US20180179583A1 - Primers for the detection and typing of carbapenemase-producing bacterial strains and detection method and kit - Google Patents

Primers for the detection and typing of carbapenemase-producing bacterial strains and detection method and kit Download PDF

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US20180179583A1
US20180179583A1 US15/877,834 US201815877834A US2018179583A1 US 20180179583 A1 US20180179583 A1 US 20180179583A1 US 201815877834 A US201815877834 A US 201815877834A US 2018179583 A1 US2018179583 A1 US 2018179583A1
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primers
pair
carbapenemase
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Gloria ROYO GARCIA
Juan Carlos Rodriguez Diaz
Antonio Jose Galiana Cabrera
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Fundacion para el Fomento de la Investigacion Sanitaria y Biomedica de la Comunitat Valenciana FISABIO
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Fundacion para el Fomento de la Investigacion Sanitaria y Biomedica de la Comunitat Valenciana FISABIO
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    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention refers to primers for rapid detection and/or typing of carbapenemase-producing bacterial strains.
  • the invention also discloses a kit for the rapid detection of carbapenemases so as to identify multi-resistant organisms producing said enzymes, to be used in clinical practice for prevention and epidemiologic control of infection outbreaks, for example hospital-acquired infection outbreaks, and to be particularly used in Intensive Care Units and in Neonatal Intensive Care Units (NICU).
  • the invention also refers to a method for the detection and/or typing of carbapenemase-producing bacterial strains using said primers.
  • Biomolecular techniques are used in epidemiology to analyze certain bacterial characteristics suitable as epidemiological markers. Techniques derived from biochemistry, immunology and genetics are included.
  • Genotypic typing methods are based on the study of chromosomal or extra-chromosomal DNA.
  • the main methods include plasmid analysis, restriction endonuclease analysis, DNA-probe fingerprinting and PCR-based fingerprinting.
  • polymerase chain reaction or PCR techniques there are several PCR variations that can be used. One of them consists of using primers complementary to repetitive regions found in the bacterial genome, and it features two modalities (REP-PCR and ERIC-PCR). Another variation is the PCR in which arbitrary primers are used (AP-PCR). Of all these techniques, REP-PCR is the one showing more discrimination and reproducibility capacity, and it may also be used both in gram-negative and gram-positive bacteria. PCR may also be combined with restriction endonucleases technique, obtaining fragments which are then electrophoretically analyzed (PCR-RFLP).
  • PCR-RFLP restriction endonucleases technique
  • PCR and other amplification techniques may have a deep effect on reducing time to and increasing certainty of diagnosis for a variety of microorganism infections.
  • the use of this technique is interesting mainly in those infections for which other alternative methods are costly and troublesome or in which a delay in the diagnosis is caused by the slow growth of microorganisms in conventional culture means.
  • RU 2415932-A describes a method for gene identification of broad-spectrum class A ⁇ -lactamases. The method is based on PCR technique and uses a DNA microchip labelled with biotin; this molecule is detected by a streptavidin-peroxidase conjugate, subsequently recognized by means of colorimetric detection of peroxidase.
  • WO2008124670-A refers to a method for identification of all known isoforms of KPC gene, which includes a new pair of primers and a new probe for enzyme detection by means of amplification methods such as PCR technique.
  • This system may be presented in the form of a kit for detection of carbapenemases in samples from patients.
  • the system described herein is the closest to that disclosed in the present invention but it is much more limited, since it only detects KPC carbapenemases.
  • the invention refers to primers and probes identified in the appended claims.
  • the primers and probes object of this invention may be used for the preparation of a kit for carbapenemase gene identification and detection, preferably carbapenemase genes contained in and produced by carbapenemase-producing bacterial strains.
  • the invention also provides a diagnosis method using said primers and probes.
  • Another object described in the present invention refers to a kit for carbapenemase gene identification and detection, preferably carbapenemase genes contained in and produced by carbapenemase-producing bacterial strains, comprising the primers and probes according to this invention.
  • the kit comprises primers and probes specific for recognizing different carbapenemase genes, for example those indicated in Table 1, included below.
  • the invention also comprises a method for detection and typing of carbapenemase-producing bacterial strains, consisting of binding and hybridizing the primers of the invention to a specific DNA sequence of the gene to be tested, performing a selective specific amplification of carbapenemase genes and identifying the amplification product with the specific probe of the genetic sequence or genetic fragment of diagnostic interest.
  • the identification method of carbapenemase genes disclosed in this invention is characterized in that it preferably uses multiplex reactions where, for example, two or three primers-probe reactions work together to detect different types of carbapenemase genes.
  • the reactions that can work together are shown in Table 1.
  • the method for carbapenemase genes identification of this invention is characterized in that each forward and reverse primer from a multiplex reaction binds to specific DNA sequences so that they can be PCR amplified, and, preferably, at the same time each specific probe labelled with a determined fluorophore binds to these amplicons which are formed in the case of carbapenemase genes presence.
  • DNA sequences are preferably subjected to PCR (polymerase chain reaction technique) which is, more preferably, Multiplex real-time PCR.
  • PCR polymerase chain reaction technique
  • the method allows detection of different targets (carbapenemases) in a single reaction and makes it possible to detect a number greater than 170 carbapenemase enzymes being comprised in, but not limited to, the following types:
  • the main contribution of the invention lies in the oligonucleotide sequences of the disclosed primers, which may be used together in a single amplification reaction allowing the detection of a great number of carbapenemase genes potentially present in a biological sample.
  • the method does not share the same technology as other commercially available systems which can detect carbapenemases (for example, the products from check-points line), and is preferably based on taqman technology, that is, it is preferably a detection method of carbapenemases by means of Taqman technology.
  • the innovation lies in using a series of oligonucleotides designed by the inventors which, combined among them and preferably together with taqman technology, can detect most of the carbapenemases described in the literature, going beyond any system described so far.
  • the technique has other additional advantages as that of being an essay with capacity to be adapted to any laboratory being provided with a PCR thermal cycler, preferably in real time, without the need of exclusive equipment or software, thus reducing costs and increasing accessibility to the technique by any laboratory.
  • Oligonucleotides disclosed are specific for each group of carbapenemases and have been designed to hybridize against DNA regions conserved for each group, such that all different variants belonging to the same group can be detected with a probe.
  • the technique used is, preferably, Multiplex real-time PCR which, as it is well known, is a technique being capable of detecting more than one target at the same time in a DNA amplification reaction (PCR). More preferably, each amplicon formed by the amplification reaction with two specific oligonucleotides is distinguished from each other thanks to a third oligonucleotide labelled with a determined fluorophore in such a way that, depending on which fluorophore detects the real-time PCR apparatus in a sample, there will be present one target or the other.
  • PCR DNA amplification reaction
  • the number of targets that can be detected at the same time is limited by two factors:
  • Detection of the target different carbapenemase genes are detected from the DNA of the strain that is thought to be carbapenemase-producing. Both genes found in plasmids and genes found in the bacteria chromosome can be detected.
  • Type of sample although in a preferred embodiment the technique is intended to carry out real-time PCR with DNA extracted from a previously culture-isolated strain, it can also be performed from DNA extracted from a biological sample of any type, e.g. a hemoculture.
  • diagnosis time is reduced to approximately 2 hours once the sample is available to be tested/assayed (for example, 35 min for extraction of the DNA contained in the sample and 1 h 20 minutes for PCR). Furthermore, sensitivity and specificity are considerably increased (coefficients very near 1).
  • the method described herein can be used for the detection of any carbapenemase-producing microorganism (gram-positive or gram-negative) during, for example, an infectious process, allowing the doctor to be able to make a better choice of the antibiotic therapy (reducing the therapeutic failure rate).
  • a first aspect of the invention refers to a pair of primers, hereinafter “pair of primers of the invention”, for detection and typing of carbapanemase-producing bacterial strains, selected from the list consisting of: SEQ ID No: 67-SEQ ID No: 68, SEQ ID No: 1-SEQ ID No: 2, SEQ ID No: 4-SEQ ID No: 5, SEQ ID No: 7-SEQ ID No: 8, SEQ ID No: 10-SEQ ID No: 11, SEQ ID No: 13-SEQ ID No: 14, SEQ ID No: 16-SEQ ID No: 17, SEQ ID No: 19-SEQ ID No: 20, SEQ ID No: 22-SEQ ID No: 23, SEQ ID No: 25-SEQ ID No: 26, SEQ ID No: 28-SEQ ID No: 29, SEQ ID No: 31-SEQ ID No: 32, SEQ ID No: 34-SEQ ID No: 35, SEQ ID No: 37-SEQ ID No: 38, SEQ ID No: 40-SEQ ID
  • Table 1 shown below, indicates what type of cabapenemase is capable of amplifying each one of these pairs of primers.
  • kit of the invention to be used in the detection of carbapenemase encoding genes, comprising at least a pair of primers of the invention and reagents required to perform an amplification reaction.
  • said kit comprises at least a probe capable of hybridization to the amplification product of said pair of primers.
  • kits required to perform an amplification reaction is meant, without limitation, the use of buffers, enzymes, polymerase enzymes, cofactors to obtain an optimal activity thereof, agents to prevent contamination, etc.
  • the kit may include all supports and recipients necessary for its operation and optimization.
  • the kit may also contain other molecules, genes, proteins or probes of interest, which are suitable as negative and positive controls.
  • the kit also comprises instructions to perform the method of the invention described above.
  • probe indicates, in the present text, the nucleotide sequence specific to recognize or detect selectively carbapenemase encoding genes, contained and produced by bacterial strains.
  • the primers are used to activate an amplification reaction of a DNA fragment, preferably by polymerase chain reaction or PCR.
  • the probe is used for specific detection of the target of interest formed by said amplification reaction, preferably PCR, and its sequence will be complementary to that of said target of interest or amplification product produced by the pair of primers of the invention.
  • Primers are sequences of oligonucleotides used as forward primer or reverse primer to activate the amplification reaction, preferably PCR, with the characteristic of binding specific regions of carbapenemases.
  • hybridizes or “hybridization” as used herein, refers to a process in which two complementary strands of nucleic acid recognize each other, and bind under appropriate astringency conditions.
  • Hybridizations to which the present invention refer to are carried out typically and preferably with nucleic acid sequences having a length between 10-100 nucleotides, more preferably between 10-50 nucleotides, even more preferably between 10-30 nucleotides.
  • the techniques and conditions of hybridization of nucleic acids are well known in the art. The average expert in the matter is capable of estimating and adjusting the rigor of the hybridization conditions.
  • the kit of the invention, the pair of primers of the invention or the probe is labelled with a fluorophore.
  • Fluorophore is understood to be any compound which can emit a light or coloured signal after being excited.
  • fluorophores include, but are not limited to: fluorescein, rhodamine, coumarin, cyanine, and derivatives thereof, GFP, YFP and RFP, oregon green, eosin, Texas red, naphthalene derivatives, coumarin derivatives, oxadiazol derivatives, pyrene derivatives, oxacin derivatives (as Nile red or Nile blue), acridine derivatives, tetrapyrrole, Alexa Fluor, DyLight Fluor, CY5, TAMRA, JOE or biotin.
  • the probe is labelled with CY5, TAMRA or JOE.
  • at least one of the primers in the pair of primers of the invention is labelled with biotin.
  • the probe comprises, more preferably consists of, the sequence:
  • the kit of the invention comprises the pairs of primers SEQ ID No: 28-SEQ ID No: 29, SEQ ID No: 46-SEQ ID No: 47, SEQ ID No: 55-SEQ ID No: 56, SEQ ID No: 67-SEQ ID No: 68 and SEQ ID No: 72-SEQ ID No: 73 and probes capable of hybridizing with the amplification products of said pairs of primers, more preferably the probes of SEQ ID No: 30, SEQ ID No: 48, SEQ ID No: 57, SEQ ID No: 69, SEQ ID No: 70, SEQ ID No: 71 and SEQ ID No: 74.
  • These pairs of primers are capable of amplifying type KPC carbapenemases (class A), OXA-48 (class D) and NDM, IMP and VIM (class B) (see Table 1).
  • the kit of the invention comprises all the pairs of primers of the invention, that is, the pairs or primers SEQ ID No: 67-SEQ ID No: 68, SEQ ID No: 1-SEQ ID No: 2, SEQ ID No: 4-SEQ ID No: 5, SEQ ID No: 7-SEQ ID No: 8, SEQ ID No: 10-SEQ ID No: 11, SEQ ID No: 13-SEQ ID No: 14, SEQ ID No: 16-SEQ ID No: 17, SEQ ID No: 19-SEQ ID No: 20, SEQ ID No: 22-SEQ ID No: 23, SEQ ID No: 25-SEQ ID No: 26, SEQ ID No: 28-SEQ ID No: 29, SEQ ID No: 31-SEQ ID No: 32, SEQ ID No: 34-SEQ ID No: 35, SEQ ID No: 37-SEQ ID No: 38, SEQ ID No: 40-SEQ ID No: 41, SEQ ID No: 43-SEQ ID No: 44, SEQ ID No: 46-SEQ ID No:
  • pairs of primers of the invention and the kit of the invention are useful for detection and/or typing of carbapenemase genes in biological samples, such that they can be used in a diagnostic method in vitro where if presence of said genes is detected, it can be stated that the tested sample is infected with bacteria producing them.
  • another aspect of the invention refers to the use in vitro of the pair of primers of the invention or the kit of the invention, for the detection and/or typing of carbapenemase-producing bacterial strains.
  • carbapenemase-producing bacterial strains include, but are not limited to, Acinetobacter baumanii, Pseduomonas sp., those bacteria that belong to the Enterobacteriaceae family (hereinafter Carbapenemase-Producing Enterobacteriaceae CPE), among which Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter spp., Serratia marcescens, Morganella moraganii , or Citrobacter spp can be cited.
  • Acinetobacter baumanii Pseduomonas sp.
  • Pseduomonas sp. those bacteria that belong to the Enterobacteriaceae family
  • Klebsiella pneumoniae Escherichia coli
  • Proteus mirabilis Enterobacter spp.
  • Serratia marcescens Enterobacter spp
  • Morganella moraganii or Citrobacter spp
  • Infection caused by carbapenemase-producing bacteria may be treated with specific antimicrobial agents or any combination thereof.
  • the preferred antimicrobial agents for treatment of CPE infections are selected from the list comprising: Carbapenems (preferably meropenem and doripenem), Aztreonam, Colistin, Aminoglycosides (preferably Amikacin), Fosfomycin, Tigecycline, Quinolones (preferably Ciprofloxacin) or Trimethoprim/Sulfamethoxazole.
  • the antimicrobial agents In order to choose the antimicrobial agents to be used, it is necessary to consider the typing of the bacterium or bacteria causing the infection, and alternatively to determine the microbiological activity thereof to the antimicrobial agent (for example, determining the minimum inhibitory concentration, MIC).
  • the choice of the treatment with the aforementioned antimicrobial agent, or with any combination of the mentioned antimicrobial agents, as well as concentrations thereof, could be determined by the expert in the art once the carbapenemase-producing bacterial species and/or the concrete strain present in the analyzed sample are identified, as it is described in the present invention.
  • the carbapenemases are selected from the list consisting of:
  • Another aspect of the invention refers to a method in vitro for detection and/or typing of carbapenemase-producing bacterial strains in an isolated biological sample, characterized in that it comprises:
  • detection of step (iii) comprises hybridization of the amplification product obtained in step (ii) to at least a probe capable of hybridizing to the amplification product of the pair of primers used in step (i).
  • the probe used in the method of the invention or present in the kit of the invention may be fixed on a support, in such a way that the product resulting from amplification of step (ii) is subsequently passed over said support on which the probe is fixed for detection thereof.
  • the term “fixed” as it is used in the present description refers to the fact that the probe can be attached to a support without losing its activity.
  • the support may be an array surface (for example a nylon array), of a membrane, a microtiter plate (for example, 96 wells) or a similar plastic support, or beads (spheres, for example, agarose spheres or small superparamagnetic microspheres consisting of biodegradable arrays).
  • the pair of primers used in step (i) or the probe used for detection in step (iii) is labelled with a fluorophore.
  • the fluorophore is selected from the list consisting of: CY5, TAMRA, JOE and biotin.
  • at least one of the primers in the pair of primers of the invention is labelled with biotin.
  • the probe comprises, more preferably consists of, the sequence:
  • step (i) the isolated biological sample is contacted with the pairs of primers SEQ ID No: 28-SEQ ID No: 29, SEQ ID No: 46-SEQ ID No: 47, SEQ ID No: 55-SEQ ID No: 56, SEQ ID No: 67-SEQ ID No: 68 and SEQ ID No: 72-SEQ ID No: 73.
  • step (i) the isolated biological sample is contacted with all the pairs of primers of the invention.
  • step (ii) is carried out by PCR, more preferably real-time PCR and even more preferably Multiplex real-time PCR.
  • the term “multiplex” indicates a mixture constitution capable of detecting different classes of carbapenemases in a single reaction (for example, “Oxa-like number”, “VIM-GIM-SPM, “KPC_SME_NMC”).
  • isolated biological sample comprising DNA refers to, but is not limited to, tissues and/or biological fluids from any origin obtained by means of any method known by an expert in the art which is suitable for such a purpose.
  • the biological sample may come from a floor, water, tissue sample (such as for example, but not limited to, skin), for example from, but not limited to, a biopsy or fine needle aspiration, or it may be a biological fluid, for example, but not limited to, blood, sputum, plasma, serum, urine or exudate (for example, rectal, pus).
  • the sample may also come from an in vitro culture, as for example, but not limited to, from a hemoculture previously inoculated with blood from an individual, preferably with bacteraemia.
  • the sample may be taken from a human, but also from non-human mammals, such as for example, but not limited to, rodents, ruminants, felines or canidae.
  • the biological sample comes from an individual, more preferably from a human individual.
  • the biological sample is blood, sputum, exudate, skin or henoculture previously inoculated with blood from an individual preferably having bacteraemia.
  • the method of the invention is a diagnosis method in vitro also comprising:
  • step (iv) assigning the individual to the group of patients infected with carbapenemase-producing bacterial strains when in step (iii) the presence of the amplification product is detected.
  • steps of the method of the invention may be completely or partially automated, for example, but not limited to, by means of robotic equipment for detection, in step (iii), of the amplification product.
  • the method of the invention may comprise other additional steps, for example, but not limited to, related to the pre-treatment of the biological sample isolated prior to the analysis thereof, for example with the purpose of extracting the genetic material from that sample.
  • the method of the invention may additionally comprise primers and probes capable of hybridizing, and amplifying in the case of primers, a DNA sequence present in the sample to be tested acting as control.
  • FIG. 1A illustrates detection of the carbapenemase groups OXA-23 and OXA-51 in samples containing three different strains of Acinetobacter baumanii.
  • FIG. 1B illustrates detection of the carbapenemase group OXA-51 in samples containing three different strains of Acinetobacter baumanii.
  • FIG. 1C illustrates detection of the carbapenemase groups OXA-23 and OXA-58 in samples containing three different strains of Acinetobacter baumanii.
  • FIG. 2 represents visualization in agarose gel of the results of the amplification of different carbapenemase genes after amplification reactions with the pairs of primers of the invention.
  • the invention is illustrated below with tests carried out by the inventors demonstrating the effectiveness of the pairs of primers of the invention in detection and/or typing of different groups of carbapenemases.
  • Primers and probes of the present invention have the following sequences selected from the group of: SEQ ID Nos: 1-77 described in Table 1 below. The primers are indicated in the two first columns and the probe is indicated in the third column. In each case, a triplet of two primers and a probe was used for detection.
  • oligonucleotides were used for carbapenemases encoding genes. Two oligonucleotides act as forward primer and reverse primer, and a third one was used being labelled with a “dye” or fluorophore and located in the middle of the amplicon which is being synthesized by PCR, and when the taq polymerase passes over it, it cleaves the “dye” which is thus released into the medium emitting its typical fluorescence.
  • the PCR amplification profile was: pre PCR: 2 min at 50° C., 2 min at 94° C. PCR: (15 seconds at 94° C., 50 seconds at 60° C.) ⁇ 40 reps. The fluorescence reading was performed in the 50 seconds step at 60° C.
  • different targets may be detected in a single reaction.
  • the experiments performed meaning by experiment each of the 10 different reactions of target detection realized in several multiplex, in reactions using 2 or 3 different probes in each well of the plate in real-time PCR, have been designed using the programme Beacon Designer v8 under the Taqman experimental profile, having the capacity of generating multiplex reactions upon introducing the different sequences of the desired genes to be amplified at the same time.
  • the DNA regions conserved among carbapenemases of the same group ej. KPC, Oxa_23 etc.
  • probes capable of detecting most of the variants inside the same group with a single PCR were designed.
  • Table 2 A second table (Table 2) is shown below with all the carbapenemases detected by the method disclosed. It should be noted that these are the carbapenemases which the system detects as a minimum, since, considering that the experiment is designed towards conserved DNA regions, it is likely that as new carbapenemases are found which have not been described in the literature yet, as long as they belong to any of the groups detected by the system, they can probably also be detected.
  • the system designed consists of 25 Taqman probes which, grouped in multiplex reactions of 2 or 3 targets, formed 8 multiplex reactions.
  • each probe was labelled with a different fluorophore, such that the 3 targets could be detected at the same time if they were present in the sample.
  • All the multiplex reactions included in this assay worked under the same temperature conditions and had a minimum score of 8 out of 10, according to the software used for its design, so all combinations worked equally well.
  • Each probe was designed to hybridize in the PCR reaction against regions highly conserved within a class of carbapenemases. So the system has the capacity to detect all variants within the same group of carbapenemases. In principle, the system is capable of detecting more than 200 known variants.
  • FIGS. 1A-1C shows the results obtained from 3 different strains of Acinetobacter baumanii from a strain collection, previously well-characterized.
  • FIG. 1A 2 types of carbapenemases are found; oxa-23 and oxa-51. In FIG. 1B only carbapenemase oxa-51 is found. In FIG. 1C oxa-58 and oxa-51 are found.
  • the system was applied to a collection of 600 bacterial strains consecutively isolated, wherein a screening for reduced sensitivity to carbapenems had been previously carried out, with the purpose of getting to know the carbapenemases most frequently found in our environment.
  • the primers described herein can also be used in a PCR amplification technology, where said primers are labelled with biotin, and a subsequent hybridization against a membrane carrying a DNA fragment complementary to the amplicon of interest. After hybridization, by means of a chemical reaction, the biotin darkens the membrane in the hybridization site, and depending on the darkening extent of the site, a software considers the reaction positive or negative.
  • a Real-time PCR system to genetically detect carbapenemase-producing strains was designed.
  • the real-time multiplex reactions (grouped in threes), were adapted in a single conventional PCR reaction, wherein instead of carrying out multiplex reactions in groups of three targets, all those reactions were put together in a single reaction, wherein all the primers designed for the real-time PCR system were present.
  • the conventional PCR programme used for amplification was as follows:
  • the most important stage of the programme is that of 45 seconds at 50° C.
  • the primers were designed to work at a range from 50 to 52 grades (theoretical data obtained from simulation programmes). However, said primers could unexpectedly work optimally in assays in temperature ranges from 57° C. to 60° C. Simulation programmes did not predict the primers could work at temperatures higher than 52° C.
  • KPC, GES, IMP, OXA-51 and SME a concentration of at least 0.5 pmol/ ⁇ l was used for both primers (forward and reverse), and for the rest of the genes (SPM, SIM, OXA-48, OXA-24, NDM, NMC, VIM, OXA-23, OXA-58) a concentration of at least 0.25 pmol/ ⁇ l, was used for both primers (forward and reverse).

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Abstract

Some pairs of primers are provided, being used for detection of carbapenemase genes and carbapenemase-producing bacterial strains, and a diagnosis method using said set of primers. Furthermore, there is also disclosed, preferably, the use of probes within each amplification reaction, more preferably multiplex PCR, for selective identification and detection of carbapenemase genes from a biological sample. The primers object of this invention may be used for preparing a kit for carbapenemase genes identification and detection, preferably carbapenemases contained in bacterial strains producing said enzymes.

Description

  • The present invention refers to primers for rapid detection and/or typing of carbapenemase-producing bacterial strains. Starting from said primers, the invention also discloses a kit for the rapid detection of carbapenemases so as to identify multi-resistant organisms producing said enzymes, to be used in clinical practice for prevention and epidemiologic control of infection outbreaks, for example hospital-acquired infection outbreaks, and to be particularly used in Intensive Care Units and in Neonatal Intensive Care Units (NICU). Finally, the invention also refers to a method for the detection and/or typing of carbapenemase-producing bacterial strains using said primers.
    • STATE OF THE ART
  • Biomolecular techniques are used in epidemiology to analyze certain bacterial characteristics suitable as epidemiological markers. Techniques derived from biochemistry, immunology and genetics are included.
  • Genotypic typing methods are based on the study of chromosomal or extra-chromosomal DNA. The main methods include plasmid analysis, restriction endonuclease analysis, DNA-probe fingerprinting and PCR-based fingerprinting. With regards to polymerase chain reaction or PCR techniques, there are several PCR variations that can be used. One of them consists of using primers complementary to repetitive regions found in the bacterial genome, and it features two modalities (REP-PCR and ERIC-PCR). Another variation is the PCR in which arbitrary primers are used (AP-PCR). Of all these techniques, REP-PCR is the one showing more discrimination and reproducibility capacity, and it may also be used both in gram-negative and gram-positive bacteria. PCR may also be combined with restriction endonucleases technique, obtaining fragments which are then electrophoretically analyzed (PCR-RFLP).
  • PCR and other amplification techniques may have a deep effect on reducing time to and increasing certainty of diagnosis for a variety of microorganism infections. The use of this technique is interesting mainly in those infections for which other alternative methods are costly and troublesome or in which a delay in the diagnosis is caused by the slow growth of microorganisms in conventional culture means.
  • Nowadays, there is intensive work being carried out on the design of reliable methods for detection of these multi-resistant strains. A bibliographic review carried out by the inventors, shows that there are several research groups working on this important clinical problem.
  • As a consequence of this research, a surveillance method has been developed so as to identify the presence of carbapenem-resistant KPC-producing enterobacteriaceae in the rectum, based on PCR detection of blakPC gene (Schechner V. Evaluation of PCR-based testing for surveillance of KPC-producing carbapenem-resistant members of the Enterobacteriaceae family. J Clin Microbiol. 2009T; 47:3261-5.). The research in question develops a rapid identification system, but it exclusively detects the blackPC gene, which represents a limitation for those media in which other carbapenemases prevail. On the other hand, the research lacks for a “gold” standard for comparison of the method, so the actual sensitivity values could be lower than those obtained experimentally.
  • Likewise, there are homologous systems for detection of the blaNDM-1 gene (Danny C. T. Ong. Rapid detection of the blaNDM-1 gene by real-time PCR. J. Antimicrob. Chemother. (2011) doi: 10.1093/jac/dkr184 y Kruttgen A. et al. Real-time PCR assay and a synthetic positive control for the rapid and sensitive detection of the emerging resistance gene New Delhi metallo-β-lactamase-1 (blaNDM-1). Med Microbiol Immunol 2011; 200:137-41).
  • Finally, there are screening methodologies which, by using boronic acid derivatives, allow the detection of class A carbapenemase-producing bacteria, including KPC, Sme, IMI, NMC-A and GES. This system that can recognize different types of carbapenemases is based, however, on phenotypic microbiological techniques and not in molecular techniques, which are generally faster than the former ones.
  • Several patents may be cited which are related to the object of the invention:
  • RU 2415932-A describes a method for gene identification of broad-spectrum class A β-lactamases. The method is based on PCR technique and uses a DNA microchip labelled with biotin; this molecule is detected by a streptavidin-peroxidase conjugate, subsequently recognized by means of colorimetric detection of peroxidase.
  • WO2008124670-A refers to a method for identification of all known isoforms of KPC gene, which includes a new pair of primers and a new probe for enzyme detection by means of amplification methods such as PCR technique. This system may be presented in the form of a kit for detection of carbapenemases in samples from patients. The system described herein is the closest to that disclosed in the present invention but it is much more limited, since it only detects KPC carbapenemases.
  • Therefore, there is a need for developing systems that allow a simple, rapid and reliable detection of carbapenemases-producing bacteria in biological samples, which would be interesting from the point of view of clinical diagnosis. It would be also desirable that this system would allow not only typing of detected carbapenemases, but also detection of the greatest number possible thereof.
    • DESCRIPTION OF THE INVENTION
  • The invention refers to primers and probes identified in the appended claims. The primers and probes object of this invention may be used for the preparation of a kit for carbapenemase gene identification and detection, preferably carbapenemase genes contained in and produced by carbapenemase-producing bacterial strains. The invention also provides a diagnosis method using said primers and probes.
  • Another object described in the present invention refers to a kit for carbapenemase gene identification and detection, preferably carbapenemase genes contained in and produced by carbapenemase-producing bacterial strains, comprising the primers and probes according to this invention.
  • In a preferred embodiment of the invention, the kit comprises primers and probes specific for recognizing different carbapenemase genes, for example those indicated in Table 1, included below.
  • The invention also comprises a method for detection and typing of carbapenemase-producing bacterial strains, consisting of binding and hybridizing the primers of the invention to a specific DNA sequence of the gene to be tested, performing a selective specific amplification of carbapenemase genes and identifying the amplification product with the specific probe of the genetic sequence or genetic fragment of diagnostic interest.
  • The identification method of carbapenemase genes disclosed in this invention is characterized in that it preferably uses multiplex reactions where, for example, two or three primers-probe reactions work together to detect different types of carbapenemase genes. The reactions that can work together are shown in Table 1.
  • In particular, the method for carbapenemase genes identification of this invention is characterized in that each forward and reverse primer from a multiplex reaction binds to specific DNA sequences so that they can be PCR amplified, and, preferably, at the same time each specific probe labelled with a determined fluorophore binds to these amplicons which are formed in the case of carbapenemase genes presence.
  • Thus, DNA sequences are preferably subjected to PCR (polymerase chain reaction technique) which is, more preferably, Multiplex real-time PCR.
  • The method allows detection of different targets (carbapenemases) in a single reaction and makes it possible to detect a number greater than 170 carbapenemase enzymes being comprised in, but not limited to, the following types:
      • Class A carbapenemase: NMC, IMI, SME, KPC and GES.
      • Class B1 carbapenemase: IMP, VIM, GIM, SPM, NDM and SIM.
      • Class D carbapenemase: OXA-23-like, 51-like, 58-like, 60a-like, 50a-like, 55-like, 62-like, 54-like, 24-like, 48-like, 133-like, 182-like, 98-like, 60c-like and 60d-like.
  • The main contribution of the invention lies in the oligonucleotide sequences of the disclosed primers, which may be used together in a single amplification reaction allowing the detection of a great number of carbapenemase genes potentially present in a biological sample.
  • The method does not share the same technology as other commercially available systems which can detect carbapenemases (for example, the products from check-points line), and is preferably based on taqman technology, that is, it is preferably a detection method of carbapenemases by means of Taqman technology.
  • Thus, the innovation lies in using a series of oligonucleotides designed by the inventors which, combined among them and preferably together with taqman technology, can detect most of the carbapenemases described in the literature, going beyond any system described so far.
  • The technique, as well as the benefits already mentioned, has other additional advantages as that of being an essay with capacity to be adapted to any laboratory being provided with a PCR thermal cycler, preferably in real time, without the need of exclusive equipment or software, thus reducing costs and increasing accessibility to the technique by any laboratory.
  • Oligonucleotides disclosed are specific for each group of carbapenemases and have been designed to hybridize against DNA regions conserved for each group, such that all different variants belonging to the same group can be detected with a probe.
  • The technique used is, preferably, Multiplex real-time PCR which, as it is well known, is a technique being capable of detecting more than one target at the same time in a DNA amplification reaction (PCR). More preferably, each amplicon formed by the amplification reaction with two specific oligonucleotides is distinguished from each other thanks to a third oligonucleotide labelled with a determined fluorophore in such a way that, depending on which fluorophore detects the real-time PCR apparatus in a sample, there will be present one target or the other.
  • The number of targets that can be detected at the same time is limited by two factors:
  • 1. By the number of spectrally different fluorophores (dyes) commercially available.
  • 2. By the number of fluorophores (“dyes”) which can be excited and detected by the apparatus used.
  • The advantages associated to the described invention are:
  • 1. Detection of the target: different carbapenemase genes are detected from the DNA of the strain that is thought to be carbapenemase-producing. Both genes found in plasmids and genes found in the bacteria chromosome can be detected.
  • 2. Type of sample: although in a preferred embodiment the technique is intended to carry out real-time PCR with DNA extracted from a previously culture-isolated strain, it can also be performed from DNA extracted from a biological sample of any type, e.g. a hemoculture.
  • 3. Quickness: diagnosis time is reduced to approximately 2 hours once the sample is available to be tested/assayed (for example, 35 min for extraction of the DNA contained in the sample and 1 h 20 minutes for PCR). Furthermore, sensitivity and specificity are considerably increased (coefficients very near 1).
  • 4. The method described herein can be used for the detection of any carbapenemase-producing microorganism (gram-positive or gram-negative) during, for example, an infectious process, allowing the doctor to be able to make a better choice of the antibiotic therapy (reducing the therapeutic failure rate).
  • 5. Regarding the control of hospital-acquired infection outbreaks, it is considered that, in the event a hospital outbreak is suspected to be taking place, applying this system could be fundamental for cost and time saving when detecting where said outbreak comes from. As it is capable of detecting and classifying the carbapenemases by classes, it could be possible to locate where the outbreak comes from and take actions quickly, reducing the potential danger being caused by said outbreak.
  • Regarding knowledge about the epidemiology of the area where a carbapenemase-producing strain research is done, it should be noted that currently, since there are not quality detection methods, the distribution and the type of carbapanemases in our environment is unknown.
  • For all that, a first aspect of the invention refers to a pair of primers, hereinafter “pair of primers of the invention”, for detection and typing of carbapanemase-producing bacterial strains, selected from the list consisting of: SEQ ID No: 67-SEQ ID No: 68, SEQ ID No: 1-SEQ ID No: 2, SEQ ID No: 4-SEQ ID No: 5, SEQ ID No: 7-SEQ ID No: 8, SEQ ID No: 10-SEQ ID No: 11, SEQ ID No: 13-SEQ ID No: 14, SEQ ID No: 16-SEQ ID No: 17, SEQ ID No: 19-SEQ ID No: 20, SEQ ID No: 22-SEQ ID No: 23, SEQ ID No: 25-SEQ ID No: 26, SEQ ID No: 28-SEQ ID No: 29, SEQ ID No: 31-SEQ ID No: 32, SEQ ID No: 34-SEQ ID No: 35, SEQ ID No: 37-SEQ ID No: 38, SEQ ID No: 40-SEQ ID No: 41, SEQ ID No: 43-SEQ ID No: 44, SEQ ID No: 46-SEQ ID No: 47, SEQ ID No: 49-SEQ ID No: 50, SEQ ID No: 52-SEQ ID No: 53, SEQ ID No: 55-SEQ ID No: 56, SEQ ID No: 58-SEQ ID No: 59, SEQ ID No: 61-SEQ ID No: 62, SEQ ID No: 64-SEQ ID No: 65, SEQ ID No: 72-SEQ ID No: 73 and SEQ ID No: 75-SEQ ID No: 76.
  • Table 1, shown below, indicates what type of cabapenemase is capable of amplifying each one of these pairs of primers.
  • Another aspect of the invention refers to a kit, hereinafter “kit of the invention”, to be used in the detection of carbapenemase encoding genes, comprising at least a pair of primers of the invention and reagents required to perform an amplification reaction. Preferably, said kit comprises at least a probe capable of hybridization to the amplification product of said pair of primers.
  • By “reagents required to perform an amplification reaction” is meant, without limitation, the use of buffers, enzymes, polymerase enzymes, cofactors to obtain an optimal activity thereof, agents to prevent contamination, etc. On the other hand, the kit may include all supports and recipients necessary for its operation and optimization. The kit may also contain other molecules, genes, proteins or probes of interest, which are suitable as negative and positive controls. Preferably, the kit also comprises instructions to perform the method of the invention described above.
  • The expression “probe”, “primer” and “diagnostic genetic fragment” indicate, in the present text, the nucleotide sequence specific to recognize or detect selectively carbapenemase encoding genes, contained and produced by bacterial strains. The primers are used to activate an amplification reaction of a DNA fragment, preferably by polymerase chain reaction or PCR. The probe is used for specific detection of the target of interest formed by said amplification reaction, preferably PCR, and its sequence will be complementary to that of said target of interest or amplification product produced by the pair of primers of the invention.
  • Primers, according to this invention, are sequences of oligonucleotides used as forward primer or reverse primer to activate the amplification reaction, preferably PCR, with the characteristic of binding specific regions of carbapenemases.
  • The term “hybridizes” or “hybridization” as used herein, refers to a process in which two complementary strands of nucleic acid recognize each other, and bind under appropriate astringency conditions. Hybridizations to which the present invention refer to are carried out typically and preferably with nucleic acid sequences having a length between 10-100 nucleotides, more preferably between 10-50 nucleotides, even more preferably between 10-30 nucleotides. The techniques and conditions of hybridization of nucleic acids are well known in the art. The average expert in the matter is capable of estimating and adjusting the rigor of the hybridization conditions.
  • In a preferred embodiment, the kit of the invention, the pair of primers of the invention or the probe is labelled with a fluorophore.
  • “Fluorophore” is understood to be any compound which can emit a light or coloured signal after being excited. Examples of fluorophores include, but are not limited to: fluorescein, rhodamine, coumarin, cyanine, and derivatives thereof, GFP, YFP and RFP, oregon green, eosin, Texas red, naphthalene derivatives, coumarin derivatives, oxadiazol derivatives, pyrene derivatives, oxacin derivatives (as Nile red or Nile blue), acridine derivatives, tetrapyrrole, Alexa Fluor, DyLight Fluor, CY5, TAMRA, JOE or biotin. In a more preferred embodiment, the probe is labelled with CY5, TAMRA or JOE. In another preferred embodiment, at least one of the primers in the pair of primers of the invention is labelled with biotin.
  • In another preferred embodiment of the kit of the invention, the probe comprises, more preferably consists of, the sequence:
      • a. SEQ ID No: 3, when the pair of primers is SEQ ID No: 1-SEQ ID No: 2,
      • b. SEQ ID No: 6, when the pair of primers is SEQ ID No: 4-SEQ ID No: 5,
      • c. SEQ ID No: 9, when the pair of primers is SEQ ID No: 7-SEQ ID No: 8,
      • d. SEQ ID No: 12, when the pair of primers is SEQ ID No: 10-SEQ ID No: 11,
      • e. SEQ ID No: 15, when the pair of primers is SEQ ID No: 13-SEQ ID No: 14,
      • f. SEQ ID No: 18, when the pair of primers is SEQ ID No: 16-SEQ ID No: 17,
      • g. SEQ ID No: 21, when the pair of primers is SEQ ID No: 19-SEQ ID No: 20,
      • h. SEQ ID No: 24, when the pair of primers is SEQ ID No: 22-SEQ ID No: 23,
      • i. SEQ ID No: 27, when the pair of primers is SEQ ID No: 25-SEQ ID No: 26,
      • j. SEQ ID No: 30, when the pair of primers is SEQ ID No: 28-SEQ ID No: 29,
      • k. SEQ ID No: 33, when the pair of primers is SEQ ID No: 31-SEQ ID No: 32,
      • l. SEQ ID No: 36, when the pair of primers is SEQ ID No: 34-SEQ ID No: 35,
      • m. SEQ ID No: 39, when the pair of primers is SEQ ID No: 37-SEQ ID No: 38,
      • n. SEQ ID No: 42, when the pair of primers is SEQ ID No: 40-SEQ ID No: 41,
      • o. SEQ ID No: 45, when the pair of primers is SEQ ID No: 43-SEQ ID No: 44,
      • p. SEQ ID No: 48, when the pair of primers is SEQ ID No: 46-SEQ ID No: 47,
      • q. SEQ ID No: 51, when the pair of primers is SEQ ID No: 49-SEQ ID No: 50,
      • r. SEQ ID No: 54, when the pair of primers is SEQ ID No: 52-SEQ ID No: 53,
      • s. SEQ ID No: 57, when the pair of primers is SEQ ID No: 55-SEQ ID No: 56,
      • t. SEQ ID No: 60, when the pair of primers is SEQ ID No: 58-SEQ ID No: 59,
      • u. SEQ ID No: 63, when the pair of primers is SEQ ID No: 61-SEQ ID No: 62,
      • v. SEQ ID No: 66, when the pair of primers is SEQ ID No: 64-SEQ ID No: 65,
      • w. SEQ ID No: 69, SEQ ID No: 70 and/or SEQ ID No: 71, when the pair of primers is SEQ ID No: 67-SEQ ID No: 68,
      • x. SEQ ID No: 74, when the pair of primers is SEQ ID No: 72-SEQ ID No: 73, or
      • y. SEQ ID No: 77, when the pair of primers is SEQ ID NO: 75-SEQ ID No: 76.
  • In another preferred embodiment, the kit of the invention comprises the pairs of primers SEQ ID No: 28-SEQ ID No: 29, SEQ ID No: 46-SEQ ID No: 47, SEQ ID No: 55-SEQ ID No: 56, SEQ ID No: 67-SEQ ID No: 68 and SEQ ID No: 72-SEQ ID No: 73 and probes capable of hybridizing with the amplification products of said pairs of primers, more preferably the probes of SEQ ID No: 30, SEQ ID No: 48, SEQ ID No: 57, SEQ ID No: 69, SEQ ID No: 70, SEQ ID No: 71 and SEQ ID No: 74. These pairs of primers are capable of amplifying type KPC carbapenemases (class A), OXA-48 (class D) and NDM, IMP and VIM (class B) (see Table 1).
  • In an even more preferred embodiment, the kit of the invention comprises all the pairs of primers of the invention, that is, the pairs or primers SEQ ID No: 67-SEQ ID No: 68, SEQ ID No: 1-SEQ ID No: 2, SEQ ID No: 4-SEQ ID No: 5, SEQ ID No: 7-SEQ ID No: 8, SEQ ID No: 10-SEQ ID No: 11, SEQ ID No: 13-SEQ ID No: 14, SEQ ID No: 16-SEQ ID No: 17, SEQ ID No: 19-SEQ ID No: 20, SEQ ID No: 22-SEQ ID No: 23, SEQ ID No: 25-SEQ ID No: 26, SEQ ID No: 28-SEQ ID No: 29, SEQ ID No: 31-SEQ ID No: 32, SEQ ID No: 34-SEQ ID No: 35, SEQ ID No: 37-SEQ ID No: 38, SEQ ID No: 40-SEQ ID No: 41, SEQ ID No: 43-SEQ ID No: 44, SEQ ID No: 46-SEQ ID No: 47, SEQ ID No: 49-SEQ ID No: 50, SEQ ID No: 52-SEQ ID No: 53, SEQ ID No: 55-SEQ ID No: 56, SEQ ID No: 58-SEQ ID No: 59, SEQ ID No: 61-SEQ ID No: 62, SEQ ID No: 64-SEQ ID No: 65, SEQ ID No: 72-SEQ ID No: 73 and SEQ ID No: 75-SEQ ID No: 76, and probes capable of hybridize to the amplification products of said pairs of primers, preferably the probes described above in sections (a) and (y).
  • The pairs of primers of the invention and the kit of the invention are useful for detection and/or typing of carbapenemase genes in biological samples, such that they can be used in a diagnostic method in vitro where if presence of said genes is detected, it can be stated that the tested sample is infected with bacteria producing them. Thus, another aspect of the invention refers to the use in vitro of the pair of primers of the invention or the kit of the invention, for the detection and/or typing of carbapenemase-producing bacterial strains. Examples of carbapenemase-producing bacterial strains include, but are not limited to, Acinetobacter baumanii, Pseduomonas sp., those bacteria that belong to the Enterobacteriaceae family (hereinafter Carbapenemase-Producing Enterobacteriaceae CPE), among which Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter spp., Serratia marcescens, Morganella moraganii, or Citrobacter spp can be cited.
  • Infection caused by carbapenemase-producing bacteria may be treated with specific antimicrobial agents or any combination thereof. The preferred antimicrobial agents for treatment of CPE infections are selected from the list comprising: Carbapenems (preferably meropenem and doripenem), Aztreonam, Colistin, Aminoglycosides (preferably Amikacin), Fosfomycin, Tigecycline, Quinolones (preferably Ciprofloxacin) or Trimethoprim/Sulfamethoxazole.
  • In order to choose the antimicrobial agents to be used, it is necessary to consider the typing of the bacterium or bacteria causing the infection, and alternatively to determine the microbiological activity thereof to the antimicrobial agent (for example, determining the minimum inhibitory concentration, MIC). The choice of the treatment with the aforementioned antimicrobial agent, or with any combination of the mentioned antimicrobial agents, as well as concentrations thereof, could be determined by the expert in the art once the carbapenemase-producing bacterial species and/or the concrete strain present in the analyzed sample are identified, as it is described in the present invention.
  • In a preferred embodiment, the carbapenemases are selected from the list consisting of:
      • Class A carbapenemase, preferably NMC, IMI, SME, KPC and/or GES,
      • Class B1 carbapenemase, preferably IMP, VIM, GIM, SPM, NDM and/or SIM, and/or
      • Class D carbapenemase, preferably OXA-23-like, 51-like, 58-like, 60a-like, 50a-like, 55-like, 62-like, 54-like, 24-like, 48-like, 133-like, 182-like, 98-like, 60c-like and/or 60d-like.
  • Another aspect of the invention refers to a method in vitro for detection and/or typing of carbapenemase-producing bacterial strains in an isolated biological sample, characterized in that it comprises:
  • i. contacting an isolated biological sample comprising DNA with at least a pair of primers of the invention,
  • ii. amplifying the DNA comprised in said isolated biological sample with the pair of primers, and
  • iii. detecting the presence of the amplification product obtained in step (ii).
  • Hereinafter, this method will be referred to as “the method of the invention”.
  • In a preferred embodiment of the method of the invention, detection of step (iii) comprises hybridization of the amplification product obtained in step (ii) to at least a probe capable of hybridizing to the amplification product of the pair of primers used in step (i).
  • In another preferred embodiment, the probe used in the method of the invention or present in the kit of the invention may be fixed on a support, in such a way that the product resulting from amplification of step (ii) is subsequently passed over said support on which the probe is fixed for detection thereof. The term “fixed” as it is used in the present description, refers to the fact that the probe can be attached to a support without losing its activity. Preferably, the support may be an array surface (for example a nylon array), of a membrane, a microtiter plate (for example, 96 wells) or a similar plastic support, or beads (spheres, for example, agarose spheres or small superparamagnetic microspheres consisting of biodegradable arrays).
  • In another preferred embodiment of the method of the invention, the pair of primers used in step (i) or the probe used for detection in step (iii) is labelled with a fluorophore. In an even more preferred embodiment, the fluorophore is selected from the list consisting of: CY5, TAMRA, JOE and biotin. In an even more preferred embodiment, at least one of the primers in the pair of primers of the invention is labelled with biotin.
  • In another preferred embodiment of the method of the invention, the probe comprises, more preferably consists of, the sequence:
      • a. SEQ ID No: 3, when the pair of primers is SEQ ID No: 1-SEQ ID No: 2,
      • b. SEQ ID No: 6, when the pair of primers is SEQ ID No: 4-SEQ ID No: 5,
      • c. SEQ ID No: 9, when the pair of primers is SEQ ID No: 7-SEQ ID No: 8,
      • d. SEQ ID No: 12, when the pair of primers is SEQ ID No: 10-SEQ ID No: 11,
      • e. SEQ ID No: 15, when the pair of primers is SEQ ID No: 13-SEQ ID No: 14,
      • f. SEQ ID No: 18, when the pair of primers is SEQ ID No: 16-SEQ ID No: 17,
      • g. SEQ ID No: 21, when the pair of primers is SEQ ID No: 19-SEQ ID No: 20,
      • h. SEQ ID No: 24, when the pair of primers is SEQ ID No: 22-SEQ ID No: 23,
      • i. SEQ ID No: 27, when the pair of primers is SEQ ID No: 25-SEQ ID No: 26,
      • j. SEQ ID No: 30, when the pair of primers is SEQ ID No: 28-SEQ ID No: 29,
      • k. SEQ ID No: 33, when the pair of primers is SEQ ID No: 31-SEQ ID No: 32,
      • l. SEQ ID No: 36, when the pair of primers is SEQ ID No: 34-SEQ ID No: 35,
      • m. SEQ ID No: 39, when the pair of primers is SEQ ID No: 37-SEQ ID No: 38,
      • n. SEQ ID No: 42, when the pair of primers is SEQ ID No: 40-SEQ ID No: 41,
      • o. SEQ ID No: 45, when the pair of primers is SEQ ID No: 43-SEQ ID No: 44,
      • p. SEQ ID No: 48, when the pair of primers is SEQ ID No: 46-SEQ ID No: 47,
      • q. SEQ ID No: 51, when the pair of primers is SEQ ID No: 49-SEQ ID No: 50,
      • r. SEQ ID No: 54, when the pair of primers is SEQ ID No: 52-SEQ ID No: 53,
      • s. SEQ ID No: 57, when the pair of primers is SEQ ID No: 55-SEQ ID No: 56,
      • t. SEQ ID No: 60, when the pair of primers is SEQ ID No: 58-SEQ ID No: 59,
      • u. SEQ ID No: 63, when the pair of primers is SEQ ID No: 61-SEQ ID No: 62,
      • v. SEQ ID No: 66, when the pair of primers is SEQ ID No: 64-SEQ ID No: 65,
      • w. SEQ ID No: 69, SEQ ID No: 70 and/or SEQ ID No: 71, when the pair of primers is SEQ ID No: 67-SEQ ID No: 68,
      • x. SEQ ID No: 74, when the pair of primers is SEQ ID No: 72-SEQ ID No: 73, or
      • y. SEQ ID No: 77, when the pair of primers is SEQ ID NO: 75-SEQ ID No: 76.
  • In another preferred embodiment of the method of the invention, in step (i) the isolated biological sample is contacted with the pairs of primers SEQ ID No: 28-SEQ ID No: 29, SEQ ID No: 46-SEQ ID No: 47, SEQ ID No: 55-SEQ ID No: 56, SEQ ID No: 67-SEQ ID No: 68 and SEQ ID No: 72-SEQ ID No: 73. In a more preferred embodiment, in step (i) the isolated biological sample is contacted with all the pairs of primers of the invention.
  • In another preferred embodiment, the amplification of step (ii) is carried out by PCR, more preferably real-time PCR and even more preferably Multiplex real-time PCR.
  • According to the present invention, the term “multiplex” indicates a mixture constitution capable of detecting different classes of carbapenemases in a single reaction (for example, “Oxa-like number”, “VIM-GIM-SPM, “KPC_SME_NMC”).
  • The term “isolated biological sample comprising DNA”, as it is used in the description, refers to, but is not limited to, tissues and/or biological fluids from any origin obtained by means of any method known by an expert in the art which is suitable for such a purpose. The biological sample may come from a floor, water, tissue sample (such as for example, but not limited to, skin), for example from, but not limited to, a biopsy or fine needle aspiration, or it may be a biological fluid, for example, but not limited to, blood, sputum, plasma, serum, urine or exudate (for example, rectal, pus). The sample may also come from an in vitro culture, as for example, but not limited to, from a hemoculture previously inoculated with blood from an individual, preferably with bacteraemia. The sample may be taken from a human, but also from non-human mammals, such as for example, but not limited to, rodents, ruminants, felines or canidae. Thus, in another preferred embodiment of this aspect of the invention, the biological sample comes from an individual, more preferably from a human individual. In another preferred embodiment, the biological sample is blood, sputum, exudate, skin or henoculture previously inoculated with blood from an individual preferably having bacteraemia.
  • In another preferred embodiment, the method of the invention is a diagnosis method in vitro also comprising:
  • (iv) assigning the individual to the group of patients infected with carbapenemase-producing bacterial strains when in step (iii) the presence of the amplification product is detected.
  • The steps of the method of the invention may be completely or partially automated, for example, but not limited to, by means of robotic equipment for detection, in step (iii), of the amplification product.
  • As well as the steps specified above, the method of the invention may comprise other additional steps, for example, but not limited to, related to the pre-treatment of the biological sample isolated prior to the analysis thereof, for example with the purpose of extracting the genetic material from that sample.
  • Furthermore, the method of the invention, as well as the kit of the invention, may additionally comprise primers and probes capable of hybridizing, and amplifying in the case of primers, a DNA sequence present in the sample to be tested acting as control.
  • Throughout the description and the claims, the term “comprise” and its variants does not exclude other technical characteristics, additives, components or steps. For experts in the field, other purposes, advantages and characteristics of the invention will derive in part from the description and in part from the practice of the invention. The following examples and figures are provided for illustration purposes, and are not intended to be limiting of the present invention.
    • DESCRIPTION OF THE FIGURES
  • FIG. 1A illustrates detection of the carbapenemase groups OXA-23 and OXA-51 in samples containing three different strains of Acinetobacter baumanii.
  • FIG. 1B illustrates detection of the carbapenemase group OXA-51 in samples containing three different strains of Acinetobacter baumanii.
  • FIG. 1C illustrates detection of the carbapenemase groups OXA-23 and OXA-58 in samples containing three different strains of Acinetobacter baumanii.
  • FIG. 2 represents visualization in agarose gel of the results of the amplification of different carbapenemase genes after amplification reactions with the pairs of primers of the invention.
    •  EXAMPLES
  • The invention is illustrated below with tests carried out by the inventors demonstrating the effectiveness of the pairs of primers of the invention in detection and/or typing of different groups of carbapenemases.
    • Example 1
  • Primers and probes of the present invention have the following sequences selected from the group of: SEQ ID Nos: 1-77 described in Table 1 below. The primers are indicated in the two first columns and the probe is indicated in the third column. In each case, a triplet of two primers and a probe was used for detection.
  • TABLE 1
    Multiplex_1 Forward Sequence (F) Reverse Sequence (R) Taqman Sequence
    Oxa_23_like TCGGTAATGCTGAAA CTGTGTATGTGCTAA AACTCTACCTCTTGA
    TTG (SEQ ID NO: 1) TTGG (SEQ ID NO: 2) ATAGGCGTAACC
    (SEQ ID NO: 3)
    Oxa_51_like CCTTCAAAATGCTTA TAGGGTCATGTCCTT CACCATAAGGCAACC
    ATGC (SEQ ID NO: 4) TTC (SEQ ID NO: 5) ACCACAG (SEQ ID
    NO: 6)
    Oxa_58_like TGAGCATAGTATGAG TGACCATCATATGTG TTCCACAAGTGAATA
    TCG (SEQ ID NO: 7) ACA (SEQ ID NO: 8) ACTCAATCATCG
    (SEQ ID NO: 9)
    Multiplex_2 F Sequence R Sequence Taqman Sequence
    Oxa_60a_like TGCAGGCTTATGTCG CTTCGAAAGCGGAAA CGCAGCCAGAACTG
    ATG (SEQ ID NO: 10) TCTC (SEQ ID NO: 11) GTCGATC (SEQ ID
    NO: 12)
    Oxa_50a_like CCTGATCGGCTTATC GTACGTTCGATGCCT TAGATCCGCCGACGA
    CAC (SEQ ID NO: 13) TGA (SEQ ID NO: 14) GGTTCT (SEQ ID NO:
    15)
    Oxa_55_like AAGCGAGTACTGACT CAGGTTAACGGCGAA CGTCCAACTCCAACC
    ATATYATTC (SEQ ID ATAG (SEQ ID NO: 17) AACCGA (SEQ ID NO:
    NO: 16) 18)
    Multiplex_3 F Sequence R Sequence Taqman Sequence
    Oxa_62_like CGATCATTCCCTGGG GAGACGACCTGATAG CCGCGACGCATCAA
    ATG (SEQ ID NO: 19) CAG (SEQ ID NO: 20) GAACTG (SEQ ID NO:
    21)
    Oxa_54_like GCTGTATCATAACAA CGATCTGAGGCTCAA CAGTCAGCGTATCGT
    GTTGC (SEQ ID NO: TTC (SEQ ID NO: 23) CAAGCAAG (SEQ ID
    22) NO: 24)
    Oxa_24_like GTGGGAGAAAGATAT TCGACCTGTGTTCCA TGTCAGCAGTTCCAG
    GACTTTA (SEQ ID ATA (SEQ ID NO: 26) TATATCAAGAGC
    NO: 25) (SEQ ID NO: 27)
    Multiplex_4 F Sequence R Sequence Taqman Sequence
    Oxa_48_like GTTGGAATGCTCACT TTCGCCCGTTTAAGA AATCCTTGCTGCTTA
    TTAC (SEQ ID NO: 28) TTA (SEQ ID NO: 29) TTCTCATTCCA (SEQ
    ID NO: 30)
    Oxa_133_like GAACGTATTGATTTC CTGTGTATGTGCCAA AACTCTACCTCTTGA
    GGTAA (SEQ ID NO: TTG (SEQ ID NO: 32) ATAGGCGTGAC
    31) (SEQ ID NO: 33)
    Oxa_182_like CCACAAGTAGGTTGG CCCTAAATTTTCTAAC AGAACCAGACATTCC
    TTAA (SEQ ID NO: 34) GACTTA (SEQ ID NO: TTGCTTCATT (SEQ ID
    35) NO: 36)
    Multiplex_5 F Sequence R Sequence Taqman Sequence
    Oxa_98_like TAGGCGAYGCTATGA CTTGGGTACCGATAT AACCAACACGCTTCA
    AAG (SEQ ID NO: 37) CTG (SEQ ID NO: 38) CTTCATTAGAC (SEQ
    ID NO: 39)
    Oxa_60c_like CATCGACGTTCAAGA CTCCCACTGCTTGTA AACAGCCTGATCGCC
    TTCC (SEQ ID NO: 40) AGG (SEQ ID NO: 41) TTCGA (SEQ ID NO:
    42)
    Oxa_60d_like GTGCCGATATACCAG CTTCGAATGCGGAAA CCACGCAGCCAGAAT
    GAG (SEQ ID NO: 43) TCTC (SEQ ID NO: 44) TGATCG (SEQ ID NO:
    45)
    Multiplex_6 F Sequence R Sequence Taqman Sequence
    VIM TCCGRCTTTACCAGAT CACGCTGTATCAATC CAACTCATCACCATC
    TG (SEQ ID NO: 46) AAA (SEQ ID NO: 47) ACGGACAA (SEQ ID
    NO: 48)
    GIM TGGAGTATATCTTCAT CTTCGTGTCTTCTTC AGGCTTGATTATTAT
    ACCTC (SEQ ID NO: 49) AGA (SEQ ID NO: 50) CCAGAACTACCA
    (SEQ ID NO: 51)
    SPM CTTCGAATGTCTTAGT TCGGCTTCATAGTCT ACCGTTGTCATTGTC
    AGC (SEQ ID NO: 52) TAG (SEQ ID NO: 53) TCTTCGC (SEQ ID
    NO: 54)
    Multiplex_7 F Sequence R Sequence Taqman Sequence
    KPC TTGGCTAAAGGGAAAC CGACGGCATAGTCAT CATACVCTCCGCAGG
    AC (SEQ ID NO: 55) TTG (SEQ ID NO: 56) TTCCG (SEQ ID NO:
    57)
    SME GGGAATGTTCTCAATG CACCTACAACCCAAT CGAGCATCACCAGTT
    CTA (SEQ ID NO: 58) CAG (SEQ ID NO: 59) GTATTACCTT (SEQ
    ID NO: 60)
    NMC ACCCATCACAACTAAA CGTTCAAGRATAATA AGCDGCAGCAGCCA
    TATAAAG (SEQ ID NO: TTAGTAGCA (SEQ ID TATCAC (SEQ ID NO:
    61) NO: 62) 63)
    Multiplex_8 F Sequence R Sequence Taqman Sequence
    GES_CARB CTCTGTGAGTCGGCTA GGCGTAGTTGTATCT AGYCGGAGATGAICG
    GA (SEQ ID NO: 64) CTGA (SEQ ID NO: 65) ACAACAC (SEQ ID
    NO: 66)
    Multiplex_9 F Sequence R Sequence Taqman Sequence
    IMP_3_like GCATCTGWATTAA CCACTACGTTATCTKG TGTGGCTTGAACCT
    CAAATG (SEQ ID AG (SEQ ID NO: 68) TACCGTCT (SEQ ID
    NO: 67) NO: 69)
    IMP_15_like AGCCAATAGCTAVC
    TCCGCT (SEQ ID NO:
    70)
    IMP_19_like TTAACTAGCCAATAR
    CTAACTCCGC (SEQ
    ID NO: 71)
    Multiplex_10 F Sequence R Sequence Taqman Sequence
    NDM GCCCAATATTATG GTCGCCAGTTTCCATT CTGAGCACCGCATT
    CACCC (SEQ ID TG (SEQ ID NO: 73) AGCCGC (SEQ ID
    NO: 72) NO: 74)
    SIM GGCTTAGTAGTTC CTTCCATTGACAGTGA ATCTCATCGACACTC
    TTGACA (SEQ ID AATC (SEQ ID NO: 76) CAGCTTCC
    NO: 75) (SEQ ID NO: 77)
  • Specific oligonucleotides were used for carbapenemases encoding genes. Two oligonucleotides act as forward primer and reverse primer, and a third one was used being labelled with a “dye” or fluorophore and located in the middle of the amplicon which is being synthesized by PCR, and when the taq polymerase passes over it, it cleaves the “dye” which is thus released into the medium emitting its typical fluorescence.
  • The PCR amplification profile was: pre PCR: 2 min at 50° C., 2 min at 94° C. PCR: (15 seconds at 94° C., 50 seconds at 60° C.)×40 reps. The fluorescence reading was performed in the 50 seconds step at 60° C.
  • In this invention, different targets may be detected in a single reaction. The experiments performed (meaning by experiment each of the 10 different reactions of target detection realized in several multiplex, in reactions using 2 or 3 different probes in each well of the plate in real-time PCR), have been designed using the programme Beacon Designer v8 under the Taqman experimental profile, having the capacity of generating multiplex reactions upon introducing the different sequences of the desired genes to be amplified at the same time.
  • In this invention, in order to increase the number of targets which can be detected with the least number of probes possible, the DNA regions conserved among carbapenemases of the same group (ej. KPC, Oxa_23 etc.) were studied, so taking a representative one from each group and focusing on said highly conserved genomic regions, probes capable of detecting most of the variants inside the same group with a single PCR were designed.
  • A second table (Table 2) is shown below with all the carbapenemases detected by the method disclosed. It should be noted that these are the carbapenemases which the system detects as a minimum, since, considering that the experiment is designed towards conserved DNA regions, it is likely that as new carbapenemases are found which have not been described in the literature yet, as long as they belong to any of the groups detected by the system, they can probably also be detected.
  • TABLE 2
    List of detected carbapenemases and fluorophores thereof
    Class Type Sub-type Labelling
    A GES 2, 4, 5, 13, 16, 14, CYS
    18, 15, 20
    A SME 1, 2, 3 TAMRA
    A KPC 1, 2, 3, 4, 5, 6, 7, 8, CYS
    9, 10, 11
    A NMC/IMI 1, 2, 3 JOE
    B IMP 1, 2, 3, 5, 6, 8, 9, 10, CYS
    11, 15, 19, 20, 21, TAMRA
    24, 25, 28, 29, 30 JOE
    B SIM 1 TAMRA
    B NDM 1, 2, 5 CYS
    B VIM 1, 2, 3, 4, 5, 6, 7, 8, 9, CYS
    10, 11, 12, 13, 14, 15,
    16, 17, 18, 19, 20, 21,
    22, 23, 24, 25, 26, 27,
    28, 29, 30, 31
    B GIM 1 TAMRA
    B SPM 1 JOE
    D OXA_23_like 23, 27, 49, 146, 73, 133, CYS
    168, 167, 166, 165, 170,
    169, 171, 225
    D OXA_51_like 77, 112, 110, 107, 51, 94, TAMRA
    92, 95, 88, 69, 65, 60, 70,
    78, 66, 76, 68, 75, 67, 82,
    90, 89, 91, 93, 84, 98, 108,
    110, 99, 107, 106, 109, 80,
    79, 83, 113, 111, 115, 116,
    117, 128, 132, 130, 131, 138,
    150, 149, 148, 144, 180, 179,
    177, 178, 176, 175, 174, 173,
    172, 208, 223, 203, 195, 200,
    206, 202, 196, 201, 197, 194
    D OXA_58_like 58, 97, 96, 164 JOE
    D OXA_48 48  CYS
    D OXA_133 133  TAMRA
    D OXA_182 182  JOE
    • Example 2 Carbapanemases Detection and Typing System Using Real-Time PCR Combined with Taqman Technology
  • The system designed consists of 25 Taqman probes which, grouped in multiplex reactions of 2 or 3 targets, formed 8 multiplex reactions. In each multiplex reaction each probe was labelled with a different fluorophore, such that the 3 targets could be detected at the same time if they were present in the sample. All the multiplex reactions included in this assay worked under the same temperature conditions and had a minimum score of 8 out of 10, according to the software used for its design, so all combinations worked equally well.
  • Each probe was designed to hybridize in the PCR reaction against regions highly conserved within a class of carbapenemases. So the system has the capacity to detect all variants within the same group of carbapenemases. In principle, the system is capable of detecting more than 200 known variants.
  • Obtained results:
  • As it is shown by the results, the system has the capacity of detecting more than one carbapenemase per reaction. FIGS. 1A-1C shows the results obtained from 3 different strains of Acinetobacter baumanii from a strain collection, previously well-characterized.
  • In FIG. 1A 2 types of carbapenemases are found; oxa-23 and oxa-51. In FIG. 1B only carbapenemase oxa-51 is found. In FIG. 1C oxa-58 and oxa-51 are found.
  • Apart from setting up the system using commercial strains, the resistance mechanisms of which were already known, additional experiments were also carried out to evaluate the system by means of 2 blind assays. Each assay had a size of 65 and 53 clinical isolates respectively (comprising Enterobacteriaceae, Pseudomonas sp. and Acinetobacter sp.), previously characterized in different origin hospitals and totally unknown for us. The results obtained by our system showed 100% coincidence with those previously obtained by those institutions.
  • Additionally, the system was applied to a collection of 600 bacterial strains consecutively isolated, wherein a screening for reduced sensitivity to carbapenems had been previously carried out, with the purpose of getting to know the carbapenemases most frequently found in our environment.
    • Example 3 Carbapanemases Detection and Typing System Using PCR Technique Without Taqman Probes
  • The primers described herein can also be used in a PCR amplification technology, where said primers are labelled with biotin, and a subsequent hybridization against a membrane carrying a DNA fragment complementary to the amplicon of interest. After hybridization, by means of a chemical reaction, the biotin darkens the membrane in the hybridization site, and depending on the darkening extent of the site, a software considers the reaction positive or negative.
  • Thus, all the primers described herein would get together (without probes in the reaction, only with the biotin labelling of said primers), in a PCR reaction to amplify the targets of interest. That is, instead of performing 8 different reactions of 3 or 2 multiplex each, a single multiplex reaction would be done with all the primers, as described below.
  • 3.1. Adapting Real-Time Amplification to Conventional PCR Amplification and Subsequent Detection of the Product by Hibridization in Biotin-Labelled Amplicon.
  • A Real-time PCR system to genetically detect carbapenemase-producing strains was designed. The real-time multiplex reactions (grouped in threes), were adapted in a single conventional PCR reaction, wherein instead of carrying out multiplex reactions in groups of three targets, all those reactions were put together in a single reaction, wherein all the primers designed for the real-time PCR system were present. So when a strain is subjected to an assay and is carrying a gene which the system can detect, this is amplified and by means of an analysis in agarose gel it can be determined if it carries or not the carbapenemases, and it is subsequently hybridized in a membrane (with the amplicons being labelled with biotin) to identify what type of carbapenemase it is carrying.
  • The conventional PCR programme used for amplification was as follows:
  • 1 cycle of 4 min at 94° C., followed by 40 cycles of 30 seconds at 94° C., 45 seconds at 50° C. and 45 seconds at 68° C., and 1 additional cycle of 7 min at 68° C.
  • The most important stage of the programme is that of 45 seconds at 50° C. The primers were designed to work at a range from 50 to 52 grades (theoretical data obtained from simulation programmes). However, said primers could unexpectedly work optimally in assays in temperature ranges from 57° C. to 60° C. Simulation programmes did not predict the primers could work at temperatures higher than 52° C. On the other hand, according to these programmes, it was not expected that in a multiplex reaction with all the primers of the invention together reliable amplification results could be obtained, since unspecific amplifications results were expected because of incompatibility among primers; however, according to the experimental data shown, the combination of all the primers in a multiplex reaction works quite well, not providing unspecific results (FIG. 2).
  • Primers concentrations used were as follows:
  • For amplification and detection of carbapenemase genes KPC, GES, IMP, OXA-51 and SME a concentration of at least 0.5 pmol/μl was used for both primers (forward and reverse), and for the rest of the genes (SPM, SIM, OXA-48, OXA-24, NDM, NMC, VIM, OXA-23, OXA-58) a concentration of at least 0.25 pmol/μl, was used for both primers (forward and reverse).

Claims (24)

1-24. (canceled)
25. A pair of primers consisting of SEQ ID NO: 1-SEQ ID NO: 2.
26. A kit comprising the pair of primers according to claim 25, and reagents required to perform an amplification reaction.
27. The kit according to claim 26, further comprising at least another pair of primers selected from the group consisting of SEQ ID NO: 4-SEQ ID NO: 5, SEQ ID NO: 7-SEQ ID NO: 8, SEQ ID NO: 10-SEQ ID NO: 11, SEQ ID NO: 13-SEQ ID NO: 14, SEQ ID NO: 16-SEQ ID NO: 17, SEQ ID NO: 19-SEQ ID NO: 20, SEQ ID NO: 22-SEQ ID NO: 23, SEQ ID NO: 25-SEQ ID NO: 26, SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 31-SEQ ID NO: 32, SEQ ID NO: 34-SEQ ID NO: 35, SEQ ID NO: 37-SEQ ID NO: 38, SEQ ID NO: 40-SEQ ID NO: 41, SEQ ID NO: 43-SEQ ID NO: 44, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 49-SEQ ID NO: 50, SEQ ID NO: 52-SEQ ID NO: 53, SEQ ID NO: 55-SEQ ID NO: 56, SEQ ID NO: 58-SEQ ID NO: 59, SEQ ID NO: 61-SEQ ID NO: 62, SEQ ID NO: 64-SEQ ID NO: 65, SEQ ID NO: 72-SEQ ID NO: 73, and SEQ ID NO: 75-SEQ ID NO: 76.
28. The kit according to claim 27, further comprising at least a probe capable of hybridizing the amplification product of any of said pair of primers.
29. The kit according to claim 28, wherein the pair of primers or the probe is labelled with a fluorophore.
30. The kit according to claim 29, wherein the fluorophore is selected from the group consisting of cyanine 5 dye, tetramethylrhodamine, 4,5-dichloro-dimethoxy-fluorescein, and biotin.
31. The kit according to claim 28, wherein the probe comprises the sequence SEQ ID NO: 3.
32. The kit according to claim 28, wherein the probe comprises the sequence:
a. SEQ ID NO: 6, when the pair of primers is SEQ ID NO: 4-SEQ ID NO: 5,
b. SEQ ID NO: 9, when the pair of primers is SEQ ID NO: 7-SEQ ID NO: 8,
c. SEQ ID NO: 12, when the pair of primers is SEQ ID NO: 10-SEQ ID NO: 11,
d. SEQ ID NO: 15, when the pair of primers is SEQ ID NO: 13-SEQ ID NO: 14,
e. SEQ ID NO: 18, when the pair of primers is SEQ ID NO: 16-SEQ ID NO: 17,
f. SEQ ID NO: 21, when the pair of primers is SEQ ID NO: 19-SEQ ID NO: 20,
g. SEQ ID NO: 24, when the pair of primers is SEQ ID NO: 22-SEQ ID NO: 23,
h. SEQ ID NO: 27, when the pair of primers is SEQ ID NO: 25-SEQ ID NO: 26,
i. SEQ ID NO: 30, when the pair of primers is SEQ ID NO: 28-SEQ ID NO: 29,
j. SEQ ID NO: 33, when the pair of primers is SEQ ID NO: 31-SEQ ID NO: 32,
k. SEQ ID NO: 36, when the pair of primers is SEQ ID NO: 34-SEQ ID NO: 35,
l. SEQ ID NO: 39, when the pair of primers is SEQ ID NO: 37-SEQ ID NO: 38,
m. SEQ ID NO: 42, when the pair of primers is SEQ ID NO: 40-SEQ ID NO: 41,
n. SEQ ID NO: 45, when the pair of primers is SEQ ID NO: 43-SEQ ID NO: 44,
o. SEQ ID NO: 48, when the pair of primers is SEQ ID NO: 46-SEQ ID NO: 47,
p. SEQ ID NO: 51, when the pair of primers is SEQ ID NO: 49-SEQ ID NO: 50,
q. SEQ ID NO: 54, when the pair of primers is SEQ ID NO: 52-SEQ ID NO: 53,
r. SEQ ID NO: 57, when the pair of primers is SEQ ID NO: 55-SEQ ID NO: 56,
s. SEQ ID NO: 60, when the pair of primers is SEQ ID NO: 58-SEQ ID NO: 59,
t. SEQ ID NO: 63, when the pair of primers is SEQ ID NO: 61-SEQ ID NO: 62,
u. SEQ ID NO: 66, when the pair of primers is SEQ ID NO: 64-SEQ ID NO: 65,
v. SEQ ID NO: 74, when the pair of primers is SEQ ID NO: 72-SEQ ID NO: 73, or
w. SEQ ID NO: 77, when the pair of primers is SEQ ID NO: 75-SEQ ID NO: 76.
33. The kit according to claim 27, comprising the pairs of primers SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 55-SEQ ID NO: 56, and SEQ ID NO: 72-SEQ ID NO: 73.
34. An in vitro method for detection and/or typing of oxa_23_like carbapenemase-producing bacterial strains in an isolated biological sample, said method comprising:
i. contacting an isolated biological sample comprising DNA with the pair of printers according to claim 25,
ii. amplifying the DNA comprised in said isolated biological sample with the pair of primers, and
iii. detecting the presence of the amplification product obtained in step (ii), wherein the presence of said amplification product is indicative of the presence of oxa_23_like carbapenemase-producing bacterial strains in the isolated biological sample.
35. The in vitro method according to claim 34, wherein step (i) additionally comprises contacting the isolated biological sample with a further pair of primers selected from the group consisting of SEQ ID NO: 4-SEQ ID NO: 5, SEQ ID NO: 7-SEQ ID NO: 8, SEQ ID NO: 10-SEQ ID NO: 11, SEQ ID NO: 13-SEQ ID NO: 14, SEQ ID NO: 16-SEQ ID NO: 17, SEQ ID NO: 19-SEQ ID NO: 20, SEQ ID NO: 22-SEQ ID NO: 23, SEQ ID NO: 25-SEQ ID NO: 26, SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 31-SEQ ID NO: 32, SEQ ID NO: 34-SEQ ID NO: 35, SEQ ID NO: 37-SEQ ID NO: 38, SEQ ID NO: 40-SEQ ID NO: 41, SEQ ID NO: 43-SEQ ID NO: 44, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 49-SEQ ID NO: 50, SEQ ID NO: 52-SEQ ID NO: 53, SEQ ID NO: 55-SEQ ID NO: 56, SEQ ID NO: 58-SEQ ID NO: 59, SEQ ID NO: 61-SEQ ID NO: 62, SEQ ID NO: 64-SEQ ID NO: 65, SEQ ID NO: 67-SEQ ID NO: 68, SEQ ID NO: 72-SEQ ID NO: 73, and SEQ ID NO: 75-SEQ ID NO: 76.
36. The in vitro method according to claim 34, wherein detection of step (iii) comprises hybridization of the amplification product obtained in step (ii) with at least a probe capable of hybridizing with the amplification product of the pair of primers used in step (i).
37. The in vitro method according to claim 36, wherein the pair of primers or the probe is labelled with a fluorophore.
38. The in vitro method according to claim 37, wherein the fluorophore is selected from the group consisting of cyanine 5 dye, tetramethylrhodamine, 4,5-dichloro-dimethoxy-fluorescein, and biotin.
39. The in vitro method according to claim 36, wherein the probe comprises the sequence SEQ ID NO: 3.
40. The in vitro method according to claim 36, wherein the probe comprises the sequence:
a. SEQ ID NO: 6, when the pair of primers is SEQ ID NO: 4-SEQ ID NO: 5,
b. SEQ ID NO: 9, when the pair of primers is SEQ ID NO: 7-SEQ ID NO: 8,
c. SEQ ID NO: 12, when the pair of primers is SEQ ID NO: 10-SEQ ID NO: 11,
d. SEQ ID NO: 15, when the pair of primers is SEQ ID NO: 13-SEQ ID NO: 14,
e. SEQ ID NO: 18, when the pair of primers is SEQ ID NO: 16-SEQ ID NO: 17,
f. SEQ ID NO: 21, when the pair of primers is SEQ ID NO: 19-SEQ ID NO: 20,
g. SEQ ID NO: 24, when the pair of primers is SEQ ID NO: 22-SEQ ID NO: 23,
h. SEQ ID NO: 27, when the pair of primers is SEQ ID NO: 25-SEQ ID NO: 26,
i. SEQ ID NO: 30, when the pair of primers is SEQ ID NO: 28-SEQ ID NO: 29,
j. SEQ ID NO: 33, when the pair of primers is SEQ ID NO: 31-SEQ ID NO: 32,
k. SEQ ID NO: 36, when the pair of primers is SEQ ID NO: 34-SEQ ID NO: 35,
l. SEQ ID NO: 39, when the pair of primers is SEQ ID NO: 37-SEQ ID NO: 38,
m. SEQ ID NO: 42, when the pair of primers is SEQ ID NO: 40-SEQ ID NO: 41,
n. SEQ ID NO: 45, when the pair of primers is SEQ ID NO: 43-SEQ ID NO: 44,
o. SEQ ID NO: 48, when the pair of primers is SEQ ID NO: 46-SEQ ID NO: 47,
p. SEQ ID NO: 51, when the pair of primers is SEQ ID NO: 49-SEQ ID NO: 50,
q. SEQ ID NO: 54, when the pair of primers is SEQ ID NO: 52-SEQ ID NO: 53,
r. SEQ ID NO: 57, when the pair of primers is SEQ ID NO: 55-SEQ ID NO: 56,
s. SEQ ID NO: 60, when the pair of primers is SEQ ID NO: 58-SEQ ID NO: 59,
t. SEQ ID NO: 63, when the pair of primers is SEQ ID NO: 61-SEQ ID NO: 62,
u. SEQ ID NO: 66, when the pair of primers is SEQ ID NO: 64-SEQ ID NO: 65,
v. SEQ ID No: 69, SEQ ID No: 70 and/or SEQ ID No: 71, when the pair of primers is SEQ ID No: 67-SEQ ID No: 68,
w. SEQ ID NO: 74, when the pair of primers is SEQ ID NO: 72-SEQ ID NO: 73, or
x. SEQ ID NO: 77, when the pair of primers is SEQ ID NO: 75-SEQ ID NO: 76.
41. The in vitro method according to claim 35, comprising in step (i) contacting an isolated biological sample with all the pairs of primers: SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 55-SEQ ID NO: 56, SEQ ID NO: 67-SEQ ID NO: 68 and SEQ ID NO: 72-SEQ ID NO: 73.
42. The in vitro method according to claim 35, comprising in step (i) contacting an isolated biological sample with all the pairs of primers SEQ ID NO: 1-SEQ ID NO: 2, SEQ ID NO: 4-SEQ ID NO: 5, SEQ ID NO: 7-SEQ ID NO: 8, SEQ ID NO: 10-SEQ ID NO: 11, SEQ ID NO: 13-SEQ ID NO: 14, SEQ ID NO: 16-SEQ ID NO: 17, SEQ ID NO: 19-SEQ ID NO: 20, SEQ ID NO: 22-SEQ ID NO: 23, SEQ ID NO: 25-SEQ ID NO: 26, SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 31-SEQ ID NO: 32, SEQ ID NO: 34-SEQ ID NO: 35, SEQ ID NO: 37-SEQ ID NO: 38, SEQ ID NO: 40-SEQ ID NO: 41, SEQ ID NO: 43-SEQ ID NO: 44, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 49-SEQ ID NO: 50, SEQ ID NO: 52-SEQ ID NO: 53, SEQ ID NO: 55-SEQ ID NO: 56, SEQ ID NO: 58-SEQ ID NO: 59, SEQ ID NO: 61-SEQ ID NO: 62, SEQ ID NO: 64-SEQ ID NO: 65, SEQ ID NO:67-SEQ ID NO:68, SEQ ID NO: 72-SEQ ID NO: 73 and SEQ ID NO: 75-SEQ ID NO: 76.
43. The in vitro method according to claim 34, wherein the amplification step (ii) is carried out by means of PCR.
44. The in vitro method according to claim 34, wherein the biological sample comes from an individual.
45. The in vitro method according to claim 44, wherein the individual is a human.
46. The in vitro method according to claim 44, wherein the biological sample is blood, sputum, exudate, skin or hemoculture previously inoculated with blood from an individual preferably having bacteraemia.
47. The method according to claim 34, further comprising:
(iv) assigning the individual to the group of patients infected with carbapenemase-producing bacterial strains when in step (iii) the presence of the amplification product is detected.
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