특이적 결합을 가능하게 하는 엄격조건 하에서 실시된다. 어닐링을 위한 엄격조건은 서열 -의존적이며 주위 환경적 변수에 따라 다양하다.  Annealing in the present invention is carried out under stringent conditions allowing specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary with ambient environmental variables.
이렇게 증폭된 타겟 유전자 (바람직하게는， mecA, SCCmec/orfX 및 16S rRNA)를 적합한 방법으로 분석하여 MRSA를 검출하는 것이다. 예를 들어, 상술한 증폭 반웅 결과물을 젤 전기영동을 하고， 그 결과 형성되는 밴드를 관찰 및 분석함으로써 타겟 유전자를 검출할 수 있다.  The amplified target genes (preferably mecA, SCCmec / orfX and 16S rRNA) are analyzed by a suitable method to detect MRSA. For example, the target gene can be detected by performing gel electrophoresis on the amplified reaction product described above, and observing and analyzing the band formed as a result.
따라서 본 발명의 방법을 미생물의 DNA를 이용하는 증폭반웅에 기초하여 실시하는 경우에는, 구체적으로 (i) SCCy7;ecA? / 뉴클레오타이드 서열에 어닐링되는 프라이머 및 프로브; mecA 뉴클레오타이드 서열에 어닐링되는 프라이머 및 프로브; 또는 16S rRNA 뉴클레오타이드 서열에 어닐링되는 프라이머를 이용하여 증폭 반웅을 실시하는 단계; 및 (Π) 상기 증폭 반웅의 산물을 형광을 통해 분석하는 단계를 포함하며 이를 통해 시료로부터 추출한 DNA에서 MRSA를 검출또는 정량할 수 있다. Therefore, the method of the present invention is applied to the amplification reaction using the DNA of the microorganism. When implementing on the basis of specific, specifically, (i) SCCy7; ecA? Primers and probes annealed to the nucleotide sequence; primers and probes annealed to the mecA nucleotide sequence; Or performing amplification reactions using primers annealed to 16S rRNA nucleotide sequences; And (Π) analyzing the product of the amplification reaction through fluorescence, through which MRSA can be detected or quantified in the DNA extracted from the sample.
본 발명의 바람직한 구현예에 따르면， 본 발명의 방법에 의해 검출될 수 있는 MRSA 스트레인은 CCARM 3792, CCARM 3795, CCARM 3798, CCARM 3803, CCARM 3805, CCARM 3877, CCARM 3897 및 CCARM 3911을 포함하지만, 이에 한정되는 것^ 아니다.  According to a preferred embodiment of the invention, MRSA strains that can be detected by the methods of the invention include, but are not limited to, CCARM 3792, CCARM 3795, CCARM 3798, CCARM 3803, CCARM 3805, CCARM 3877, CCARM 3897 and CCARM 3911 It is not limited ^
본 명세서 용어 "흔성화 (hybridization)" 는 2개의 단일 가닥 핵산이 상보적인 염기 서열들의 페어링 (pairing)에 의하여 이합체 구조 (duplex structure)를 형성하는 것을 의미한다. 혼성화는 단일 가닥 핵산 서열 간의 상보성이 완전할 경우 (per feet match) 일어나거나 일부 미스매치 (mismatch) 염기가 존재하여도 일어날 수 있다. 흔성화에 필요한 상보성의 정도는 흔성화 반웅 조건에 따라 달라질 수 있으며， 특히 온도에 의하여 조절될 수 있다. ? 용어 "어닐링" 과 "흔성화" 는 차이가 없으며， 본 명세서에서 흔용된다. As used herein, the term "hybridization" means that two single stranded nucleic acids form a duplex structure by pairing complementary base sequences. Hybridization can occur when the complete complement between single stranded nucleic acid sequences (per feet match) or even when some mismatch base is present. The degree of complementarity required for the shake may vary depending on the shake reaction conditions, and in particular, may be controlled by temperature. ? The terms "annealing" and "animation" do not differ and are commonly used herein.
본 발명의 바람직한 구현예에 따르면， 본 발명의 방법 및 키트는 MRSA를 다른 미생물들과 구분하기 위해 3개의 유전자 0»eo4, SCOec/ or/ 및 16S rRNA)를 동시에 검출하는 멀티플렉스 실 -시간 PCR을 통해 검출할 수 있다.  According to a preferred embodiment of the present invention, the method and kit of the present invention is a multiplex real-time PCR that simultaneously detects three genes 0 »eo4, SCOec / or / and 16S rRNA to distinguish MRSA from other microorganisms. Can be detected through.
본 발명의 바람직한 구현예에 따르면, 본 발명의 방법 및 키트는 서열목록 제 1서열 내지 서열목록 제 6서열로 구성된 군으로부터 선택된 하나 이상의 정방향 프라이머， 서열목톡 제 7서열 및 서열목록 제 8서열로 구성된 군으로부터 선택된 하나의 역방향 프라이머 및 서열목록 제 9서열의 프로브; 서열목록 제 10서열 및 서열목록 제 11서열의 프라이머쌍 및 서열목록 제 12서열의 프로브; 및 서열목록 제 13서열 및 서열목록 제 14서열의 프라이머쌍 및 서열목록 제 15서열의 프로브를 포함한다.  According to a preferred embodiment of the present invention, the method and kit of the present invention consists of at least one forward primer selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 One reverse primer selected from the group and a probe of SEQ ID NO: 9; Primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 and probes of SEQ ID NO: 12; And a primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 and a probe of SEQ ID NO: 15.
본 발명의 보다 바람직한 구현예에 따르면， 본 발명의 방법 및 키트에서 이용되는 타겟 유전자는 서열목록 게 1서열 내지 서열목록 제 6서열로 구성된 군으로부터 선택된 하나 이상와 정방향 프라이머， 서열목톡 제 7서열 및 서열목톡 계 8서열로 구성된 군으로부터 선택된 하나의 역방향 프라이머 및 서열목록 제 9서열의 프로브에 의해 검출되는 SCCmec/orfX 유전자; 서열목록 제 10서열 및 서열목록 제 11서열의 프라이머쌍 및 서열목록 제 12서열의 프로브에 의해 검출되는 mecA 유전자; 및 서열목록 제 13서열 및 서열목록 제 14서열의 프라이머쌍 및 서열목록 제 15서열의 프로브에 의해 검출되는 16S rRNA유전자를 포함한다. According to a more preferred embodiment of the present invention, the target gene used in the method and kit of the present invention is SEQ ID NO: 1 to SEQ ID NO: An SCCmec / orfX gene detected by at least one forward primer selected from the group consisting of the sixth sequence, one reverse primer selected from the group consisting of SEQ ID NO: 7 sequence, and sequence 8 sequence; A mecA gene detected by a primer pair of SEQ ID NO: 10 and SEQ ID NO: 11 and a probe of SEQ ID NO: 12; And 16S rRNA genes detected by primer pairs of SEQ ID NO: 13 and SEQ ID NO: 14 and probes of SEQ ID NO: 15.
실 -시간 PCR은 PCR 증폭 산물의 증가를 실-시간으로 모니터링하여 분석하는 기술이다 (Levak KJ, et a\.,PCR Methods Appl. , 4(6)： 357-62(1995)). PCR생산물의 증가가타겟 템플레이트의 초기 양과비례하는지수기 (exponential phase) 동안 각사이클에서 형광 방출량을 기특하여 PCR 반웅을 모니터링할 수 있다. 핵산 타겟의 출발 카피 수가높을수록， 형광 증가가 더 빨리 관찰되고 더 낮은 값 (threshold cycle)을가지게 된다. 3-15사이클사이에서 측정된 기준값보다 높은 형광의 뚜렷한 증가는 축적된 PCR 생산물의 검출을 의미한다. 종래의 PCR방법에 비해， 실 -시간 PCR은다음과같은장점을가진다: (a) 종래의 PCR은 정체 상태 (plateau)에서 측정되는 반면에， 실 -시간 PCR은 지수성장기 (exponential growth phase) 동안 데이터를 얻을 수 있다; (b) 리포터 형광 시그널의 증가는 발생된 앰플리콘 (amp Π cons)의 수와 직접적으로 비례한다; (c) 분해된 프로브는 앰플리콘의 영구적인 기록 증폭 (record amplification)을쎄공한다; (d) 검출 범위의 증가; (e) 종래 PCR 방법에 비해 1,000배 이상 적은 핵산을 필요로 한다; (f) 전기영동을통한분리 없이 증폭된 DNA의 검출이 가능하다; (g) 작은 앰플리콘크기를이용하여 증가된 증폭효을을 획득할수 있다; 및 (h) 오염 위험성이 적다.  Real-time PCR is a technique for monitoring and analyzing the increase of PCR amplification products in real time (Levak KJ, et a \., PCR Methods Appl., 4 (6): 357-62 (1995)). PCR reactions can be monitored by characterizing the amount of fluorescence emission in each cycle during the exponential phase, if the increase in PCR product is proportional to the initial amount of the target template. The higher the starting copy number of the nucleic acid target, the faster the fluorescence increase is observed and the lower the threshold cycle. A marked increase in fluorescence above the reference value measured between 3-15 cycles means detection of accumulated PCR product. Compared to conventional PCR methods, real-time PCR has the following advantages: (a) Conventional PCR is measured in the plateau, whereas real-time PCR is used during the exponential growth phase. Data can be obtained; (b) the increase in the reporter fluorescence signal is directly proportional to the number of amp π cons generated; (c) the cleaved probe provides permanent record amplification of the amplicon; (d) increase the detection range; (e) requires at least 1,000 times less nucleic acid than conventional PCR methods; (f) detection of amplified DNA without separation via electrophoresis is possible; (g) small amplicon sizes can be used to obtain increased amplification effects; And (h) the risk of contamination is low.
PCR 증폭 산물량은 형광으로 검출 가능한 양에 도달하면 증폭곡선이 일어나기 시작해 지수적으로 시그널이 상승하다가 정체 상태에 도달한다. 초기 DNA량이 많을수록 증폭 산물량이 검출 가능한 양에 달하는 사이클 수가 적어지므로 증폭곡선이 빨리 나타난다. 따라서， 단계적으로 회석한 표준시료를 사용하여 실 -시간 PCR 반응을 하면 초기 DNA량이 많은 순서로 같은 간격으로 늘어선 증폭 곡선이 얻어진다. 여기서 적당한 지점에 한계치 (threshold)를 설정하면 한계치와 증폭 곡선이 교차하는 지점 Cf 값이 산출된다. 실 -시간 PCR에서는 PCR 증폭 산물을 형광을 통해 검출한다. 검출 방법은 크게 interchelating 방법 (SYBR 그린 I 방법), 형광 표지 프로브를 이용하는 방법 (TaqMan 프로브 방법) 등이 있다. interchelating 방법은 이중 가닥 DNA를 모두 검출하기 때문에 유전자별 프로브를 준비할 필요하 없어 저렴한 비용으로 반웅계를 구축할 수 있다. 형광 표지 프로브를 이용하는 방법은 고비용이 드는 반면에 검출 특이성이 높아 유사 서열까지도 구별해서 검출할 수 있다. When the amount of PCR amplification products reaches a detectable amount by fluorescence, an amplification curve begins to occur, and the signal rises exponentially and reaches a steady state. The larger the initial DNA amount, the fewer cycles the amount of amplification products can detect, resulting in faster amplification curves. Therefore, a real-time PCR reaction using a step-diluted standard sample yields amplification curves arranged at equal intervals in order of increasing initial DNA content. Setting a threshold at an appropriate point here yields the point C f at which the threshold intersects the amplification curve. In real-time PCR, PCR amplification products are detected by fluorescence. Detection methods include interchelating methods (SYBR Green I method) and fluorescent labeling probes (TaqMan probe method). The interchelating method detects both double-stranded DNA, so there is no need to prepare gene-specific probes, so the counterunggy system can be constructed at low cost. While the method using the fluorescent labeling probe is expensive, the detection specificity is high and even similar sequences can be detected.
먼저， interchelating 방법은 이중 가닥 DNA 결합 다이를 이용하는 방법으로, 비 -서열 특이적 형광 intercalating 시약 (SYBR 그린 I 또는 ethidium bromide)을 이용하여 비-특이적 증폭 및 프라이머-다이머 복합체를 포함하는 앰풀리콘 생산을 정량하는 것이다. 상기 시약은 ssDNA와는 결합하지 않는다. SYBR 그린 I은 이중 가닥 DNA의 마이너 그루브 (minor groove)에 결합하는 형광성 다이로， 용액 상에서는 거의 형광을 보이지 않지만 이중 가닥 DNA와 결합하면 강한 형광을 나타내는 시약 (interchelator)이다 (Morrison TB, Biotechniques. , 24(6)： 954-8, 960, 962(1998)). 따라서 SYBR 그린 I과 이중 가닥 DNA 간의 결합을 통해 형광을 방출하기 때문에 증폭 산물의 생성량을 측정할 수 있다. SYBR 그린 실 -시간 PCR은 앰플리콘 동정을 위해 융해점 (melting point) 또는 해리 곡선 (dissociation curve) 분석과 같은 최적화 과정을 동반한다. 정상적으로 SYBR 그린은 싱글플렉스 (singleplex) 반응에 이용되지만, 융해곡선 (melting curve) 분석이 동반되면 멀티플렉스 (multiplex) 반응에 이용될 수 있다 (Siraj AK, et al.Xlin Cancer Res. , 8(12)： 3832- 40(2002)； 및 Vrettou C. , et al., Hum Mutat., Vol 23(5)： 513-521(2004)).  First, the interchelating method uses a double-stranded DNA-binding die, and uses a non-sequence specific fluorescent intercalating reagent (SYBR Green I or ethidium bromide) to produce an amplicon including non-specific amplification and primer-dimer complexes. To quantify. The reagent does not bind to ssDNA. SYBR Green I is a fluorescent die that binds to the minor groove of double-stranded DNA and is a reagent that shows little fluorescence in solution but shows strong fluorescence when combined with double-stranded DNA (Morrison TB, Biotechniques., 24 (6): 954-8, 960, 962 (1998). Therefore, fluorescence is emitted through the binding between SYBR Green I and double-stranded DNA, and thus the amount of amplified products can be measured. SYBR Green Real-Time PCR is accompanied by optimization procedures such as melting point or dissociation curve analysis for amplicon identification. Normally, SYBR green is used for singleplex reactions, but it can be used for multiplex reactions when accompanied by melting curve analysis (Siraj AK, et al. Xlin Cancer Res., 8 (12). ): 3832-40 (2002); and Vrettou C., et al., Hum Mutat., Vol 23 (5): 513-521 (2004)).
Cf(threshold cycle) 값은 반응에서 발생된 형광이 역치 (threshold)를 넘어서는 사이클 수를 의미하며， 이는 초기 카피 수의 대수에 반비례한다. 그러므로, 특정 웰에 할당된 값은 반웅에서 앰플리콘의 층분한 수가 축적된 사이클의 수를 반영한다. t 값은 Δϋη의 증가가 처음으로 검출된 사이클이다. Rn은 각 시점에서 PCR 동안 발생된 형광 시그널의 크기를 의미하며， ARn은 레퍼런스 다이의 형광 방출 강도로 나위어진 리포터 다이의 형광방출 강도 (표준화된 리포터 시그널)를 의미한다. 값은 LightCycler에서는 Cp(crossing point)로도 명명된다. Ct 값은 시스템이 로그 -선형 단계 (log-linear phase)에서 PCR 생산물의 지수성장과 관련된 형광 시그널의 증가를 검출하기 시작하는 시점을 나타낸다. 아 시기는 반웅에 대한 가장 유용한 정보를 제공한다. 로그- 선형 단계의 기을기는 증폭 효율 (amplification efficiency, Eff)을 나타낸다 (http://冒 w.appl iedbiosys terns, co.kr/) . The threshold cycle (C f ) value refers to the number of cycles in which the fluorescence generated in the reaction exceeds the threshold, which is inversely proportional to the logarithm of the initial copy number. Therefore, the value assigned to a particular well reflects the number of cycles in which the stratified number of amplicons in the reaction accumulates. The t value is the cycle in which the increase in Δϋη was first detected. Rn refers to the magnitude of the fluorescence signal generated during PCR at each time point, and ARn refers to the fluorescence emission intensity (standardized reporter signal) of the reporter die divided by the fluorescence emission intensity of the reference die. The value is also named Cp (crossing point) in LightCycler. The C t value represents the point in time when the system begins to detect an increase in the fluorescence signal associated with the exponential growth of the PCR product in a log-linear phase. The period provides the most useful information about reaction. The log-linear phase creeping represents the amplification efficiency (Eff) (http: // www.appl iedbiosysterns, com /).
한편， TaqMan 프로브는 전형적으로 5' -말단에 형광물질 ( uorophore) 및 3' -말단에 뭔처 (quencher; 예컨대, TAMRA 또는 비 -형광 뭔처 (NFQ))를 포함하는 프라이머 (예컨대 , 2으 30 뉴클레오타이드) 보다 더 긴 을리고뉴클레오타이드이다. 여기된 형광물질은 형광올 내기 보다는 근처의 퀀처에 에너지를 전달한다 (FRET = Forster or fluorescence resonance energy transfer； Chen, X. , et al . ,Proc Natl Acad Sci USA, 94(20)： 10756-61(1997)). 그러므로， 프로브가 정상인 경우， 어떠한 형광도 발생되지 않는다. TaqMan 프로브는 PCR 생산물의 내부 부위에 어닐링할 수 있도톡 고안된다. 바람직하게는, TaqMan 프로브는 서열목록 제 13서열 및 제 14서열에 의해 증폭되는 16S rRNA 유전자 절편의 내부서열로 고안될 수 있다.  TaqMan probes, on the other hand, typically contain primers (eg, 2-30 nucleotides) comprising a 5'- uorophore and a 3'-quencher (e.g., TAMRA or non-fluorescent something (NFQ)). It is longer than l) nucleotides. The excited fluorescent material transfers energy to nearby quencher rather than to fluorescence (FRET = Forster or fluorescence resonance energy transfer; Chen, X., et al., Proc Natl Acad Sci USA, 94 (20): 10756-61 (1997)). Therefore, no fluorescence is generated when the probe is normal. TaqMan probes are designed to anneal to internal sites of PCR products. Preferably, the TaqMan probe may be designed as an internal sequence of a 16S rRNA gene segment that is amplified by SEQ ID NO: 13 and 14.
TaqMan 프로브는 어닐링 단계에서 템풀레이트 DNA에 특이적으로 흔성화하지만, 프로브 상에 ¾처에 의해 형광 발색이 억제된다. 연장 반웅 시에 TaQ DNA 폴리머라제가 갖는 5' to 3' 뉴클레아제 활성에 의해 템플레이트에 흔성화한 TaqMan 프로브가 분해되어 형광 색소가 프로브로부터 유리되면서 퀀처에 의한 억제가 해제되어 형광은 나타낸다. 이 때, TaqMan 프로브의 5' -말단은 상기 연장 프라이머의 3' -말단의 다운스트림에 위치하여야 한다. 즉， 연장 프라이머의 3' -말단이 주형- 의존성 핵산 중합효소에 의해 연장되는 경우, 이 중합효소의 5' to 3' 뉴클레아제 활성에 의해 TaqMan 프로브의 5' -말단이 절단되어 리포터 분자의 형광 시그널이 발생하게 된다.  TaqMan probes specifically specific for templatated DNA in the annealing step, but fluorescence is inhibited by ¾ on the probe. Upon prolonged reaction, TaqMan probes that were localized in the template were degraded by the 5 'to 3' nuclease activity of TaQ DNA polymerase, and the fluorescent dye was released from the probe, thereby suppressing the inhibition by the quencher, indicating fluorescence. At this time, the 5'-end of the TaqMan probe should be located downstream of the 3'-end of the extension primer. That is, when the 3'-end of the extension primer is extended by the template-dependent nucleic acid polymerase, the 5'-end of the TaqMan probe is cleaved by the 5 'to 3' nuclease activity of the polymerase to The fluorescent signal is generated.
TaqMan 프로브에 결합되어 있는 상기 리포터 분자 및 뭔처 분자는 모두 형광성 물질이다. 본 발명에 이용될 수 있는 형광성 리포터 분자 및 퀀처 분자는 당업계에 공지되어 있는 어떠한 것도 이용할 수 있으며， 그 예는 다음과 같다 (괄호의 숫자는 나노미터 단위로 표시한 발광 최대 파장이다): Cy2™ (506)， Y0-PR0™-! (509), Y0Y0™- 1 (509)， Calcein (517), FITC (518) FluorX™ (519), Alexa™(520), Rhodamine 110 (520), 5-FAM (522)， Oregon Green™ 500 (522) , Oregon Green™ 488 (524), RiboGreen™ (525), Rhodamine Green™ (527) , Rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™ (533), T0-PR0™-1 (533), T0T01 (533), JOE (548)， B0DIPY530/550 (550), Dil (565), B0DIPY TMR (568)， B0DIPY558/568 (568), B0DIPY564/570 (570), Cy3™ (570)， Alexa™ 546 (570)， TRITC (572) , Magnesium Orange™ (575) , Phycoerythrin R&B (575)， Rhodamine Phal loidin (575) , Calcium Orange™ (576) , Pyronin Y (580), Rhodamine B (580), TAMRA (582)， Rhodamine Red™ (590), Cy3.5™ (596), R0X (608) , Calcium Crimson™ (615) , Alexa™ 594 ( 615)， Texas Red(615), Nile Red (628) , Y0- PR0™-3 (631), Y0Y0™— 3 (631)， R-phycocyanin (642), C-Phycocyanin (648) , T0-PR0™-3 (660), T0T03 (660), DiD DilC(5) (665), Cy5™ (670), Thiadicarbocyanine (671), Cy5.5(694), HEX (556), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544) , CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637) , FAM (520) , Fluorescein (520)， Fluorescein— C3 (520)， Pulsar 650 (566) , Quasar 570 (667) , Quasar 670 (705) 및 Quasar 705 (610). 괄호의 숫자는 나노미터 단위로 표시한 발광 최대 파장이다. 바람직하게는, 리포터 분자 및 뭔처 분자는 HEX, FAM및 Cy5.5ᅳ기반 표지를 포함한다. Both the reporter molecule and the molecule somewhere bound to the TaqMan probe are fluorescent materials. Fluorescent reporter molecules and quencher molecules that can be used in the present invention can be any known in the art, for example: (The number in parentheses is the maximum emission wavelength in nanometers): Cy2 ™ 506, Y0-PR0 ™-! (509), Y0Y0 ™ -1 (509), Calcein (517), FITC (518) FluorX ™ (519), Alexa ™ (520), Rhodamine 110 (520), 5-FAM (522), Oregon Green ™ 500 (522), Oregon Green ™ 488 (524), RiboGreen ™ (525), Rhodamine Green ™ (527), Rhodamine 123 (529), Magnesium Green ™ (531), Calcium Green ™ (533), T0-PR0 ™ -1 (533), T0T01 (533), JOE (548), B0DIPY530 / 550 (550) , Dil (565), B0DIPY TMR (568), B0DIPY558 / 568 (568), B0DIPY564 / 570 (570), Cy3 ™ (570), Alexa ™ 546 (570), TRITC (572), Magnesium Orange ™ (575) , Phycoerythrin R & B (575), Rhodamine Phal loidin (575), Calcium Orange ™ (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red ™ (590), Cy3.5 ™ ( 596), R0X (608), Calcium Crimson ™ (615), Alexa ™ 594 (615), Texas Red (615), Nile Red (628), Y0- PR0 ™ -3 (631), Y0Y0 ™ — 3 (631 ), R-phycocyanin (642), C-Phycocyanin (648), T0-PR0 ™ -3 (660), T0T03 (660), DiD DilC (5) (665), Cy5 ™ (670), Thiadicarbocyanine (671) , Cy5.5 (694), HEX (556), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Or ange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM 520, Fluorescein (520), Fluorescein— C3 (520), Pulsar 650 ( 566), Quasar 570 (667), Quasar 670 (705) and Quasar 705 (610). The numbers in parentheses are the maximum wavelengths emitted in nanometers. Preferably, the reporter molecule and something molecule comprises HEX, FAM and Cy5.5 ′ based labels.
본 발명에서, 광범위 파장 또는 특정 파장의 형광을 ¾칭 할 수 있는 비 -형광 블랙 퀀처 분자가 이용될 수 있다는 것은 주목할 만하다. 비-형광 블랙 ¾처 분자의 예는 BHQ 및 DABCYL를 포함하지만, 이에 한정되는 것은 아니며, 가장 바람직하게는 BHQ1 및 BHQ2이다.  In the present invention, it is noteworthy that non-fluorescent black quencher molecules can be used which can fluoresce a wide range of wavelengths or a particular wavelength. Examples of non-fluorescent black quarter molecules include, but are not limited to, BHQ and DABCYL, most preferably BHQ1 and BHQ2.
본 발명의 바람직한 구현예에 따르면, 본 발명의 프로브에서 이용되는 리포터 -퀀처 쌍은 HEX, FAM, Cy5.5, BHQ1 및 BHQ2—기반 표지를 포함한다. 본 발명의 보다 바람직한 구현예에 따르면, 본 발명의 서열목록 제 9서열은 5' -말단의 형광물질로 HEX 및 3' -말단에 퀀처로 BHQ1을 이용하고， 본 발명의 서열목록 제 12서열은 5' -말단의 형광물질로 FAM 및 3' -말단에 뭔처로 BHQ1을 이용하며, 본 발명의 서열목톡 제 15서열은 5' -말단의 형광물질로 Cy5.5 및 3' —말단에 뭔처로 BHQ2를 이용한다. 적합한 리포터 -뭔처 쌍 (pairs)은 많은 문헌에 개시되어 있다: Pesce et al . , editors, FLUORESCENCE SPECTR0SC0PY(Marcel Dekker , New York, 1971)； White et al., FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker , New York, 1970)； Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2nd EDITION (Academic Press, New York, 1971)； Griffiths, COLOUR AND CONSTITUTION OF ORGANIC MOLECULESC Academic Press, New York, 1976)； Bishop, editor, INDICATORSCPergamon Press, Oxford, 1972)； Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS(Molecular Probes, Eugene, 1992)； Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Inter science Publishers, New York, 1949)； Haugland, R. P., HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Sixth Edition, Molecular Probes, Eugene, Or eg., 1996； U.S. Pat. Nos. 3,996,345 and 4,351,760. According to a preferred embodiment of the invention, the reporter-quencher pair used in the probes of the invention comprises HEX, FAM, Cy5.5, BHQ1 and BHQ2—based labels. According to a more preferred embodiment of the present invention, the sequential sequence of the ninth sequence of the present invention uses BHQ1 as the quencher at the HEX and 3'-ends as the 5'-terminal fluorescent substance, Using FHQ and 5'-end BHQ1 as the fluorescent material at the 5'-end, the sequence 15 of the sequence Moktok of the present invention is Cy5.5 and 3'-something at the 5'-end. Use BHQ2. Suitable reporter-something pairs are disclosed in many literatures: Pesce et al. , editors, FLUORESCENCE SPECTR0SC0PY (Marcel Dekker, New York, 1971); White et al., FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York, 1970); Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2nd EDITION Press, New York, 1971); Griffiths, COLOUR AND CONSTITUTION OF ORGANIC MOLECULESC Academic Press, New York, 1976); Bishop, editor, INDICATORSCPergamon Press, Oxford, 1972); Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes, Eugene, 1992); Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Inter science Publishers, New York, 1949); Haugland, RP, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Sixth Edition, Molecular Probes, Eugene, Or eg., 1996; US Pat. Nos. 3,996,345 and 4,351,760.
본 발명에서 이용되는 타겟 핵산은 특별하게 제한되지 않으며, DNA(gDNA 또는 cDNA) 또는 RNA 분자를 모두 포함하며 , 보다 바람직하게는 gDNA이다. 타겟 핵산이 RNA 분자인 경우에는 cDNA로 역전사 하여 사용한다. 타겟 핵산은 예컨대， 원핵세포 핵산, 진핵세포 (예컨대， 원생동물과 기생동물, 균류, 효모, 고등 식물, 하등 동물 및 포유동물과 인간을 포함하는 고등동물) 핵산, 바이러스 (예컨대， 헤르페스 바이러스, HIV, 인플루엔자 바이러스， 엡스타인-바 바이러스, 간염 바이러스, 폴리오바이러스 등) 핵산또는 비로이드 핵산을 포함한다.  The target nucleic acid used in the present invention is not particularly limited and includes all DNA (gDNA or cDNA) or RNA molecules, more preferably gDNA. If the target nucleic acid is an RNA molecule, reverse transcription to cDNA is used. Target nucleic acids include, for example, prokaryotic nucleic acids, eukaryotic cells (eg, protozoa and parasites, fungi, yeast, higher plants, lower animals and higher animals, including mammals and humans) nucleic acids, viruses (eg, herpes virus, HIV) , Influenza virus, Epstein-Barr virus, hepatitis virus, poliovirus, etc.) nucleic acid or non-loid nucleic acid.
타겟 핵산을 연장 프라이머 및 프로브에 어닐링 또는 흔성화 시키는 방법은 당업계에 공지된 혼성화 방법에 의해 실시할 수 있다. 본 발명에서, 적합한 흔성화 조건은 최적화 절차에 의하여 일련의 과정으로 결정될 수 있다. 이런 절차는 연구실에서 사용을 위한 프로토콜을 수립하기 위하여 당업자에 의하여 일련의 과정으로 실시된다. 예를 들어， 온도， 성분의 농도， 흔성화 및 반응 시간， 완층액 성분 및 이들의 pH 및 이온세기 등의 조건은 올리고뉴클레오타이드의 길이 및 GC 양 및 타깃 뉴클레오타이드 서열 등의 다양한 인자에 의존한다. 흔성화를 위한 상세한 조건은 Joseph Sambrook, et al . , Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N.Y.(2001); 및 M.L.M. Anderson, Nucleic Acid Hybridization, Springerᅳ Verlag New York Inc. N.Y.(1999)에서 확인할 수 있다. The method of annealing or hybridizing the target nucleic acid to the extension primers and probes can be carried out by hybridization methods known in the art. In the present invention, suitable isomerization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, shake and reaction time, complete liquid components and their pH and ionic strength depend on various factors such as the length and GC amount of the oligonucleotide and the target nucleotide sequence. Detailed conditions for the shake are described by Joseph Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer® Verlag New York Inc. NY (1999).
본 발명에 이용되는 주형-의존성 핵산 중합효소는 5' to 3' 뉴클레아제 활성을 가지는 효소이다. 본 발명에 이용되는 주형-의존성 핵산 중합효소는 바람직하게는 DNA 중합효소이다. 통상적으로 DNA 중합효소들은 5' to 3' 뉴클레아제 활성을 가지고 있다. 본 발명에 이용되는 주형-의존성 핵산 증합효소는 E. coli DNA 중합효소 I, 열안정성 DNA 중합효소 및 박테리오파아지 T7 DNA 중합효소를 포함한다. 바람직하게는， 주형-의존성 핵산 중합효소는 다양한 박테리아 종으로부터 얻을 수 있는 열안정성 DNA 중합효소이고, 이는 Therms aquat i cusi ^) , Thermus ther ophi lusilt ) , Ther us filiform is, Therm is flavus, Thermococcus literal is, Pyrococcus furiosusCPiu) , Thermus antranikiani i , Thermus caldophi lus, Thermus chl iarophi lus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai , Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17、 Thermus thermophi lus, Thermotoga maritima, Thermotoga neapolitana 및 Thermos ipho africanus^\ DNA 중합효소를 포함한다,  The template-dependent nucleic acid polymerase used in the present invention is an enzyme having 5 'to 3' nuclease activity. The template-dependent nucleic acid polymerase used in the present invention is preferably a DNA polymerase. DNA polymerases typically have 5 'to 3' nuclease activity. Template-dependent nucleic acid polymerases used in the present invention include E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the template-dependent nucleic acid polymerase is a thermostable DNA polymerase obtained from various bacterial species, which include Therms aquat i cusi ^), Thermus ther ophi lusilt), Ther us filiform is, Therm is flavus, Thermococcus literal is, Pyrococcus furiosusCPiu), Thermus antranikiani i, Thermus caldophi lus, Thermus chl iarophi lus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scmus ductus, Thermus silotoductus 17 、 Contains Thermus thermophi lus, Thermotoga maritima, Thermotoga neapolitana and Thermos ipho africanus ^ \ DNA polymerase,
주형ᅳ의존성 핵산 증합효소에 의해 촉매되는 "주형-의존성 연장반웅" 은 주형의 서열에 상보적인 뉴클레오타이드 서열을 합성하는 반웅을 의미한다.  "Template-dependent extension reaction" catalyzed by a template-dependent nucleic acid polymerase means a reaction that synthesizes a nucleotide sequence complementary to the sequence of the template.
본 발명의 바람직한 구현예에 따르면, 본 발명의 실 -시간 PCR은 TaqMan프로브 방법으로 실시된다. 【발명의 효과】  According to a preferred embodiment of the present invention, the real-time PCR of the present invention is carried out by TaqMan probe method. 【Effects of the Invention】
본 발명의 특징 및 이점을 요약하면 다음과 같다:  The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 다양한 프라이머 및 프로브를 이용한 3개의 타겟 유전자를 동시에 증폭하여 분석하여 MRSA를 검출하는 방법 및 이를 이용한 진단키트에 관한 것이다.  (a) The present invention relates to a method for detecting MRSA by amplifying and analyzing three target genes simultaneously using various primers and probes, and a diagnostic kit using the same.
(b) 본 발명은 타겟 유전자 (바람직하게는, mecA, ^Xmec/ orfX 및 (b) the present invention is directed to a target gene (preferably mecA, ^ Xmec / orfX and
16S rRNA)-특이적인 프라이머와 프로브를 이용한 멀티플렉스 실 -시간 PCR( real -time polymerase chain reaction)을 통해 MRSA를 효과적으로 검출하고 이를 다른 스트레인들과 용이하게 구별할 수 있다. Multiplex real-time using 16S rRNA) -specific primers and probes Real-time polymerase chain reaction (PCR) can effectively detect MRSA and easily distinguish it from other strains.
(c) 또한， 본 발명의 진단키트는 시료 내 타겟 유전자들을 멀티플렉스 실 -시간 PCR을 통해 간편하고 효율적으로 검출할 수 있다.  (c) In addition, the diagnostic kit of the present invention can easily and efficiently detect target genes in a sample through multiplex real-time PCR.
(d) 따라서, 본 발명의 방법 및 키트는 MRSA의 정확한 진단 뿐 아니라 감염을 검출할 수 있으며， 이를 기반으로 보다 정확하게 질환의 예후를 예측하고 치료에 적용될 수 있다.  (d) Thus, the methods and kits of the present invention can detect infections as well as accurate diagnosis of MRSA, and can be used to more accurately predict the prognosis of the disease and apply it to treatment based on this.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 209개의 MRSA 및 74개의 MRCoNS 분리주들에서 mecA, 1 shows mecA in 209 MRSA and 74 MRCoNS isolates,
SCOec/or/ 및 16S rRNA의 t 값들 간의 상관관계를 보여주는 결과이다. Results show the correlation between t values of SCOec / or / and 16S rRNA.
【발명을 실시하기 위한 구체적인 내용】 [Specific contents to carry out invention]
이하， 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. 실시예  Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . Example
실험결과 Experiment result
본 발명의 어세이 방법은 다양한 국제적 모집물 (international col lections)로부터 얻어진 레퍼런스 스트레인들 (reference strains) 및 실험실로부터 얻어진 임상 분리주들 모두를 포함하는 444개의 스트레인들을 이용하여 평가되었다. 상기 레퍼런스 스트레인들은 8개의 MRSA 스트레인들 (CCARM 3792, CCARM 3795, CCARM 3798, CCARM 3803, CCARM 3805, CCARM 3877, CCARM 3897 및 CCARM 3911), 4개의 MSSA 스트레인들 (KCTC 1621 KCTC 1916, KCTC 1928 및 ATCC 29213)， 그리고 11개의 코아귤라제- 음성 (coagulase-negative) 스타필로코커스 스트레인들
Figure imgf000016_0001
The assay method of the present invention was evaluated using 444 strains including both reference strains obtained from various international collections and clinical isolates obtained from the laboratory. The reference strains include eight MRSA strains (CCARM 3792, CCARM 3795, CCARM 3798, CCARM 3803, CCARM 3805, CCARM 3877, CCARM 3897 and CCARM 3911), four MSSA strains (KCTC 1621 KCTC 1916, KCTC 1928 and ATCC). 29213), and 11 coagulase-negative Staphylococcus strains
Figure imgf000016_0001
epidermidis, KCCM 35494； Staphylococcus simulans, KCCM 41686； Staphylococcus capitis, KCCM 41466； Staphylococcus warneri, KCTC 3340； Staphylococcus haemolyticus, KCTC 3341； Staphylococcus xylosus, KCTC 3342； Staphylococcus intermedius, KCTC 3344； Staphylococcus saprophyticus, KCTC 3345； Staphylococcus cohnii, KCTC 3574； Staphylococcus caprae, KCTC 3583； 및 Staphylococcus auricular is, KCTC 3584)을 포함하였다, SCOec 타이핑에 의해 확인된 (17, 18) SCCmec 앨리먼트를 운반하고 있는 29개의 MSSA 분리주들이 대조군 스트레인들로 테스트되었다. 209개의 MRSA 스트레인들， 109개의 MSSA 스트레인들， 그리고 74개의 MRCoNS 스트레인들로 구성된 임상 분리주들은 대부분 상처， 타액 (sputum), 혈액 및 소변 시료들로부터 회수되었다. 상술한 스타필로코커스 분리주들의 동정 및 민감성 (susceptibility) 테스팅은 MicroScan WalkAway 96(Siemens Healthcare Diagnostics Inc. , West Sacramento, CA, USA) 및 VITEK 2(bioMerieux Inc. , Durham, NC, USA)- 자동화된 동정 및 민감성 테스팅 시스템을 이용하여 실시하였다. epidermidis, KCCM 35494; Staphylococcus simulans, KCCM 41686; Staphylococcus capitis, KCCM 41466; Staphylococcus warneri, KCTC 3340; Staphylococcus haemolyticus, KCTC 3341; Staphylococcus xylosus, KCTC 3342; Staphylococcus intermedius, KCTC 3344; Staphylococcus saprophyticus, KCTC 3345; Staphylococcus cohnii, KCTC 3574; Staphylococcus caprae, KCTC 3583; and Staphylococcus auricular is, KCTC 3584), confirmed by SCOec typing (17, 18) Twenty nine MSSA isolates carrying the elements were tested with control strains. Clinical isolates consisting of 209 MRSA strains, 109 MSSA strains, and 74 MRCoNS strains were mostly recovered from wound, saliva, blood and urine samples. The identification and susceptibility testing of the Staphylococcus isolates described above were performed by MicroScan WalkAway 96 (Siemens Healthcare Diagnostics Inc., West Sacramento, CA, USA) and VITEK 2 (bioMerieux Inc., Durham, NC, USA) And sensitivity testing systems.
레퍼런스 스트레인들과 임상 분리주들은 혈액 -아가 플레이트 (Asan Pharmaceutical, seoul, Korea)에서 37°C로 24시간 동안 배양하였다. 레퍼런스 스트레인들과 분리주들의 약 2-3개의 박테리아 콜로니들이 1 μ ΐ 루프 (loop)로 수득하여 0.5 ml의 증류수에 현탁하였다. 상기 현탁액이 끓는 워터 배스에서 10분 동안 가열시킨 후, 13,000 X g에서 5분 동안 원심분리시켰다. 상층액이 실 -시간 PCR에 이용되었다. Reference strains and clinical isolates were incubated for 24 hours at 37 ° C in blood-agar plates (Asan Pharmaceutical, seoul, Korea). About 2-3 bacterial colonies of the reference strains and isolates were obtained in 1 μΐ loop and suspended in 0.5 ml of distilled water. The suspension was heated in a boiling water bath for 10 minutes and then centrifuged at 13,000 X g for 5 minutes. Supernatants were used for real-time PCR.
SCCmec/ or fX정션， mecA 및 스타필로코커스 16S rR A 유전자들의 염기 서열은 NCBI GenBank로부터 얻어서 Sequencher 5.0 소프트웨어 (Gene Codes Co., Ann Arbor, MI, USA)를 이용하여 정렬 (align)하였다. 서열 정렬에 기초하여, 본 발명자들은 목적 부위 (regions of interest)를 찾고 the Primer 3 program(http://f rodo.wi .mit .edu/pr imer 3/) 또는 수작업으로 프라이머들 및 프로브들을 제작하였다. 상기 프라이머 서열의 해당 유전자 내 위치는 다음과 같다: (a) SCCmec, staphylococcus의 0RF(open reading frame); 및 (b) orfX, 3' 말단 근처의 attBscc 부위. 본 연구에서 고안되어 이용된 실 -시간 PCR 프라이머 및 프로브들은 표 1에 제시되어 있다. [표 1】 The base sequences of the SCCmec / or fX junction, mecA and Staphylococcus 16S rR A genes were obtained from NCBI GenBank and aligned using Sequencher 5.0 software (Gene Codes Co., Ann Arbor, MI, USA). Based on the sequence alignment, we find regions of interest and fabricate primers and probes by the Primer 3 program (http: // f rodo.wi.mit.edu/pr imer 3 /) or by hand. It was. The position in the gene of interest of the primer sequence is as follows: (a) open reading frame (0RF) of SCCmec, staphylococcus; And (b) orfX, the attBscc site near the 3 'end. The real-time PCR primers and probes designed and used in this study are shown in Table 1. TABLE 1
MRSA검출용 실 -시간 PCR프라이머 및 프로브 . Real-Time PCR Primers and Probes for MRSA Detection.
Figure imgf000018_0001
Figure imgf000018_0001
a슷자는프라이머에의해증폭된 SCOec타입을나타낸다. 약어: F, 정방향; B, 역방향; 및 P, 프로브. 실 -시간 PCR 반응은 Rotor-gene Q 실 -시간 PCR instrument (QIAGEN Inc. , Germantown, MD, USA)을 이용하여 실시하였다. PCR 흔합물은 0.5 μΐ의 프라이머-프로브 믹스 (mix), 5 μ 1의 2x Rotor-Gene 멀티플렉스 PCR 마스터 믹스 (QIAGEN Inc., Germantown, MD, USA) 및 1.0 μ 1의 템플레이트 DNA를 포함하는 총 10 μΐ의 반옹액으로 구성된다. PCR 조건은 다음과 같다: (a) PCR 사이클, (i) 전 -변성 (pre-denaturign) 단계， 95°C에서 5분; 및 (Π) 40 사이클, 95°C에서 15초 및 60°C에서 15초로 구성된 1사이클; 및 (b) 검출 단계， 녹색， 황색 및 진흥색 (crimson) 형광을 검출. PCR 완료 후, mecA, SCCmec/orfX 및 16S rRNA 유전자들의 Ct 값은 Rotor- gene Q 소프트웨어를 아용하여 기록하였다. 상관계수 (r) 및 기술통계학을 포함하는 통계적 테스트들은 SPSS 13.0 소프트웨어 (SPSS Inc., Chicago, IL, USA)* 이용하여 실시하였다. 5% 레벨 이하의 P 값은 통계적으로 유의한 것으로 간주되었다. The pair indicates the SCOec type amplified by the primer. Abbreviation: F, forward; B, reverse direction; And P, probe. Real-time PCR reactions were performed using a Rotor-gene Q real-time PCR instrument (QIAGEN Inc., Germantown, MD, USA). The PCR mixture was prepared with a total of 0.5 μl primer-probe mix, 5 μl of 2 × Rotor-Gene multiplex PCR master mix (QIAGEN Inc., Germantown, MD, USA) and 1.0 μl of template DNA. It consists of 10 μΐ of the repellent solution. PCR conditions were as follows: (a) PCR cycle, (i) pre-denaturign step, 5 min at 95 ° C .; And (Π) 40 cycles, 15 seconds at 95 ° C and 15 seconds at 60 ° C 1 cycle; And (b) a detection step, detecting green, yellow and crimson fluorescence. After PCR completion, C t values of mecA, SCCmec / orfX and 16S rRNA genes were recorded using Rotor-Q Q software. Statistical tests including correlation coefficients (r) and descriptive statistics were conducted using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) *. P values below the 5% level were considered statistically significant.
실 -시간 PCR의 분석 민감도 (sensitivity)는 10배씩 연속적으로 희석된 (10-fold serial dilutions) MRSA 스트레인 CCARM 3792의 계대배양액으로 결정하였다. 상기 스트레인이 혈액 아가 폴레이트에서 하룻밤 동안 배양되어 0.5의 맥팔란드 탁도 (McFarland turbidity number)와 동일한 밀도로 염 용액에 현탁시키고, 102에서 107까지 10배씩 연속적으로 회석하였다ᅳ DNA는 QIAamp® DNA mini Kit 내 QIAcube(QIAGEN Inc., Germantown, MD, USA)를 아용하여 박테리아 희석액 (200 μ ΐ)으로부터 추출하여 50 μ ΐ에 녹였다. 동시에, 상기 박테리아 희석액 (200 μ ΐ)이 혈액 아가에 플레이팅되어 24시간 동안 37°C에서 배양되었다. 이후, CFU( colony- forming units)가 카운팅되었다. Assay sensitivity of real-time PCR was determined by subculture of 10-fold serial dilutions of MRSA strain CCARM 3792. The strain was incubated overnight in blood agar folate, suspended in a salt solution at the same density as McFarland turbidity number of 0.5, and serially dilute in 10-fold from 102 to 107 ᅳ DNA was QIAamp® DNA mini Kit My QIAcube (QIAGEN Inc., Germantown, MD, USA) was used to extract from bacterial dilutions (200 μΐ) and dissolved in 50 μΐ. At the same time, the bacterial dilutions (200 μΐ) were plated on blood agar and incubated at 37 ° C for 24 hours. Thereafter, colony-forming units (CFUs) were counted.
우선ᅳ 실—시간 PCR 어세이는 23개꾀 레퍼런스 스트레인들 및 29개의 MSSA 대조군 스트레인들에 대해 실싱하였다. 예상된 PCR 산물은 레퍼런스 스트레인에서만 증폭되었다. 하지만， SCCmec/orfX는 29개의 대조군 스트레인들 중 6개에서 검출되지 않았다.  Priority real-time PCR assays were subjected to 23 strain reference strains and 29 MSSA control strains. The expected PCR product was only amplified at the reference strain. However, SCCmec / orfX was not detected in 6 of 29 control strains.
392개의 임상 분리주들에 대한 결과들은 다음과 같았다: 3개의 타겟들이 209개의 모든 MRSA(100%) 분리주들 및 4개의 MRCoNS(5.4¾) 분리주들에서 동시에 검출되었다. 109개의 MSSA 분리주들 중， mecA 및 16S rRNA가 2개의 분리주들 (1.8%)에서 동시에 검출되었으며, meclorfX 및 16S rRNA는 11개의 분리주들 (10.1%)에서 모두 검출되었다. ec4의 Ct 값과 mecl orfX 및 16S rRNA의 Ct 값이 비교되었다. mecA Ct 값과 XC ec/orf Ct 값 간의 상관계수는 MRSA 분리주들에서 높았고 (r = 0.959； P < 0.0001), mecA Ct 값과 16S rRNA Ct 값 간의 상관계수는 MRSA 분리주들 (r = 0.970； P < 0.0001) 및 MRCoNS 분리주들 (r = 0.963； P < 0.0001)에서 높았다. 상술한 결과들은 도 1 및 표 2에 나타나 있다. 종The results for 392 clinical isolates were as follows: Three targets were detected simultaneously in all 209 MRSA (100%) isolates and four MRCoNS (5.4¾) isolates. Of the 109 MSSA isolates, mecA and 16S rRNA were detected in both isolates (1.8%) simultaneously, and meclorfX and 16S rRNA were detected in all 11 isolates (10.1%). The C t value and mecl C t values of the orfX and 16S rRNA of ec4 compared. The correlation coefficient between mecA C t value and XC ec / orf Ct value was high in MRSA isolates (r = 0.959; P <0.0001), and the correlation coefficient between mecA C t value and 16S rRNA C t value was found in MRSA isolates (r = 0.970; P <0.0001) and MRCoNS isolates (r = 0.963; P <0.0001). The above results are shown in FIGS. 1 and 2. Bell
Η수M¾의 κ i! . Η number M¾ κ i! .
.ffiS 209 MSSA 109 MRCoNS ?4.ffiS 209 MSSA 109 MRCoNS? 4
^ : ¾( cycle t ^ : ¾ (cycle t
00 00
Figure imgf000020_0001
Figure imgf000020_0001
그 결과, 얗 mecl orfX mecA 간의 Ct 값 차이 (Ctscc), 그리고 16S rRNA와 mecA Ct 값 차이 (Ctl6S)는 MRSA의 존재를 평가하는 데 이용되었다. Ctscc(≥ 4.7； mean+4SD)은 MRSA와 MRSA 이외의 스타필로코커스가 동시에 존재한다는 것을 나타내는 반면에， Ctl6S(≤ -1.72； mean-4SD)은 MRSA와 mecA 유전자가 없는 스타필로코커스가 함께 존재한다는 것을 의미하였다. As a result, the C t value difference (C tscc ) between the white mecl orfX mecA, and the 16S rRNA and mecA C t value difference (C tl6S ) were used to assess the presence of MRSA. C t scc (≥ 4.7; mean + 4SD) indicates simultaneous presence of MRSA and non-MRSA Staphylococcus, whereas C tl6S (≤ -1.72; mean-4SD) shows Staphylococcus without MRSA and mecA genes Meant that they exist together.
어세이의 검출 한계 (detection limit)는 MRSA 스트레인 CCARM 3792 스탁 (stock) 용액의 1:104 회석액으로부터 정제된 게놈 (genomic) DNA를 이용하여 실시하였으며， PCR 반웅 당 20 CFU( colony forming unit)인 것으로 측정되었다ᅳ The detection limit of the assay was performed using genomic DNA purified from 1:10 4 dilution of MRSA strain CCARM 3792 stock solution, 20 CFU (colony forming unit) per PCR reaction. Was measured to be
현재， S. aureus^ orfX 및 SCCsec 앨리먼트의 오른쪽 최외곽 정션 (right extremity junction)을 타겟팅하는 실 -시간 PCR 어세이는 감염 대조군을 위해 이용되고 있다 (19-22). 이전 연구들은 상술한 단일-유전자 위치 어세이에서 각각 0.0%부터 7.3%까지의 거짓 음성 결과들 및 0.0%부터 5.4%까지의 거짓 양성 결과들을 보고하였다 (4， 6， 23-25) . 거짓 음성 및 양성 결과들은 전체 감염 조절 프로그램에 영향을 미쳐 병원 내 MRSA 전파 및 불필요한 동정 및 보균제거 (decolonization) 과정을 실시하게 한다. 알려지지 않은 mec 타입을 가진 MRSA가 시료 내에 존재한다면， 거짓 음성 결과가 나을 수 있다. 현재까지, 11개의 다른 타입의 SCOec들이 5. a /s에 알려져 있다 (http://w丽. sccmec.org/). 이에， 본 발명자는 알려진 모든 타입의 SCOec들을 검출하기 위해 6개의 정방향 프라이머, 2개의 역방향 프라이머 및 1개의 프로브를 제작하였다.  Currently, real-time PCR assays targeting the right extremity junctions of S. aureus ^ orfX and SCCsec elements are used for infection control (19-22). Previous studies have reported false negative results from 0.0% to 7.3% and false positive results from 0.0% to 5.4%, respectively, in the single-gene position assay described above (4, 6, 23-25). False negative and positive results affect the overall infection control program, leading to in-hospital MRSA propagation and unnecessary identification and decolonization processes. If MRSA with unknown mec type is present in the sample, false negative results may be obtained. To date, eleven different types of SCOecs are known at 5.a / s (http://www.sccmec.org/). Accordingly, the inventors have constructed six forward primers, two reverse primers and one probe to detect all known types of SCOecs.
본 발명자가 아는 한， 한국에서 거짓 양성의 경우가 meclorfX 정션을 타겟팅하는 실 -시간 PCR 어세이를 통해 보고되지 않았다. 본 발명자는 낮은 MRSA 풍토병을 나타내는 나라들에서보다 더 높은 거짓 양성 결과들을 예측하였다. 예측한 대로， 거짓 양성 결과들이 3개월 이상 본 발명자의 실험실에서 3개월 이상 수집된 스타필로코커스의 임상 분리주들에서 10.1%였다. 거짓 양성 결과들의 비율이 높다면， 단일- 유전자 위치에 대한 실 -시간 PCR 어세이의 진단값은 높은 MRSA 풍토성 (endemicity)에 대한 실험에 적합하지 않다. 따라서, 본 발명자는 SCCmec 잔여물을 운반하고 mecA 유전자가 없는 MSSA 분리주들을 제외하기 위해 mecA 유전자 및 mecl orfX 정션의 동시 증폭을 고려하였다. 하지만， 상기 동시 증폭 방법도 mec 잔여물을 포함하는 MRCoNS 및 MSSA가 임상 시료에 모두 존재하는 경우에 거짓 양성 결과를 초래할 수 있다. 따라서, 거짓 양성 결과들을 감소시키기 위해 스타필로코커스 16S rRNA 유전자가 타겟으로 부가되었다. 진짜 MRSA에서， Ct 값으로 표현되는 상기 3개의 타겟 유전자들의 카피 수는 모두 동일한 반면에, mec 잔여물을 포함하는 MRCoNS 및 MSSA이 복합된 개체들 (populations)의 경우, 상기 유전자들의 카피 수가 대부분 다를 것이다. To the best of our knowledge, no false positive cases have been reported in real-time PCR assays targeting meclorfX junctions in Korea. We predicted higher false positive results than in countries with low MRSA endemic disease. As expected, false positive results were 10.1% in Staphylococcus clinical isolates collected in our laboratory for at least 3 months. If the rate of false positive results is high, the diagnostic value of a real-time PCR assay for single-gene locations is not suitable for experiments with high MRSA endemicity. Thus, we considered simultaneous amplification of the mecA gene and the mecl orfX junction to exclude MSSA isolates carrying SCCmec residues and lacking the mecA gene. However, the simultaneous amplification method can also lead to false positive results when both MRCoNS and MSSA containing mec residues are present in the clinical sample. Thus, Staphylococcus 16S rRNA gene was added to the target to reduce false positive results. In true MRSA, the number of copies of the three target genes expressed in C t values are all the same, whereas for populations with MRCoNS and MSSA containing mec residues, the number of copies of the genes is mostly Will be different.
본 발명자는 SCOec 잔여물을 포함하는 MRCoNS 및 MSSA이 복합된 개체들이 상기 3개의 타겟들의 상대적인 정량을 통해 구별될 수 있는 지 여부를 테스트하였다. MRSA 및 MRSA 이외의 스타필로코커스로부터 유래한 게놈 DNA의 흔합물 및 SCOec 잔여물을 포함하는 MRCoNS 및 MSSA의 흔합물을 포함하는 스타필로코커스 게놈 DNA 시료들의 복합 칵테일이 상기 3개의 타겟을 증폭하는 데 이용되었다. 이후， 증폭된 시료들아 분석되었다 (결과를 보이지 않음). 본 발명자들은 SCOec 잔여물을; 포함하는 MRCoNS 및 MSSA는 CtSCC 값이 > 4.7이고 Ctl6S 값이 < L.72인 경우에만 동시에 존재하였고, MRSA 및 MRCoNS의 흔합 개체군들은 CtSCC 값이 > 4.7이고 Ctl6S 값이 ≥ -1.기인 경우에 SCOec 잔여물을 포함하는 MRCoNS 및 MSSA와 구별할 수 없다는 것을 확인하였다. We tested whether MRCoNS and MSSA complexes comprising SCOec residues can be distinguished through relative quantification of the three targets. A composite cocktail of Staphylococcus genomic DNA samples comprising a mixture of MRCoNS and MSSA, including a mixture of genomic DNA derived from MRSA and non-MRSA and Staphylococcus and a SCOec residue, was used to amplify the three targets. Was used. The amplified samples were then analyzed (no results shown). We have found SCOec residues ; Including MRCoNS and MSSA existed only when the C tSCC value was> 4.7 and the C tl6S value was <L.72, and the mixed populations of MRSA and MRCoNS had a C tSCC value> 4.7 and a C tl6S value ≧ −1. It was confirmed that it was indistinguishable from MRCoNS and MSSA containing SCOec residues.
본 연구에서, 4개의 MRCoNS 분리주들은 3개의 타겟들의 동시 증폭물을 생산하였다: Ctscc 값은 각각 19.69, 17.50, 18.55 및In this study, four MRCoNS isolates produced simultaneous amplifications of three targets: C tscc values were 19.69, 17.50, 18.55 and
15.37이었으며; Ctl6S 값은 각각 0.61, -0.97， -0.31 및 -0.09이었다. 상술한 경우들은 MRSA 및 MRCoNS의 복합물 또는 SC wec 잔여물을 포함하는 MRCoNS 및 MSSA의 복합물로부터 얻은 결과였다. 대한민국에서， SCC/sec 잔여물을 포함하는 MSSA는 임상 표본들로부터 회수된 aureus^ 약 3%에 달할 것으로 추정되는데, 이는 약 30%의 5. aureus 분리주들이 MSSA 및 10%의 SCOec 잔여물을 포함하는 MSSA이기 때문이다, Becker의 연구에 따르면， MSSA와 MRCoNS에 의한 비강 공동 -군체 (cocolonization)는 약 3. 의 환자들에서 관찰되었다 (8). 더욱이, mec 잔여물을 포함하는 MSSA가 15 이상의 Ctscc 값을 가지는 흔합된 개체군들 중 90% 이상이라는 것을 분석된 시료들로부터 확인하였다. 따라서, mec 잔여물을 포함하는 MRCoNS 및 MSSA의 흔합 개체군들로부터 상기 3개의 타켓들이 동시 증폭될 가능성이 매우 낮다. 15.37; Ctl6S values were 0.61, -0.97, -0.31 and -0.09, respectively. The cases described above were from a combination of MRSA and MRCoNS, or a combination of MRCoNS and MSSA, including SC wec residues. In Korea, MSSA containing SCC / sec residues is estimated to be about 3% of aureus ^ recovered from clinical samples, with approximately 30% of 5. aureus isolates containing MSSA and 10% SCOec residues. This is because MSSA, according to Becker's study, showed that nasal colonization by MSSA and MRCoNS was observed in about 3 patients (8). Moreover, it was confirmed from the analyzed samples that the MSSA containing mec residues was at least 90% of the mixed populations with C tscc values of 15 or more. Thus, the three targets are simultaneously available from mixed populations of MRCoNS and MSSA containing mec residues. It is very unlikely to be amplified.
결론적으로， 본 발명자는 본 발명의 멀티플렉스 실 -시간 PCR 어세이 방법이 !!neclorfX 정션을 증폭하는 단일ᅳ유전자 위치 실 -시간 PCR 어세이와 비교하여 매우 낮은 거짓 양성 결과들을 초래한다는 것을 증명하였다. 비록 임상 표본들의 직접적 MRSA 스크리닝에 적용되기 전에 추가적인 평가가 필요할 지라도， 본 발명의 어세이 방법은 높은 MRSA 풍토병을 가진 임상 실험실에서 유용할 것이다. 이상으로 본 발명의 특정한 부분을 상세히 기술하였는바， 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며， 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다. 참고문헌  In conclusion, we have demonstrated that the multiplex real-time PCR assay method of the present invention results in very low false positive results compared to a single gene gene real-time PCR assay that amplifies !! neclorfX junctions. . Although further evaluation may be necessary before applying to direct MRSA screening of clinical specimens, the assay method of the present invention will be useful in clinical laboratories with high MRSA endemic diseases. The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that these specific technologies are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof. references
1. Bootsma MC, Diekmann 0， and Bonten MJ. 2006. Controlling methici 11 in-resistant Staphylococcus aureus: quantifying the effects of inter vent ions and rapid diagnostic testing. Proc Natl Acad Sci U S A 103:5620-5625.  Bootsma MC, Diekmann 0, and Bonten MJ. 2006. Controlling methici 11 in-resistant Staphylococcus aureus: quantifying the effects of inter vent ions and rapid diagnostic testing. Proc Natl Acad Sci U S A 103: 5620-5625.
2. Harbarth S, Masuet-Aumatell C, Schrenzel J, Francois P, Akakpo C, Renzi G, Pugin J, Ricou B, and Pittet D. 2006. Evaluation of rapid screening and pre-emptive contact isolation for detecting and controlling methici 11 in-resistant Staphylococcus aureus in critical care: an interventional cohort study. Crit Care 10:R25. Harbarth S, Masuet-Aumatell C, Schrenzel J, Francois P, Akakpo C, Renzi G, Pugin J, Ricou B, and Pittet D. 2006. Evaluation of rapid screening and pre-emptive contact isolation for detecting and controlling methici 11 in-resistant Staphylococcus aureus in critical care: an interventional cohort study. Crit Care 10: R25.
3. Cho ers MY, Pa it an Y, Gottesman BS, Gerber B, Ben-Nissan Y, and Shitrit P. 2009. Hospital-wide methici 11 in-resistant Staphylococcus aureus control program'- A 5-year followᅳ up. Infect Control Hosp Epidemiol 30:778—781. 3.Cho ers MY, Pa it an Y, Gottesman BS, Gerber B, Ben-Nissan Y, and Shitrit P. 2009. Hospital-wide methici 11 in-resistant Staphylococcus aureus control program ' -A 5-year follow ᅳ up. Infect Control Hosp Epidemiol 30: 778—781.
4. Huletsky A, Giroux R, Rossbach V, Gagnon M, Vaillancourt M, Bernier M Gagnon F, Truchon K, Bast i en M, Pi card FJ, van Belkum A, Ouellette M, Roy PH, and Bergeron MG. 2004. New Real-Time PCR Assay for Rapid ω Huletsky A, Giroux R, Rossbach V, Gagnon M, Vaillancourt M, Bernier M Gagnon F, Truchon K, Bast i en M, Pi card FJ, van Belkum A, Ouellette M, Roy PH, and Bergeron MG. 2004.New Real-Time PCR Assay for Rapid ω
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Claims

【특허청구범위】  Patent Claim
【청구항 U [Claim port U
다음의 단계를 포함하는 MRSACmethici 11 in-resistant Staphylococcus aureus)^] 또는 정량방법 :  MRSACmethici 11 in-resistant Staphylococcus aureus ^] or quantification method comprising the following steps:
(a) 시료를 준비하는 단계;  (a) preparing a sample;
(b) 서열목록 제 1서열 내지 서열목록 제 6서열로 구성된 군으로부터 선택된 하나 이상의 정방향 프라이머， 서열목록 제 7서열 및 서열목록 제 8서열로 구성된 군으로부터 선택된 하나의 역방향 프라이머 및 서열목톡 제 9서열의 프로브; 서열목록 제 10서열 및 서열목록 제 11서열의 프라이머쌍 및 서열목록 제 12서열의 프로브; 및 서열목록 제 13서열 및 서열목록 제 14서열의 프라이머쌍 및 서열목톡 제 15서열의 프로브를 이용하여 상기 시료 내의 뉴클레오타이드 서열을 증폭하는 단계； 및  (b) at least one forward primer selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, one reverse primer selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8 Probes; Primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 and probes of SEQ ID NO: 12; And amplifying the nucleotide sequence in the sample using the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 and the probe of SEQ ID NO: 15; and
(c) 상기 증폭 결과를 형광으로 확인하는 단계 .  (c) confirming the amplification result by fluorescence.
【청구항 2】 [Claim 2]
제 1 항에 있어세 상기 시료는 혈액, 타액 또는 소변인 것을 특징으로 하는 방법.  The method of claim 1 wherein said sample is blood, saliva or urine.
【청구항 3】 [Claim 3]
제 1 항에 있어서, 상기 증폭은 PCR( polymerase chain reaction)에 따라 실시되는 것을 특징으로 하는 방법.  The method of claim 1, wherein the amplification is performed according to a polymerase chain reaction (PCR).
【청구항 4】 [Claim 4]
제 1 항에 있어서, 상기 증폭은 실 -시간 (real-time) PCR에 따라 실시되는 것을 특징으로 하는 방법.  The method of claim 1, wherein said amplification is performed according to real-time PCR.
【청구항 5】 [Claim 5]
제 4 항에 있어서, 상기 실 -시간 PCR은 TaqMan 프로브 방법으로 실시되는 것을 특징으로 하는 방법. 5. The method of claim 4, wherein said real-time PCR is performed by TaqMan probe method.
【청구항 6] [Claim 6]
제 1 항에 있어서, 상기 MRSA 스트레인은 CCARM 3792, CCARM 3795, CCARM 3798, CCARM 3803, CCARM 3805, CCARM 3877, CCARM 3897 또는 CCARM 3911을 포함하는 것을 특징으로 하는 방법.  The method of claim 1, wherein the MRSA strain comprises CCARM 3792, CCARM 3795, CCARM 3798, CCARM 3803, CCARM 3805, CCARM 3877, CCARM 3897 or CCARM 3911.
【청구항 7】 [Claim 7]
제 1 항에 있어서， 상기 실 -시간 PCR에 의한 MRSA의 정량 범위는 102-107 세포인 것을 특징으로 하는 방법 .  The method of claim 1, wherein the quantitative range of MRSA by real-time PCR is 102-107 cells.
【청구항 8】 [Claim 8]
서열목록 제 1서열 내지 서열목톡 제 6서열로 구성된 군으로부터 선택된 하나이상의 정방향 프라이머, 서열목록 제 7서열 및 서열목록 제 8서열로 구성된 군으로부터 선택된 하나의 역방향 프라이머 및 서열목록 제 9서열의 프로브; 서열목톡 제 10서열 및 서열목록 제 11서열의 프라이머쌍 및 서열목록 제 12서열의 프로브; 및 서열목록 제 13서열 및 서열목록 제 14서열의 프라이머쌍 및 서열목록 제 15서열의 프로브를 포함하는 MRSA(methici 11 in-resistant Staphylococcus aureus) 검출■ 또는 진단용' 키트, At least one forward primer selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, a reverse primer selected from the group consisting of SEQ ID NO: 7, and SEQ ID NO: 8, and a probe of SEQ ID NO: 9; Primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 and probes of SEQ ID NO: 12; And a method for detecting or diagnosing ' methici 11 in-resistant Staphylococcus aureus (MRSA) ' comprising a primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 and a probe of SEQ ID NO: 15
【청구항 9】 [Claim 9]
제 8 항에 있어서, 상기 키트는 유전자 증폭에 의해 실시되는 것을 특징으로 하는 키트.  The kit of claim 8, wherein the kit is performed by gene amplification.
【청구항 10] [Claim 10]
제 9 항에 있어서, 상기 증폭은 실—시간 PCR에 따라 실시되는 것을 특징으로 하는 키트.  10. The kit of claim 9, wherein said amplification is performed according to real-time PCR.
【청구항 111 [Claim 111]
제 10 항에 있어서, 상기 실 -시간 PCR은 TaqMan 프로브 방법으로 실시되는 것을 특징으로 하는 키트.  The kit of claim 10, wherein said real-time PCR is performed by TaqMan probe method.
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