WO2023107058A2 - Direct carbapenemase triple (kpc-2, oxa-48, ndm-1) pcr diagnostic kit - Google Patents
Direct carbapenemase triple (kpc-2, oxa-48, ndm-1) pcr diagnostic kit Download PDFInfo
- Publication number
- WO2023107058A2 WO2023107058A2 PCT/TR2022/051394 TR2022051394W WO2023107058A2 WO 2023107058 A2 WO2023107058 A2 WO 2023107058A2 TR 2022051394 W TR2022051394 W TR 2022051394W WO 2023107058 A2 WO2023107058 A2 WO 2023107058A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- carbapenemase
- oxa
- pcr
- oxacillinase
- diagnostic kit
- Prior art date
Links
- 108010068385 carbapenemase Proteins 0.000 title claims abstract description 30
- 238000009007 Diagnostic Kit Methods 0.000 title claims abstract description 8
- 101100026178 Caenorhabditis elegans egl-3 gene Proteins 0.000 title description 4
- 238000001514 detection method Methods 0.000 claims abstract description 10
- 108090000790 Enzymes Proteins 0.000 claims description 21
- 102000004190 Enzymes Human genes 0.000 claims description 21
- 238000007399 DNA isolation Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 9
- 241000588747 Klebsiella pneumoniae Species 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 5
- 241000191967 Staphylococcus aureus Species 0.000 claims description 5
- 230000003115 biocidal effect Effects 0.000 claims description 5
- 238000009396 hybridization Methods 0.000 claims description 5
- 101150114434 vanA gene Proteins 0.000 claims description 5
- 101150037181 vanB gene Proteins 0.000 claims description 5
- 108020001019 DNA Primers Proteins 0.000 claims description 4
- 239000003155 DNA primer Substances 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 241000194032 Enterococcus faecalis Species 0.000 claims description 3
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 claims description 3
- 108010059993 Vancomycin Proteins 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 229960003085 meticillin Drugs 0.000 claims description 3
- 229960003165 vancomycin Drugs 0.000 claims description 3
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 claims description 3
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 claims description 3
- 241000588626 Acinetobacter baumannii Species 0.000 claims description 2
- 108010078777 Colistin Proteins 0.000 claims description 2
- 241000194031 Enterococcus faecium Species 0.000 claims description 2
- 206010036790 Productive cough Diseases 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 claims description 2
- 229960003346 colistin Drugs 0.000 claims description 2
- JORAUNFTUVJTNG-BSTBCYLQSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Chemical compound CC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O.CCC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O JORAUNFTUVJTNG-BSTBCYLQSA-N 0.000 claims description 2
- XDJYMJULXQKGMM-UHFFFAOYSA-N polymyxin E1 Natural products CCC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O XDJYMJULXQKGMM-UHFFFAOYSA-N 0.000 claims description 2
- KNIWPHSUTGNZST-UHFFFAOYSA-N polymyxin E2 Natural products CC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O KNIWPHSUTGNZST-UHFFFAOYSA-N 0.000 claims description 2
- 210000003802 sputum Anatomy 0.000 claims description 2
- 208000024794 sputum Diseases 0.000 claims description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 claims 2
- 238000009825 accumulation Methods 0.000 claims 1
- 239000000523 sample Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 4
- 241000588921 Enterobacteriaceae Species 0.000 description 3
- 238000009004 PCR Kit Methods 0.000 description 3
- 238000007403 mPCR Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 241000589291 Acinetobacter Species 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000194033 Enterococcus Species 0.000 description 2
- 101150004219 MCR1 gene Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 229940041011 carbapenems Drugs 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000013615 primer Substances 0.000 description 2
- 108020004256 Beta-lactamase Proteins 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 101000740455 Klebsiella pneumoniae Metallo-beta-lactamase type 2 Proteins 0.000 description 1
- 101100111649 Klebsiella pneumoniae blaNDM-1 gene Proteins 0.000 description 1
- -1 OXA-48 Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229940041009 monobactams Drugs 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000010807 real-time PCR kit Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Definitions
- the present invention relates to a PCR-based rapid diagnostic kit for the detection of carbapenemases causing antibiotic (carbapenem) resistance.
- Carbapenemases are P-lactamases that hydrolyze penicillin, that hydrolyze cephalosporins in most cases and hydrolyze carbapenems and monobactams to a certain extent.
- carbapenemases in the Enterobacteriaceae family worldwide are Klebsiella pneumoniae, Acinetobacter baumannii carbapenemase (KPC), New Delhi metallo-P- lactamase (NDM), and oxacillinase-48 OXA-48; E. coli carbapenemases KPC, OXA, NDM, VIM and IMP; Enterococcus spp. common Vancomycin resistance genes Van A, VanB, and Staphylococcus aureus Methicillin resistance gene MecA enzymes.
- the carbapenems are the antibiotics with the broadest spectrum in the beta-lactam class with rapid bactericidal action and are widely used in infections caused by many aerobic and anaerobic microorganisms with their broad antibacterial spectrum.
- Carbapenemases are identified in routine microbiology laboratories by traditional culture and antibiogram determination.
- the bacteria are first cultured from the patient and after 24 hours of incubation, the bacteria are named using automated systems.
- the working principle of the systems is based on the metabolites used by the bacteria during reproduction and the resulting enzymes and pH changes are basically named based on their biochemical properties.
- the antibiogram uses automated systems to determine which antibiotic the said bacteria is susceptible to.
- the said procedure while the presence of the resistance enzyme is indicated, no definitive judgment can be made about the details and type of resistance.
- it is not possible to prevent infectiousness and thus outbreaks by identifying the type of plasmid-mediated enzyme that is dangerous and spreads rapidly among patients and isolating a patient from other patients.
- Phenotypic Carba NP and the Modified Hodge Test are also used for the identification of carbapenemases in the known state of the art. Although MHT is highly susceptible in Klebsiella pneumoniae, it does not perform well in other Enterobacteriaceae members. Furthermore, in the state of the art, these tests confirm the presence of a general carbapenemase but cannot identify the precise name of the enzyme causing resistance. Although the Carba NP test is reliable, it is not preferred due to its excessive cost.
- the present invention aims to design a PCR-based rapid diagnostic kit for the detection of antibiotic-resistant carbapenemases.
- the PCR-based rapid diagnostic kit of the invention enables rapid (2-3 hours) identification of enzymes that cause resistance to antibiotics and detects which enzyme causes resistance.
- a plasmid-mediated enzyme the patient is rapidly isolated, and the spread of resistance is prevented.
- kits to be used for diagnostic purposes in microbiology routine laboratories can be produced based on probe hybridization technique, while those to be used in screening tests and epidemiological research studies can be produced with Conventional Single or Multiplex Sybr- Green, Probe-Hybridization techniques.
- the kit of the invention enables rapid diagnosis (2-3 hours) of important (Kpc-2, Oxa-48-23-24- 58, Ndm-1, IMP, VIM, VanA, VanB, mecA, mcr-1, BIC) resistance genes, which are frequently encountered in our country and which pose a danger since their spread is plasmid-mediated, and has a very low cost.
- Bacteria reference strains such as carbapenemase-positive Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus f aecium, Staphylococcus aureus, etc.
- the PCR-based diagnostic kit of the invention for the detection of antibiotic-resistant carbapenemases comprises; DNA primer pairs NO: 1, NO:2 and NO:3, NO:4, NO:5, NO:6, NO:7, NO:8, NO:9, NO:10, NO: 11, NO:12, NO:13, NO:14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO:20, NO:21, NO:22, NO:23, NO:24, NO:25, NO:26, NO:27, NO:28, NO:29, NO:30 to detect enzyme types from the carbapenemase group, including Klebsiella pneumoniae carbapenemase (KPC) belonging to the carbapenemase group, New Delhi metallo-P-lactamase (NDM), oxacillinase-48 (Oxa-48), Colistin Resistance (MCR), Oxacillinase-23 (Oxa-23), Oxacillinase-24 (Oxa- 24), Oxacillinas
- the invention is firstly produced by accumulating bacteria (1) such as Carbapenemase-positive Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., Pseudomonas aeruginosa, Enterococcus fecalis, E. Faecium and Staphylococcus aures etc. at -80 °C.
- bacteria (1) such as Carbapenemase-positive Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., Pseudomonas aeruginosa, Enterococcus fecalis, E. Faecium and Staphylococcus aures etc. at -80 °C.
- clinical samples (2) are prepared in routine diagnostic laboratories as recommended in the selected DNA isolation kit and bacterial culture (3) is produced from clinical samples such as blood, endotracheal aspirate, urine, and sputum sent to the laboratory.
- Direct DNA isolation from clinical samples (4) and DNA isolation from bacterial culture (5) are performed simultaneously with a DNA isolation kit or boiling method. If the PCR step is not to be performed immediately, the samples are stored at +4°C for a short time (a few weeks) and at -80°C for a period of 6 months.
- kits that enable the detection of the appropriate number of enzymes for the study objects can be produced by Conventional Single (7) or Multiplex (8) and Multiplex Sybr-Green (9), Probe-Hybridization (10) and measurements can be performed.
- multiplex PCR can be performed with any two or three of the blaKPC-2, blaOXA-48, blaXDM , blaOXA-23, blaOXA-24, blaOXA-58, blaOXA-204, blaOXA- 181, /W/VIM, blaAMP, blaBIC, mecA, vanA, vanB blaMCR-1 gene regions or in a single reaction depending on the number of PCR device channels, and probe hybridization can be performed with single and/or multiple enzymes.
- PCR kits are produced that enable the amplification of the selected DNA regions ready to be designed in different types suitable for the objects of the study.
- Table 1 shows the DNA primer pairs included in the kit that are specific for the detection of different carbapenemase genes.
- Table 1 DNA primer pairs included in the kit that are specific for the detection of different carbapenemase genes
- kits contains a solution and materials called master mix, which includes Taq DNA polymerase enzyme and primers specific to the region to be amplified, as well as tubes prepared as control.
- master mix which includes Taq DNA polymerase enzyme and primers specific to the region to be amplified, as well as tubes prepared as control.
- the reaction is completed in a short time (approximately 3-4 hours) in a thermal cycling device.
- DNA sequence and variant analysis (10), PCR samples are run on a 1% agarose gel at 100V for 30 minutes if the traditional PCR kit is selected. If a Real-Time PCR kit is selected, Melt-Curve analysis and accurate melting points as well as internal amplification controls (IAK) are evaluated, and results are recorded.
- the enzyme variation will also be statistically evaluated by examining the nucleotide differences according to the DNA Sequence Analysis result of the selected samples.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Measurement And Recording Of Electrical Phenomena And Electrical Characteristics Of The Living Body (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a PCR-based rapid diagnostic kit for the detection of carbapenemases.
Description
DIRECT CARBAPENEMASE TRIPLE (KPC-2, OXA-48, NDM-1) PCR DIAGNOSTIC
KIT
Technical Field of Invention
The present invention relates to a PCR-based rapid diagnostic kit for the detection of carbapenemases causing antibiotic (carbapenem) resistance.
State of the Art Related to the Invention (Background)
Carbapenemases are P-lactamases that hydrolyze penicillin, that hydrolyze cephalosporins in most cases and hydrolyze carbapenems and monobactams to a certain extent.
Infections caused by extended-spectrum beta-lactamases (ESBLs) and carbapenemase-producing Gram-negative bacteria in recent years have posed serious therapeutic problems, especially in hospitalized patients and those with underlying immunosuppressive conditions.
The most commonly detected carbapenemases in the Enterobacteriaceae family worldwide are Klebsiella pneumoniae, Acinetobacter baumannii carbapenemase (KPC), New Delhi metallo-P- lactamase (NDM), and oxacillinase-48 OXA-48; E. coli carbapenemases KPC, OXA, NDM, VIM and IMP; Enterococcus spp. common Vancomycin resistance genes Van A, VanB, and Staphylococcus aureus Methicillin resistance gene MecA enzymes.
The carbapenems are the antibiotics with the broadest spectrum in the beta-lactam class with rapid bactericidal action and are widely used in infections caused by many aerobic and anaerobic microorganisms with their broad antibacterial spectrum.
In the state of the art; Carbapenemases are identified in routine microbiology laboratories by traditional culture and antibiogram determination.
Using the traditional method of bacterial identification and antibiogram used in routine microbiology laboratories, the bacteria are first cultured from the patient and after 24 hours of incubation, the bacteria are named using automated systems. The working principle of the systems is based on the metabolites used by the bacteria during reproduction and the resulting enzymes and pH changes are basically named based on their biochemical properties. Although there is no technical problem in the use of these methods for the said Enterobacteriaceae member bacteria, at least 48 hours are needed for detection and during this time, resistance enzymes spread rapidly from one patient to another.
As a secondary procedure, the antibiogram uses automated systems to determine which antibiotic the said bacteria is susceptible to. During the said procedure, while the presence of the resistance enzyme is indicated, no definitive judgment can be made about the details and type of resistance. Thus, it is not possible to prevent infectiousness and thus outbreaks by identifying the type of plasmid-mediated enzyme that is dangerous and spreads rapidly among patients and isolating a patient from other patients.
Phenotypic Carba NP and the Modified Hodge Test (MHT) are also used for the identification of carbapenemases in the known state of the art. Although MHT is highly susceptible in Klebsiella pneumoniae, it does not perform well in other Enterobacteriaceae members. Furthermore, in the state of the art, these tests confirm the presence of a general carbapenemase but cannot identify the precise name of the enzyme causing resistance. Although the Carba NP test is reliable, it is not preferred due to its excessive cost.
Usually, conventional PCR or internationally produced kits are used for the determination of enzymes in our country. The cost of these kits is quite high as they contain many enzymes other than the important (Kpc-2, Oxa-48, Ndm-1, IMP, VIM, VanA, VanB, mecA, mcr-1, BIC) enzymes that are frequently seen in our country and cause problems and are produced abroad. The determination of resistance also takes a longer time.
The patent numbered US 9,914,980 B2 relates to a kit for the identification of carbapenemase genes, which determines more than 170 carbapenemase enzyme types. Therefore, resistance determination takes a considerably long time.
Brief Description and Objects of the Invention
The present invention aims to design a PCR-based rapid diagnostic kit for the detection of antibiotic-resistant carbapenemases.
The PCR-based rapid diagnostic kit of the invention enables rapid (2-3 hours) identification of enzymes that cause resistance to antibiotics and detects which enzyme causes resistance. In the case of a plasmid-mediated enzyme, the patient is rapidly isolated, and the spread of resistance is prevented.
The rapid spread of resistance enzymes from one patient to another, which is important for patients with high comorbidity hospitalized in intensive care units, is prevented and the patient is isolated from other patients, and infectiousness is prevented.
The contents of the kit of the invention can be simply designed to correspond to the intended use. For example, kits to be used for diagnostic purposes in microbiology routine laboratories can be produced based on probe hybridization technique, while those to be used in screening tests and epidemiological research studies can be produced with Conventional Single or Multiplex Sybr- Green, Probe-Hybridization techniques.
The kit of the invention enables rapid diagnosis (2-3 hours) of important (Kpc-2, Oxa-48-23-24- 58, Ndm-1, IMP, VIM, VanA, VanB, mecA, mcr-1, BIC) resistance genes, which are frequently encountered in our country and which pose a danger since their spread is plasmid-mediated, and has a very low cost.
Descriptions of the Figures Illustrating the Invention
Figure 1: Workflow diagram
Descriptions of the elements/sections/parts that constitute the invention
1. Bacteria reference strains such as carbapenemase-positive Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus f aecium, Staphylococcus aureus, etc.
2. Clinical sample
3. Bacterial culture from a clinical sample
4. Direct DNA isolation from a clinical sample
5. DNA isolation from bacterial culture
6. Standardization of DNA isolation, determination of specificity and susceptibility
7. Conventional PCR (blaKPC-2, blaOXA-48, blaNDM-1, blaOXA-23, blaOXA-24, blaOXA-58, blaOXA-204, blaOXA- 181, blaVLM, blaIMP, blaBIC, mecA, vanA, vanB)
8. Multiplex PCR
9. Quantitative Sybr-Green Multiplex PCR
10. Quantitative Prob-Hibridizasyon
11. DNA sequence and variant analysis
Detailed Description of the Invention
The PCR-based diagnostic kit of the invention for the detection of antibiotic-resistant carbapenemases comprises; DNA primer pairs NO: 1, NO:2 and NO:3, NO:4, NO:5, NO:6, NO:7, NO:8, NO:9, NO:10, NO: 11, NO:12, NO:13, NO:14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO:20, NO:21, NO:22, NO:23, NO:24, NO:25, NO:26, NO:27, NO:28, NO:29, NO:30 to detect enzyme types from the carbapenemase group, including Klebsiella pneumoniae carbapenemase (KPC) belonging to the carbapenemase group, New Delhi metallo-P-lactamase (NDM), oxacillinase-48 (Oxa-48), Colistin Resistance (MCR), Oxacillinase-23 (Oxa-23), Oxacillinase-24 (Oxa- 24), Oxacillinase-58 (Oxa-58), Pseudomonas aeruginosa carbapenemases (blaVIM, blaTMP, and blaBIC), enterococcal Vancomycin resistance genes (vanA, vanB), Staphylococcus aureus methicillin resistance gene (MecA).
According to the process step sequence shown in Figure 1; the invention is firstly produced by accumulating bacteria (1) such as Carbapenemase-positive Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., Pseudomonas aeruginosa, Enterococcus fecalis, E. Faecium and Staphylococcus aures etc. at -80 °C. Afterward, clinical samples (2) are prepared in routine diagnostic laboratories as recommended in the selected DNA isolation kit and bacterial culture (3) is produced from clinical samples such as blood, endotracheal aspirate, urine, and sputum sent to the laboratory. Direct DNA isolation from clinical samples (4) and DNA isolation from bacterial culture (5) are performed simultaneously with a DNA isolation kit or boiling method. If the PCR step is not to be performed immediately, the samples are stored at +4°C for a short time (a few weeks) and at -80°C for a period of 6 months. After standardization of DNA isolation, determination of specificity and susceptibility (6), kits that enable the detection of the appropriate number of enzymes for the study objects can be produced by Conventional Single (7) or Multiplex (8) and Multiplex Sybr-Green (9), Probe-Hybridization (10) and measurements can be performed. While single conventional PCR can be performed with any of blaKPC-2, blaOXA-48, bla DM-1, blaOXA-23, blaOXA-24, blaOXA-58, blaOXA-204, blaOXA- 181, blaVLM, blaIMP, blaB C, mecA, vanA, vanB /Vc/MCR-k, multiplex PCR can be performed with any two or three of the blaKPC-2, blaOXA-48, blaXDM , blaOXA-23, blaOXA-24, blaOXA-58, blaOXA-204, blaOXA- 181, /W/VIM, blaAMP, blaBIC, mecA, vanA, vanB blaMCR-1 gene regions or in a single reaction depending on the number of PCR device channels, and probe hybridization can be performed with single and/or multiple enzymes. In this art, by using primers targeting the DNA regions encoding the problematic resistance enzymes, PCR kits are produced that enable the amplification of the selected DNA regions ready to be designed in different types suitable for the objects of the study. Table 1 shows the DNA primer pairs included in the kit that are specific for the detection of different carbapenemase genes.
Table 1 : DNA primer pairs included in the kit that are specific for the detection of different carbapenemase genes
20
The content of these kits contains a solution and materials called master mix, which includes Taq DNA polymerase enzyme and primers specific to the region to be amplified, as well as tubes prepared as control. With the PCR kit selected according to the suspected resistance enzyme, the reaction is completed in a short time (approximately 3-4 hours) in a thermal cycling device. For the final step, DNA sequence and variant analysis (10), PCR samples are run on a 1% agarose gel at 100V for 30 minutes if the traditional PCR kit is selected. If a Real-Time PCR kit is selected, Melt-Curve analysis and accurate melting points as well as internal amplification controls (IAK) are evaluated, and results are recorded. The enzyme variation will also be statistically evaluated by examining the nucleotide differences according to the DNA Sequence Analysis result of the selected samples.
Claims
CLAIMS PCR-based diagnostic kit for detection of antibiotic-resistant carbapenemase, characterized in that it comprises DNA primer pairs NO:1, NO:2 and NO:3, NO:4, NO:5, NO:6, NO:7, NO:8, NO:9, NO: 10, NO: 11, NO: 12, NO: 13, NO: 14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO:20, NO:21, NO:22, NO:23, NO:24, NO:25, NO:26, NO:27, NO:28, NO:29, NO:30 including Klebsiella pneumoniae carbapenemase (KPC) belonging to the carbapenemase group, New Delhi metallo-P-lactamase (NDM), oxacillinase-48 (Oxa-48), Colistin Resistance (MCR), Oxacillinase-23 (Oxa-23), Oxacillinase-24 (Oxa- 24), Oxacillinase-58 (Oxa-58), Pseudomonas aeruginosa carbapenemases (blaVIM, blaTMP, and blaBIC), enterococcal Vancomycin resistance genes (vanA, vanB), Staphylococcus aureus methicillin resistance gene (MecA) to detect enzyme types from the carbapenemase group, and as Internal Controls Klebsiella pneumoniae 16S-rRNA Internal Control KPIC and Acinetobacter baumannii 16S-rRNA (ABIC). The method of operation of the PCR-based diagnostic kit according to claim 1, characterized in that it comprises the following process steps;
• producing bacteria (1) such as carbapenemase-positive Escherichia coli and Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecium, Enterococcus fecalis, Staphylococcus aureus by accumulation at -80 °C,
• obtaining bacterial cultures (3) from clinical samples (2), such as blood, endotracheal aspirate, or sputum,
• direct DNA isolation from clinical specimens (4) and DNA isolation from bacterial culture (5),
• standardization of DNA isolation, determination of specificity and susceptibility (6),
• producing kits for enzyme detection by Conventional Single (7) or Multiplex (8) and Multiplex Sybr-Green (9), Probe-Hybridization (10) techniques,
• DNA sequence and variant analysis (11).
8
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP22904821.0A EP4413152A2 (en) | 2021-12-04 | 2022-12-01 | Direct carbapenemase triple (kpc-2, oxa-48, ndm-1) pcr diagnostic kit |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TR2021/019090 | 2021-12-04 | ||
| TR2021019090 | 2021-12-04 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2023107058A2 true WO2023107058A2 (en) | 2023-06-15 |
| WO2023107058A3 WO2023107058A3 (en) | 2023-08-03 |
Family
ID=86731435
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/TR2022/051394 WO2023107058A2 (en) | 2021-12-04 | 2022-12-01 | Direct carbapenemase triple (kpc-2, oxa-48, ndm-1) pcr diagnostic kit |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2023107058A2 (en) |
-
2022
- 2022-12-01 WO PCT/TR2022/051394 patent/WO2023107058A2/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023107058A3 (en) | 2023-08-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6258360B2 (en) | Methods and kits for detecting antibiotic-resistant bacteria | |
| AU2008242250B2 (en) | Methods, compositions and kits for detection and analysis of antibiotic-resistant bacteria | |
| Kommedal et al. | Direct 16S rRNA gene sequencing from clinical specimens, with special focus on polybacterial samples and interpretation of mixed DNA chromatograms | |
| Zhang et al. | Evaluation of a micro/nanofluidic chip platform for the high-throughput detection of bacteria and their antibiotic resistance genes in post-neurosurgical meningitis | |
| Stefaniuk et al. | Evaluation of the BD Phoenix automated identification and susceptibility testing system in clinical microbiology laboratory practice | |
| Mencacci et al. | Comparison of conventional culture with SeptiFast real-time PCR for microbial pathogen detection in clinical specimens other than blood | |
| WO2018074762A1 (en) | Quantamatrix assay platform-based diagnostic method and kit capable of detecting and identifying gram-positive and gram-negative bacteria and candida species and determining resistance to antibiotics simultaneously | |
| Barraud et al. | Shotgun metagenomics for microbiome and resistome detection in septic patients with urinary tract infection | |
| CN110669851A (en) | Primer and/or probe composition for detecting cocci causing bloodstream infections and use thereof | |
| CN111593131A (en) | Primer probe system and kit capable of simultaneously detecting multiple drug-resistant genes | |
| Wong et al. | Disruption of adeB gene has a greater effect on resistance to meropenems than adeA gene in Acinetobacter spp. isolated from University Malaya Medical Centre. | |
| Deutch et al. | Diagnosis of ventricular drainage-related bacterial meningitis by broad-range real-time polymerase chain reaction | |
| CN113584191B (en) | Primer, probe and kit for multiplex PCR detection of 7 drug-resistant genes | |
| CN110656188A (en) | Primer and/or probe composition for detecting bacillus causing bloodstream infection and application thereof | |
| EP2614155A1 (en) | Method for detecting the presence of bacterial strains resistant to antibiotics in a biological sample | |
| Shaker et al. | Evaluation of combined use of temocillin disk and mastdisks inhibitor combination set against polymerase chain reaction for detection of carbapenem-resistant Enterobacteriaceae | |
| Zalas-Więcek et al. | Rapid detection of genes encoding extended-spectrum beta-lactamase and carbapenemase in clinical Escherichia coli isolates with eazyplex SuperBug CRE system | |
| WO2023107058A2 (en) | Direct carbapenemase triple (kpc-2, oxa-48, ndm-1) pcr diagnostic kit | |
| EP4413152A2 (en) | Direct carbapenemase triple (kpc-2, oxa-48, ndm-1) pcr diagnostic kit | |
| Marbjerg et al. | Commercial bacterial and fungal broad-range PCR (Micro-Dx™) used on culture-negative specimens from normally sterile sites: diagnostic value and implications for antimicrobial treatment | |
| Rajni et al. | Report on carbapenemase-producing rare sequence types of Escherichia coli and Enterobacter hormaechei | |
| US20220145358A1 (en) | Direct carbapenemase triple (kpc-2, oxa-48, ndm-1) pcr diagnostic kit | |
| Mohamed et al. | Phylogenetic Analysis of Klebsiella pneumoniae Isolated from Nosocomial and Community Infection in Diyala, Iraq | |
| Huletsky et al. | Bacterial genotypic drug resistance assays | |
| Nordmann et al. | Development of rapid diagnostic tests and screening culture media by the NARA for detection of multidrug resistance |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WWE | Wipo information: entry into national phase |
Ref document number: 2022904821 Country of ref document: EP |
|
| ENP | Entry into the national phase |
Ref document number: 2022904821 Country of ref document: EP Effective date: 20240509 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |