KR102250629B1 - A composition for detecting antibiotics-resistant bacteria and a method for detecting antibiotics-resistant bacteria using the same - Google Patents

Abstract

The present invention provides one or more primer pairs selected from the group consisting of a primer pair of SEQ ID NOs: 7 and 8, a primer pair of SEQ ID NOs: 13 and 14, a primer pair of SEQ ID NOs: 31 and 32, and a primer pair of SEQ ID NOs: 33 and 34;
At least one primer pair selected from the group consisting of a primer pair of SEQ ID NOs: 53 and 54, a primer pair of SEQ ID NOs: 89 and 90, and a primer pair of SEQ ID NOs: 91 and 92; And/or
It relates to a kit for detecting multiple antibiotic-resistant Staphylococcus aureus, multiple antibiotic-resistant pneumococci and/or Salmonella typhimurium including primer pairs of SEQ ID NOs: 127 and 128, and a detection method using the same. The primer pair of the present invention can be usefully used to detect these multiple antibiotic resistant strains.

Description

A composition for detecting antibiotic-resistant strains and its method {A COMPOSITION FOR DETECTING ANTIBIOTICS-RESISTANT BACTERIA AND A METHOD FOR DETECTING ANTIBIOTICS-RESISTANT BACTERIA USING THE SAME}

The present invention relates to a composition and a method for detecting antibiotic-resistant strains.

Antibiotics are medicines used to prevent and treat bacterial infections, and have long been widely used because they have many beneficial effects in controlling infectious diseases, which are the major causes of morbidity and mortality in many humans for a long time. However, the recent misuse and abuse of antibiotics has led to the development of antibiotic-resistant bacteria. As reported in some countries, antibiotic-resistant bacteria have developed because the antibiotics used to control bacteria in animals or to produce more meat are much higher than those in humans. Because of the emergence and spread of multiple resistance and cross resistance in pathogenic bacteria, the mechanism between antibiotic use and development of resistant bacteria must be understood, which helps to find possible solutions for the treatment of multiple drug resistant bacteria.

The pathogen Klebsiella pneumoniae is mainly involved in pathogenic infections with high morbidity and mortality because multidrug resistant K. pneumoniae strains can cause treatment failures of current antibiotic therapy.

Staphylococcus aureus is one of the top 5 pathogen food category pairs that cause outbreak-related diseases in the United States, according to the Centers for Disease Control and Prevention 2016 (CDC 2016).

Repeated exposure to the same and different antibiotics has been reported to isolate methicillin-resistant S. aureus (MRSA), causing serious clinical and public health problems. Salmonellosis caused by Salmonella is one of the infections that become difficult to treat because the antibiotics used for treatment are less effective (WHO 2018). Multidrug resistant Salmonella typhimurium poses a serious threat to public health.

Infection by multidrug-resistant Gram-negative bacteria resistant to β-lactam antimicrobial agents has recently increased. These infections increase mortality and morbidity, increase the treatment period, and cause medical expenses to rise, making it difficult to manage nosocomial infections.

Therefore, in order to prevent, manage, and properly treat infectious diseases, multidrug resistant bacteria must be quickly detected. The antibiotic susceptibility test is a basic test to detect multi-drug resistant bacteria. This test takes a certain amount of time to cultivate the bacteria, and the degree of expression of the β-lactamase gene is varied and there is no specific inhibitor, making it difficult to accurately identify the mechanism of resistance. On the other hand, a test that detects the β-lactamase gene by a molecular biological method can quickly and accurately reveal the mechanism of resistance, so it is useful for the control of nosocomial infections and epidemiological studies of resistant bacteria.

[Prior patent literature]

Korean Patent Registration 10-1473444

The present invention was conceived by the necessity of the above, and an object of the present invention is to provide a composition for detecting antibiotic-resistant strains.

Another object of the present invention is to provide a method for detecting antibiotic resistant strains.

In order to achieve the above object, the present invention is selected from the group consisting of a primer pair of SEQ ID NOs: 7 and 8, a primer pair of SEQ ID NOs: 13 and 14, a primer pair of SEQ ID NOs: 31 and 32, and a primer pair of SEQ ID NOs: 33 and 34. It provides a kit for detection of multiple antibiotic-resistant Staphylococcus aureus ( Staphylococcus aureus) comprising one or more primer pairs.

In addition, the present invention is a multi-antibiotic resistant yellow color comprising a primer pair consisting of a primer pair of SEQ ID NOs: 7 and 8, a primer pair of SEQ ID NOs: 13 and 14, a primer pair of SEQ ID NOs: 31 and 32, and a primer pair of SEQ ID NOs: 33 and 34 A kit for detecting Staphylococcus aureus is provided.

In one embodiment of the present invention, the antibiotic is preferably one or more antibiotics selected from the group consisting of erythromycin, ciprofloxacin, norprosacin, and tetracycline, but is not limited thereto.

In addition, the present invention extracts DNA from a specimen containing bacteria and uses a primer pair of SEQ ID NOs: 7 and 8, a primer pair of SEQ ID NOs: 13 and 14, a primer pair of SEQ ID NOs: 31 and 32, and a primer pair of SEQ ID NOs: 33 and 34 provides a method of detecting a multi-antibiotic-resistant Staphylococcus aureus (Staphylococcus aureus) which is selected includes the step of performing at least one primer pair was treated polymerase chain reaction from the group consisting of.

In addition, the present invention is a multi-antibiotic-resistant pneumonia rodent comprising at least one primer pair selected from the group consisting of a primer pair of SEQ ID NOs: 53 and 54, a primer pair of SEQ ID NOs: 89 and 90, and a primer pair of SEQ ID NOs: 91 and 92 ( Klebsiella pneumoniae ) provides a kit for detection.

In another aspect, the present invention SEQ ID NO: 53, and primer pair 54, SEQ ID NO: 89, and primer pair 90, and SEQ ID NO: 91 and a multi-antibiotic resistant pneumonia rod bacterium comprising a pair of primers consisting of primer pair 92 (Klebsiella pneumoniae) for detecting Kits are provided.

In one embodiment of the present invention, the antibiotic is preferably one or more antibiotics selected from the group consisting of tobobramycin, chromphenicol, erythromycin, ciprofloxacin, norprosacin, ampicillin, and cytotasim, but limited thereto. It doesn't.

In addition, the present invention extracts DNA from a sample containing bacteria, and at least one primer pair selected from the group consisting of a primer pair of SEQ ID NOs: 53 and 54, a primer pair of SEQ ID NOs: 89 and 90, and a primer pair of SEQ ID NOs: 91 and 92. It provides a method for detecting multiple antibiotic-resistant pneumonia bacillus (Klebsiella pneumoniae ) comprising the step of performing a polymerase chain reaction by treating.

In another aspect, the present invention provides a multi-antibiotic resistant Salmonella typhimurium (Salmonella Typhimurium) detection kit comprising the primer pair of SEQ ID NO: 127 and 128.

In one embodiment of the present invention, the antibiotic is preferably at least one antibiotic selected from the group consisting of tobobramycin, erythromycin, tetracycline, and ampicillin, but is not limited thereto.

In addition, the present invention is to detect multiple antibiotic-resistant Salmonella Typhimurium (Salmonella Typhimurium) comprising the step of performing a polymerase chain reaction by extracting DNA from a sample containing bacteria and processing the primer pairs of SEQ ID NOs: 127 and 128. Provides a way.

Hereinafter, the present invention will be described.

The present inventors set a dual focus: First, the present inventors observed the expression of specific genes designed in proteomic studies in antibiotic-resistant strains to detect antibiotic-resistant strains, and secondly, S. aureus, K in antibiotics. pneumoniae and Salmonella thiphimurium can be exposed followed by gene expression studies to understand the mechanism of resistance and provide a possible way to treat these bacteria.

According to one embodiment of the present invention, the amplification of the present invention is carried out according to PCR (polymerase chain reaction). According to certain embodiments of the present invention, the primers of the present invention are used in gene amplification reactions.

The term “amplification reaction” as used herein refers to a reaction to amplify a nucleic acid molecule. Various amplification reactions have been reported in the art, including polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (RT-PCR; Sambrook et al., Molecular Cloning A Laboratory Manual, 3rd ed Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C et al. (EP 329,822), multiplex PCR (McPherson and Moller, 2000 ), ligase chain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA; WO 88) /10315), self sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence priming polymerase chain Reaction (consensus sequence primed polymerase chain reaction, CP-PCR; U.S. Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR; U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid base Nucleic acid sequence based amplification (NASBA; U.S. Patent Nos. 5,130,238, 5,409,818, 5,554,517 and 6,063, 603), strand displacement 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 09/854,317.

The term “primer” as used herein refers to an oligonucleotide, under conditions under which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of a polymerization agent such as nucleotide and DNA polymerase, and It can act as a starting point for synthesis under conditions of suitable temperature and pH. Specifically, the primer is deoxyribonucleotide and is single-chain. Primers used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also include a ribonucleotide.

The primer should be long enough to prime the synthesis of the extension product in the presence of a polymerizing agent. The suitable length of a primer depends on a number of factors, such as temperature, application and source of the primer.

As used herein, the term “annealing” or “priming” means that oligodioxynucleotides or nucleic acids are juxtaposed to a template nucleic acid, and the juxtaposition means that a polymerase polymerizes nucleotides to be complementary to the template nucleic acid or a portion thereof. To form a typical nucleic acid molecule.

PCR is the most well-known nucleic acid amplification method, and its many modifications and applications 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 chain reaction ( Inverse polymerase chain reaction (IPCR), vectorette PCR and TAIL-PCR (thermal asymmetric interlaced PCR) have been developed for specific applications.

For more information on PCR, see McPherson, MJ, and Moller, SG PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

When the method of the present invention is performed using a primer, a gene amplification reaction may be performed to simultaneously detect target genes in an analysis target (eg, a clinical sample-derived sample). Therefore, the method of the present invention performs a gene amplification reaction using a primer that binds to DNA extracted from a sample.

According to one embodiment of the present invention, a sample that can be used in the method of the present invention is a clinical specimen-derived sample, and the clinical specimen includes sputum, saliva, blood, and urine, but is not limited thereto.

Extraction of DNA from a sample can be carried out according to a conventional method known in the art (see Sambrook, J et al, Molecular Cloning A Laboratory Manual, 3rd ed Cold Spring Harbor Press (2001); Tesniere, C et al. al, Plant Mol Biol Rep, 9:242 (1991); Ausubel, FM et al, Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P et al, Anal Biochem 162:156 (1987)) The primer used in the present invention is hybridized or annealed at one site of the template to form a double-chain structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are 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 the amplification of the present invention, and include the "Klenow" fragment of E coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Specifically, the polymerase is a thermostable DNA polymerase that can be obtained from various bacterial species, and it includes Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Includes.

When carrying out the polymerization reaction, it is preferable to provide the reaction vessel with the components necessary for the reaction in excess. The excessive amount of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the component. It is desirable to provide cofactors such as Mg2+, dATP, dCTP, dGTP and dTTP to the reaction mixture to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer allows all enzymes to approach optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

In the present invention, annealing is performed under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary according to environmental variables.

The term "hybridization" as used herein means that two single-stranded nucleic acids form a duplex structure by pairing of complementary nucleotide sequences. Hybridization may occur when the complementarity between single-stranded nucleic acid sequences is complete (perfect match) or even in the presence of some mismatch bases. The degree of complementarity required for hybridization may vary depending on the conditions of the hybridization reaction, and in particular, may be controlled by temperature. There is no difference between the terms “annealing” and “hybridization” and are used interchangeably in this specification.

According to certain embodiments of the present invention, the method and kit of the present invention can simultaneously detect various antibiotic-resistant strains through real-time PCR.

Real-time PCR is a technique that monitors and analyzes the increase of PCR amplification products in real-time (Levak KJ, et al., PCR Methods Appl., 4(6): 357-62(1995)). The PCR reaction can be monitored by recording the amount of fluorescence emission in each cycle during the exponential phase where 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 increase in fluorescence is observed and has a lower Ct value (cycle threshold).

A distinct increase in fluorescence above the baseline measured between 3-15 cycles indicates the detection of accumulated PCR products.

Compared to conventional PCR methods, real-time PCR has the following advantages: (a) Conventional PCR is measured in a plateau, whereas real-time PCR is performed during the exponential growth phase. Data can be obtained; (b) the increase in reporter fluorescence signal is directly proportional to the number of amplicons generated; (c) the disassembled probe provides permanent record amplification of the amplicon; (d) an increase in detection range; (e) requires at least 1,000 times less nucleic acid than conventional PCR methods; (f) detection of amplified DNA without separation through electrophoresis is possible; (g) increased amplification efficiency can be obtained by using a small amplicon size; And (h) the risk of contamination is low.

When the amount of PCR amplification product reaches the amount detectable by fluorescence, an amplification curve starts to occur, the signal rises exponentially, and then reaches a stagnation state. As the initial amount of DNA increases, the number of cycles for the amount of amplification product to reach the detectable amount decreases, so the amplification curve appears faster. Therefore, when a real-time PCR reaction is performed using a stepwise diluted standard sample, an amplification curve arranged at equal intervals in the order of a large amount of initial DNA is obtained. Here, if a threshold is set at an appropriate point, the Ct value at which the threshold and the amplification curve intersect is calculated.

In real-time PCR, PCR amplification products are detected through fluorescence. The detection method is largely an interchelating method (SYBR Green I method), a method using a fluorescently labeled probe (TaqMan probe method), and the like. Since the interchelating method detects all double-stranded DNA, there is no need to prepare a probe for each gene, so it is possible to construct a reaction system at low cost. While the method using a fluorescently labeled probe is expensive, it has high detection specificity, so that even similar sequences can be distinguished and detected. According to certain embodiments of the present invention, the methods and kits of the present invention utilize the TaqMan probe method.

First, the interchelating method is a method using a double-stranded DNA binding die, and non-specific amplification using a non-sequence-specific fluorescent intercalating reagent (SYBR Green I or ethidium bromide) and amplicon production including a primer-dimer complex Is to quantify. This reagent does not bind to ssDNA. SYBR Green I is a fluorescent die that binds to the minor groove of double-stranded DNA. It shows almost no fluorescence in solution, but is an interchelator that shows strong fluorescence when it binds to double-stranded DNA (Morrison TB, Biotechniques., 24(6): 954-8, 960, 962(1998)). Therefore, since fluorescence is emitted through the binding between SYBR Green I and double-stranded DNA, the amount of amplification product produced can be measured. SYBR Green real-time PCR is accompanied by an optimization process such as melting point or dissociation curve analysis for amplicon identification. Normally, SYBR green is used for a singleplex reaction, but can be used for a multiplex reaction when accompanied by melting curve analysis (Siraj AK, et al., Clin Cancer Res., 8( 12): 3832-40 (2002); And Vrettou C., et al., Hum Mutat., Vol 23 (5): 513-521 (2004)).

The Ct (cycle threshold) value refers to the number of cycles in which the fluorescence generated in the reaction exceeds the threshold value, which is inversely proportional to the logarithm of the initial copy number. Therefore, the Ct value assigned to a particular well reflects the number of cycles in which a sufficient number of amplicons have accumulated in the reaction. The Ct value is the cycle in which an increase in ΔRn is first detected. Rn refers to the size of the fluorescence signal generated during PCR at each time point, and ΔRn refers to the fluorescence emission intensity (normalized reporter signal) of the reporter die divided by the fluorescence emission intensity of the reference die. The Ct value is also called Cp (crossing point) in LightCycler.

The Ct value represents the point at which the system begins to detect an increase in the fluorescence signal associated with the exponential growth of the PCR product in the log-linear phase. This period provides the most useful information about the reaction. The slope of the log-linear step represents the amplification efficiency (Eff) (http://www.appliedbiosystems.co.kr/).

On the other hand, the TaqMan probe is typically a primer (e.g., 20-30 nucleotides) comprising a fluorophore at the 5'-end and a quencher at the 3'-end (e.g., TAMRA or non-fluorescent quencher (NFQ)). ) Is a longer oligonucleotide. Excited fluorescent material transfers energy to a nearby quencher rather than emitting fluorescence (FRET = For fluorescence resonance energy transfer; Chen, X., et al., Proc Natl Acad Sci USA, 94(20): 10756-61( 1997)). Therefore, when the probe is normal, no fluorescence is generated. TaqMan probes are designed to anneal to the internal sites of PCR products. Specifically, the TaqMan probe can be designed with the internal sequence of the target gene segment of the present invention.

The TaqMan probe specifically hybridizes to the template DNA in the annealing step, but fluorescence is inhibited by quencher on the probe. During the extension reaction, the TaqMan probe hybridized to the template is decomposed by the 5'to 3'nuclease activity of the Taq DNA polymerase, and the fluorescent dye is released from the probe, and inhibition by the quencher is released, resulting in fluorescence. In this case, 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 a template-dependent nucleic acid polymerase, the 5'-end of the TaqMan probe is cleaved by the 5'to 3'nuclease activity of this polymerase, A fluorescent signal is generated.

The reporter molecule and quencher molecule bound to the TaqMan probe include a fluorescent substance and a non-fluorescent substance.

Fluorescent reporter molecules and quencher molecules that can be used in the present invention may be any known in the art, and examples thereof are as follows (the number in parentheses is the maximum emission wavelength expressed in nanometers): Cy2 ™ (506), YOPRO™-1 (509), YOYO™-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), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3™ (570), Alexa™ 546 (570), TRITC (572), Magnesium Orange™ (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange™ (576) , Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red™ (590), Cy3.5™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594 (615) ), Texas Red (615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631), Rphycocyanin (642), C-Phycocyanin (648), TO-PRO™-3 (660), TOTO3 (660), DiD DilC(5) (665), Cy5™ (670), Thiadicarbocyanine ( 671), Cy5.5 (694), HEX (556), TET (536), VIC (546), BHQ-1 (534), BHQ-2 (579), BHQ-3 (672), 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) and Quasar 705 (610). The numbers in parentheses indicate the maximum emission wavelength expressed in nanometers. According to certain embodiments of the present invention, the reporter molecule and the quencher molecule comprise a Cy5, ROX, HEX, FAM, BHQ-1, BHQ-2 or Cy5.5-based label.

Suitable reporter-quencher pairs are disclosed in many literatures: Pesce et al., editors, FLUORESCENCE SPECTROSCOPY (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 MOLECULES (Academic Press, New York, 1976); Bishop, editor, INDICATORS (Pergamon Press, Oxford, 1972); Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes, Eugene, 1992); Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Interscience Publishers, New York, 1949); Haugland, R.P., HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Sixth Edition, Molecular Probes, Eugene,Oreg., 1996; U.S. Pat. Nos. 3,996,345 and 4,351,760.

According to certain embodiments of the present invention, the target nucleic acid of the present invention includes a nucleic acid sample derived from a clinical specimen. The method of annealing or hybridizing the target nucleic acid to the extension primer and the probe may be performed by a hybridization method known in the art. In the present invention, suitable hybridization conditions can be determined in a series of processes by an optimization procedure.

This procedure is performed as a series of procedures by a person skilled in the art to establish a protocol for use in the laboratory.

For example, conditions such as temperature, concentration of components, hybridization and reaction time, buffer components, and their pH and ionic strength depend on various factors such as the length of the oligonucleotide and the amount of GC and the target nucleotide sequence. Detailed conditions for hybridization include 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. Typically, DNA polymerases 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. Specifically, template-dependent nucleic acid polymerases are thermostable DNA polymerases that can be obtained from various bacterial species, which are Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Pyrococcus furiosus ( Pfu), Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus rubens, Thermus scotoductus, Thermus thermophilus 17, Thermus species Z05, Thermus species Z05, Thermus thermophilus , Thermotoga neapolitana and Thermosipho africanus DNA polymerase.

The "template-dependent extension reaction" catalyzed by a template-dependent nucleic acid polymerase refers to a reaction for synthesizing a nucleotide sequence complementary to the sequence of a template.

As can be seen through the present invention, the primer pair of the present invention can be used as a useful biomarker for detecting antibiotic-resistant Staphylococcus aureus, Klebsiella pneumoniae, and/or Salmonella Typhimurium, so it is usefully used to detect these multiple antibiotic-resistant strains. I can.

Figure 1 shows the qPCR threshold of Staphylococcus aureus ATCC 15564 (SAWT), ciprofloxacin-induced Staphylococcus aureus ATCC 15564 (SACIP), oxacillin-induced Staphylococcus aureus ATCC 15564 (SAOXA) and Staphylococcus aureus CCARM 3080 (SACCARM)) graph.
Figure 2 is a graph showing the qPCR threshold (Ct) values of Klebsiella pneumoniae ATCC 23357 (KPWT), ciprofloxacin-induced Klebsiella pneumoniae ATCC 23357 (KPCIP) and Klebsiella pneumoniae CCARM 10237 (KPCCARM),
Figure 3 is a graph showing the qPCR threshold (Ct) values of Salmonella Typhimurium ATCC 19585 (STWT), ciprofloxacin-induced Salmonella Typhimurium ATCC 23357 (STCIP) and Salmonella Typhimurium CCARM 8009 (STCCARM).
Figure 4 shows Staphylococcus aureus CCARM 3080 (SACCARM), Staphylococcus aureus ATCC 15564 (SAWT), ciprofloxacin-induced Staphylococcus aureus ATCC 15564 (SACIP), and oxacilloccus aureus ATCC 15564 exposed to ciprofloxacin for 30 minutes with 8 * MIC. A graph showing the qPCR threshold (Ct) value of (SAOXA),
5 is a graph showing the qPCR threshold (Ct) of Klebsiella pneumoniae CCARM 10237 (KPCCARM), Klebsiella pneumoniae ATCC 23357 (KPWT), and ciprofloxacin-induced Klebsiella pneumoniae ATCC 23357 (KPCIP) exposed to ciprofloxacin.
Figure 6 shows the qPCR threshold (Ct) values of Salmonella Typhimurium CCARM 8009 (STCCARM), Salmonella Typhimurium ATCC 19585 (STWT), and ciprofloxacin-induced Salmonella Typhimurium ATCC 23357 (STCIP) exposed to ciprofloxacin for 30 minutes with 8 * MIC. Indicating graph.

Wild-type (WT), ciprofloxacin (CIP) and/or oxacillin (OXA) induction and clinically isolated tolerance (CCARM) S. aureus (SA ^WT , SA ^CIP , SA ^OXA , and SA ^CCARM ), K. pneumoniae ( KP ^WT , KP ^CIP , and KP ^CCARM ), and S. Typhimurium (ST ^WT , ST ^CIP , and ST ^CCARM ) were determined after induction of resistance by ciprofloxacin or oxacillin (Table 1).

The wild-type strain was sensitive to all antibiotics used in the present invention except for erythromycin and novobiosin ^{for ST WT} , ampicillin, erythromycin and novobiosin for ^{KP WT , and cytotaxime and polymyxin B for SA WT.} . SA ^CCARM , KP ^CCARM and ST ^CCARM showed high resistance to most antibiotics, and showed MIC of 512 μg/ml or more (Table 1).

Antibiotic SA ^WT SA ^CIP SA ^OXA SA ^CCARM KP ^WT KP ^CIP KP ^CCARM ST ^WT ST ^CIP ST ^CCARM Tobramycin 4 4 16 >512 4 (S) 2 (S) 256(R) 4(S) 2(S) 16(R) Chloramphenicol 8(S) 16(I) 8(S) 16(I) 8(S) 256(R) 512(R) 8(S) 32(R) 4(S) Erythromycin 0.5(S) 0.5(S) 0.5(S) >512(R) 256(R) 256(R) 256(R) 128(R) 128(R) 128(R) Ciprofloxacin 0.5(S) 32(R) 16(R) 32(R) 0.125(S) 8(R) 16(R) 0.0156(S) 4(R) 0.0156(S) Norfloxacin 1(S) 32(R) 16(R) 32(R) 0.25(S) 16(R) 16(R) 0.0625(S) 16(R) 0.125(S) Tetracycline 1(S) 0.5(S) 0.5(S) 64(R) 4(S) 8(I) 4(S) 1(S) 4(S) 512(R) Ampicillin 16 16 128 64 >512(R) >512(R) >512(R) 8(S) 16(I) >512 (R) Cefotaxime 2 2 16 >512 0.125(S) 0.5(S) 8(R) 0.125(S) 0.5(S) 0.125(S) Novobiocin 0.5 0.25 0.25 One 128 >512 256 512 >512 512 Polymyxin B 256 256 128 256 2 8 8 2 2 2

Table 1 shows Staphylococcus aureus (SA ^WT , SA ^CIP , SA ^OXA , and SA ^CCARM ) ^* , Klebsiella pneumoniae (KP ^WT , KP ^CIP , and KP ^CCARM ) ^* , and Salmonella Typhimurium (ST ^WT , ST ^CIP , and ST ^CCARM ). ^{* It} is a table showing the minimum inhibitory concentration (MICs, μg /ml) of. In the table, ST ^WT , KP ^WT , and SA ^WT are S. Typhimurium ATCC 19585, K. pneumoniae ATCC 23357, and S. aureus ATCC 15564 wild-type strains, respectively. , ST ^CIP , KP ^CIP , SA ^CIP , and SA ^OXA represent ciprofloxacin-induced S. Typhimurium, K. pneumoniae , S. aureus , and oxacillin-induced S. aureus , respectively, ST ^CCARM , KP ^CCARM , and SA OXA, respectively, And SA ^CCARM represent clinically obtained antibiotic resistance S. Typhimurium, K. pneumoniae , and S. aureus , respectively, and S, I, and R represent susceptible, intermediate, and resistant strains, respectively.

Proteins that are uniquely expressed and highly expressed in antibiotic resistant strains have been targeted to detect antibiotic resistant pathogens. The primers were designed based on the proteomics results and their sensitivity and accuracy were verified. The target genes are as follows:

Staphylococcus aureus-

.chaperone protein (dnaK), glyceraldehyde-3-phosphate dehydrogenase (gapA), chaperonin GroEL protein (groEL), leucyl-tRNA synthetase (leuS), formate acetyltransferase (pflB), DNA-directed RNA polymerase beta subunit (rpoB), threonyl -tRNA synthetase 1 (thrS), elongation factor TS (tsf), elongation factor G (fusA), peptidylprolyl isomerase (prsA), Alkyl hydroperoxide reductase protein F (ahpF), phosphoglucosamine-mutase (GlmM), cell division protein (FtsZ) , And alkyl hydroperoxide reductase protein C (ahpC),

Klebsiella pneumoniae-

alcohol dehydrogenase (KPHS_20050), GTP-binding elongation factor family protein (KPHS_39660), heat shock protein 90 (KPHS_07240), succinyl-CoA synthetase alpha subunit (KPHS_15690), 50S ribosomal protein L23 (KPHS_48650), cysteine 10350), cysteine transport protein (KPHS_10) outer membrane protein A precursor (KPHS_18650), putative glycerol dehydrogenase (KPHS_46250), putative small heat shock protein (pKPNCZ_0056), and ferrous iron transporter B, partial (KPHS_49310), and

Salmonella Typhimurium-

outer membrane protein X (ompX), molecular chaperone (dnaK), Rho factor (rho), DNA starvation/stationary phase protection protein (dps), posphopyruvate hydrtase (eno), succinyl-CoA ligase (sucC), ribose-phosphate pyrophosphokinase ( prsA), adenylate kinase (adk), Mn-superoxide dismutase (sodB), DNA-directed RNA polymerase subunit beta (rpoB), alcohol dehydrogenase (adhE), catalase hpII (KatE), pyruvate formate lyase-activating protein (pflE), succinate--CoA ligase subunit alpha (sucC), alkyl hydroperoxide reductase subunit F (ahpF), anaerobic glycerol-3-phosphate dehydrogenase subunit A (glpA), serine protein kinase PrkA (YeaG), and putative oxidoreductase (yhhX).

In the present invention, the relative expression level of the gene used in the present invention was calculated using a comparison method. The CT values of the target genes were compared between antibiotic-sensitive and antibiotic-resistant strains, respectively.

In S. aureus, reference gene (16s rRNA), chaperone protein (dnaK), glyceraldehyde-3-phosphate dehydrogenase (gapA), chaperonine GroEL protein (groEL), leucyl-tRNA synthetase (leuS) , DNA-directed RNA polymerase beta subunit (rpoB), threonyl-tRNA synthetase 1 (thrS), elongation factor TS (tsf), elongation factor G (fusA), peptidylprolyl isomerase (prsA), Alkyl hydroperoxide reductase protein F (ahpF), phosphoglucosamine-mutase (GlmM), cell division protein (FtsZ), and alkyl hydroperoxide reductase protein C (ahpC) genes were transferred between antibiotic sensitive and antibiotic resistant strains. It was compared (Fig. 1).

Stress-related genes (dnaK, groEL, alhF, and ahpC) were relatively overexpressed in multidrug-resistant staphylococcus aureus CCARM 3080 (SACCARM) (Figure 1), indicating that activation of stress-related genes may be due to the development of antibiotic resistance. Suggests. Overexpression of GlmM in SACCARM helps catalyze the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is an early protoplasmic step in peptidoglycan biosynthesis. Upregulated GlmM can lead to high levels of methicillin resistance.

Overexpression of fusA is involved in the resistance of S. aureus to methicillin, linezolide and daptomycin. No significant changes in LeuS, rpoB, ahpF and tsf in gene expression were observed between antibiotic sensitive and antibiotic resistant strains.

The prsA gene is highly overexpressed in multidrug resistant Staphylococcus aureus CCARM 3080 (SACCARM), followed by oxacillin-induced Staphylococcus aureus ATCC 15564 (SAOXA) and ciprofloxacin-induced Staphylococcus aureus ATCC 15564 ( SACIP) and wild-type Staphylococcus aureus ATCC 15564 (SAWT) followed (Figure 1). prsA is a membrane-immobilized peptidyl-prolyl cis-trans isomerase protein capable of stabilizing PPB2a. PBP2a is encoded by the mecA gene on the mobile SCCmec cassette chromosome. Acquisition and stabilization of PBP2a results in the low affinity of the β-lactam antibiotic in methicillin-resistant S. aureus (MRSA). prsA is also detected in oxacillin- and glycopeptide-resistant S. aureus. The expression of prsA is a unique phenomenon in antibiotic resistant Staphylococcus aureus. Thus, the prsA gene can be used as a resistance marker for the detection of multidrug resistant Staphylococcus aureus.

Alcohol dehydrogenase (KPHS_20050), GTP-binding elongation factor family protein (KPHS_39660), heat shock protein 90 (KPHS_07240), succinyl-CoA synthase alpha subunit (KPHS_15690), 50S ribosomal protein L23 (KPHS_48650), cysteine transport protein (KPHS_10350) ), outer membrane protein A precursor (KPHS_18650), putative glycerol dehydrogenase (KPHS_46250), putative small heat shock protein (pKPNCZ_0056) and iron ion transporter B, relative expression of moiety (KPHS_49310) was observed in KPATCC, KPCIP and KPCCARM (Fig. 2).

The expression patterns of target genes varied between antibiotic sensitive and antibiotic resistant strains. The expression of the gene KPHS_39660 was relatively overexpressed in KPCCARM compared to KPWT (Fig. 2). Bacteria need high energy to repair damaged proteins and DNA, or to pump antibiotics from cells. The expressions of the genes KPHS_07240, KPHS_48650, KPHS_10350 and KPHS_49310 did not show a significant difference between antibiotic susceptibility and antibiotic resistance strains. Genes KPHS_20050 and KPHS_18650 did not show significant expression for KPCCARM. However, the putative small heat shock protein (pKPNCZ_0056) was expressed only in KPCCARM. The highly upregulated gene (pKPNCZ_0056) in KPCCARM can be used as a biomarker for detecting antibiotic resistance K. pneumoniae.

Outer membrane protein X (ompX), molecular chaperone (dnaK), Rho factor (rho), DNA starvation/normal phase protection protein (dps), phosphopyruvate hydrase (eno), succinyl-CoA ligase (sucC), Ribose-phosphate pyrophosphokinase (prsA), adenylate kinase (adk), Mn-superoxide dismutase (sodB), DNA-directed RNA polymerase subunit beta (rpoB), alcohol dehydrogenase (adhE), catalase hpII (KatE), pyruvate formate degrading enzyme-activated protein (pflE), succinate-CoA ligase subunit alpha (sucC), alkyl hydroperoxide reductase subunit F (ahpF), anaerobic glycerol-3-phosphate dehydrogenation The relative expression of the enzyme subunit A (glpA), serine protein kinase PrkA (YeaG) and putative oxidoreductase (yhhX)) genes was observed in STWT, STCIP and STCA (Fig. 3).

No significant difference in gene expression was observed between STWT, STCIP and STCCARM (Fig. 3). In order to detect a biomarker for antibiotic-resistant Salmonella Typhimurium, the present inventors performed an antibiotic exposure experiment.

In the present invention, the relative expression of genes was investigated after exposure to 8*MIC ciprofloxacin for 30 minutes to help detect antibiotic-resistant bacteria. Stress-related genes (dnaK and groEL) were overexpressed in multidrug-resistant staphylococcus aureus CCARM 3080 (SACCARM) after exposure to ciprofloxacin (Figure 4), suggesting that activation of stress-related proteins may be due to the development of antibiotic resistance. do.

However, the leuS gene is overexpressed in multidrug resistant Staphylococcus aureus CCARM 3080 (SACCARM) compared to wild-type Staphylococcus aureus ATCC 15564 (SAWT), oxacillin-induced Staphylococcus aureus ATCC 15564 (SAOXA) and ciprofloxacin-induced Staphylococcus aureus. The sequence was ATCC 15564 (SACIP) (Figure 4). The differentially expressed leuS gene after exposure to antibiotics can be used as a biomarker for detecting antibiotic-resistant Staphylococcus aureus. The expression of rpoB, rpsE, thrS and tsf remained almost the same in all strains after exposure to ciprofloxacin. The antibiotic resistance genes aac(6')/aph(2”), qacA and qacB are only detected in antibiotic-resistant Staphylococcus aureus (SACIP, SAOXA and SACCARM) after exposure to antibiotics and thus as detection markers for antibiotic-resistant Staphylococcus aureus. Can be used.

In addition, relative expression of the efflux pump-related genes (acrA and acrB), β-lactamase-related genes (CTX-M), and iron transporter B gene (KPHS_49310) were relatively overexpressed in KPCIP and KPCCARM compared to KPWT (Fig. 5). ) After exposure to ciprofloxacin with 8 * MIC for 30 minutes. The genes arcA and acrB involved in efflux pump activity enhance resistance to antibiotics. Increased CTX-M gene is associated with decreased sensitivity to β-lactam antibiotics in KPCIP and KPCCARM. However, expression of toxicity-related genes (vagC and vagD) and putative small heat shock genes (pKPNCZ_0056) were observed only in KPCCARM after exposure to ciprofloxacin. Therefore, genes highly expressed (vagC, vagD and pKPNCZ_0056) in KPCCARM after exposure to antibiotics can be used as biomarkers for detecting multidrug resistant K. pneumoniae.

Genes related to efflux pumps (acrA, acrB and tolC), outer membrane proteins (ompC and ompX), stress response (rpoS and sodB), protein folding (dnaK and prsA) and energy production (eno, sucC, adK, yhhX and yeaG) are After exposure to ciprofloxacin with 8 * MIC for 30 minutes, it was relatively upregulated in STCCARM and STCIP compared to STWT (FIG. 6 ). ArcAB-TolC is an efflux pump-related gene belonging to the RND family involved in the resistance of bacteria to multiple antibiotics. Changes in the foreign-related genes were directed by the plasmid encoding the antibiotic resistance gene. Interestingly, the yhhX gene is highly overexpressed in multidrug resistant Salmonella typhimurium CCARM 8009 (STCCARM) compared to wild-type Salmonella typhimurium ATCC 19585 (STWT), followed by ciprofloxacin-induced Salmonella typhimurium ATCC 23357 (STCIP) sequence. Was (Fig. 5). Highly upregulated genes (yhhX) in STCCARM and STCIP can be used as biomarkers to detect multidrug resistant Salmonella typhimurium.

Tables 2 and 3 are primer sequences used in real-time analysis for S. aureus

^* F, forward; R, reverse

Tables 4 and 5 are primer sequences used in real-time analysis for K. pneumoniae

^* F, forward; R, reverse

Tables 6 and 7 are primer sequences used in real-time analysis for Salmonella Typhimurium.

^* F, forward; R, reverse

<110> University-Industry Cooperation Foundation, Kangwon National University <120> A COMPOSITION FOR DETECTING ANTIBIOTICS-RESISTANT BACTERIA AND A METHOD FOR DETECTING ANTIBIOTICS-RESISTANT BACTERIA USING THE SAME <130> P19-0147HS <160> 144 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cagaagaagc accggctaac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 aatgcagttc ccaggttgag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cgctgcatta gcgttcaaag 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 accgcagcag cgatatctc 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aggtgacatt gaaacaggtg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tgagcgtgtt actttagttg g 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gggaagatgg aacaatgaca ac 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ttagctagtg gagactcacc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 atacacctgg acacgtagac 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cgatacgtgg aacaccataa g 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gtgacgttca ctactctcac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gctatcttct tcgtcagctg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tcaatggata ctggttcagc 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 acaacctctg atacttcacc a 21 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gtcctgctag tggtagtgc 19 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tgaagttctc aataccagca g 21 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 aagcgcttga acaagaagg 19 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gtccagattg ttctccacc 19 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 aagatgctgc agatgaagac g 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ttccgaatcc agtgctacca g 21 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 tatcgaccca gacggtgttg 20 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 agctgggcat acttcgcca 19 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 acttacacat gggtggacgc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gcaacgtctc ttgctgcttc 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 ggaccaccag cgataactcc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 ccacacacaa ttactggccg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 ttcggaccat agaatgccgc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 taaagaggca gcggatgagc 20 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 tgccacacta tcataaccac tacc 24 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 acatgaatta cacgagggca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 cgcagaaagt gcagagttcg 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 agctaatgca gtggcccttt 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 caattgcacc cggattagcg 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 gccgtaccag ctccaactaa 20 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 ggacgggtga gtaatgtc 18 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 tctcagacca gctagggatc g 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 gaaagcctgg tagaagacct g 21 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 tctgcaccat cggcatacg 19 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 tcttgagtac gatccgaacc 20 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 tgcctgcttt gattgcagc 19 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 gtgctatgat acgctgctgg 20 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 aaaccgttgt ggattgcatg g 21 <210> 43 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 tcgcgtcctc tcctatgac 19 <210> 44 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 cggttcgcct ttcttcacg 19 <210> 45 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 tgttgttctg ggctacacc 19 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 cctttagcaa ccaggtagtc 20 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 cagctcatca acgcatacc 19 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 cagcttcatc gtgtctttac c 21 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 actgagcctt aacgatgcct 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 ggtgatcagg tcgagatcgt 20 <210> 51 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 gattgtggcg aataccgac 19 <210> 52 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 acgcttacgt ccaatcgct 19 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 tgtctgcatt gactccgtgg 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 aatgcgttct catcctcgct 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 gagcagattg cctgccacta 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 agcgtcagat agaggttgcg 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 57 cggtgggaag aagaaaccga 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 58 cttggttccc aagcctttgc 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 59 atgtgacgat aaaccggctc 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 60 ctggcagttc ggtggttatt 20 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 61 cgataacctg atgtacatgt cc 22 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 ccgacaacca tcaggaagct 20 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 63 atttgctgat ttcgctcggc 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 64 cctgcttggc ccgaataaca 20 <210> 65 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 65 tcgatcaaac cgttcagtta gg 22 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 66 accgtagtgt cgcaatcatc 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 67 tgtgagaaaa acggcaacga 20 <210> 68 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 68 ctggcggcga aaagca 16 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 69 gaatggccgg tctctttctt 20 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 70 cctgtcgctg gaaaaagtgt 20 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 71 gcagcaccag taaagtgatg g 21 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 72 gcgatatcgt tggtggtgcc 20 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 73 tcgtctatcc cgacgtcttt 20 <210> 74 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 74 ccagtcgtag gaggtgtact ta 22 <210> 75 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 75 tccctgccct gctggtag 18 <210> 76 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 76 ctggtgtcgc cattggtgg 19 <210> 77 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 77 cgaagaaaga cctccctacc c 21 <210> 78 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 78 cgccgccaat gagataca 18 <210> 79 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 79 tgatgggctt tgtggcttc 19 <210> 80 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 80 cttgctgtca tagtcgct 18 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 81 gcataagcaa caccgacagg 20 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 82 ggcgaagtaa tcgcaacatc c 21 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 83 ctgcaacggc tgtttttaca 20 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 84 gtggttctct ttgcggtagg 20 <210> 85 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 85 aaggcgaatc cagcttgttc agc 23 <210> 86 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 86 tgacgttgca tgttcgcacc catca 25 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 87 gcaggcggcg ctggcgaata 20 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 88 agtcgccaga aagtcaggat 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 89 gctcaaagcg tccttcatcg 20 <210> 90 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 90 gggaagggga cagcatcatt c 21 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 91 cgaagcgcat ctcggcatac 20 <210> 92 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 92 gatgctcgac accaacatct g 21 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 93 aggccttcgg gttgtaaagt 20 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 94 gttagccggt gcttcttctg 20 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 95 caagcatcta cggtgtagtc 20 <210> 96 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 96 gtaagagaag tccagagcaa c 21 <210> 97 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 97 atggtgaaac tctggttggt c 21 <210> 98 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 98 gtacggcatg atagaaacgt c 21 <210> 99 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 99 atcatcaacg aaccgactgc 20 <210> 100 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 100 tcaggtgctt ctactggttc 20 <210> 101 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 101 tcgcgtcctc tcctatgac 19 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 102 ctggtcttta gcgtctttgc 20 <210> 103 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 103 cgcgattgaa cagattgtct c 21 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 104 acaggcataa cggatgatcg 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 105 aaccaacgac aacctgatgg 20 <210> 106 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 106 acaggaagtc agcgacaac 19 <210> 107 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 107 tgtgaaagac gtactgctg 19 <210> 108 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 108 cagtagagaa cacctggct 19 <210> 109 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 109 actctcctgc cataactgc 19 <210> 110 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 110 cgttacgttc ggtgatttcg 20 <210> 111 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 111 aagatgctgc agatgaagac g 21 <210> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 112 tatcaccagt gcggaggttg 20 <210> 113 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 113 agacctgagc gacatcacg 19 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 114 cacgtacggt atcagcagac 20 <210> 115 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 115 ctcattcgtg aaggcgcaac 20 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 116 ccagatacct gccgcattga 20 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 117 cccaggaaga ctgccgtaac 20 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 118 atccacgaca atacccgctt 20 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 119 ctttaaacag ctctgccgcc 20 <210> 120 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 120 gatgagatca tggcgacggt 20 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 121 ttcccacaca tcaaccgtca 20 <210> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 122 cggatggaca tggctggtaa 20 <210> 123 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 123 aagcgaagcg ccgtaaaatc 20 <210> 124 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 124 acccttgtta ccgtgacgac 20 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 125 accggctaac ttccttgacg 20 <210> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 126 ccagaaccgc tttcacgttg 20 <210> 127 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 127 ggacgccaag tccgtagaaa 20 <210> 128 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 128 attgagagcg ggaaactggg 20 <210> 129 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 129 aaaacggcaa agcgaaggt 19 <210> 130 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 130 gtaccggact gcgggaatt 19 <210> 131 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 131 tgaaaaaaat ggaaccgttc ttc 23 <210> 132 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 132 cgaacggcgt ggtgtca 17 <210> 133 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 133 gcccgtgcgc aatatgat 18 <210> 134 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 134 ccgcgttatc caggttgttg 20 <210> 135 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 135 tcgcagcctg ctgaaccaga ac 22 <210> 136 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 136 acgggttgcg ttataggtct gag 23 <210> 137 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 137 catcgtcaaa tggttgttcg 20 <210> 138 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 138 atcttccagt gtcgcagctt 20 <210> 139 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 139 ttgcgagtct gatgtttgtc g 21 <210> 140 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 140 cacgctcacc ggagtaggat 20 <210> 141 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 141 accggctaac ttccttgacg 20 <210> 142 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 142 ccagaaccgc tttcacgttg 20 <210> 143 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 143 cccaggaaga ctgccgtaac 20 <210> 144 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 144 atccacgaca atacccgctt 20

Claims (3)

Multi-antibiotic resistant Salmonella typhimurium (Salmonella Typhimurium) detection kit comprising the primer pair of SEQ ID NO: 127 and 128. The method of claim 1, wherein the antibiotic is tobo bra azithromycin, erythromycin, tetracycline, and multi-antibiotic resistant Salmonella typhimurium (Salmonella Typhimurium) detection kit for, characterized in that one or more antibiotics selected from the group consisting of ampicillin. A method for detecting multiple antibiotic-resistant Salmonella Typhimurium (Salmonella Typhimurium) comprising the step of performing a polymerase chain reaction by extracting DNA from a sample containing bacteria and treating a primer pair of SEQ ID NOs: 127 and 128.

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