KR101775406B1 - A method for detecting both Antibiotic resistance related genes and gram negative bacteria and A composition therefor - Google Patents

Abstract

The present invention relates to a method for simultaneously detecting gram negative bacteria and genes related to resistance to antibiotic agents and a composition thereof. More specifically, the present invention relates to development of PCR-reverse blot hybridization assay (REBA) for simultaneously detecting gram negative bacteria and genes of ESBL, AmpC -lactamase, carbapenemase related to resistance to antibiotic agents. According to the present invention, the REBA method for distinguishing species of gram negative bacteria and identifying whether each gene of ESBLs (TEM-, CTX-M-, SHV-type, OXA-type), AmpC (ACT, CMY2, DHA, SPM, CMY-1 like/MOX, ACC-1, and FOX), and carbapenemases (OXA-48 like, IMP, VIM, NDM, and KPC) has resistance was developed.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method and a composition for simultaneous detection of genes related to Gram-negative bacteria and antibiotic resistance,

The present invention relates to a method and a composition for detecting Gram-negative bacteria and antibiotic resistance-related genes, and more particularly, to a method for detecting Gram-negative bacteria and antibiotic resistance-related genes, and more particularly to a method for the detection of ESBL, AmpC &bgr; -lactamase and carbapenemase genes associated with Gram- Reverse blot hybridization assay.

Antimicrobial resistance of pathogens is a common problem in both advanced and underdeveloped countries, as it is defined as a serious threat to public health by WHO. However, Asian countries including Korea have a higher overall resistance rate of major bacteria than Western countries (Gottlieb T, Nimmo GR, Med J Aust 2012; 194: 281-283.). Recently, antibiotic resistance, which is the most problematic in pneumococcal bacteria and Escherichia coli, is the generation of extended-spectrum β-lactamase (ESBL), which is resistant to a wide range of cephalosporin antibiotics (Song JH, .

ESBL is an enzyme that inactivates the extended-spectrum β-lactam antibiotics such as cefotaxime, ceftazidime, and aztreonam. Recently, the frequency of isolating Enterobacteriaceae in this enzyme has been increasing. These strains are also difficult to detect, and mainly Klebsiella spp. And Escherichia coli. In addition to these two strains, ESBL-producing bacteria such as Citrobacter, Enterobacter, Serratia, Morganella, Pseudomonas, and Salmonella have also been reported. It has been reported that 1.3-8.6% of isolated K. pneumoniae and E. coli produce ESBL, and 7.5-15% and 22.8-38% of E. coli and K. pneumoniae are ESBL-producing strains in Korea (Espinar MJ , Rocha R, Ribeiro M, Goncalves Rodrigues A, Pina-Vaz C. Extended-spectrum β-lactamases of Escherichia coli and Klebsiella pneumoniae screened by the VITEK 2 system J Med Microbiol. 60.).

ESBLs form new enzymes mediated by plasmids. TEM, SHV and AmpC types are well known. In addition, the genes belonging to the 2be group of the Bush et al. Group (1995) were mutated by mutation of TEM-1, TEM-2 and SHV-1 (Bush K, Jacoby G. Updated functional classification of beta-lactamases. Agents Chemother. 2010 Mar; 54 (3): 969-76). TEM-degenerating enzymes caused by one or a few amino acid variations of the TEM-1 protein amino acid sequence have been found to date. Extended-spectrum TEM and SHV enzymes differ from TEM-1, TEM-2 and SHV-1 in that they degrade to third generation cephalosporins and monobactams. Carbapenem, cephamycin and temocillin are not degraded. These enzymes degrade one of the three generations depending on the species. That is, TEM-3 and SHV-2 are resistant to all third-generation cephalosporins, while TEM-10 and TEM-26 degrade ceftazidime better. Since Escherichia coli was first reported to produce SHV-2 in Germany in 1983, many cases of ESBL-producing strains have been reported in Europe and the United States, and many cases have been reported in Korea. ESBL-producing strains are considered to be resistant to all cephalosporin antibiotics because they are likely to show clinical failure even if they are susceptible to cephalosporin in the laboratory. According to recently published domestic data, the ceftazidime resistance rate of pneumococcal bacillus was 25 ~ 35% and the quinolone resistance rate was 30% in 2004 (Lee K, Lim JH, Lee WG, Uh Y, Kim HJ, Yong D, Chong Y. a KONSAR program in 2004. Yonsei Med J 47: 634-645, 2006). In Korea, ESBL-producing pneumococcal bacteremia has been reported to have a mortality rate of 23.3% in the case of ESBL-producing bacteria infection and 20.0% in the case of non-ESBL strain infection (Kim BN, Woo JH , Kim MN, Ryu J, Kim YS J Hosp Infect 52: 99-106, 2002). In the case of mild to moderate infections due to ESBL-producing bacteria, β-lactam / β-lactamase inhibitor or fluoroquinolone may be used depending on the susceptibility results. However, even if they are susceptible to cephalosporin and aztreonam, they are clinically resistant to these antimicrobial agents It is well known that cephalosporin treatment is unsuccessful and is resistant to other antimicrobial agents such as aminoglycoside and cotrimoxazole. Therefore, it is not easy to select antimicrobial agents for the treatment of infectious diseases, but carbapenem is recommended as a selective agent because it is most stable in this enzyme . However, when carbapenem resistance is present, there is virtually no antibiotic that can be safely and effectively treated, and carbapenem-resistant Enterobacteriaceae (CRE) have emerged and this is becoming a global problem (Miriagou V, Cornaglia G, Edelstein M et al. Clin Microbiol Infect 2010; 16: 112-122). For this reason, rapid detection of ESBL-producing strains and resistance to β-lactamase and carbapenem drugs will help to minimize the risks of antibiotic resistance and the use of indiscriminate drugs.

In particular, bacteremia patients are in a very critical condition, so rapid results play a major role in saving the patient's life. Because sepsis can be accompanied by various infectious diseases and is caused by various kinds of bacteria, blood cultures are mainly used to identify causative organisms. Analysis of blood culture results, Identification has provided important information for the treatment of patients. Blood culture is a very important method of bacteremia testing and is an indispensable test to determine the diagnosis, treatment guidelines and prognosis of disease (Aronson MD and Bor DH Blood culture. Ann Intern Med 1987; 106: 246-53). However, blood culture takes more than 5 days. If positive culture signal is detected, it takes more than 10 days to perform Gram stain, identification of the species, and antibiotic susceptibility test. If Gram negative bacteria are detected, especially Enterobacteriaceae The presence of ESBL resistance is confirmed.

The detection of ESBL-producing bacteria is very important for the treatment of infectious diseases, but is not easy. In other words, these ESBL-producing bacteria may not show tolerance to all cefotaxime, ceftazidime, and aztreonam 30 g discs commonly used in microbiology laboratories. In the 1998 revision of NCCLS, cefpodoxime, aztreonam, ceftazidime (22mm), ceftazidime (22mm), cefotaxime (27mm) and ceftriaxone (25mm) The sensitivity of the disc was higher than that of the cefotaxime and ceftriaxone discs. If isolates suspected of ESBL production are isolated, ESBL-producing bacteria can be identified as TEM and SHV by using double disc synergy test, three dimensional test, E test, Vitek ESBL card (Thomson KS, Cornish NE, Hong SG, Hemrick K, Herdt C, Moland ES, J Clin Microbiol 2007 Aug; 45 (8): 2380-4.).

Recently, a method for detecting ESBL-related genes using molecular diagnostic methods such as Real-time PCR, Microarray, and MALDI-TOF MS has been introduced. However, these tests can only detect genes associated with ESBL, AmpC, or carbapenemase, and do not detect all related genes at the same time. Although ESBL tests are available at hospitals in Korea, resistance to antibiotics such as AmpC β-lactamases and carbapenems is low and the tests for detecting them are complex, A rapid detection method is required.

The present invention has been made in view of the above needs, and it is an object of the present invention to provide a method for simultaneous detection of genes associated with novel Gram-negative bacteria and antibiotic resistance.

Another object of the present invention is to provide a composition for simultaneous detection of novel Gram-negative bacteria and genes associated with antibiotic resistance.

In order to accomplish the above object, the present invention provides a method for detecting an antibiotic resistance, comprising the steps of: a) separating DNA from a specimen sample; b) determining whether the antibiotic resistance of the extended-spectrum β- CMY2, DHA, CMY-1-like / MOX, ACC-1, and the like that can be confirmed to be resistant to the antibiotics TEM-, CTX-M-, SHV-type and OXA-type genes, AmpC beta lactamase antibiotics PCR amplification of OXA-48-like, IMP, VIM, NDM, SPM, and KPC genes, 16s rRNA and Gram-negative bacteria-specific gene fragments capable of confirming FOX gene, carbapenem resistance, and c) An extended-spectrum beta-lactamase (ESBL) oligomer probe for gene detection, an oligomer probe for gene detection to confirm the resistance to AmpC beta lactamase antibiotics, a carbapenem resistance Oligo probe for gene detection, oligo probe for detecting Gram-negative bacteria gene, oligo probe for detecting E. coli or Shigella genus gene, oligo probe for detecting Shigella gene, oligo probe for detecting Salmonella genus gene, gene for Klebsiella pneumoniae strain An oligo probe for detection of Klebsiella oxytoca strain gene, an oligo probe for detection of Pseudomonas aeruginosa strain gene, an oligo probe for detection of a strain of Serratia marcescens, an oligo probe for detection of Morganella genus gene, an oligo probe for detection of a strain of Acinetobacter baumannii, And a PCR-reverse blot hybridization step of hybridizing a solid support to which an oligo probe for detecting Haemophilus influenzae strain gene is attached and the PCR amplification product obtained in step b)

Herein, primers for amplifying a gene fragment capable of confirming antibiotic resistance with extended-spectrum beta-lactamase (ESBL) using the primers from the DNA include SEQ ID NOs: 1 to 2, 4 to 5, 7 to 8, 10 to 11, 13 to 14, 16 to 17 and 19 to 20,

 The primers for amplifying the gene fragment capable of confirming resistance to the AmpC beta lactamase antibiotic are primers of SEQ ID NOs: 22 to 23, 25 to 26, 28 to 29, 31 to 32, 34 to 35, and 37 to 38,

The primer for amplifying the gene fragment capable of confirming the carbapenem resistance is a primer of SEQ ID NOs: 40 to 41, 43 to 44, 46 to 47, 49 to 50, 52 to 53, and 55 to 56,

 The primers for amplifying the Gram-negative bacteria-specific gene fragment are the primers of SEQ ID NOs: 60 to 61,

Wherein the primer for amplifying the 16s rRNA gene fragment is a primer set forth in SEQ ID NOS: 62 to 63. The present invention also provides a method for simultaneously diagnosing Gram-negative bacteria, ESBL, AmpC beta lactamase antibiotic and carbapenem resistance.

In one embodiment of the present invention, the oligomer probe for gene detection which can confirm the antibiotic resistance of the extended-spectrum beta-lactamase (ESBL) is SEQ ID NOS: 3, 6, 9, 12, 15 , 18 and 21, respectively,

24, 27, 30, 33, 36 and 39 are oligonucleotide probes for gene detection that can confirm the resistance to AmpC beta lactamase antibiotics,

42, 45, 48, 51, 54, and 57, wherein the oligopeptide probe for gene detection that can confirm the carbapenem resistance is a probe of SEQ ID NOS: 42, 45, 48,

The Gram negative bacterial gene detection probe is the probe of SEQ ID NOs: 64 to 65,

The oligo probe for detecting E. coli or Shigella genus gene is SEQ ID NO: 66, the oligopeptide for detecting Shigella gene is SEQ ID NO: 67, the oligopeptide for detecting Salmonella genus gene is SEQ ID NO: 68, the oligo probe for detecting Klebsiella pneumoniae strain gene SEQ ID NO: 69, Klebsiella oxytoca strain gene detection oligo probe is SEQ ID NO: 70, Pseudomonas aeruginosa strain gene detection oligo probe is SEQ ID NO: 71, Oligonucleotide probe for detecting Serratia marcescens gene is SEQ ID NO: 72, Morganella sp. The oligonucleotide probe of SEQ ID NO: 73, the oligonucleotide probe of the Acinetobacter baumannii gene, and the probe of SEQ ID NO: 75, respectively, for the gene probe for detecting the gene of Haemophilus influenzae strain gene are not limited thereto.

In another embodiment of the present invention, the oligomer probe for gene detection which can confirm the antibiotic resistance of the extended-spectrum beta-lactamase (ESBL) is SEQ ID NO: 78 to 80, 83 to 84, 89 And at least one probe selected from the group consisting of probes having a nucleotide sequence of 92 to 97, but is not limited thereto.

In another embodiment of the present invention, it is preferable, but not limited, that the method further comprises primers of SEQ ID Nos. 58 to 59 as an internal control primer.

1 to 2, 4 to 5, 7 to 8, 10 to 11 for amplifying a gene fragment capable of confirming resistance to an antibiotic resistance of extended-spectrum beta-lactamase (ESBL) , SEQ ID NOs: 22 to 23, 25 to 26, 28 to 29, 31 to 31, SEQ ID NO: 23 to 25, 26 to 28 and 29 to 31 for amplifying a gene fragment capable of confirming resistance to AmpC beta lactamase antibiotics SEQ ID NOS: 40 to 41, 43 to 44, 46 to 47, 49 to 50, 52 to 53 for amplifying the primer pair, 32, 34 to 35, and 37 to 38 primer pairs, And primer pairs 55 to 56, primer pairs of SEQ ID Nos. 60 to 61 for amplifying Gram-negative bacteria-specific gene fragments, and primer pairs of SEQ ID NOS: 62 to 63 for amplifying 16s rRNA gene fragment, And ESBL, AmpC beta lactamase antibiotic, and carbapenem resistance.

In one embodiment of the present invention, the composition comprises an oligomer probe for gene detection which can confirm the resistance to ESBL antibiotics, AmpC beta lactamase antibiotic resistance test An oligo probe for detecting a Gram-negative bacterium gene, an oligo probe for detecting E. coli or Shigella genus gene, an oligo probe for detecting a Shigella germ gene, a gene probe for gene detection, Oligo probe for detecting Salmonella spp. Gene, oligo probe for detecting Klebsiella pneumoniae gene, oligo probe for detecting Klebsiella oxytoca strain gene, oligo probe for detecting Pseudomonas aeruginosa strain gene, oligo probe for detecting gene of Serratia marcescens, Morganella spp. But are not limited to, an oligo probe for self-detection, an oligo probe for detecting Acinetobacter baumannii strain gene, and an oligo probe for detecting Haemophilus influenzae strain gene.

In another embodiment of the present invention, the oligomer probe for gene detection which can confirm the antibiotic resistance of the extended-spectrum beta-lactamase (ESBL) is SEQ ID NOS: 3, 6, 9, 12, 15 , 18, and 21, wherein the oligonucleotide probe for detecting a gene capable of confirming resistance to the AmpC beta lactamase antibiotic is a probe of SEQ ID Nos. 24, 27, 30, 33, 36, and 39, The oligomer probe for identifying a gene is a probe of SEQ ID NOS: 42, 45, 48, 51, 54, and 57, the probe for detecting a Gram negative organism gene is a probe of SEQ ID NOS: 64 to 65, and the E. coli or Shigella The oligo probe for genetic gene detection is SEQ ID NO: 66, the oligopeptide for Shigella gene detection is SEQ ID NO: 67, the oligopeptide for detecting Salmonella genus gene is SEQ ID NO: 68, the Klebsiell the oligo probe for the detection of a pneumoniae strain gene is SEQ ID NO: 69, the oligo probe for Klebsiella oxytoca strain gene detection is SEQ ID NO: 70, the oligo probe for Pseudomonas aeruginosa strain gene detection is SEQ ID NO: 71 and the oligo probe for detecting Serratia marcescens strain gene is SEQ ID NO: 72, an oligo probe for detecting Morganella genus strain gene is SEQ ID NO: 73, an oligo probe for detecting Acinetobacter baumannii strain gene is SEQ ID NO: 74, and an oligo probe for detecting Haemophilus influenzae strain gene is a probe of SEQ ID NO: 75 .

In another embodiment of the present invention, the oligomer probe for gene detection which can confirm the antibiotic resistance of the extended-spectrum beta-lactamase (ESBL) is SEQ ID NO: 78 to 80, 83 to 84, 89 And at least one probe selected from the group consisting of probes having a nucleotide sequence of 92 to 97, but is not limited thereto.

1 to 2, 4 to 5, 7 to 8, 10 to 11 for amplifying a gene fragment capable of confirming resistance to an antibiotic resistance of extended-spectrum beta-lactamase (ESBL) , SEQ ID NOs: 22 to 23, 25 to 26, 28 to 29, 31 to 31, SEQ ID NO: 23 to 25, 26 to 28 and 29 to 31 for amplifying a gene fragment capable of confirming resistance to AmpC beta lactamase antibiotics SEQ ID NOS: 40 to 41, 43 to 44, 46 to 47, 49 to 50, 52 to 53 for amplifying the primer pair, 32, 34 to 35, and 37 to 38 primer pairs, And primer pairs 55 to 56, primer pairs of SEQ ID Nos. 60 to 61 for amplifying Gram-negative bacteria-specific gene fragments, and primer pairs of SEQ ID NOS: 62 to 63 for amplifying 16s rRNA gene fragment, And ESBL, AmpC beta lactamase antibiotic and carbapenem resistance.

In one embodiment of the present invention, the kit comprises an oligomer probe for detecting a gene capable of confirming resistance to ESBL antibiotics, AmpC beta lactamase antibiotic resistance test An oligo probe for detecting a Gram-negative bacterium gene, an oligo probe for detecting E. coli or Shigella genus gene, an oligo probe for detecting a Shigella germ gene, a gene probe for gene detection, , Oligo probe for detecting Salmonella spp. Gene, oligo probe for detecting Klebsiella pneumoniae gene, oligo probe for detecting Klebsiella oxytoca strain gene, oligo probe for detecting Pseudomonas aeruginosa strain gene, oligo probe for detecting Serratia marcescens strain gene, An oligope probe for detection, an oligo probe for detecting Acinetobacter baumannii strain gene, and an oligo probe for detecting Haemophilus influenzae strain gene,

3, 6, 9, 12, 15, 18 and 21, the oligomer probe for gene detection which can confirm the antibiotic resistance of the extended-spectrum beta-lactamase (ESBL) 24, 27, 30, 33, 36, and 39, the probe for detecting a gene capable of confirming resistance to AmpC beta lactamase antibiotics is a probe of SEQ ID NOS: 24, 27, 30, 33, 36 and 39. The oligomer probe for gene detection, Wherein said probe for Gram negative bacterial gene detection is a probe of SEQ ID NOS: 64 to 65, said oligo probe for detecting E. coli or Shigella genus gene is selected from the group consisting of SEQ ID NOs: 42, 45, 48, 51, 54, No. 66, Oligonucleotide probe for Shigella gene detection is SEQ ID NO: 67, Oligo probe for detecting Salmonella genus strain gene is SEQ ID NO: 68, Oligonucleotide for detecting Klebsiella pneumoniae strain gene SEQ ID NO: 69 for the high probe, SEQ ID NO: 70 for the Klebsiella oxytoca strain gene detection oligonucleotide, SEQ ID NO: 71 for the Pseudomonas aeruginosa strain gene detection oligonucleotide, SEQ ID NO: 72 for the Oligo probe for the Serratia marcescens strain gene, The oligo probe for detection is preferably SEQ ID NO: 73, the oligope probe for Acinetobacter baumannii strain gene detection is SEQ ID NO: 74, and the oligo probe for detecting Haemophilus influenzae strain gene is a probe of SEQ ID NO: 75.

In another embodiment of the present invention, the oligomer probe for gene detection which can confirm the antibiotic resistance of the extended-spectrum beta-lactamase (ESBL) is SEQ ID NO: 78 to 80, 83 to 84, 89 And at least one probe selected from the group consisting of probes having a nucleotide sequence of 92 to 97, but is not limited thereto.

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

The term " amplification reaction " as used herein refers to a reaction to amplify a nucleic acid molecule. A variety of amplification reactions have been reported in the art and include polymerase chain reaction (PCR) (US Patent Nos. 4,683,195, 4,683,202, and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR; The method of Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), the multiplex PCR (McPherson and Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press Moller, 2000), ligase chain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification , 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 p 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 sequence-based amplification 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 may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617, and U.S. Patent No. 09 / 854,317.

As used herein, the term " primer " means an oligonucleotide in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of a polymerizing agent such as a nucleotide and a DNA polymerase, It can act as a starting point for synthesis at suitable temperature and pH conditions. Specifically, the primer is a deoxyribonucleotide and a single strand. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides.

The primer should be long enough to be able to prime the synthesis of the extension product in the presence of the polymerizing agent. The appropriate length of the primer is determined by a number of factors, such as temperature, application, and the source of the primer.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof 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, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

When the method of the present invention is carried out using a primer, target genes can be simultaneously detected in an analysis target (for example, a clinical specimen-derived sample) by performing a gene amplification reaction. Therefore, in the method of the present invention, a gene amplification reaction is performed using a primer that binds to DNA extracted from a sample.

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

Extraction of DNA from the sample can be carried out according to conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); (1987) and Chomczynski, P. et < RTI ID = 0.0 > et al., & 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-stranded structure. Nucleic acid hybridization conditions suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization < RTI ID = 0.0 > (" , A Practical Approach, IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used in the amplification of the present invention and include "Klenow" fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Specifically, the polymerase is a thermostable DNA polymerase from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus .

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desirable to provide the reaction mixture with a joinder such as Mg2 +, dATP, dCTP, dGTP and dTTP 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, buffers make all enzymes close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reaction without changing conditions such as the addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

Hereinafter, the present invention will be described.

The present invention distinguishes between species of Gram-negative bacteria and can be used for ESBLs (TEM-, CTX-M-, SHV-type), OXA-type beta-lactamase, AmpC (ACT, CMY2, DHA, CMY- 1, and FOX), and carbapenemases (OXA-48 like IMP, VIM, NDM, SPM, and KPC) And evaluated its usefulness.

Hereinafter, the present invention will be described in detail.

PCR  Amplification result

The 16s rRNA that can identify Gram-negative bacteria was 280 bp, TEM 360 bp, CTX-M1 120 bp, CTX-M9 117 bp, SHV 130 bp, ACT 140 bp, CMY-2 140 bp, DHA 135 bp, CMY-1 / MOX 128bp, OXA-1 124bp, OXA-2 127bp, OXA-48 120bp, IMP 126bp and KPC 125bp, respectively.

Sensitivity specificity results using standard strains

PCR-REBA was performed to confirm the reactivity of the bacterial-specific oligonucleotide probes prepared using the PCR products amplified through the standard strains (Table 1). As a result, it was confirmed that each DNA sample reacted only to the probe of the target and no other cross reaction occurred (Fig. 2).

p T-AADAOXA-48 VIM IMP NDM KPC B s- - - - - Bi - - - - - - - - - - - - - - - Bn - - - - - - - - - - - - - - - - - - - - Bn - - - - - Bn - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Xs- - - - - - - - - - - - - - - - - - - - Cp- - - - - Cp- - - - - - - - - - pi - - - - - pi - - - - - - - - - - - - - - - . - - - - - el + - - - - 4+ - - - - r - + - - - r - + - - - . - - - + - - - - - + shrciEclEcl NNNNNNNNNNNND ND ND ND ND EclEcl NNNNNNNNNNNND ND ND ND ND a. eoeeEtrbcesp NNNNNNNNNNNND ND ND ND ND irbceCfeni GangtvNNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND SfenrSielsp NNNNNNNNNNNND ND ND ND ND SsneSielsp NNNNNNNNNNNND ND ND ND ND iND ND ND ND ND ml. yhSloelsp NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND i ND ND ND ND ND ND ND ND ND ND iND ND ND ND ND Pvlai GangtvNNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND PargnsPargns NNNNNNNNNNNND ND ND ND ND ampiuHifunaHifuna NNNNNNNNNNNND ND ND ND ND b.amni NNNNNNNNNNNND ND ND ND ND elriLaeabxlt GangtvNNNNNNNNNNNND ND ND ND ND eND ND ND ND ND ND ND ND ND ND Saru NNNNNNNNNNNNND ND ND ND ND Saru NNNNNNNNNNNNND ND ND ND ND Saru NNNNNNNNNNNNND ND ND ND ND Saru NNNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Sheoyiu NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND o. ia NNNNNNNNNNNND ND ND ND ND ND Erfiou NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND Edrn NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Efeai NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Efaecn NNNNNNNNNNNND ND ND ND ND ND Egliau NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND Trimmed NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND tetccuSpemna NNNNNNNNNNNND ND ND ND ND ND Saaata NNNNNNNNNNNND ND ND ND ND ND Spoee NNNNNNNNNNNND ND ND ND ND ND Sslvru NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND irccuMltu NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND Mceoa NNNNNNNNNNNND ND ND ND ND ND Mgsr NNNNNNNNNNNND ND ND ND ND ND Mitaellr NNNNNNNNNNNND ND ND ND ND ND Mknai NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND Mnnhooeiu NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND Msrflcu NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND Mtra NNNNNNNNNNNND ND ND ND ND ND Mtiil NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND Mglu NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Mfaecn NNNNNNNNNNNND ND ND ND ND ND Mprgiu NNNNNNNNNNNND ND ND ND ND ND Mspiu NNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NNNNNNNNNNNNNNNND ND ND ND Tmnarpye NNNNNNNNNNNNNNNND ND Ttusrn NNNNNNNNNNNNNNNND ND Tvruou NNNNNNNNNNNNNNNND ND ND ND Mgsu NNNNNNNNNNNNNNNND ND ND ND dot. lcou NNNNNNNNNNNNNNNND ND addCabcn NNNNNNNNNNNNNNNND ND Cabcn NNNNNNNNNNNNNNNND ND Ctoiai NNNNNNNNNNNNNNNND ND Cgart NNNNNNNNNNNNNNNND ND Cprpioi NNNNNNNNNNNNNNNND ND Clstna NNNNNNNNNNNNNNND ND ND ND ND ND Ckue NNNNNNNNNNNNNNND ND ND eiiluPcmmet NNNNNNNNNNNNNNND ND ND Ppnu NNNNNNNNNNNNNNND ND ND ND ND ND ND ND ND Acaau NNNNNNNNNNNNNNND ND ND ND ND ND ND ND ND Afau NNNNNNNNNNNNNNND ND ND Atmr NNNNNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND ND ND ND oaeKomr NNNNNNNNNNNNNNND ND ND ND ND ND ND ND ND ND ND ND bii Arpn NNNNNNNNNNNNNNND ND ND euei Bbsin NNNNNNNNNNNNNNND ND ND

Table 1 shows the specificity of PCR-REBA for detecting ESBL resistance in 21 ESBL, 7 AmpC β-lactamases, and 6 carbapenemases-producing strains and 119 standard strains

In order to examine the sensitivity of PCR-REBA to 18 probes (4 ESBL, 2 β-lactamase, 6 AmpC β-lactamase, 6 carbapenemases), each Escherichia coli-ESBL strain-resistant DNA was diluted 10 times , 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg). As a result, the detection limit of PCR-REBA was 100 to 10 fg / reaction (Fig. 3).

To further investigate the sensitivity of real-time PCR to 18 probes (4 ESBL, 2 β-lactamase, 6 AmpC β-lactamase, 6 carbapenemases), each strain of Escherichia coli-ESBL strain was diluted 10-fold 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg). The results were confirmed with SYBR green. As a result, the detection limit of PCR-REBA was about 100 fg / reaction (Fig. 4).

To further confirm the specificity of PCR-REBA, ESBL-resistant primers and probes were tested for total 153 (34 ESBL producing strains, 84 gram negative-positive strains, and 35 fungal strains) The remaining standard strains were not detected in resistant genes (Table 1).

β- lactamase  Clinical Generating The solid culture strain  Used PCR - Of REBA  Usability evaluation

The PCR-REBA assay was performed with 327 strains identified as ESBL-producing isolates by VITEK 2 system and boronic acid (BA) disk test (Table 2). Among the strains producing β-lactamase, 196 (60%) were E. coli, 120 (36.7%) were Klebsiella pneumonia, 4 (1.2%) were Proteus mirabilis, 3 (0.9%) were Klebsiella oxytoca, 2 %) Enterobacter cloacae, 1 (0.3%) Enterobacter aerogenes, and 1 (0.3%) Providencia stuartii, respectively. PCR-REBA results for ESBL detection showed that 327-β-lactamase-producing strains were detected in the strain. Multidrug resistance (MDR, two or more β-lactamases genes) was detected in 56.3% (n = 184) between ESBL-producing strains (109 (55.6%) E. coli and 75 (62.5%) Klebsiella spp. Respectively.

The detection rate of ESBL-producing strains was 85.4% in ESBL-related genes (TEM, CTX-M groups, and SHV) and 13.7% of AmpC β-lactamase genes (DHA, CMY-2, and ACT ), and 0.9% carbapenemases genes (NDM, VIM, IMP, and KPC), respectively.

Of the 196 E. coli strains, 185 (94.4%) were positive for the ESBL gene, 7 (3.6%) for the ESBL and AmpC β-lactamase genes, and 4 (2%) for the ESBL and carbapenemases genes Respectively. In 120 K. pneumoniae strains, 67 (55.8%) were assigned to the ESBL and AmpC β-lactamase genes, 35 (25%) to the ESBL gene, 16 (13.3%) to the AmpC β-lactamase gene and 1 %) Were resistant to ESBL and carbapenemases, and 1 (0.8%) were resistant to all genes. 4 (100%) of P. milabilis and 3 (100%) of K. oxytoca were found to be ESBL genes (Fig. 5). In particular, the peculiarity of PCR-REBA-ESBL is the ability to distinguish ESBLs from TEM-SHV β-lactamases. TEM and SHV were detected in E. coli and Klebsiella spp. It is important to distinguish between genes that are commonly found among species, or because TEM-1 and SHV-1 bases are replaced by other types, as some of these types represent ESBLs. In this test, the resistance regions of each gene were searched for and found in the TEM with a high frequency, and Gln39Lys (CAG ==> AAG), Glu104Lys (GAG ==> AAG), Arg164Ser (CGT ==> AGT), Arg164His ), Gly238Ser (GGT ==> AGT), Glu240Lys (GAG ==> AAG) and SHI in Leu35Gln (CTA ==> CAA), Asp179 (GAC ==> GCC, AAC, GGC), Gly238Ser AGC) / Ala (GGC ==> GCC), Glu240Lys (GAG ==> AAG, GAA ==> AAA) probes were added. In the case of TEM, the types classified as ESBL were TEM-52, -66, and so on. Most of Glu104Lys and Gly238Ser were found in the resistant region of the gene.

In addition, in the case of SHV, SHV-12 and 2a were the most abundant type of ESBL. Gly238Ser was the resistance region of the gene, and the same results were confirmed by sequencing.

In addition, 25 isolates from 101 strains that were positive for TEM in E. coli 196 strain producing 327-β-lactamase were identified as non-ESBL, and Klebsiella pneumoniae Of the 119 strains that were positive for SHV in 120 strains, 90 strains were identified as non-ESBL strains.

In addition, the test was carried out by real-time PCR using SYBR green and a specific primer, another molecular diagnostic method, to confirm the results of PCR-REBA. First, real-time PCR was performed with an internal control to confirm the amplification of all strains. As a result, Ct values were within the range of 26.21-33.38 (SD ± 1.4) in all strains, and ESBL, AmpC β-lactamase, The carbapenemases gene was found to be 94.8% (310/327) positive and less sensitive than PCR-REBA (Table 2).

Table 2 shows the results of PCR-REBA and real-time-PCR for detecting ESBL, AmpCβ-lactamases, and carbapenemases genes in 327 ESBLs-producing strains

Usefulness of PCR-REBA for detection of β-lactamase gene in blood culture

A total of 400 (200 blood culture positive and 200 blood culture negative) blood cultures from patients suspected of blood infection were used to evaluate the usefulness of PCR-REBA (Figure 6). (N = 120) gram-positive bacteria, 36.5% (n = 73) Gram-negative bacteria and 3.5% (n = 7) Candida were isolated by conventional methods in 200 blood culture positive samples and 200 Blood culture negative, 120 Gram positive, and 7 Candida were not detected and showed 100% specificity. For Gram-negative bacteria, 33 E. coli, 13 K. pneumonia, 6 Pseudomonas aeruginosa and Acinetobacter baumannii , 4 Enetrobacter cloacae, 1 Citrobacter freundii , Providencia stuartii , Enetobacter aerogenes , Serratia marcescens , Providencia The isolates were divided into two groups , namely , rettgeri , Klebsiella oxytoca , Proteus mirabilis, Campylobacter spp., Acinetobacter lowffii , Sphingomonas paucimobilis and Burkholderia cepacia . All of them except Sphingomonas paucimobilis showed the same results.

In addition, the detection of ESBL-producing genes was 12 (16.4%) by the VITEK 2 system, and 11 (91.7%) of them were positive by PCR-REBA. .

The CTX-M (CTX-M1, CTX-M9) genotypes were involved in both the VITEK 2 system and PCR-REBA positive specimens. VITEK 2 system, but 5 additional samples (3 E. cloacae, 1 C. freundii and P. stuartii) in PCR-REBA were positive for AmpCβ-lactamases, but the hospital tested for AmpCβ-lactamases No results were obtained and sequence analysis showed the same results. SHV-11 (n = 5) and SHV-115 (n = 5) were detected in 11-K pneumoniae and 10 E. coli TEM, respectively, 3), SHV-151 (n = 2), and SHV-61 (n = 1) and TEM-1 genotypes were confirmed and the PCR-REBA confirmed the ESBL genotype of TEM-SHV, It is possible to detect all the genes that produce β-lactamase.

Genus and species Totalno.
of samples (%) Detection of b-lactamase gene, n (%) Microscan PCR-REBA Real-time PCR Sequence analysis Positive Negative Positive Negative Positive Negative Positive Negative E. coli 33 (45.2) 11 (33.3) 22 (66.7) 10 (30.3) 23 (69.7) 20 (60.6) 13 (39.4) 10 (30.3) 23 (69.7) Klebsiellaspp. 14 (19.1) 1 (7.1) 13 (92.9) 1 (7.1) 13 (92.9) 12 (85.7) 2 (14.3) 1 (7.1) 13 (92.9) Acinetobacterspp. 7 (9.6) NA NA 0 (0) 7 (100) 0 (0) 7 (100) 0 (0) 7 (100) Pseudomonas aeruginosa 6 (8.2) NA NA 0 (0) 6 (100) 0 (0) 6 (100) 0 (0) 6 (100) Enetrobacterspp. 5 (6.8) 0 (0) 5 (100) 3 (60) 2 (40) 3 (60) 2 (40) 3 (60) 2 (40) Providenciaspp. 2 (2.7) 0 (0) 2 (100) 1 (50) 1 (50) 1 (50) 1 (50) 1 (50) 1 (50) Citrobacter freundii 1 (1.4) 0 (0) 1 (100) 1 (100) 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) Serratia marcescens 1 (1.4) 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 1 (100) Proteus mirabilis 1 (1.4) 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 1 (100) Campylobacterspp. 1 (1.4) NA NA 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 1 (100) Sphingomonas paucimobilis 1 (1.4) NA NA 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 1 (100) Burkholderia cepacia 1 (1.4) NA NA 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 1 (100) Total 73 (100) 12 (16.4) 45 (61.6) 16 (21.9) 57 (78.1) 37 (50.7) 36 (49.3) 16 (21.9) 57 (78.1)

Table 3 is a comparison table of the results of conventional methods and molecular diagnostics for ESBL detection in blood cultures containing 73 Gram-negative bacteria, and the abbreviation NA is not available (not available)

Target and primer name Necleotide sequence (5'-3 ') Size ESBLs CTXM1-210-F (SEQ ID NO: 1) GATGTGCAGCACCAGTAAAGTGATG 122bp CTXM1-332-R (SEQ ID NO: 2) ATCGGATTATAGTTAACMARGTCAGATTT CTXM-M15-258P (SEQ ID NO: 3) AAGTGAAAGCGAACCGAATCTGTTA CTXM2-15-213F (SEQ ID NO: 4) GTGCAGYACCAGTAARGTGATG 116bp CTXM2-261P (SEQ ID NO: 5) AA CGAGAGCGATAAGCACCTGC CTXM2-329R (SEQ ID NO: 6) GGGATTGTAGTTAACCAGGTCGCT CTXM9-210-F (SEQ ID NO: 7) TGTGCAGTACCAGTAAAGTTATGGCG 117bp CTXM9-327-R (SEQ ID NO: 8) ATTGTAGTTAACCAGATCGGCAGGC CTXM9-261P (SEQ ID NO: 9) GAAACGCAAAAGCAGCTGCTT TEM 127-F (SEQ ID NO: 10) GGTTACATCGAGCTGGATCTCAACA 360bp TEM 488-R (SEQ ID NO: 11) CAACGATCAAGGCGGGTTACAT TEM-441-P (SEQ ID NO: 12) GTGCGGTATTATCCCGTGTTGA SHV147-F (SEQ ID NO: 13) GCAAATTAAACAAAGCGAAAGCCA 133bp SHV280-R (SEQ ID NO: 14) GCACTACTTTAAAGGTGCTCATCATGG SHV-189-P (SEQ ID NO: 15) CATGATAGAAATGGATCTGGCCAG OXA1-65-F (SEQ ID NO: 16) AGCGCCAGTGCATCAACAGAT 136bp OXA1-201-R (SEQ ID NO: 17) CATTTGCGTTGCACACTTTGCT OXA1-30-138P (SEQ ID NO: 18) TTACGATGCATCCACAAACGCT OXA2-131-F (SEQ ID NO: 19) TAGTTGTGGCAGACGAACGCC 127bp OXA2-258-R (SEQ ID NO: 20) GCCTGCATCAAGTGCAAAAAGTG OXA2-176P (SEQ ID NO: 21) TTGATCCTGTGCGATCGAAGAA AmpC ACT-MIR-416-F (SEQ ID NO: 22) TACAGGTRCCGGATGAGGTCACG 141bp ACT-MIR-557-R (SEQ ID NO: 23) GAAGGTTTGACCGCCAGCGC ACT-MIR-464-P (SEQ ID NO: 24)   ATCAAAACTGGCAGCCGCAG CMY2-261-F (SEQ ID NO: 25) AAGACGTTTAACGGCGTGTTGG 140bp CMY2-401-R (SEQ ID NO: 26) GCCGTATAGGTGGCTAAGTGCAG CMY2-328-P (SEQ ID NO: 27) ACGAAATACTGGCCAGAACTGACA DHA-261-F (SEQ ID NO: 28) TTTCACAGGTGTGCTGGGTGC 135bp DHA-396-R (SEQ ID NO: 29) GGTATAGGTAGCCAGATCCAGCAATG DHA-299-P (SEQ ID NO: 30) AAGAGATGGCGCTGAATGATCC ACC-1-397F (SEQ ID NO: 31) ACGT AGCGTAACAAGTCGCTGGAG 125bp ACC-1-522R (SEQ ID NO: 32) TGACCAGATGGAAAACTATGCGTG ACC-1-439p (SEQ ID NO: 33) GCTTCGTTACCCAAAATCTCCATATTC CMY1 / MOX-128F (SEQ ID NO: 34)  CTGCTCAAGGAGCACMGGATC 127bp CMY1 / MOX-255R (SEQ ID NO: 35) CTCGAACAGGGTCTGCTCGCT CMY1 / MOX-172P (SEQ ID NO: 36) A AAGGATGGCAAGGCCCACTA Fox-182F (SEQ ID NO: 37) ATTTCAACTATGGGGTTGCCAACC 147bp Fox-315R (SEQ ID NO: 38) TGGCTCACCTTGTCATCCAGC Fox-239P (SEQ ID NO: 39) TGTTCGAGATTGGCTCGGTCAG Carbapenemases IMP-263F (SEQ ID NO: 40) TAAAAGGCAGTATYTCCTCWCATTT 126bp IMP-389R (SEQ ID NO: 41) ACCTTACCGTCTTTTTTAAGMAGTTCA IMP-307P (SEQ ID NO: 42) GGAATAGAGTGGCTTAATTCTCRATCTAT VIM-136F (SEQ ID NO: 43) CTTTACCAGATTGCCGATGGTGTT 128bp VIM-264R (SEQ ID NO: 44) ACCCCACGCTGTATCAATCAAAAG VIM-198P (SEQ ID NO: 45) CTACCCGTCCAATGGTCTCATTGT KPC-269F (SEQ ID NO: 46) TGGACACACCCATCCGTTACG 127bp KPC-396R * (SEQ ID NO: 47) GGCGTTATCACTGTATTGCACGG KPC-329-1P (SEQ ID NO: 48) AATATCTGACAACAGGCATGACGGT NDM-327F (SEQ ID NO: 49) AGATCCTCAACTGGATCAAGCAGG 134bp NDM-461R * (SEQ ID NO: 50) ACGCATTGGCATAAGTCGCAAT NDM-385P (SEQ ID NO: 51) TCACGCGCATCAGGACAAGA OXA48-131F (SEQ ID NO: 52) TTGTGCTCTGGAATGAGAATAAGCAG 118bp OXA48-249R (SEQ ID NO: 53) CAAATCGAGGGCGATCAAGCT OXA48-186P (SEQ ID NO: 54) GAACCAAGCATTTTTACCCGCA SPM-50-F (SEQ ID NO: 55) CGGATCATGTCGACTTGCCCT 118bp SPM-197R (SEQ ID NO: 56) TCAAACGGCGAAGAGACAATGAC SPM-107-P (SEQ ID NO: 57) TCGTCACAGACCGCGATTTCT

No. name Sequence Species 20 IC-1218F (SEQ ID NO: 58) CCTTACCAAGCCTTGTCTCTAA Brassica rapa IC-1313R (SEQ ID NO: 59) CATGGAAATTGGATCGGAACTG 21 120F (SEQ ID NO: 60) AGYKGCGRACGGGTGAGTAA Gram negative 280R * (SEQ ID NO: 61) TGT GGC YGR TCR YCC TCT CAG Primer 22 407F (SEQ ID NO: 62) CTACGGGAGGCAGCAGTRGGGAAT 16S rRNA 608R * (SEQ ID NO: 63) TATTACCGCGGCTGCTGGCA Probe 23 GN350-5-1 (SEQ ID NO: 64)     AACKACGATCCCTAGCTGGTC Gram negative 24 AbaNei-350 (SEQ ID NO: 65) AACGACGATCHGTAGCGGGT 25 Eco-5 (SEQ ID NO: 66)  GGAAGGGAGTAAAGTTAATACCTTTGCTCA E. coli / Shigella spp. 26 shi (SEQ ID NO: 67)  AGTTCAGTAAGATGGTTGTGCGCA Shigella spp. 27 sal-ent-523 (SEQ ID NO: 68) TGT TGT GGT TAA TAA CCG CAGCA Salmonella spp. 28 kpn-11 (SEQ ID NO: 69) AAAAAAA GGTTAATAACCTCATCGATTGAC K. pneumoniae 29 K. oxytoca (SEQ ID NO: 70) GGAAGGGAGTGAGGTTAATAACCTTATTC K. oxytoca 30 Pae-2 (SEQ ID NO: 71)   ATAACGTCCGGAAACGGGC P. aeruginosa 31 serr-1 (SEQ ID NO: 72) GTGATGAGCTTAATACGCTCATCAAT Serratia marcescens 32 Mor-2 (SEQ ID NO: 73) AAG GTG TCA AGG TTA ATA ACC TTG GC Morganella spp. 33 Aba-479 (SEQ ID NO: 74) GAGGCTACTTTAGTTAATACCTAGAGATAGTGG A.baumannii 34 Hin-469 (SEQ ID NO: 75) CGGTATTGAGGAAGGTTGATGTGT H. influenzae

Tables 4 and 5 show the relationship between the primer used for PCR-REBA and the probe

In the present invention, Gram-negative bacterial species are discriminated and ESBLs (TEM-, CTX-M-, SHV-type, OXA-type), AmpC (ACT, CMY2, DHA, SPM, CMY- (Reverse blot hybridization assay) ESBL-ID was developed to confirm the resistance of each gene corresponding to carbapenemases (OXA-48 like, IMP, VIM, NDM, and KPC)

1, Results of PCR amplification products for REBA ESBL-ID. Lanes M, 100-bp DNA ladder (Bioneer, Daejeon, South Korea); lane 1, TEM (360 bp); lane 2, CTX-M1 (120 bp); lane 3, CTX-M9 (117 bp); lane 4, SHV (130 bp); lane 5, ACT (140 bp); lane 6, CMY-2 (140 bp); lane 7, DHA (135 bp); lane 8, CMY-1 / MOX (128 bp) lane 9, OXA-1 (124 bp); lane 10, OXA-2 (127 bp); lane 11, OXA-48 (120 bp); lane 12, IMP (126 bp); lane 13, KPC (125 bp); lane 14, set 1 mixture for ESBLs gene; lane 15, Set 2 mixture for AmpC? -lactamase genes; And lane 16, set 3 mixture for carbapenemases gene,
Figure 2 shows the specificity results of PCR-REBA for 18 ESBL resistance probes and 7 Gram negative bacteria. Lanes: 1, CTX M1-2 group; 2, CTX M9 group; 3, TEM; 4, SHV; 5, OXA-1-30 group; 6, OXA-2 group; 7, ACT; 8, CMY-2 like; 9, DHA; 10, ACC-1; 11, CMY-1 like / MOX; 12, FOX; 13, IMP; 14, VIM; 15, NDM; 16, KPC; 17, OXA-48; 18, SPM; 19, E. coli ; 20, E. aerogenes ; 21, K. pneumoniae ; 22, P. aeruginosa ; 23, A. baumannii ; N, negative control; ESBLs, extended spectrum beta-lactamase; AmpC, plasmid-mediated cephalosporinases,
3, Detection limits for 18 resistant probes from 10-fold diluted DNA samples of Escherichia coli-ESBL-producing bacteria. CTX M1-2 group (A), CTX M9 cluster (B), TEM (C), SHV (D), OXA-1 (E), OXA- ), DHA (I), ACC-1 (J), CMY1 / MOX (K), FOX (L), IMP (M), VIM Q), and 10 ng / 10 fg DNA per reaction SPM (R) were used to determine the detection limit of PCR-REBA. Lane 1, 10ng; Lane 2, 1 ng; Lang 3, 100 pg; Lane 4, 10 pg; Lane 5, 1 pg; Lane 6, 100fg; Lane 7, 10fg, Lane 8, negative control. The total detection limit of this assay for 18 target probes is 10 fg DNA at about 100 fg per reaction,
Figure 4 Detection limits for 18 resistant probes from 10-fold diluted DNA samples of Escherichia coli-ESBL-producing bacteria. CTX M1-2 group (A), CTX M9 cluster (B), TEM (C), SHV (D), OXA-1 (E), OXA- ), DHA (I), ACC-1 (J), CMY1 / MOX (K), FOX (L), IMP (M), VIM Q), and 10 ng / 10 fg DNA per reaction SPM (R) were used to determine the detection limit of PCR-REBA. Lane 1, 10ng; Lane 2, 1 ng; Lang 3, 100 pg; Lane 4, 10 pg; Lane 5, 1 pg; Lane 6, 100fg; Lane 7, 10fg, Lane 8, negative control. The total detection limit of this assay for 18 target probes is about 100 fg DNA per reaction,
Figure 5 compares the positive rates of ESBLs, AmpC β-lactamase, and carbapenemases in Enterobacteriaceae. A, antibiotic resistance rates of ESBLs, co-producing ESBLs or AmpCs- β-lactamases, ESBLs or carbapenemases, and all ESBLs, AmpCs- β-lactamases, carbapenemases in Enterobacteriaceae. The positive rates of ESBL gene (B), AmpC β-lactamases gene (C), and carbapenemases gene (D) in Enterobacteriaceae,
Figure 6 shows PCR-REBA results for the detection of mycobacteria and ESBLs in positive blood cultures. Lanes: 1-13, 18, 19, 22, 23, ESBL resistance; lanes 14-17, 20, 21, ESBL sensitivity; And N, a negative control,
FIG. 7 shows the results of a comparative experiment for the CTXM-1 target,
FIG. 8 shows the results of a comparative experiment for the CTXM-9 target,
FIG. 9 shows a result of a comparison test for an SHV target,
FIG. 10 shows the results of a comparative experiment using the sequences described in the existing articles and the sequences of the present invention,
Fig. 11 is a schematic diagram of a probe for distinguishing TEM-ESBL from a mutation site target due to amino acid substitution at some gene sites in TEM-1; Fig.
FIG. 12 is a schematic diagram of a probe for distinguishing an SHV-ESBL from a mutation site target due to amino acid substitution of some gene regions in SHV-1,
Fig. 13 is a Leva-test for distinguishing TEM- and SHV-ESBL. Lane 1: E. coli ; 2, TEM-1; 3, TEM-73; 4, TEM-ESBL (Glu104Lys); 5, TEM-ESBL (Gly238Ser); 6-7, TEM-52 and TEM-66 (Glu104Lys and Gly238Ser mixed); 8, K. pneumonia ; 9, SHV-1; 10, SHV-ESBL (Asp179Ala); 11-13, SHV-12 and SHV-2a (Gly238Ser).
FIG. 14 shows the result of sequencing in order to confirm the amino acid substitution site shown in FIG. * Indicates that TEM Glu104Lys was substituted with lysine (AAG) for glutamic acid (GAG) at position 104 and TEM Gly238Ser was substituted for serine (AGT) at glycine (GGT) , SHV Gly238Ser shows the result (E) of 238 glycine (GGC) replaced with serine (AGC).

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Example  1. Materials

20 ESBL, 8 AmpC β-lactamases, 6 carbapenemases producing strains, 119 bacterial standard strains and 327 ESBL clinical strains were used to confirm the specificity of PCR-REBA. All strains were provided through ATCC (American Type Culture Collection, LGC Promochem GmbH, Wesel, Germany), Yonsei University Diagnostic Microbiology Laboratory (Korea Wonju), Hallym University Medical Center (Seoul DLMH) or KCMF (Korean Collection of Medical Fungi, Korea Daejeon) received. In addition, blood culture specimens were submitted to Wonju Christian Hospital, Department of Diagnostic Imaging. The specimens were positive for blood culture and specimens with antibiotic susceptibility. Blood cultures were inoculated with 5 to 10 ml of blood each in a pair of BACTEC Standard 10 Aerobic / F and BACTEC PLUS Anaerobic / F bottles in patients suspected of bacteremia. All blood culture bottles were received 24 hours a day and two blood culture automation equipment, BACTEC 9240 system (BD, Franklin Lakes, NJ, USA) and BacT / Alert 3D system (BioMerieux, Durham, NC, Lt; RTI ID = 0.0 > 37 C < / RTI > for 5 days. Gram stain, identification of isolates, and antimicrobial susceptibility test were carried out by subculture when the culture positive signal was detected. Biochemical methods using the Vitek II system (BioMerieux, Marcy, 1'Eltoile, France) were used to identify bacteria. Candida albicans was identified as Candida albicans after positive germination test and identified by ATB ID 32C (bioMerieux SA, Marcy-1'Etoile, France).

Example  2. Standard strains and Of clinical isolates genomic  DNA extraction

A portion of the culture was cultured on a Bactec 9240 system (Becton Dickinson Microbiology System, Sparks, Md., USA). 100 μl of the DNA extraction solution was added, vortexed for 1 minute, heated at 100 ° C. for 10 minutes, and the supernatant obtained by centrifugation for 3 minutes at rpm was used to isolate the nucleic acid. The isolated nucleic acid was used as a template for PCR to perform REBA sepsis-ID.

Example  3. REBA ESBL -ID gene collection and analysis

The PCR primers and oligonucleotide probes targeted at Genbank include 16s rRNAs for accurate identification of Gram-negative bacteria (see, eg, National Center for Biotechnology Information, NCBI, http://www.ncbi.nlm.nih.gov ) CMY2, DHA, SPM, CMY-1, and OXA-2 genes, which are known to be susceptible to AmpC, and TEM, CTX-M1, CTX-M9, SHV, OXA- 1, like / MOX, search for ACC-1, FOX gene, carbazole penem antibiotics can be seen the resistance Status (OXA-48 like, IMP, VIM, NDM, KPC gene was collected sequencing. multi-aligned (http : //multalin.toulouse.inra.fr/multalin ) and then two pairs of primers were designed at the base sequence region in which each of the target genes is common. Among them, reverse primer was used for the PCR-REBA method biotin was attached to the 5 'end, and the bacterial specific sites inside the two pairs of primers An oligonucleotide probe capable of detecting each target species was designed, and an amino group was attached to the 5 'end of each oligonucleotide probe so that a thin membrane and a peptide bond could be formed. The primer and oligonucleotide probe designed were biona , Korea) (see Table 5).

Example  4. one-tube nested PCR

PCR was performed using Prime Taq Premix (2X) (Genet Bio, Nonsan, Korea), which was used as a template for the genomic DNA of each strain. The composition of Prime Taq Premix (2X) is 1 unit / 10 μl of primer Taq polymerase, 2X reaction buffer, 4 mM MgCl2, enzyme stabilizer, sediment, loading dye, pH 9.0, 0.5 mM each dATP, dCTP, dGTP and dTTP. For each PCR, 10 μl of Prime Taq premix (2X), 1 μl each of a pair of 10 pmole primers, 3 μl of ultrapure water, and 5 μl of genomic DNA of each strain were used to make a total amount of 20 μl. The PCR reaction was repeated 10 times at 95 ° C for 5 minutes for the first denaturation, 30 seconds at 95 ° C for 30 seconds at 65 ° C for 30 seconds, 30 seconds at 95 ° C for the second amplification, 40 times After repeating, the reaction was carried out at 72 ° C for 10 minutes for a complete elongation reaction. After PCR, the PCR product was electrophoresed in 290 bolts at 2% TBE (Tris-borate-ethylenediaminetetraacetic acid disodium salt dihydrate) agarose gene (W / V ratio) for 20 minutes. After staining with ethidium bromide for 10 minutes, Respectively.

Example  5. REBA ESBL Performing-ID

The REBA ESBL-ID using the amplified PCR product was performed using the experimental conditions suggested by the manufacturer. The PCR product was reacted with the same amount of denaturation solution (0.2N NaOH, 0.2 mM EDTA) at room temperature for 5 minutes to denature the double stranded PCR reaction product into a single strand. During the reaction, the membranes were diluted with 2X SSPE / 0.1% SDS in a membrane REBA ESBL-ID membrane (Optipharm, Korea) with oligonucleotide probes attached to the MN45 miniblotter system (immunetics, Boston, Mass., USA) (Roche, Mannheim, Germany) diluted 1: 2000 (v / v), followed by washing with WS (washing solution) at 62 ° C for 10 min. And reacted at room temperature for 30 minutes. After the reaction was completed, the membrane was washed with 2X SSPE twice at room temperature for 5 minutes. The plate was exposed to CDP-star (GE healthcare, UK) for alkaline phosphatase reaction for 10 minutes and then exposed to Chemi doc (VILBER LOURMAT, To determine whether or not to detect it.

Example 6: Korea  Patent Registration No. 10-1473444 primer  And With probe  Comparative experiment

The method described in the above-mentioned prior patent and the comparative test result performed by the method of the present invention are shown in FIGS.

As can be seen from the figure, there is a difference between the primer and probe sequence of the present invention and the primer and probe sequence of the present invention. As a result of performing this difference and the PCR-REBA of the present invention, Although there is no difference, it can be confirmed that the base sequence used in the present invention is further amplified at a lower concentration, and it can be confirmed that the sensitivity is higher when REBA is performed in PCR.

In addition, as for the discrimination from the prior art described in the existing paper, there is a difference in sensitivity depending on which part of the ESBL, AmpC β-lactamase, and carbapenemases genes are commonly used in the world, . On the left side of FIG. 10 of the present invention, the result of PCR using the nucleotide sequence of the existing (on paper) region is poor in sensitivity. However, the right case shows the sensitivity result using the nucleotide sequence of the newly created site of the present invention. Therefore, even in the case of the same gene (for example, TEM), there is a difference in sensitivity. Therefore, in the test method which considers sensitivity, it is necessary to use the nucleotide sequence of the present invention rather than the existing nucleotide sequence.

Example 7: TEM -1 and SHV -1 Mutant Site Target Addition Due to Amino Acid Substitution of Some Gene Sites Additional TEM-ESBL and SHV-ESBL Distinguishing

In TEM-1, due to the amino acid substitution of some gene sites, each type is transformed into ESBL type. A probe was designed so that the amino-acid substitution at the red box portion of FIG. 11 is most frequently observed and this portion is targeted to further discriminate the TEM-ESBL.

Likewise, some amino acid substitutions in SHV-1 lead to the transformation of ESBL type into each type.

The most abundant amino acid substitution at the red box in FIG. 12 was designed so that the additional SHV-ESBL could be discriminated by targeting this portion (see Table 6).

TEM Size Gln39Lys-66F (SEQ ID NO: 76) TGCTCACCCAGAAACGCTGGT Gln39Lys-154R * (SEQ ID NO: 77) CGCTGTTGAGATCCAGTTCGATGTA 88bp Gln39Lys-P (SEQ ID NO: 78) AAAGATGCTGAAGAT A AGTTGGGTG Glu104Lys-P (SEQ ID NO: 79) CCAAC T ATCAAGGCG G GTTACA Gly164His-P (SEQ ID NO: 80) TTCCCAA TG ATCAAGGCG A GTTA Glu104Lys-268F (SEQ ID NO: 81) GGTCGCCGCATACACTATTCTCA Glu104Lys-531R * (SEQ ID NO: 82) GTCACGCTCGTCGTTTGGTATG 263bp Gly238Ser-P (SEQ ID NO: 83) GAGA T CCA T GCTCACCGGCTC Glu240Lys-P (SEQ ID NO: 84) GAGATCCA T GCTCAC C GGCTC Gly238-668F (SEQ ID NO: 85) AACGGCAGCTGCTGCAGTG Gly238-806R * (SEQ ID NO: 86) CCAAGCAGGGCGACAATCC 138bp SHV Leu39Gln-243F (SEQ ID NO: 87) TATTATCTCCCTGTTAGCCACCCT Leu39Gln-436R (SEQ ID NO: 88) GCACTACTTTAAAGGTGCTCATCATGG 193bp Leu39Gln-P (SEQ ID NO: 89) GGCTTTCGCTTTGTTTAATTTGCTC 556-F (SEQ ID NO: 90) GAACTGAATGAGGCGCTTCCC 806R * (SEQ ID NO: 91) CCAAGCAGGGCGACAATCC 250bp Asp179Ala (SEQ ID NO: 92) GCCCGCG C CACCACTA Asp179Asn (SEQ ID NO: 93) CCCGC A ACACCACTACCC Asp179Gly (SEQ ID NO: 94) GGGGTAGTGGTG C CGCG Gly238Ser (SEQ ID NO: 95) CCGCTCGCTAGCTCCGGT Gly238Ala (SEQ ID NO: 96) GCTCG G CAGCTCCGGTC Glu240Lys (SEQ ID NO: 97) G AGCTGGC A R R CGGGGT

The underlined part in Table 6 above is devised.

<110> Yonsei University Wonju Industry-Academic Cooperation Foundation <120> A method for detecting both Antibiotic resistance related genes          and gram-negative bacteria <130> HY160049 <160> 97 <170> Kopatentin 2.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gatgtgcagc accagtaaag tgatg 25 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atcggattat agttaacmar gtcagattt 29 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 3 aagtgaaagc gaaccgaatc tgtta 25 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtgcagyacc agtaargtga tg 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aacgagagcg ataagcacct gc 22 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 6 gggattgtag ttaaccaggt cgct 24 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tgtgcagtac cagtaaagtt atggcg 26 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 attgtagtta accagatcgg caggc 25 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 9 gaaacgcaaa agcagctgct t 21 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ggttacatcg agctggatct caaca 25 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 caacgatcaa ggcgggttac at 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 12 gtgcggtatt atcccgtgtt ga 22 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gcaaattaaa caaagcgaaa gcca 24 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gcactacttt aaaggtgctc atcatgg 27 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 15 catgatagaa atggatctgg ccag 24 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 agcgccagtg catcaacaga t 21 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 catttgcgtt gcacactttg ct 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 18 ttacgatgca tccacaaacg ct 22 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 tagttgtggc agacgaacgc c 21 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gcctgcatca agtgcaaaaa gtg 23 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 21 ttgatcctgt gcgatcgaag aa 22 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 tacaggtrcc ggatgaggtc acg 23 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gaaggtttga ccgccagcgc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 24 atcaaaactg gcagccgcag 20 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 aagacgttta acggcgtgtt gg 22 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 gccgtatagg tggctaagtg cag 23 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 27 acgaaatact ggccagaact gaca 24 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 tttcacaggt gtgctgggtg c 21 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 ggtataggta gccagatcca gcaatg 26 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 30 aagagatggc gctgaatgat cc 22 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 acgtagcgta acaagtcgct ggag 24 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 tgaccagatg gaaaactatg cgtg 24 <210> 33 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 33 gcttcgttac ccaaaatctc catattc 27 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 ctgctcaagg agcacmggat c 21 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 ctcgaacagg gtctgctcgc t 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 36 aaaggatggc aaggcccact a 21 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 atttcaacta tggggttgcc aacc 24 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 tggctcacct tgtcatccag c 21 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 39 tgttcgagat tggctcggtc ag 22 <210> 40 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 taaaaggcag tatytcctcw cattt 25 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 accttaccgt cttttttaag magttca 27 <210> 42 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 42 ggaatagagt ggcttaattc tcratctat 29 <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 ctttaccaga ttgccgatgg tgtt 24 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 accccacgct gtatcaatca aaag 24 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 45 ctacccgtcc aatggtctca ttgt 24 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 tggacacacc catccgttac g 21 <210> 47 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 ggcgttatca ctgtattgca cgg 23 <210> 48 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 48 aatatctgac aacaggcatg acggt 25 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 agatcctcaa ctggatcaag cagg 24 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 acgcattggc ataagtcgca at 22 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 51 tcacgcgcat caggacaaga 20 <210> 52 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 ttgtgctctg gaatgagaat aagcag 26 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 caaatcgagg gcgatcaagc t 21 <210> 54 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 54 gaaccaagca tttttacccg ca 22 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 cggatcatgt cgacttgccc t 21 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 tcaaacggcg aagagacaat gac 23 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 57 tcgtcacaga ccgcgatttc t 21 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 58 ccttaccaag ccttgtctct aa 22 <210> 59 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 59 catggaaatt ggatcggaac tg 22 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 60 agykgcgrac gggtgagtaa 20 <210> 61 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 61 tgtggcygrt crycctctca g 21 <210> 62 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 ctacgggagg cagcagtrgg gaat 24 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 63 tattaccgcg gctgctggca 20 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 64 aackacgatc cctagctggt c 21 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 65 aacgacgatc hgtagcgggt 20 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 66 ggaagggagt aaagttaata cctttgctca 30 <210> 67 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 67 agttcagtaa gatggttgtg cgca 24 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 68 tgttgtggtt aataaccgca gca 23 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 69 aaaaaaaggt taataacctc atcgattgac 30 <210> 70 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 70 ggaagggagt gaggttaata accttattc 29 <210> 71 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 71 ataacgtccg gaaacgggc 19 <210> 72 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 72 gtgatgagct taatacgctc atcaat 26 <210> 73 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 73 aaggtgtcaa ggttaataac cttggc 26 <210> 74 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 74 gaggctactt tagttaatac ctagagatag tgg 33 <210> 75 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 75 cggtattgag gaaggttgat gtgt 24 <210> 76 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 76 tgctcaccca gaaacgctgg t 21 <210> 77 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 77 cgctgttgag atccagttcg atgta 25 <210> 78 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 78 aaagatgctg aagataagtt gggtg 25 <210> 79 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 79 ccaactatca aggcgggtta ca 22 <210> 80 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 80 ttcccaatga tcaaggcgag tta 23 <210> 81 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 81 ggtcgccgca tacactattc tca 23 <210> 82 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 82 gtcacgctcg tcgtttggta tg 22 <210> 83 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 83 gagatccatg ctcaccggct c 21 <210> 84 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 84 gagatccatg ctcaccggct c 21 <210> 85 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 85 aacggcagct gctgcagtg 19 <210> 86 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 86 ccaagcaggg cgacaatcc 19 <210> 87 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 87 tattatctcc ctgttagcca ccct 24 <210> 88 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 88 gcactacttt aaaggtgctc atcatgg 27 <210> 89 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 89 ggctttcgct ttgtttaatt tgctc 25 <210> 90 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 90 gaactgaatg aggcgcttcc c 21 <210> 91 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 91 ccaagcaggg cgacaatcc 19 <210> 92 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 92 gcccgcgcca ccacta 16 <210> 93 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 93 cccgcaacac cactaccc 18 <210> 94 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 94 ggggtagtgg tgccgcg 17 <210> 95 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 95 ccgctcgcta gctccggt 18 <210> 96 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 96 gctcggcagc tccggtc 17 <210> 97 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 97 gagctggcaa rcggggt 17

Claims (12)

a) separating DNA from a specimen sample;
b) TEM-, CTX-M-, SHV-type, and OXA-type genes capable of confirming antibiotic resistance of extended-spectrum beta-lactamase (ESBL) AmpC Beta lactamase OXA-48 analogs, IMP, VIM, NDM to confirm resistance to carbapenem, ACT, CMY2, DHA, CMY-1 mimic / MOX, ACC-1 and FOX genes to confirm antibiotic resistance , SPM, and KPC gene, 16s rRNA and Gram-negative bacteria-specific gene fragments; and
(c) an oligomer probe for gene detection which can confirm the antibiotic resistance of the extended-spectrum beta-lactamase (ESBL), an oligomer probe for gene detection for confirming resistance to AmpC beta lactamase antibiotics, Oligonucleotide probes for gene detection, genetic probe for detecting Gram-negative bacteria gene, oligo probe for detecting E. coli or Shigella genus gene, oligo probe for detecting Shigella gene, oligo probe for detecting Salmonella genus gene , Oligo probe for detecting Klebsiella pneumoniae gene, oligo probe for detecting Klebsiella oxytoca strain gene, oligo probe for detecting Pseudomonas aeruginosa strain gene, oligo probe for detecting Serratia marcescens strain gene, oligo probe for detecting Morganella genus gene, Acinetobacter baumann reverse blot hybridization in which a solid support having an oligo probe for detecting a strain gene and a oligo probe for detecting Haemophilus influenzae strain gene is hybridized with the PCR amplification product obtained in step b)
Herein, primers for amplifying a gene fragment capable of confirming antibiotic resistance with extended-spectrum beta-lactamase (ESBL) using the primers from the DNA include SEQ ID NOs: 1 to 2, 4 to 5, 7 to 8, 10 to 11, 13 to 14, 16 to 17, and 19 to 20, and the primers for amplifying the gene fragment capable of confirming resistance to the AmpC beta lactamase antibiotic are SEQ ID NOs: 22 to 23 , 25 to 26, 28 to 29, 31 to 32, 34 to 35 and 37 to 38, and the primers for amplifying the gene fragment capable of confirming the carbapenem resistance are SEQ ID NOs: 40 to 41 and 43 To 44, 46 to 47, 49 to 50, 52 to 53, and 55 to 56, and the primer for amplifying the Gram-negative bacteria-specific gene fragment is a primer pair of SEQ ID NOS: 60 to 61 Primers for amplifying the 16s rRNA gene fragment are the primer pairs of SEQ ID NOS: 62 to 63,
3, 6, 9, 12, 15, 18 and 21, the oligomer probe for gene detection which can confirm the antibiotic resistance of the extended-spectrum beta-lactamase (ESBL) 24, 27, 30, 33, 36, and 39, the probe for detecting a gene capable of confirming resistance to AmpC beta lactamase antibiotics is a probe of SEQ ID NOS: 24, 27, 30, 33, 36 and 39. The oligomer probe for gene detection, Wherein said probe for Gram negative bacterial gene detection is a probe of SEQ ID NOS: 64 to 65, said oligo probe for detecting E. coli or Shigella genus gene is selected from the group consisting of SEQ ID NOs: 42, 45, 48, 51, 54, No. 66, Oligonucleotide probes for Shigella gene detection, SEQ ID NO: 67 for Oligonucleotide probes, Oligonucleotide probes for Salmonella genus gene detection are SEQ ID NO: 68, Olly for detection of Klebsiella pneumoniae strain gene SEQ ID NO: 69, Klebsiella oxytoca strain gene detection oligo probe is SEQ ID NO: 70, Pseudomonas aeruginosa strain gene detection oligo probe is SEQ ID NO: 71, Oligonucleotide probe for detecting Serratia marcescens gene is SEQ ID NO: 72, Morganella sp. SEQ ID NO: 73 for the oligonucleotide probes, SEQ ID NO: 74 for the oligonucleotides for the Acinetobacter baumannii strain gene detection, and SEQ ID NO: 75 for the Haemophilus influenzae strain gene-detecting oligonucleotides. The extended- lactamase (ESBL) The oligomer probe for gene detection, which can confirm the antibiotic resistance, further comprises at least one probe selected from the group consisting of probes consisting of the nucleotide sequences of SEQ ID NOS: 78 to 80, 83 to 84, 89 and 92 to 97 Gram-negative bacteria and ESBL, AmpC Beta lactamase Antibiotic and carbapenem-resistant methods for simultaneous diagnosis. delete delete The method according to claim 1, wherein the method further comprises primer pairs of SEQ ID NOS: 58 to 59 as internal control primers. Delivery method. SEQ ID NOS: 1 to 2, 4 to 5, 7 to 8, 10 to 11, 13 to 14 for amplifying a gene fragment capable of confirming resistance to antibiotic resistance to extended-spectrum beta-lactamase (ESBL) , 16 to 17, and 19 to 20 primer pairs,
A pair of primers of SEQ ID NOS: 22 to 23, 25 to 26, 28 to 29, 31 to 32, 34 to 35, and 37 to 38 for amplifying a gene fragment capable of confirming AmpC beta lactamase antibiotic resistance,
A primer pair of SEQ ID NOS: 40 to 41, 43 to 44, 46 to 47, 49 to 50, 52 to 53, and 55 to 56 for amplifying a gene fragment capable of confirming carbapenem resistance,
A primer pair of SEQ ID NOS: 60 to 61 for amplifying a Gram-negative bacteria-specific gene fragment, and
A composition for simultaneous diagnosis of Gram-negative bacteria and ESBL, AmpC beta lactamase antibiotic and carbapenem resistance including primer pairs of SEQ ID NOS: 62 to 63 for amplifying a 16s rRNA gene fragment. [Claim 6] The composition according to claim 5, wherein the composition further comprises an oligomer probe for detecting genes susceptible to extended-spectrum beta-lactamase (ESBL) antibiotic resistance, a gene capable of confirming resistance to AmpC beta lactamase antibiotics An oligo probe for detecting a Gram-negative bacterium, an oligo probe for detecting E. coli or Shigella genus gene, an oligo probe for detecting Shigella gene, a genomic DNA probe for detecting Salmonella genus An oligo probe for detecting a strain gene, an oligo probe for detecting a Klebsiella pneumoniae strain gene, an oligo probe for detecting a Klebsiella oxytoca strain gene, an oligo probe for detecting a Pseudomonas aeruginosa strain gene, an oligo probe for detecting a strain of Serratia marcescens, The present invention further provides an oligodeoxynucleotide probe for detecting an antibiotic resistance of an extended-spectrum beta-lactamase (ESBL) gene, which further comprises a high probe, an oligo probe for detecting a gene of Acinetobacter baumannii strain, and an oligo probe for detecting Haemophilus influenzae strain gene. 3, 6, 9, 12, 15, 18 and 21, and the oligonucleotide probe for gene detection which can confirm the resistance to the AmpC beta lactamase antibiotic is SEQ ID NO: 24, 27, 30 42, 45, 48, 51, 54, and 57, wherein the oligopeptide probe for gene detection that can confirm the resistance to carbapenem is a probe of SEQ ID NOS: 42, 45, 48, 51, 54, Wherein the probe is a probe of SEQ ID NOS: 64 to 65, and the oligo probe for detecting E. coli or Shigella genus gene is SEQ ID NO: 66, The oligo probe for the Salmonella genus strain gene detection is SEQ ID NO: 68, the Klebsiella pneumoniae strain gene detection oligo probe is SEQ ID NO: 69, the Klebsiella oxytoca strain gene detection oligo probe is SEQ ID NO: 70, the Pseudomonas aeruginosa strain The oligo probe for gene detection is SEQ ID NO: 71, the Serratia marcescens strain gene detection oligo probe is SEQ ID NO: 72, the Morganella genus strain gene detection oligo probe is SEQ ID NO: 73, the Acinetobacter baumannii strain gene detection oligo probe is SEQ ID NO: 74, an oligope probe for detecting an influenzae strain gene is a probe of SEQ ID NO: 75, and an oligomer probe for detecting a gene capable of confirming resistance to the extended-spectrum beta-lactamase (ESBL) antibiotic is SEQ ID NO: 78 to 80 , 83 to 84, 89 and 92 97; and a composition for simultaneous diagnosis of Gram-negative bacteria and ESBL, AmpC beta lactamase antibiotic and carbapenem resistance. delete delete SEQ ID NOS: 1 to 2, 4 to 5, 7 to 8, 10 to 11, 13 to 14 for amplifying a gene fragment capable of confirming resistance to antibiotic resistance to extended-spectrum beta-lactamase (ESBL) , 16 to 17, and 19 to 20 primer pairs,
A pair of primers of SEQ ID NOS: 22 to 23, 25 to 26, 28 to 29, 31 to 32, 34 to 35, and 37 to 38 for amplifying a gene fragment capable of confirming AmpC beta lactamase antibiotic resistance,
A primer pair of SEQ ID NOS: 40 to 41, 43 to 44, 46 to 47, 49 to 50, 52 to 53, and 55 to 56 for amplifying a gene fragment capable of confirming carbapenem resistance,
A primer pair of SEQ ID NOS: 60 to 61 for amplifying a Gram-negative bacteria-specific gene fragment, and
Kit for the simultaneous diagnosis of Gram-negative bacteria including the primer pair of SEQ ID NOS: 62 to 63 for amplifying a 16s rRNA gene fragment and ESBL, AmpC beta lactamase antibiotic and carbapenem resistance. [Claim 11] The kit according to claim 9, wherein the kit comprises an oligomer probe for gene detection that can confirm the resistance to ESBL antibiotics, AmpC beta lactamase antibiotic resistant gene An oligo probe for detecting a Gram-negative bacterium gene, an oligo probe for detecting E. coli or Shigella genus gene, an oligo probe for detecting Shigella gene, a genomic DNA probe for detection of Salmonella genus An oligo probe for detecting a strain gene, an oligo probe for detecting a Klebsiella pneumoniae strain gene, an oligo probe for detecting a Klebsiella oxytoca strain gene, an oligo probe for detecting a Pseudomonas aeruginosa strain gene, an oligo probe for detecting a strain of Serratia marcescens, The An oligo probe for detecting a strain of Acinetobacter baumannii strain, and an oligo probe for detecting a strain of Haemophilus influenzae strain,
3, 6, 9, 12, 15, 18 and 21, the oligomer probe for gene detection which can confirm the antibiotic resistance of the extended-spectrum beta-lactamase (ESBL) 24, 27, 30, 33, 36, and 39, the probe for detecting a gene capable of confirming resistance to AmpC beta lactamase antibiotics is a probe of SEQ ID NOS: 24, 27, 30, 33, 36 and 39. The oligomer probe for gene detection, Wherein said probe for Gram negative bacterial gene detection is a probe of SEQ ID NOS: 64 to 65, said oligo probe for detecting E. coli or Shigella genus gene is selected from the group consisting of SEQ ID NOs: 42, 45, 48, 51, 54, No. 66, Oligonucleotide probes for Shigella gene detection, SEQ ID NO: 67 for Oligonucleotide probes, Oligonucleotide probes for Salmonella genus gene detection are SEQ ID NO: 68, Olly for detection of Klebsiella pneumoniae strain gene SEQ ID NO: 69, Klebsiella oxytoca strain gene detection oligo probe is SEQ ID NO: 70, Pseudomonas aeruginosa strain gene detection oligo probe is SEQ ID NO: 71, Oligonucleotide probe for detecting Serratia marcescens gene is SEQ ID NO: 72, Morganella sp. SEQ ID NO: 73 for the oligonucleotide probes, SEQ ID NO: 74 for the oligonucleotides for the Acinetobacter baumannii strain gene detection, and SEQ ID NO: 75 for the Haemophilus influenzae strain gene-detecting oligonucleotides. The extended- lactamase (ESBL) The oligomer probe for gene detection, which can confirm the antibiotic resistance, further comprises at least one probe selected from the group consisting of probes having the nucleotide sequences of SEQ ID NOS: 78 to 80, 83 to 84, 89 and 92 to 97 Gram-negative bacteria and ESBL, Am pC Beta lactamase kit for simultaneous diagnosis of antibiotic and carbapenem resistance. delete delete

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