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WO2016038351A1 - Procédés de détection de bactéries multirésistantes - Google Patents

Procédés de détection de bactéries multirésistantes Download PDF

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Publication number
WO2016038351A1
WO2016038351A1 PCT/GB2015/052586 GB2015052586W WO2016038351A1 WO 2016038351 A1 WO2016038351 A1 WO 2016038351A1 GB 2015052586 W GB2015052586 W GB 2015052586W WO 2016038351 A1 WO2016038351 A1 WO 2016038351A1
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WO
WIPO (PCT)
Prior art keywords
probe
gene
blandm
dna
biochemical
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PCT/GB2015/052586
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English (en)
Inventor
Grace HENIHAN
Holger Schulze
Jimmy Ming-Yuan HUANG
Daniel Macdonald
Till T. Bachmann
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The University Court Of The University Of Edinburgh
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Publication of WO2016038351A1 publication Critical patent/WO2016038351A1/fr

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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

Definitions

  • the invention relates to the field of multidrug resistant bacteria and methods of detecting the same.
  • Organisms harbouring the gene bldNDM tend to be multidrug resistant and some are only sensitive in vitro to agents of uncertain efficacy such as tigecycline and colistin, leaving few treatment options.
  • the blaNDM gene was first isolated from a Swedish patient previously hospitalised in India in 2008, and has disseminated to broad geographical locations, 4 predominantly linked to treatment in the Indian region, though also through independent routes. 5
  • blaNDM metallo beta-lactamase
  • ESBL extended-spectrum beta-lactamase
  • carbapenemase producing bacteria are culture based, with a time-to-result (TTR) incompatible with a rapid treatment decision (e.g. ChromID ESBL 6,7 Etest ESBL 8 , Vitek (bioMerieux) 9 . etc.)
  • TTR time-to-result
  • Some molecular diagnostic approaches for detection of beta-lactam resistance in gram negative bacteria, such as Check-MDR (Checkpoints) 10 , Evigene (AdvanDx) 11 , Hyplex SuperBug ID (Amplex) 12 etc, are not suited for true point of care detection as they are based on sophisticated optical detection systems and require demanding sample preparation and preanalytics.
  • Electrochemical impedance spectroscopy is a technique that detects changes in the resistance to charge transfer (Ret) between two electrodes via a redox mediator. Binding events at the electrode surface changes this Ret value and therefore allows the detection of analytes such as DNA 13 14 , and has been described in WO 2013/076143 (ITI Scotland Limited), for example, the contents of which are hereby incorporated by way of reference. Whilst EIS has been successfully used to detect the presence of a limited number of pathogens, each potential pathogen requires a separate probe at the electrode surface to bind to a specific DNA sequence of that pathogen.
  • probes for every known such bacteria would need to be present at the electrode surface, and even then, such a method of detection would not be able to detect bacteria that have newly acquired multidrug resistance.
  • a biochemical probe for the detection of the blaNDM gene comprising a nucleic acid recognition sequence operable to hybridise under stringent conditions with a specific DNA sequence of the blaNDM gene complementary to the nucleic acid recognition sequence.
  • bladeNDM gene we refer to the gene for encoding the New Delhi metallo beta- lactamase, including all known variants of the blaNDM gene, for example bldNDM-i gene, blaNDM- 2 gene, blaNDM-3 gene, blaNDM-4 gene, blaNDMs gene, and bldNDM- ⁇ gene (sequences of which are given below), and variants identified in the future.
  • telomere sequence of the bldNDM gene or “target sequence” we refer to a DNA sequence that is unique to the bldNDM gene, such that a nucleic acid recognition sequence that is operable to hybridise with a specific DNA sequence of the bldNDM gene and will only substantially hybridise with DNA when the bldNDM gene is present.
  • This may be a specific DNA sequence that is unique to the bldNDM gene generally.
  • This may be a specific DNA sequence that is unique to an individual variant of the bldNDM gene, including gene, blaNDM-2 gene, blaNDM-3 gene, blaNDM-4 gene, bldNDMs gene, and blaNDM-e gene and any variant identified in the future.
  • the nucleic acid recognition sequence may be operable to hybridise with a specific DNA sequence adjacent to the bldNDM gene and which is closely genetically associated with the bldNDM gene.
  • the nucleic acid recognition sequence may be operable to hybridise with a specific DNA sequence adjacent to the bldNDM gene and a specific DNA sequence of the bldNDM gene.
  • the nucleic acid recognition sequence may be operable to overlap the bldNDM gene and an adjacent DNA sequence.
  • the nucleic acid recognition sequence may be operable to hybridise with a consecutive specific DNA sequence comprising a DNA sequence of the bldNDM gene and a DNA sequence adjacent to that DNA sequence of the bldNDM gene.
  • the promotor regions or after the transcription termination signal sequence we refer to a sequence of DNA that is non-coding and within 500 bases of the bldNDM gene, 200 bases of the bldNDM gene or 50 bases of the bldNDM gene, for example.
  • probe we refer to a species comprising a nucleic acid or derivative thereof of known sequence to which nucleic acids from a sample (e.g. biological samples such as wound fluid) can hybridise if the nucleic acids from the sample (or derivatives thereof) are of complementary or substantially complementary sequence (target sequences) through one or more types of chemical bonds.
  • probes comprise nucleic acid recognition sequences of 10 to 100 bases in length, preferably, 10 to 50 bases in length, more preferably, 10 to 30 bases in length.
  • the hybridisation of the probe to the sample nucleic acid is typically detected in an assay to thereby indicate the presence and/or the concentration of the target sequence.
  • nucleic acid we refer to a deoxyribonucleotide polymer, a ribonucleotide polymer, or a derivative thereof, in either single- or double- stranded form, and unless otherwise stated, encompass known analogues of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • the terms encompass nucleic acid-like structures with synthetic backbones (such as, for example, peptide nucleic acids or Morpholinos), as well as amplification products.
  • a nucleic acid is obtained from a larger nucleic acid molecule, e.g., by fragmentation (whether chemical, physical, enzymatic, or any combination thereof and whether artificially or naturally or both).
  • hybridise and “hybridisation” we refer to a process where oligonucleotides and their analogs hybridise by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases.
  • nucleic acid molecules consist of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).
  • base pairing bonds between a pyrimidine and a purine
  • base pairing More specifically, A will hydrogen bond to T or U, and G will bond to C.
  • “Complementary” refers to nucleic acid sequences that base-pair according to the standard Watson-Crick complementary rules outlined above, or that are capable of hybridising to a particular nucleic acid segment under relatively stringent conditions. Nucleic acid polymers may be complementary across only portions of their entire sequences.
  • Hybridisation conditions resulting in particular degrees of stringency will vary depending upon the nature of the chosen hybridisation method and the composition and length of the hybridising nucleic acid sequences. Generally, the temperature of hybridisation and the ionic strength (especially the Na + and/or Mg 2+ concentration) of the hybridisation buffer will contribute to the stringency of hybridisation, though wash times also influence stringency. Calculations regarding hybridisation conditions required for attaining particular degrees of stringency are discussed in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, chs. 9 and 1 1.
  • stringent conditions we encompass conditions under which hybridisation will only occur if there is less than 50% mismatch between the hybridisation molecule and the DNA target.
  • Stringent conditions include further particular levels of stringency.
  • “moderate stringency” conditions are those under which molecules with more than 50% sequence mismatch will not hybridise;
  • conditions of "high stringency” are those under which sequences with more than 20% mismatch will not hybridise;
  • conditions of "very high stringency” are those underwhich sequences with more than 10% mismatch will not hybridise.
  • stringent conditions can include hybridisation at 65 °C, followed by washes at 65 °C with 0.1 x SSC / 0.1 % SDS for 40 minutes.
  • the recognition sequence and the specific DNA sequence of the bldNDM gene complementary to the recognition sequence can remain bound under very high stringency hybridisation conditions. In these and further embodiments, the recognition sequence and the specific DNA sequence of the bldNDM gene complementary to the recognition sequence can remain bound under high stringency hybridisation conditions. In these and further embodiments, the recognition sequence and the specific DNA sequence of the bldNDM gene complementary to the recognition sequence can remain bound under moderate stringency hybridisation conditions.
  • Methods of detecting pathogens known in the art typically rely on the detection of analytes that are specific to that pathogen or culturing the microorganism. However, it is often necessary to be able to quickly determine whether a patient has a bacterial infection and whether that bacterial infection is multidrug resistant. For example, if a patient has a multidrug resistant gram negative bacterial infection, administering typical antibiotics may be ineffective and may do more harm than good.
  • Multidrug resistant gram negative bacteria typically comprise the bldNDM gene. Therefore, a biochemical probe that is operable to detect the presence of the bldNDM gene within a biological sample via hybridisation of the probe with a sequence of the DNA specific to the bldNDM gene (or adjacent sequences) allows the detection of multidrug resistant gram negative bacteria within the biological sample, without necessarily determining the identity of that bacteria. Accordingly, if the bldNDM gene is detected, the appropriate treatment can be prescribed immediately, without first subjecting the patient to standard antibiotics to which the bacteria are resistant.
  • the probe may be operable to detect the presence of the blaNDM gene by hybridising with a specific sequence of DNA within the intact plasmid containing the blaNDM gene.
  • the probe may hybridise with a sequence of the blaNDM gene (or adjacent sequences) that is located on the exterior of the blaNDM gene in its folded state.
  • the probe may hybridise with a sequence of the blaNDM gene (or adjacent sequences) that is within the interior of the blaNDM gene in its folded state and which only becomes available for hybridisation under specific conditions. For example, the sequence may only become available when the blaNDM gene has been denatured and has at least partially become unfolded.
  • the probe may hybridise with a sequence of the blaNDM gene (or adjacent sequences) that is on the interior of the £>/a «D gene in its folded state, and the sequence may only become available for hybridisation after the blaNDM gene has been fragmented.
  • the probe detects a specific fragment of the blaNDM gene.
  • the DNA fragments of the blaNDM gene may be formed by any or any combination of methods, including, but not limited to, enzymatic digestion (such as, for example, restriction enzyme digestion) and/or physical fragmentation (e.g., sonication, acoustic shearing, nebulising, point-sink shearing, needle shearing, passing through a French press, etc.).
  • DNA fragments of the blaNDM gene are formed by restriction enzyme digestion.
  • the DNA fragments of the blaNDM gene are formed by sonication.
  • the fragmentation method or methods can be performed on, e.g., plasmid DNA comprising the blaNDM gene, larger fragments containing the blaNDM gene, and/or polymerase chain reaction (PCR) products containing the blaNDM gene.
  • the DNA fragments of the blaNDM gene may be polymerase chain reaction (PCR) products.
  • the probe is operable to be used in an assay where a hybridisation event of the recognition sequence of the probe with the specific target DNA sequence is detected.
  • the probe may be adapted for any of a number of assays that rely on such hybridisation events.
  • the probe is used in solution. Examples of assays in which the probe may be used in solution include, but are not limited to, blot hybridisation assays (including Northern blot and Southern blot assays), in situ hybridisation assays (including fluorescence in situ hybridisation (FISH)), PCR-based assays (including real-time PCR and PCR clamping), and fluorescence resonance energy transfer (FRET)-based assays.
  • FISH fluorescence in situ hybridisation
  • FRET fluorescence resonance energy transfer
  • the probe is operable to be immobilised on a solid substrate.
  • the probe may be immobilised on a solid substrate during use.
  • the probe is immobilized on a solid substrate after the probe is synthesised; in some embodiments, the probe is synthesised in situ on a solid substrate, so that it is already attached. Any of a variety of different solid substrates can be used, depending on the assay chosen.
  • suitable solid substrates include substrates made of silica, silicone, glass, plastic (such as, e.g., polypropylene, polyacrylamide, polyamide (nylon), polydimethylsiloxane (PDMS), etc.), cellulose, nitrocellulose, and metals (as further detailed below).
  • the solid substrate can take the form of, for example, slides, chips or microarrays, beads (including magnetic beads), and electrodes.
  • the probe is immobilised on an electrode for use in, e.g., an electrochemical assay.
  • Electrodes used in electrochemical assays can be made of any of a variety of materials, including, for example, gold, silver, platinum, carbon, mercury, steel, and graphite.
  • the probe may be suitable for use in electrochemical impedance spectroscopy (EIS).
  • the probe may be suitable for use in surface plasmon resonance spectroscopy (SPRS).
  • the concentration of DNA fragments within the biological sample may be amplified.
  • the concentration of DNA fragments within the biological sample may be amplified using PCR or PCR-based methods.
  • the recognition sequence may be 7-50 base pairs long.
  • the recognition sequence may be 15-40 base pairs long.
  • the recognition sequence is 15-30 base pairs long.
  • the recognition sequence may be 18, 20 or 22 base pairs long.
  • the recognition sequence may comprise any one of SEQ ID NO 1-36 (Table 1) or variants thereof.
  • the recognition sequence comprises SEQ ID NO 7 or SEQ ID NO 25 or variants thereof.
  • Such variants preferably retain the ability to hybridise.
  • the recognition sequence may comprise at least 75% of any one of SEQ ID NOs 1-36.
  • the recognition sequence may comprise any one of SEQ ID NOs 1-36 plus one or more additional bases.
  • the recognition sequence may be thirty bases long and the first twenty bases from the 3' end may comprise SEQ ID NOs 1 or 2, the first twenty bases from the 5' end may comprise SEQ ID NOs 7 or 25, or any other twenty base sequence within the recognition sequence may comprise SEQ ID NOs 7 or 25. Accordingly, the recognition sequence may be longer than any one of SEQ ID NOs 1-36. However, in some embodiments, the recognition sequence may be shorter than any one of SEQ ID NOs 1-36, and may comprise a subset of any one of SEQ ID NOs 1-36.
  • a sequence of bases within the recognition sequence which may be the whole recognition sequence or a subset of the recognition sequence, is preferably identical to that of any one of SEQ ID NOs 1-36.
  • probes comprising recognition sequences SEQ ID NOs 7 and 25 are surprisingly suitable for use in assays such as EIS to detect the presence of the blaNDM gene.
  • assays such as EIS
  • the location of the complementary sequences to SEQ ID NOs 7 and 25 within the blaNDM gene may be on the exterior of the blaNDM gene and therefore, these complementary sequences may be readily available to hybridise with the probes on the sensing surface.
  • Probes according to the present aspect of the invention have been found to be particularly suitable for use in ElS-based assays.
  • the impedance of a system is generally determined by applying a voltage perturbation with a small amplitude and detecting the current response.
  • Faradaic EIS measures the resistance to charge transfer (Ret) between two electrodes via a redox mediator that has been added to the biological sample.
  • a working electrode is typically covered in a self-assembled monolayer (SAM) comprising probes and a blocking species.
  • SAM self-assembled monolayer
  • probes typically comprise complementary strands of DNA fixed to the surface of the working electrode that hybridise with any target DNA within the biological sample for detection.
  • DNA has a negatively charged phosphate backbone and therefore, the DNA probe sequences on the working electrode repel the anionic redox mediators thereby reducing the concentration of redox mediators at the surface of the working electrode, leading to a reduction in the sensitivity of the working electrode to a change in the Ret.
  • the nucleic acid recognition sequence may comprise a naturally occurring nucleic acid, such as DNA, or RNA, for example.
  • the nucleic acid recognition sequence comprises a synthetic nucleic acid. More preferably, the synthetic nucleic acid comprises an uncharged synthetic backbone in ambient conditions.
  • the nucleic acid recognition sequence may be a Morpholino recognition sequence comprising a backbone of morpholine rings linked by phosphorodiamidate groups.
  • the nucleic acid recognition sequence may be a peptide nucleic acid (PNA) recognition sequence comprising a peptide backbone.
  • PNA peptide nucleic acid
  • the nucleic acid recognition sequence is a PNA recognition sequence.
  • a probe that comprises an uncharged backbone rather than the charged backbone of DNA minimises the repulsion of redox mediators from the working electrode surface, and therefore maximises the sensitivity of the working electrode to changes in resistance to charge transfer.
  • the recognition sequence is a PNA recognition sequence
  • PNA has a higher binding strength than DNA and therefore, probes for use in the detection of DNA may have a shorter complementary PNA sequence than equivalent probes comprising DNA.
  • the PNA/DNA helix resulting from the hybridisation of the target DNA sequence to the probe PNA is more sensitive to base mismatches.
  • probes comprising PNA sequences are less likely to hydridise with DNA other than the target sequence, when compared to an equivalent probe comprising DNA, and therefore, provide a more reliable result.
  • the probe comprises an anchor operable to immobilise the probe to a sensing surface.
  • the anchor may be attached to the 3' end of the recognition sequence.
  • the anchor may be attached to the 5' end of the recognition sequence. Accordingly, the 5' or 3' end of the recognition sequence may be available for hybridisation with the target DNA sequence.
  • the sensing surface comprises a gold surface and the anchor comprises at least one terminal sulphur containing group to bind to the gold surface of the sensing surface.
  • the at least one terminal sulphur containing group is a thiol group.
  • the sensing surface may be a gold electrode and the probe may be immobilised onto the surface of the gold electrode by an anchor comprising a thiol group.
  • the anchor may be, for example, a mercapto-alkane group.
  • the anchor may be a mercapto-undecan group, such as C1 1 M.
  • the sensing surface may comprise an epoxy-treated glass surface and the anchor may comprise at least one terminal amine group.
  • the sensing surface may comprise glass and the anchor may comprise an alkylchlorosilane, alkylalkoxysilane or alkylaminosilane, for example.
  • a mixed SAM may be immobilised onto the sensing surface, and may comprise a population of probes of the present aspect of the invention and a population of at least one blocking species.
  • the blocking species typically minimises non-specific binding to the sensing surface by analyte, such that at least the majority of binding events at the sensing surface correspond to binding/hybridisation events between the recognition sequence of the probe and the target DNA sequence.
  • the sensing surface comprises a gold layer
  • the blocking species comprises a thiol group.
  • the blocking species may be a mercapto alcohol, or a mercapto alkane, for example.
  • the probe may comprise a linker connecting the anchor to the recognition sequence such that the recognition sequence is available for hybridisation with the target DNA sequence.
  • the linker may space the recognition sequence away from the sensing surface to minimise nonspecific interactions of the recognition sequence with the sensing surface.
  • the linker may comprise one or more ether groups.
  • the linker is an ethylene glycol such as [2-(2- Amino-ethoxy)-ethoxy]-acetic acid (AEEA) or ⁇ 2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy ⁇ -acetic acid (AEEEA).
  • the linker may be a polyethylene glycol (PEG), or a functionalised PEG.
  • the linker may extend between the anchor and the 3' end of the recognition sequence.
  • the linker may extend between the anchor and the 5' end of the recognition sequence. Accordingly, the 5' or 3' end of the recognition sequence may be available for hybridisation with the target DNA sequence.
  • the probe may have the sequence (5' to 3'): GTGCTGCCAGACATTCGGTG- Lys-AEEEA-C11 M (SEQ ID NO 37).
  • the probe may have the sequence (5' to 3'): C1 1 M- A E E E A- ATC AG G C AG CC ACC A AA AG C (SEQ ID NO 38).
  • the probe may be labelled. In some embodiments, a mixture of labelled and unlabelled probes is used.
  • the probes may be labelled in such a way that the detectability of the probe is altered when the probe is bound to the target sequence.
  • the invention extends in a second aspect to a diagnostic kit for the detection of the blaNDM gene, the kit comprising at least one biochemical probe according to the first aspect of the invention.
  • the kit may comprise suitable reagents to allow the at least one biochemical probe to bind to a sensing surface.
  • the kit may comprise a solvent, and the at least one biochemical probe according to the first aspect of the invention may be solubilised within the solvent.
  • the kit may comprise one or more suitable buffers for the storage of the at least one biochemical probe.
  • the kit may comprise one or more suitable buffers for the immobilisation of the at least one biochemical probe to a surface, such as a sensing surface.
  • the kit may comprise at least one blocking species.
  • the at least one blocking species may be mixed with the at least one biochemical probe.
  • the mixture of the blocking species and the probe may be operable to form a self-assembled monolayer (SAM) on a sensing surface.
  • SAM self-assembled monolayer
  • the kit may comprise a sensing surface to which the at least one biochemical probe is immobilised.
  • a SAM comprising the at least one probe and the blocking species may be formed on the sensing surface.
  • the sensing surface may comprise a gold electrode and be suitable for use in electrochemical impedance spectroscopy (EIS).
  • EIS electrochemical impedance spectroscopy
  • SPRS surface plasmon resonance spectroscopy
  • a method of detection of the blaNDM gene comprising the steps:
  • the sensing apparatus may be a surface plasmon resonance spectroscopy (SPRS) apparatus and hybridisation of DNA within the biological sample with the at least one probe on the sensing surface may be determined by a change in the wavelength of light absorbed by the sensing surface, measured as a change in angle of reflection minimum (corresponding to the maximum absorption), for example.
  • SPRS surface plasmon resonance spectroscopy
  • the sensing apparatus is an electrochemical impedance spectroscopy (EIS) apparatus comprising at least one working electrode comprising the sensing surface.
  • EIS electrochemical impedance spectroscopy
  • Hybridisation of DNA within the biological sample with the at least one probe on the sensing surface may be determined by an increase in the resistance to charge transfer (Ret) of a working electrode, wherein an increase in the Ret of the working electrode is indicative of the presence of the blaNDM gene.
  • EIS electrochemical impedance spectroscopy
  • the increase in the Ret of the working electrode may be determined relative to a reference electrode.
  • the reference electrode may be a Ag/AgCI (silver/silver chloride) reference electrode.
  • the reference electrode may be a platinum electrode or a gold electrode.
  • the Ret of the working electrode may be measured at room temperature and the biological sample may comprise at least 30% formamide by volume.
  • the biological sample comprises at least 50% formamide by volume.
  • the biological sample may comprise 50% formamide by volume.
  • the amount of formamide by volume is sufficient when the binding of the probe to substrates other than the target sequence is prevented at room temperature.
  • the Ret of the working electrode may be measured at room temperature and the biological sample may comprise at least 30% by volume of urea, formaldehyde or dimethylsulfoxide (DMSO), for example.
  • DMSO dimethylsulfoxide
  • the Ret of the working electrode may be measured at an elevated temperature, and the biological sample may comprise at least 30% by volume of formamide, urea, formaldehyde or DMSO, for example.
  • the method may not comprise the step of amplifying the DNA within the biological sample.
  • methods or assays that utilise the detection of DNA to determine the presence of a medically relevant species are not sufficiently sensitive to detect the typical concentrations (e.g. 100 nM) of specific sequences of DNA within biological samples, and therefore, these methods or assays require any DNA within a sample to be analysed to have that DNA amplified, using techniques such as PCR.
  • the amplification process takes time and so increases the time required to determine whether a given sample comprises the specific sequence of DNA of interest.
  • the longer it takes to determine whether the DNA (and therefore the biological entity from which the DNA originates) is present delays treatment. Indeed, if the time to result of the assay is too long, the assay may be deemed inappropriate for use at the point of care and therefore, the treatment provided may be inappropriate, ineffective, or at best delayed.
  • a method of detection of the blaNDM gene that does not require the DNA within the biological sample to be amplified will typically have a shorter time to result and therefore be a more appropriate method to be used as a true point of care assay, thereby allowing the appropriate treatment to be provided more rapidly.
  • the step of treating the biological sample to extract and/or fragment the DNA within the biological sample may be carried out after the biological sample is introduced into the sample chamber.
  • the step of treating the biological sample may be carried out before the biological sample is introduced into the sample chamber, or the step of treating the biological sample may be carried out both before and after the biological sample is introduced into the sample chamber.
  • one or more steps in a treatment protocol may be carried out before introducing the biological sample into the sample chamber, and one or more steps may be carried out after introducing the biological sample into the sample chamber.
  • the biological sample may be taken from a subject.
  • the biological sample may be a preexisting sample taken from a subject for an alternative purpose, and/or may be taken for the purpose of performing a method as described herein.
  • the subject may be a mammal.
  • the subject is a human.
  • the biological sample may comprise any solid or fluid (or combination thereof) sample obtained from, excreted by, exuded by, and/or secreted by any living cell or organism or tissue.
  • the biological sample may comprise cells.
  • the biological sample comprises nucleic acids and/or proteins. In some such embodiments, at least some of the nucleic acids and/or proteins contained in the biological sample are contained within cells.
  • the biological sample may comprise a bodily fluid.
  • the biological sample may include, for example, wound fluid, blood, plasma, serum, urine, stool, saliva, cord blood, chorionic villus samples, amniotic fluid, transcervical lavage fluid, or any combination thereof.
  • the biological sample is or comprises, a blood sample.
  • the biological sample may be obtained from a subject before, during, and/or after a course of treatment.
  • the biological sample itself may be treated (e.g., subject to a process) or it may be untreated.
  • the biological sample may be a treated blood sample.
  • the biological sample may be a swab sample.
  • a body part is swabbed, the swab is then incubated in a solution for a period of time, and then the solution is used as a sample.
  • a solution for a period of time, and then the solution is used as a sample.
  • Any kind of swab sample can be used, including, but not limited to cheek swabs, oral swabs, nasal swabs, armpit swabs, perineal swabs, wound site swabs, skin lesions swabs, and other skin swabs.
  • Similar "swab"-like samples can also be used, e.g., samples from used medical devices such as wound dressings, which may contain wound exudates.
  • the biological sample may be a tissue sample, or the biological sample may comprise DNA extracted from cells obtained from the subject.
  • the biological sample may be obtained directly from the subject; however, embodiments in which the biological sample is obtained indirectly from the subject are also contemplated.
  • the biological sample may be obtained by culturing a sample obtained from the subject.
  • cell cultures and/or supernatants or other fluids obtained from cultures can also be used as biological samples.
  • the step of treating the biological sample may include the step of lysing cells in the biological sample to extract the DNA from the cells within the biological sample.
  • the extracted DNA may be treated to fragment the DNA by the application of an enzyme, such as DNase, or by sonication, or by the application of heat, for example.
  • the biological sample comprises cells, such as blood samples
  • the biological sample is treated to extract the DNA from cells within the biological sample, and then treated to fragment the extracted DNA.
  • the biological sample may be treated to fragment the extracted DNA.
  • the DNA within the biological sample may have been extracted in a prior step. For example, where the biological sample has been previous obtained for an alternative purpose, cells within the biological sample may have already been lysed.
  • the biological sample may be treated to break down at least some of the tissue into cells before the biological sample is lysed to extract DNA from those cells.
  • the EIS apparatus may comprise a second working electrode that does not comprise biochemical probes. In this way, the second working electrode may be used to take into account any non-specific binding to the sensing surface by the target analyte, or by other species that may be present in the biological sample, and thereby provide a more accurate measurement of whether the blaNDM gene is present in the biological sample, and allow reliable detection of lower minimum concentrations of the blaNDM gene (i.e. a lower limit of detection).
  • the method may provide a level of detection of less than 500 nM target analyte.
  • the method may provide a level of detection of less than 200 nM target analyte.
  • the method may provide a level of detection of less than 100 nM target analyte. That is, it may be possible to detect concentrations of less than 500 nM, 200 nM or preferably, 100 nM blaNDM gene DNA within a biological sample using the method of the present aspect.
  • the method may provide a level of detection of 20 nM target analyte, 50 nM target analyte, 100 nM target analyte or 200 nM target analyte.
  • the step of fragmenting the DNA within the biological sample may be adapted to provide a population of DNA fragments between 20 and 1000 base pairs in length.
  • the majority of the DNA fragments within the population of DNA fragments may be between 20 and 1000 base pairs in length, between 20 and 750 base pairs in length, or between 20 and 500 base pairs in length.
  • incubation of plasmid DNA for 1.5 minutes with 0.8 U/ng DNase may result in a population of DNA fragments that are 25-50 base pairs long.
  • an electrochemical sensor for the detection of bacteria having the blaNDM gene comprising a sensing surface and a sample chamber; the sensing surface defining an interior surface of the sample chamber; the sensing surface comprising a plurality of biochemical probes according to the first aspect of the invention such that the addition of a biological sample comprising blaNDM gene DNA to the sample chamber results in the detection of hybridisation between the recognition sequence of the biochemical probes within the plurality of biochemical probes and the blaNDM gene DNA.
  • the electrochemical sensor may be an electrochemical impedance spectroscopy sensor, and the sensing surface may be a surface of a working electrode.
  • the electrochemical sensor may be a surface plasmon resonance spectroscopy (SPRS) sensor, and the sensing surface may be the sensing surface of the SPRS sensor.
  • SPRS surface plasmon resonance spectroscopy
  • hybridisation of blaNDM gene DNA to the recognition sequence of the biochemical probes within the plurality of biochemical probes on the sensing surface results in a change in the wavelength of light absorbed by the sensing surface, measured as a change in angle of reflection minimum (corresponding to the maximum absorption), for example.
  • the nomenclature signifies probe number followed by sense (S)/ anti-sense (AS) of sequence output by UPS software, with 57 3' end immobilised on the microarray;
  • the nomenclature signifies probe number followed by sense (S)/ anti-sense (AS) of sequence output by UPS software, with 5V 3' end immobilised on the microarray;
  • Figure 3 is a dose-response curve of EIS detection of short synthetic oligonucleotide using PNA probe P7 construct using Ret value at 60 minutes (52 minutes post sample addition) normalized to baseline Ret values;
  • Figure 4 is an online EIS detection assay plot showing Ret changes on P7 functionalised electrodes over time post addition of 10 nM PCR product treated with Lambda exonuclease for different incubation times (0-25 minutes) normalized to baseline Ret values.
  • a synthetic ssDNA oligonucleotide non-complementary to the probe was included as a negative control (nc ssDNA), showing response comparable to that of dsDNA without Lambda exonuclease treatment (0 minutes).
  • Figure 6 shows Ret changes over time upon hybridisation with 10 nM ssDNA blaNDM PCR products and non-complementary mecA PCR products (negative control) under ambient conditions in the presence and absence of 50% formamide in the EIS buffer normalized to baseline Ret values (target addition after 8 minutes).
  • the figure shows the enhancement of specificity of blaNDM PCR product detection by addition of formamide during hybridisation;
  • Figure 7 shows direct detection of NDM plasmid DNA. Online EIS detection assay plot showing Ret change on blaNDM specific PNA P7 functionalised electrodes normalised to Ret change on negative control PNA functionalised electrode over time (n ⁇ 2);
  • Figure 8 shows an example of a specificity check on blaNDM probes on DNA microarray. Hybridisation of 4 ng/ ⁇ fluorescent Pseudomonas aeruginosa 16S rDNA PCR product on DNA microarray functionalized with blaNDM specific probes and controls. The only probes producing a fluorescence signal are positive control P. aerugionosa probes, hybridisation and spotting controls. This indicates very good specificity with all blaNDM probes tested;
  • Figure 9 shows an example of EIS control experiment (left) whereby 1 ⁇ synthetic perfect match oligonucleotide was hybridised on P7 functionalised and blocked PNA-free electrodes to verify electrode functionality. Right: As a control on linearisation of plasmid sample an agarose gel was run. Lane 1 : 1 kb ladder, lane 2: Intact plasmid, lane 3: S1 nuclease treated plasmid; and
  • Figure 10 is a schematic drawing of the relative position of the selected NDM probes P7 and P25, and length and relative position of the four different tested PCR products.
  • DNA oligonucleotides were purchased from Metabion (Martinsried, Germany). PNA oligonucleotides were ordered via Cambridge Research Biochemicals (Cleveland, UK) from Panagene (Daejeon, South Korea). PCR kit and Qiaspin Miniprep kits were purchased from Qiagen (Crawley, UK). Potassium ferricyanide, potassium ferrocyanide, phosphate buffered saline, monosodium phosphate, disodium phosphate and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (Poole, UK).
  • blaNDM-i specific probes of 20 nt in length were designed in silico with an online tool named UPS Unique Probe Selector (UPS) (http://array.iis.sinica.edu.tw/ups/).
  • UPS UPS Unique Probe Selector
  • the UPS algorithm considers percent guanine-cytosine (%GC) content, the secondary structure, melting temperature (Tm), the stability of the probe-target duplex estimated by the thermodynamic model, sequence complexity, similarity of probes to non-target sequences, and other empirical parameters used in the laboratory when selecting probes.
  • %GC percent guanine-cytosine
  • Tm melting temperature
  • sequence complexity sequence complexity
  • similarity of probes to non-target sequences and other empirical parameters used in the laboratory when selecting probes.
  • the option to select probes at a 'pangenomic level' was selected and the 826 bp blaNDM-i sequence (accession no. FN396876.1 , sequence given below) was entered in FASTA format.
  • a salt concentration of 330 mM was specified and subsequently 10 probes, 20 nucleotides in length, were generated.
  • the candidate set of probes were further scrutinized by considering the %GC content, melting temperatures (Tm) and the free energy ( ⁇ ) values of possible secondary structures such as self-dimer and hairpin structures. These values were produced using OligoAnalyzer 3.1 (available at http://eu.idtdna.com/analyzer/applications/oligoanalyzer/).
  • the selection process allowed ten optimal probe sequences to be selected of which seven were selected.
  • the complete probe set chosen for further experiments consisted of two previously identified primers (probes numbered 29-32 17 and 33-36 18 ) and seven UPS generated probe sequences (Table 2).
  • probes were ordered with amino modification at 5' or 3' of the sequence for immobilisation of both orientations.
  • Sense the strand corresponding to mRNA sequence
  • anti-sense the strand complementary to the mRNA sequence
  • oligonucleotides were spotted in 1x Schott Nexterion spot buffer (20 ⁇ ) in replicates of three within each array on Schott Nexterion Slides E (epoxysilane modified surface; Schott, Jena, Germany) with four 200 ⁇ (diameter) split pins and a MicroGrid II (BioRobotics, Cambridge, UK) at 40-50% relative humidity at room temperature. Epoxysilane slides were immediately immobilized at a relative humidity of 75% at room temperature for one hour followed by storage overnight at room temperature under dry conditions. This generated spots with a diameter of approximately 200 ⁇ . Each oligonucleotide was equipped with a 12-thymidine spacer and an amino modification at the 5' end.
  • the slides were then washed with 0.1 % TritonX-100 solution under constant mixing for five minutes at room temperature, with 1 mM HCI solution for four minutes, with 100 mM KCI solution for ten minutes, and with deionized water for one minute.
  • the slides were blocked with 50 mM ethanolamine and 0.1 % sodium dodecyl sulfate (SDS) in 0.1 M Tris buffer (pH 9) for fifteen minutes at 50°C. After blocking the slides were washed in deionized water for one minute and then dried by centrifugation (two minutes at 800g).
  • Hybridisation was done using Agilent 8 gasket slides and hybridisation chambers (Agilent Technologies, Stockport, UK). 50 ⁇ _ hybridisation solution (100nM hybridisation control plus bldNDM-i PCR product), 2x SSC (300mM NaCI and 30mM sodium citrate) were added to each of the gaskets. The printed slide which had been washed and blocked was placed array facing down on top of the hybridisation solution. The sandwich slides were then sealed using a hybridisation chamber and rotated in a pre-heated oven at 55°C for two hours.
  • Epoxysilane slides were washed with 2x saline sodium citrate buffer (SSC) and 0.1 % SDS for ten minutes, 2x SSC for ten minutes, 0.2x SSC for ten minutes. Each slide was then dipped in distilled water for a few seconds and dried by centrifugation for two minutes at 800g.
  • SSC 2x saline sodium citrate buffer
  • Fluorescence images were generated with a Tecan LS Reloaded fluorescence scanner (Tecan, Maennedorf, Switzerland) with excitation at 532nm and emission at 575nm. Target sequences were labeled with the Cy3 fluorophore as described below. Quantification of fluorescence signal intensities was performed with the Quantarray software (Perkin Elmer, Waltham, MA) using the histogram quantification method. For further analysis, the mean signal intensity minus local background intensity was processed with Excel (Microsoft Corp., Redmond, USA) and the mean and standard deviation of all replicates were calculated. DNA extraction and PCR
  • Plasmid DNA was extracted from an overnight Luria-Bertani Broth culture of an NDM-1 producing Citrobacterfreundii isolate using Qiaspin Miniprep Kit (Qiagen Crawley, UK).
  • 5 ⁇ _ Citrobacter freundii NDM-1 plasmid DNA was mixed with 4 ⁇ bldNDM-i specific primers, 0.1 mM deoxynucleotide triphosphates (dNTPs), 1x Taq buffer (50 mM KCI, 10 mM Tris HCI (pH 9.0 at 25°C), 1.5 mM MgCI 2 and 0.1 % Trtion X-100), 1x Q- solution, 1.0 mM MgC and 0.1 U HotstarTaq polymerase (Qiagen, Hilden, Germany) in a final volume of 25 ⁇ _.
  • dNTPs deoxynucleotide triphosphates
  • 1x Taq buffer 50 mM KCI, 10
  • the blaNDM-i specific forward primer was 5' phosphate modified for Lambda exonuclease treatment to produce single-stranded products.
  • fluorescent PCR products were required for microarray dNTPs were substituted with 0.1 mM dATP, dGTP and dTTP were added, in combination with 0.6 mM dCTP and 0.4 mM dCTP-Cy3 (GE healthcare, Buckinghamshire, UK).
  • PCR products used in microarray work, TEM, SHV, KPC and 16S PCR were produced using the same reagent concentrations and conditions and primers.
  • the amplification was performed in a Techne TC-512 thermal cycler (Bibby Scientific Limited, Stone, UK) using the following protocol: 95°C for fifteen minutes; 30 cycles at 95°C for one minute, 50°C for one minute and 72°C for one minute; followed by a final elongation at 72°C for ten minutes.
  • Upon completion of the reaction amplicon was pooled and Lambda exonuclease treated. The length and relative size in the bla gene of each amplicon used (1-4) are shown in Figure 10.
  • PCR product was incubated with 15 U Lambda exonuclease (EURx, Gdansk, Tru) in 1x exonuclease buffer for twenty five minutes at 37°C. In optimisation experiments incubation times of five, fifteen and twenty five minutes were applied at 37°C. The enzyme was then deactivated at 95°C for five minutes followed by cooling on ice. DNase treated plasmid was prepared by incubating the DNA with 0.8 mU DNase I (Promega, Mannheim, Germany) per ng plasmid in 1x DNase buffer for one and a half minutes at room temperature.
  • DNase treated plasmid was prepared by incubating the DNA with 0.8 mU DNase I (Promega, Mannheim, Germany) per ng plasmid in 1x DNase buffer for one and a half minutes at room temperature.
  • EGTA ethylene glycol tetraacetic acid
  • EIS measurements were performed on screen printed sensors connected to an Autolab PGSTAT12 potentiostat (Metrohm Autolab, Herisau, Switzerland) at open circuit potential at an amplitude of 10mV rms at fifteen frequencies in the range 100,000 Hz - 0.1 Hz.
  • 15 Hybridisation and measurement were performed in 0.1 mM K 4 [Fe(CN)6] + 0.1 mM K 3 [Fe(CN) 6 ] + pH 7.0 10 mM phosphate buffer + 20 mM NaCI.
  • DNase treated plasmid DNA sample was heated to 95°C for five minutes, and transferred to ice for two minutes prior to measurement. PCR products and synthetic DNA were measured without preheating.
  • Uncharged PNA probes were used in place of polyanionic DNA which allow a low background signal to be achieved resulting in improved resolution upon binding of charged DNA target as described previously. 22"25
  • Probe density within an alkane-thiol monolayer of 5% mole fraction was employed for optimal sensitivity.
  • Enhanced EIS signal transduction is expected resulting from the abundant negative charges of the phosphate backbone of the target DNA hybridised with the PNA probe at the electrode surface causing a higher degree of repulsion of anionic redox mediators.
  • a large DNA target may result in a lower accessibility of the 20 nucleotides (nt) complementary to the immobilised probe.
  • nt nucleotides
  • blaNDM specific probes of 20 nt in length were designed in silico with an online tool named UPS Unique Probe Selector. Since the hybridisation efficiency of long PCR products with probes immobilised on solid supports is influenced by probe orientation and relative probe/target position both 3' and 5' immobilised sense and antisense probes were tested with regard to their affinity for blaNDM PCR product.
  • fluorescence-based DNA microarrays for pre-selection of in silico designed probes enabled the test of a large number of probes in parallel which could not have been done on the electrochemical platform in the same time frame.
  • This new approach of combining fluorescence-based DNA microarrays with electrochemical detection platforms for assay development has the potential to significantly enhance the assay performance of nucleic acid based, electrochemical in vitro diagnostic tests.
  • the probes were also tested for their cross reactivity towards other relevant antibiotic resistance gene specific PCR products, including genes encoding the beta-lactamases TEM, SHV and KPC, and a P. aeruginosa 16S rDNA PCR product. 29"32 As can be seen in Figure 8 there was virtually no cross-hybridisation of any of the tested probes to P. aeruginosa 16S rDNA PCR product. Similar results were obtained with blajEM, blasHv and blaKPc PCR products (data not shown). A thiol-terminated PNA probe of equivalent sequence to probe 7 was obtained for EIS biosensor development.
  • PNA probe P7 was designed to bind the blaNDM PCR product target with a short (32 nt) 3' and long (571 nt) 5' overhang.
  • the long overhang was expected to be facing towards the bulk solution, which suits EIS application in terms of accessibility and signal transduction.
  • Alternative linkers, or spacers, are well known to the person skilled in the art and are described on pages 10 to 16 of WO 2013/076143 (ITI Scotland Limited), for example.
  • the applied EIS setup enabled the direct, label-free detection of target hybridisation to the immobilized probe. EIS measurements prior to and after target addition allowed the hybridisation to be monitored over the course of ten subsequent EIS spectra, each plotted as Nyquist Plots for capture of charge transfer resistance values (Ret).
  • Figure 3 shows the standard curve which was established based on the dRct after sixty minutes (fifty two minutes after target addition, respectively the tenth consecutive EIS measurement after target addition).
  • the mean increase in Ret above the baseline was plotted relative to time post sample addition for each synthetic oligonucleotide concentration tested.
  • the limit of detection of hybridisation of a synthetic oligonucleotide to immobilised PNA probes was determined to be 10nM. EIS detection of PCR product
  • PCR products were generated using plasmid DNA isolated from an NDM-1 producing Citrobacter freundii strain as template and published primers. Initially, investigations into hybridisation of double stranded PCR products on PNA probe 7 functionalised electrode chip were carried out. However, the Ret change yielded upon hybridisation of 10nM blaNDM-i PCR product (620 bp, dsDNA) was in the range of non-specific absorption observed on the PNA- negative control electrode and upon hybridisation of non-complementary DNA (see Figure 4).
  • Figure 4 shows the signal change over time caused by target binding to the probe under ambient conditions without mixing of the solution.
  • Optimisation of enzyme digestion time over a range of five to twenty five minutes allowed a further enhancement of this detection, with a 20% increase in sensitivity when twenty five minute incubation time was applied (see Figure 4).
  • This optimized Lambda exonuclease treatment protocol applied to 10nM blaNDM PCR product generated a Ret value increase of 170% (dRct 2.7) after twenty eight minute incubation.
  • Figure 4 shows that there is a significant signal change within the first ten minutes after target addition.
  • the 620 bp blaNDM ssPCR product was tested over a concentration range from 0.1 to 50 x 10 " 9 M and an EIS standard curve was constructed using Ret data derived from Nyquist plots of baseline EIS spectra (before target addition) and after ten consecutive EIS measurements following sample addition (Figure 5).
  • a LOD of 100 pM (0.1x10-9M; 0.05 ng/ ⁇ -.) was achieved.
  • This very sensitive detection of long single-stranded PCR products and a 100X lower LOD compared to the detection of a short complementary target was attributed to the enhancement of EIS signal transduction resulting from the large number of negative charges at the electrode surface repelling anionic redox mediators.
  • NDM PCR products of 100pM can be generated from 10 3 gene copies/mL as determined by colony counting. This LOD of 10 3 gene copies/mL is similar to the LOD described by a commercial molecular assay for ESBL and carbapenem resistances in Gram negative bacteria.
  • DNase treatment is routinely used in hybridisation assays to improve accessibility of target sequences to probes. Success of DNase treatment in EIS depends on generating a target of a length with which the balance between accessibility of the complementary probe sequence to the target sequence and signal enhancement is optimum. The incubation of the plasmid DNA for one and a half minutes with 0.8 U/ng DNase resulted in 25-50 bp long fragments. These DNase treated plasmid samples could be detected directly by EIS at ambient conditions at the low nanomolar range (Figure 7). Data represented in Figure 7 show the specific signal increase normalised to Ret change on a negative control PNA functionalised electrode (sequence detailed in Table 2). blaNDM Sequences

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Abstract

La présente invention concerne une sonde biochimique pour la détection du gène blaNDM, la sonde comprenant une séquence de reconnaissance de l'acide nucléique capable de s'hybrider dans des conditions stringentes avec une séquence d'ADN spécifique du gène blaNDM complémentaire à la séquence de reconnaissance de l'acide nucléique. L'invention concerne en outre une trousse comprenant la sonde biochimique, un procédé de détection utilisant la sonde biochimique et un capteur électrochimique comprenant le capteur biochimique.
PCT/GB2015/052586 2014-09-08 2015-09-08 Procédés de détection de bactéries multirésistantes WO2016038351A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020072858A1 (fr) * 2018-10-04 2020-04-09 T2 Biosystems, Inc. Procédés et compositions pour la détection à haute sensibilité de marqueurs de pharmacorésistance
WO2021111353A1 (fr) * 2019-12-03 2021-06-10 Ramja Genosensor Private Limited Dispositif biocapteur, système et kit pour détecter une infection et une résistance antimicrobienne
CN114560914A (zh) * 2022-03-09 2022-05-31 丽水市中心医院 一种抑制blaNDM基因表达的肽核酸及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999037670A1 (fr) * 1998-01-27 1999-07-29 Boston Probes, Inc. Polymeres synthetiques et procedes, trousses ou compositions permettant de moduler leur solubilite
US20050202492A1 (en) * 2004-03-12 2005-09-15 Nam Yun-Sun Microarray having probe polynucleotide spots capable of binding to the same target polynucleotide fragment maximally separated from each other and a method of producing the same
CN202297608U (zh) * 2011-10-12 2012-07-04 中国检验检疫科学研究院 一种细菌耐药基因的检测试剂盒
CN102534014A (zh) * 2012-01-18 2012-07-04 泰普生物科学（中国）有限公司 一种ndm-1泛耐药基因pcr检测试剂盒
WO2013076143A1 (fr) 2011-11-22 2013-05-30 Iti Scotland Limited Détection d'analytes
CN103614465A (zh) * 2013-11-06 2014-03-05 南方医科大学 用于检测革兰阴性杆菌常见碳青霉烯酶基因的lamp引物、试剂盒及其检测方法
CN104237353A (zh) * 2014-10-20 2014-12-24 中国人民解放军第三军医大学第一附属医院 Ndm-1 锁核酸探针修饰电极及其制备方法和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999037670A1 (fr) * 1998-01-27 1999-07-29 Boston Probes, Inc. Polymeres synthetiques et procedes, trousses ou compositions permettant de moduler leur solubilite
US20050202492A1 (en) * 2004-03-12 2005-09-15 Nam Yun-Sun Microarray having probe polynucleotide spots capable of binding to the same target polynucleotide fragment maximally separated from each other and a method of producing the same
CN202297608U (zh) * 2011-10-12 2012-07-04 中国检验检疫科学研究院 一种细菌耐药基因的检测试剂盒
WO2013076143A1 (fr) 2011-11-22 2013-05-30 Iti Scotland Limited Détection d'analytes
CN102534014A (zh) * 2012-01-18 2012-07-04 泰普生物科学（中国）有限公司 一种ndm-1泛耐药基因pcr检测试剂盒
CN103614465A (zh) * 2013-11-06 2014-03-05 南方医科大学 用于检测革兰阴性杆菌常见碳青霉烯酶基因的lamp引物、试剂盒及其检测方法
CN104237353A (zh) * 2014-10-20 2014-12-24 中国人民解放军第三军医大学第一附属医院 Ndm-1 锁核酸探针修饰电极及其制备方法和应用

Non-Patent Citations (48)

* Cited by examiner, † Cited by third party
Title
"Molecular Cloning: A Laboratory Manual", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY PRESS
"The bacterial challenge - time to react", EJHP PRACTICE, vol. 15, no. 5, 2009, pages 15
ANONYMOUS: "nrs conference 2012 bachmann - Google Search", 23 November 2015 (2015-11-23), XP055230433, Retrieved from the Internet <URL:https://www.google.de/search?q=nrs+conference+2012+bachmann&biw=2001&bih=877&source=lnt&tbs=cdr%3A1%2Ccd_min%3A%2Ccd_max%3A9%2F7%2F2014&tbm=> [retrieved on 20151123] *
ARPIN, C.; NOURY, P.; BORAUD, D.; COULANGE, L.; MANETTI, A.; ANDRE, C.; M'ZALI, F.; QUENTIN, C: "NDM-1-Producng Klebsiella pneumoniae Resistant to Colistin in a French Community Patient without History of Foreign Travel", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 56, no. 6, 2012, pages 3432 - 3434
BRINKER, A.; SCHULZE, H.; BACHMANN, T.; MOLLER, R.: "Lambda exonuclease pre-treatment for improved DNA-chip performance depends on the relative probe-target position", BIOSENSORS & BIOELECTRONICS, vol. 26, no. 2, 2010, pages 898 - 902
CARRER, A.; FORTINEAU, N.; NORDMANN, P.: "Use of ChromID Extended-Spectrum beta-Lactamase Medium for Detecting Carbapenemase-Producing Enterobacteriaceae", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 48, no. 5, 2010, pages 1913 - 1914
CHIHARA S; OKUZUMI K; YAMAMOTO Y; OIKAWA S; HISHINUMA A: "First Case of New Delhi Metallo-{beta}-Lactamase 1-Producing Escherichia coli Infection in Japan", CLINICAL INFECTIOUS DISEASES : AN OFFICIAL PUBLICATION OF THE INFECTIOUS DISEASES SOCIETY OF AMERICA, vol. 52, no. 1, 2011, pages 153 - 4
CHIHARA, S.; OKUZUMI, K.; YAMAMOTO, Y.; OIKAWA, S; HISHINUMA, A: "First Case of New Delhi Metallo-beta-Lactamase 1-Producing Escherichia coli Infection in Japan", CLINICAL INFECTIOUS DISEASES, vol. 52, no. 1, 2011, pages 153 - U170
CORRIGAN, D. K.; SCHULZE, H.; HENIHAN, G.; CIANI, I.; GIRAUD, G.;; TERRY, J. G.; WALTON, A. J.; PETHIG, R; GHAZAL, P.; CRAIN, J.: "Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates", BIOSENSORS & BIOELECTRONICS, vol. 34, no. 1, 2012, pages 178 - 184
D. K. CORRIGAN ET AL: "Development of a PCR-free electrochemical point of care test for clinical detection of methicillin resistant Staphylococcus aureus (MRSA)", THE ANALYST, vol. 138, no. 22, 21 November 2013 (2013-11-21), GB, pages 6997, XP055231082, ISSN: 0003-2654, DOI: 10.1039/c3an01319g *
DEGEFA, T. H.; KWAK, J.: "Electrochemical impedance sensing of DNA at PNA self assembled monolayer", JOURNAL OF ELECTROANALYTICAL CHEMISTRY, vol. 612, no. 1, 2008, pages 37 - 41
ELANDER RP: "Industrial production of b-lactam antibiotics", APPL MICROBIOL BIOTECHNOL, 2003, pages 385 - 392
FARBER, J.; MODER, K. A.; LAYER, F.; TAMMER, I; KONIG, W.; KONIG, B.: "Extended-Spectrum Beta-Lactamase Detection with Different Panels for Automated Susceptibility Testing and with a Chromogenic Medium", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 46, no. 11, 2008, pages 3721 - 3727
FOSHEIM, G. E.; CAREY, R. B.; LIMBAGO, B. M.: "Evaluation of the AdvanDx VRE EVIGENE assay for detection of vanA in vancomycin-resistant Staphylococcus aureus", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 45, no. 5, 2007, pages 1611 - 1613
G. ZARFEL; M. HOENIGL EL ET AL.: "Emergence of New Delhi Metallo- p-Lactamase, Austria", EMERGING INFECTIOUS DISEASES, vol. 17, no. 1, 2011, pages 129 - 130
GAZIN, M.; PAASCH, F.; GOOSSENS, H.; MALHOTRA-KUMAR, S.: "Current Trends in Culture-Based and Molecular Detection of Extended-Spectrum-beta-Lactamase-Harboring and Carbapenem-Resistant Enterobacteriaceae", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 50, no. 4, 2012, pages 1140 - 1146
GERNOT ZARFEL ET AL: "Emergence of New Delhi Metallo- [beta]-Lactamase, Austria", EMERGING INFECTIOUS DISEASES, vol. 17, no. 1, 1 January 2011 (2011-01-01), pages 129 - 130, XP055230818 *
HIGUCHI, R. G.; OCHMAN, H.: "Production of Single-Stranded-Dna Templates by Exonuclease Digestion Following the Polymerase Chain-Reaction", NUCLEIC ACIDS RESEARCH, vol. 17, no. 14, pages 5865
JIMMY MING ET AL: "Supporting Information Rapid electrochemical detection of New Delhi metallo-beta- lactamase genes to enable point of care testing of carbapenem resistant Enterobacteriaceae", 29 June 2015 (2015-06-29), XP055230484, Retrieved from the Internet <URL:http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.5b01270/suppl_file/ac5b01270_si_001.pdf> [retrieved on 20151123] *
JIMMY MING-YUAN HUANG ET AL: "Rapid electrochemical detection of multi-drug resistance NDM-1 producing bacteria", 9 October 2012 (2012-10-09), XP055230430, Retrieved from the Internet <URL:http://www.nrsconference.org.uk/nrsconference2012/documents/PosterWinners2012forwebsite.pdf> [retrieved on 20151123] *
JIMMY MING-YUAN HUANG ET AL: "Rapid Electrochemical Detection of New Delhi Metallo-beta-lactamase Genes To Enable Point-of-Care Testing of Carbapenem-Resistant Enterobacteriaceae", ANALYTICAL CHEMISTRY, vol. 87, no. 15, 29 June 2015 (2015-06-29), pages 7738 - 7745, XP055207712, ISSN: 0003-2700, DOI: 10.1021/acs.analchem.5b01270 *
JIN-YOUNG PARK ET AL: "DNA Hybridization Sensors Based on Electrochemical Impedance Spectroscopy as a Detection Tool", SENSORS, vol. 9, no. 12, 1 January 2009 (2009-01-01), pages 9513 - 9532, XP055022249, ISSN: 1424-8220, DOI: 10.3390/s91209513 *
KAASE, M.; SZABADOS, F.; WASSILL, L.; GATERMANN, S. G: "Detection of Carbapenemases in Enterobacteriaceae by a Commercial Multiplex PCR", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 50, no. 9, 2012, pages 3115 - 3118
KEIGHLEY, S. D.; ESTRELA, P.; LI, P.;; MIGHORATO, P.: "Optimization of label-free DNA detection with electrochemical impedance spectroscopy using PNA probes", BIOSENSORS & BIOELECTRONICS, vol. 24, no. 4, 2008, pages 906 - 911
KEIGHLEY, S. D.; LI, P.; ESTRELA, P.; MIGHORATO, P.: "Optimization of DNA immobilization on gold electrodes for label-free detection by electrochemical impedance spectroscopy", BIOSENSORS & BIOELECTRONICS, vol. 23, no. 8, 2008, pages 1291 - 1297
KUMARASAMY, K. K.; TOLEMAN, M. A.; WALSH, T. R.; BAGARIA, J.; BUTT, F.; BALAKRISHNAN, R.; CHAUDHARY, U.; DOUMITH, M.; GISKE, C. G.: "Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study", LANCET INFECTIOUS DISEASES, vol. 10, no. 9, 2010, pages 597 - 602
KURAKIN, A.; LARSEN, H. J.; NIELSEN, P. E.: "Cooperative strand displacement by peptide nucleic acid (PNA", CHEMISTRY & BIOLOGY, vol. 5, no. 2, 1998, pages 81 - 89
LEINBERGER, D. M.; GRIMM, V.; RUBTSOVA, M.; WEILE, J.; SCHROPPEL, K.; WICHELHAUS, T. A.; KNABBE, C.; SCHMID, R. D.; BACHMANN, T. T: "Integrated Detection of Extended-Spectrum-Beta-Lactam Resistance by DNA Microarray-Based Genotyping of TEM, SHV, and CTX-M Genes", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 48, no. 2, 2010, pages 460 - 471
LINSCOTT, A. J.; BROWN, W. J.: "Evaluation of four commercially available extended-spectrum beta-lactamase phenotypic confirmation tests", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 43, no. 3, 2005, pages 1081 - 1085
LISDAT, F.; SCHAFER, D: "The use of electrochemical impedance spectroscopy for biosensing", ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 391, no. 5, 2008, pages 1555 - 1567
LIU, J. Y.; TIAN, S. J.; NIELSEN, P. E.; KNOLL, W.: "In situ hybridisation of PNA/DNA studied label-free by electrochemical impedance spectroscopy", CHEMICAL COMMUNICATIONS, 2005, pages 2969 - 2971
LOY, A.; ARNOLD, R.; TISCHLER, P.; RATTEI, T.; WAGNER, M.; HORN, M.: "probecheck - a central resource for evaluating oligonucleotide probe coverage and specificity", ENVIRONMENTAL MICROBIOLOGY, vol. 10, no. 10, 2008, pages 2894 - 2898
MALIC, S.; HILL, K. E.; HAYES, A.; PERCIVAL, S. L.; THOMAS, D. W.; WILLIAMS, D. W.: "Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridisation (PNA FISH", MICROBIOLOGY-SGM, vol. 155, 2009, pages 2603 - 2611
MARTIN KAASE ET AL: "Detection of carbapenemases in Enterobacteriaceae by a commercial multiplex PCR", vol. 50, no. 9, 1 September 2012 (2012-09-01), pages 3115 - 3118, XP002710012, ISSN: 0095-1137, Retrieved from the Internet <URL:http://jcm.asm.org/content/50/9/3115> [retrieved on 20120711], DOI: 10.1128/JCM.00991-12 *
NAAS, T.; CUZON, G.; BOGAERTS, P.; GLUPCZYNSKI, Y.; NORDMANN, P: "Evaluation of a DNA Microarray (Check-MDR CT102) for Rapid Detection of TEM, SHV, and CTX-M Extended-Spectrum beta-Lactamases and of KPC, OXA-48, VIM, IMP, and NDM-1 Carbapenemases", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 49, no. 4, 2011, pages 1608 - 1613
NORDMANN, P.; POIREL, L.; CARRER, A.; TOLEMAN, M. A.; WALSH, T. R.: "How To Detect NDM-1 Producers", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 49, no. 2, 2011, pages 718 - 721
PARK, J. Y.; PARK, S. M.: "DNA Hybridisation Sensors Based on Electrochemical Impedance Spectroscopy as a Detection Tool", SENSORS, vol. 9, no. 12, 2009, pages 9513 - 9532
PERRY-O'KEEFE, H.; RIGBY, S.; OLIVEIRA, K.; SORENSEN, D.; SLENDER, H.; COULL, J.; HYLDIG-NIELSEN, J. J.: "Identification of indicator microorganisms using a standardized PNA FISH method", JOURNAL OF MICROBIOLOGICAL METHODS, vol. 47, no. 3, 2001, pages 281 - 292
PETER, H.; BERGGRAV, K.; THOMAS, P.; PFEIFER, Y.; WITTE, W.; TEMPLETON, K.; BACHMANN, T. T.: "Direct Detection and Genotyping of KPC Carbapenemases from Urine using a new DNA Microarray Test", J. CLIN. MICROBIOL., 2012
PEYTAVI, R.; TANG, L. Y.; RAYMOND, F. R.; BOISSINOT, K.; BISSONNETTE, L.; BOISSINOT, M; PICARD, F. J.; HULETSKY, A.; OUELLETTE, M.: "Correlation between microarray DNA hybridisation efficiency and the position of short capture probe on the target nucleic acid", BIOTECHNIQUES, vol. 39, no. 1, 2005, pages 89 - 96
POIREL, L.; REVATHI, G.; BERNABEU, S.; NORDMANN, P: "Detection of NDM-1-Producing Klebsiella pneumoniae in Kenya", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 55, no. 2, 2011, pages 934 - 936
REGLIER-POUPET, H; NAAS, T.; CARRER, A.; CADY, A.; ADAM, J. M.; FORTINEAU, N.; POYART, C.; NORDMANN, P: "Performance of chromlD ESBL, a chromogenic medium for detection of Enterobacteriaceae producing extended-spectrum beta-lactamases", JOURNAL OF MEDICAL MICROBIOLOGY, vol. 57, no. 3, 2008, pages 310 - 315
S. CHIHARA ET AL: "First Case of New Delhi Metallo-beta-Lactamase 1-Producing Escherichia coli Infection in Japan", CLINICAL INFECTIOUS DISEASES, vol. 52, no. 1, 1 January 2011 (2011-01-01), US, pages 153 - 154, XP055230822, ISSN: 1058-4838, DOI: 10.1093/cid/ciq054 *
SHAKEEL, S.; KARIM, S.; ALI, A.: "Peptide nucleic acid (PNA) - a review", JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, vol. 81, no. 6, 2006, pages 892 - 899
STEICHEN, M.; DECREM, Y.; GODFROID, E.; BUESS-HERMAN, C: "Electrochemical DNA hybridisation detection using peptide nucleic acids and [Ru(NH3)(6)](3+) on gold electrodes", BIOSENSORS & BIOELECTRONICS, vol. 22, no. 9-10, 2007, pages 2237 - 2243
STROMMENGER, B.; KETTLITZ, C.; WERNER, G.; WITTE, W.: "Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 41, no. 9, 2003, pages 4089 - 4094
TULLMAN, J.; GUNTAS, G.; DUMONT, M.;; OSTERMEIER, M.: "Protein Switches Identified From Diverse Insertion Libraries Created Using S1 Nuclease Digestion of Supercoiled-Form Plasmid DNA", BIOTECHNOLOGY AND BIOENGINEERING, vol. 108, no. 11, 2011, pages 2535 - 2543
ZARFEL, G.; HOENIGL, M.; WURSTL, B.; LEITNER, E.; SALZER, H. J. F.; VALENTIN, T.; POSCH, J.; KRAUSE, R; GRISOLD, A. J.: "Emergence of carbapenem-resistant Enterobacteriaceae in Austria, 2001-2010", CLINICAL MICROBIOLOGY AND INFECTION, vol. 17, no. 11, 2011, pages E5 - E8

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