WO2014073858A1 - Procédé de détection de staphylococcus aureus résistant à la méthicilline (mrsa), et trousse à l'aide de celui-ci - Google Patents

Procédé de détection de staphylococcus aureus résistant à la méthicilline (mrsa), et trousse à l'aide de celui-ci 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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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
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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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
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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
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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.

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Abstract

La présente invention concerne un procédé de détection de MRSA par l'amplification et l'analyse simultanées de trois gènes cibles à l'aide de diverses amorces et sondes, et une trousse de diagnostic à l'aide de celui-ci. La présente invention peut détecter efficacement MRSA et facilement distinguer celui-ci à partir d'autres souches par l'intermédiaire d'une réaction en chaîne de la polymérase (PCR) en temps réel multiplexe à l'aide d'amorces et de sondes spécifiques de gènes cibles (de préférence mecA, SCCmec/orfX et ARNr 16S). De plus, la trousse de diagnostic de la présente invention peut détecter de façon pratique et efficace des gènes cibles dans un échantillon par l'intermédiaire de la PCR en temps réel multiplexe. Par conséquent, le procédé et la trousse de la présente invention peuvent détecter une infection par MRSA et diagnostiquer précisément MRSA, et plus précisément prédire le pronostic de maladies et traiter les maladies sur la base du résultat.
PCT/KR2013/010025 2012-11-09 2013-11-06 Procédé de détection de staphylococcus aureus résistant à la méthicilline (mrsa), et trousse à l'aide de celui-ci WO2014073858A1 (fr)

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