WO2026018002A1 - Procédé de purification d'adn de pathogène bactérien ou fongique issu d'un échantillon sanguin - Google Patents

Procédé de purification d'adn de pathogène bactérien ou fongique issu d'un échantillon sanguin

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Publication number
WO2026018002A1
WO2026018002A1 PCT/GB2025/051582 GB2025051582W WO2026018002A1 WO 2026018002 A1 WO2026018002 A1 WO 2026018002A1 GB 2025051582 W GB2025051582 W GB 2025051582W WO 2026018002 A1 WO2026018002 A1 WO 2026018002A1
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WIPO (PCT)
Prior art keywords
protease
pathogen
sample
dna
produce
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Pending
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PCT/GB2025/051582
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English (en)
Inventor
Josh DYER
Sarah PALMER
Clio ANDREAE
Daniel ROGERSON
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Genomic Labs Ltd
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Genomic Labs Ltd
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Publication date
Priority claimed from GBGB2410515.7A external-priority patent/GB202410515D0/en
Priority claimed from GBGB2410516.5A external-priority patent/GB202410516D0/en
Application filed by Genomic Labs Ltd filed Critical Genomic Labs Ltd
Publication of WO2026018002A1 publication Critical patent/WO2026018002A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • 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
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/125—Specific component of sample, medium or buffer
    • 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
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/122—Massive parallel sequencing

Definitions

  • the present invention concerns methods for purifying DNA. More particularly, but not exclusively, this invention concerns differential lysis of cells in order to extract DNA. More particularly, this invention concerns the use of cyclodextrins in a method of purifying DNA. The invention also concerns related methods including methods of medical diagnosis, and related products, including kits, for carrying out methods of the invention.
  • the challenge of detecting low copy -number pathogen DNA in a sample swamped by host DNA is not simply a problem of sensitivity. This is because simply increasing sensitivity of the detection of pathogen DNA without regard to other requirements of the detection method may also inadvertently increase the sensitivity of detection of host DNA which does not resolve the “swamping” problem. This challenge may not be so pressing when the detection of pathogenic DNA targets a particular sequence using a sequence-specific primer-annealing and amplification method if primer annealing to host DNA can be successfully minimised. It may, however, be more problematic when the method comprises whole genome amplification - for example whole genome amplification using random primers which are essentially non-discriminating for target DNA.
  • pathogen DNA is present in pathogen cells which may be of a different type and have a different structure, size, or durability to host cells.
  • bacterial pathogens of a eukaryotic host contain DNA which is present in a bacterial cell of a different size and chemical composition to the host cell
  • fungal pathogens of a mammalian host contains DNA which is present in fungal cells which have a different chemical composition to the mammalian host cell.
  • amplify and/or detect the DNA it may be necessary to first free it from the cell in which it is contained, for example by lysing the cell.
  • Different cell types may have different susceptibility to lysis and this can be exploited to differentially free DNA from one cell type versus another cell type.
  • differential lysis Whilst differential lysis is potentially useful in protocols for differentially isolating, amplifying, and/or detecting pathogen DNA versus host DNA, it presents a number of problems which the present invention seeks to overcome or mitigate.
  • the products of lysis comprise compounds with the potential to inhibit down-stream reactions such as amplification and sequencing. Conventional clean-up methods may result in loss of yield.
  • US20110256592A1 sets out to solve problems in nucleic acid amplification due to primers annealing to non-target sequences. This is a long-recognised problem which is typically addressed by using “hot-start” amplification reagents whereby an essential component of the amplification reaction - for example the polymerase enzyme - is bound to an inhibitor and only released from said inhibitor when a sufficiently-high temperature so as to minimise non-target sequence annealing of primers has been reached.
  • US20110256592A1 discloses the use of cyclodextrins to sequester hot- amplification inhibitors of the polymerase enzyme in order to solve problems of amplification specificity rather than amplification sensitivity.
  • US2014/0322781A1 discloses the use of cyclodextrins to suppress alterations of polymerase enzymatic activity resulting from freeze-drying of the polymerase.
  • the disclosure is not an attempt to increase amplification sensitivity, rather it is an attempt to make polymerase more easily stored without loss of activity.
  • WO2016074822A1 discloses the amplification of nucleic acid from microbes present in a faecal sample in order to characterise the gut microbiome. Cyclodextrin is used to sequester compounds present in faeces and to sequester the detergent used in processing. Again, this is not carried out to improve the sensitivity of the application in order to detect very rare organisms, rather the method is intended to quantify the microbes in faeces of which there are very many.
  • US20200140924 discloses the use of cyclodextrin to alleviate inhibition of a real-time PCR reaction resulting from the presence of sodium cholate, a cholesterol derivative, and CN111363793 discloses a combination of trehalose, L-carnitine and cyclodextrin.
  • the present invention is based not only on a realisation that cyclodextrins can be used to relieve inhibition of nucleic acid amplification, but that they can, surprisingly, be used to in method so the invention to improve the sensitivity of whole genome amplification of microbial pathogen DNA in a sample which is swamped by host DNA.
  • the present invention seeks to mitigate one or more of the above-mentioned problems.
  • the present invention provides a method of obtaining DNA of a bacterial or fungal pathogen from a mammalian blood sample comprising the steps of a, bulk removal of mammalian cells from the mammalian blood sample to produce a depleted blood sample, b, optionally, contacting the depleted blood sample with a first protease to produce a protease-treated sample, c, optionally, reducing the volume of the protease-treated sample, by filtration to produce a first filtrate and a first retentate, d, contacting the first retentate, or where step c is absent the protease-treated sample, or where step b and c are absent the depleted blood sample, with DNase and one or more cholesterol-targeting surfactant, in order to differentially lyse mammalian cells and differentially degrade mammalian DNA to produce a pathogen-enriched sample, e, optionally, contacting the pathogen-enriched sample with a
  • a method of obtaining a DNA sequence of a bacterial or fungal pathogen from a mammalian blood sample comprising the steps of: a, bulk removal of mammalian cells from the mammalian blood sample to produce a depleted blood sample, b, optionally, contacting the depleted blood sample with a first protease to produce a protease-treated sample, c, optionally, reducing the volume of the protease-treated sample, by filtration to produce a first filtrate and a first retentate, d, contacting the first retentate, or where step c is absent the protease-treated sample, or where step b and c are absent the depleted blood sample, with DNase and one or more cholesterol-targeting surfactant, in order to differentially lyse mammalian cells and differentially degrade mammalian DNA to produce a pathogen-enriched sample, e, optionally, contacting the pathogen
  • the invention provides a kit for carrying out a method according to the first aspect of the invention or according to the second aspect of the invention, said kit comprising, a cholesterol-targeting surfactant, a protease, one or more cyclodextrins, a DNase, one or more filtration devices.
  • Figure 1 shows data demonstrating that the use of proteinase K treatment prevents clogging of filtration membranes.
  • Figure 2 shows data demonstrating that the absence of saponin treatment relieves inhibition of phi29 amplification.
  • Figure 3 shows data demonstrating that the absence of saponin increases the amplification of human DNA and decreases the amplification of target bacteria DNA.
  • Figure 4 shows data demonstrating that a second proteinase K treatment following saponin lysis of human cells enhances subsequent DNA amplification by phi29 DNA polymerase.
  • Figure 5 shows data demonstrating that cyclodextrin treatment of sample increases whole genome amplification.
  • Figure 6 shows data demonstrating that DIMEB and RAMEB use increases whole genome amplification.
  • Figure 7 shows data demonstrating that methods of the invention improve the detection of microbial organisms in blood samples when present at very low levels.
  • Figure 8 shows data demonstrating that inclusion of cyclodextrin in a method of the invention improves the resulting amplification of low-CFU E. coli nucleic acid.
  • Figure 9 shows data demonstrating that improvements in sample filtration can be achieved using a protease.
  • the present invention provides a method of obtaining DNA of a bacterial or fungal pathogen from a mammalian blood sample comprising the steps of: a, bulk removal of mammalian cells from the mammalian blood sample to produce a depleted blood sample, b, optionally, contacting the depleted blood sample with a first protease to produce a protease-treated sample, c, optionally, reducing the volume of the protease-treated sample, by filtration to produce a first filtrate and a first retentate, d, contacting the first retentate, or where step c is absent the protease-treated sample, or where steps b and c are absent the depleted blood sample, with DNase and one or more cholesterol-targeting surfactant, in order to differentially lyse mammalian cells and differentially degrade mammalian DNA to produce a pathogen-enriched sample, e, optionally contacting the pathogen-enriched sample with a
  • Methods of the invention include methods of providing DNA of a bacterial or fungal pathogen.
  • said DNA is of sufficient quality and/or sufficient purity to permit sequencing of at least 80% of the total genome.
  • the DNA may be of sufficient quality and/or of sufficient purity to permit sequencing, for example of at least 80% of the total genome, by use of a highly- progressive strand-displacement DNA polymerase, for example a highly-progressive strand displacement DNA polymerase as described herein.
  • the amplification step of a method of the invention comprises the use of a mixture of essentially random primers or primers otherwise designed to anneal to a range of target amplification sequences.
  • the amplification step of a method of the invention may comprise the use of random oligo nucleotide primers, for example random hexamer primers.
  • the pathogen is a bacterial pathogen, according to certain other embodiments, the pathogen is a fungal pathogen.
  • the pathogen is a bacterium with the potential to causes bacterial sepsis in a human or non-human mammal, for example Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, or Klebsiella spp.
  • the pathogen is a fungus with the potential to cause fungal sepsis - for example, a Candida species of yeast.
  • the mammalian blood sample contains fewer than 1000 viable pathogen cells per 1ml, for example fewer than 500, fewer than 200, fewer than 100, fewer than 50, fewer than 20, fewer than 10, fewer than 5 viable pathogen cells per ml, fewer than 2 viable pathogen cells per ml, or fewer than 1 viable pathogen cell per ml.
  • the mammalian blood sample may only be of a few millilitres (see further details below), meaning that the total mammalian blood sample may contain fewer than 10,000, fewer than 5,000, fewer than 2,000, fewer than 1,000, fewer than 500, fewer than 200, fewer than 100, fewer than 50, fewer than 20, fewer than 10, fewer than 5 , fewer than 4, fewer than 3 or fewer than 2 viable pathogen cells in total.
  • CFU colony -forming unit
  • pathogen cells and pathogen DNA is “swamped” in the blood sample by mammalian cells and mammalian DNA. More specifically, the ratio of mammalian cells to pathogen cells is at least 100: 1, at least 1000:1, at least 10000:1, or at least 100000: 1 (all ratios expressed as relative cell counts) according to certain embodiments. According to certain embodiments, the ratio of mammalian DNA to pathogen DNA may be at least 100: 1, at least 1000: 1, at least 10000: 1, or at least 100000:1 (all ratios expressed as relative DNA weights). Enrichment ratios
  • the method significantly improves on the ratios given immediately above by enriching for pathogen cells and/or pathogen DNA.
  • the number of pathogen cells is enriched relative to the number of mammalian cells by a factor of at least 100, at least 1000, at least 10000 or at least 100000.
  • the weight of pathogen DNA relative to mammalian DNA is enriched by a factor of at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000 or at least 2,000,000,000.
  • the pathogen DNA may still be potentially swamped by mammalian host DNA, for example at one of the ratios given above.
  • the blood sample is a mammalian blood sample. It is preferred that the mammal is human. However, in other embodiments, it may be a non-human mammal, for example a research animal such as a rodent (for example mouse, rat or rabbit), a livestock animal (for example cattle, sheep, pigs, or goats) or another animal of economic or social value (for example a cat, dog, horse).
  • a rodent for example mouse, rat or rabbit
  • livestock animal for example cattle, sheep, pigs, or goats
  • another animal of economic or social value for example a cat, dog, horse.
  • the human is a human who is suspected of having sepsis or a human displaying one or more symptoms of suspected sepsis. According to certain embodiments, the human is a human exhibiting one or more symptoms of sepsis. According to certain embodiments, the human is a human previously diagnosed as having sepsis.
  • the blood sample may be from a non-mammalian animal. In particular, it may be from a non-mammalian animal which is a domestic or farm animal, for example it may be from poultry (ducks, turkeys, geese, chickens) or from farmed fish
  • the blood sample is a sample of whole blood. It may optionally be a minimally processed sample of whole blood, for example it may be a sample of whole blood to which an anti-coagulant such as K2 EDTA or heparin or another anti -coagulant has been added. It may be a recently-drawn blood sample (i.e., drawn within the previous 30, 60 or 90 minutes) or a previously stored
  • the blood sample may have a volume of between 0.1 ml and 500 ml, for example between 0.1 ml and 200 ml, or between 0.5 ml and 200 ml, for example between 1ml and 200 ml, between 0.1 ml and 100 ml, between 1 ml and 100 ml, between 1 ml and 50 ml, between 1 ml and 20 ml, between 1 ml and 10 ml, for example between 1 ml and 5 ml, between 2 ml and 5 ml, or between 0.1 ml and 20 ml, between 0.1 ml and 10 ml, between 0.1 ml and 5 ml, or between 0.1 ml and 2 ml.
  • the blood sample volume may be expressed scaled to the estimated total blood volume of the animal, so that the blood sample volume is between 0.01% and 0.5 %, between 0.01% and 0.2%, between 0.01% and 0.1%, between 0 01% and 0.05%, for example between 0.01% and 0 02% or between 0.02% and 0.05% of the mammal’s total blood volume.
  • red cells may be depleted by using a reagent to aggregate red cells followed by centrifugation or filtration of the aggregated red cells. Alternatively, a method such as density gradient centrifugation may be used. Alternatively, or additionally, red blood cells may be removed by an immunomagnetic depletion (the details of which may correspond to immunomagnetic depletion methods discussed below). Suitable antigens for targeting by immunodepletion of red blood cells include the erythrocyte antigen.
  • an antibody or functional derivative thereof
  • the antibody is immobilized onto a substrate for example a surface or magnetic bead.
  • a mixture of antibodies is used such that the antibodies mixture as a whole has affinity for antibodies on all, or the vast majority of all, white blood cells.
  • Suitable mixtures of antibodies, including antibodies conjugated to magnetic beads may optionally be used to deplete all or the vast majority of red blood cells.
  • white blood cells may be depleted using a centrifugation method or a specialist white blood cell depletion membrane such as the membranes described in Yaprak I, Yercen N, Ak ⁇ it S, Akdeniz F, Tiirker M, Caglayan S. “A comparison of different filters for white cell reduction”. Turk J Pediatr. 1998 Jan-Mar;40(l):89-95 PMID: 9673534.
  • mammalian DNA “swamping” pathogen DNA are therefore likely to remain and require a further selection in favour of pathogen cells by means of a differential lysis step as described herein in relation to the present invention.
  • step c involves filtration. Filtration in certain configurations may be frustrated or slowed by protein-clogging of the filtration membrane. Therefore methods of the invention (in both its first and second aspect) make use of step b in which the depleted blood sample is contacted with a first protease.
  • the first protease is a broadspectrum serine protease, for example proteinase K or a derivative thereof.
  • proteases include trypsin and trypsin-like proteases, chymotrypsin and chymotrypsin-like proteases, thrombin and thrombin-like proteases, elastase, and elastase-like proteases, subtilisin and subtili sin-like proteases.
  • the protease is used as part of a mixture of more than one protease, for example two or more of the proteases disclosed above.
  • the protease is a cysteine protease, a threonine protease, and aspartic-acid protease, a glutamic acid protease, a metalloprotease, or an asparagine peptide lyase.
  • the contacting of the depleted blood sample with the first protease to produce the protease-treated blood sample is optionally for sufficient time and under sufficient conditions for at least 50% of the protein present in the depleted blood sample to be digested to peptides of 20 or fewer residues.
  • the material may be at a temperature of between 10°C and 50°C (for example between 25°C and 45°C, for example 33°C to 42°C, for example between 35°C and 39°C, for example about 37°C) for between 1 and 20 minutes (for example for between 2 and 10 minutes, for example between 1 and 5 minutes, for example for about 5 minutes). According to certain embodiments it may be for between 2 and 8 minutes at between 30°C and 45°C.
  • the material may be briefly heated to 100 °C to facilitate lysis.
  • the use of protease is carried out in the presence of one or more cyclodextrins. Additionally or alternatively, subsequent filtration may be carried out in the presence of one or more cyclodextrins. Further details of cyclodextrins for use in this step and subsequent filtration steps are provided below.
  • Method of the invention include methods having filtration steps.
  • steps c and step e of method according to the first or second aspect of the invention include filtration steps.
  • Filtration steps are carried out using one or more filtration devices for example one or more filtration devices comprising filtration membranes.
  • Step c of method of the invention provide for a reduction in sample volume by means of filtration using a first filtration membrane.
  • the volume may be reduced by at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. Percentage reduction in volume can be calculated by dividing the filtrate volume by the starting volume of the protease treated-sample.
  • the first filtration membrane is provided in a first filtration device. First filtration membrane
  • the purpose of the first filtration membrane is to retain pathogen cells, whilst allowing smaller substances to pass through the filtration membrane. Because mammalian cells may tend to be of similar size to pathogen cells or larger than pathogen cells, the first filtration membrane may also, optionally, be expected to retain mammalian cells. That is not a problem.
  • the pore size of the first filtration membrane may be at less than about 0.5 pm. The pore size can be considerably smaller than that. However, smaller pore sizes tend to produce slower filtration because they have a greater tendency to clog and so a compromise pore size may be selected in preferred embodiment of the invention.
  • the pore size is less than 0.5 pm, less than 0.4pm, less than 0.3 pm, or less than 0.2 pm.
  • the pore size may be larger, for example less than 0.7 pm, less than 1pm, less than 2 pm or less than 5 pm.
  • the minimum pore size is a factor in filtration speed and so is preferably 0.01 pm, 0.02 pm, 0.05 pm.
  • Any suitable filtration membrane may be used, for example a nylon, PES, MES, PVDF or PCTE membrane.
  • the pore size is less than 1 pm, less than 0.8pm, less than 0.6 pm, or less than 0.5 pm. In certain embodiments, the pore size may be between 0.1 pm, and 1.0 pm, between 0.2 pm, and 0.8 pm, or between 0.3 pm and 0.6 pm. Any suitable filtration membrane may be used, for example a nylon, PES, MES, PVDF or PCTE membrane.
  • Step d of methods of both the first and second aspect of the invention provide for contact of the first retentate with DNA and one or more cholesterol-targeting surfactant.
  • Step d may optionally include suspending the retentate in an aqueous medium if required, for example by backwashing the retentate from the surface of the filtration membrane.
  • step d contacts the first retentate with DNase and one or more cholesterol-targeting surfactants simultaneously.
  • the invention also encompasses methods wherein the retentate is first contacted with one or more cholesterol-targeting surfactant and then subsequently contacted with DNase.
  • the contact time with the DNase is between 1 minute and 10 minutes (for example between 2 minutes and 8 minutes, for example between 3 minutes and 7 minutes, for example for 4 minutes to 6 minutes, for example for about 5 minutes.
  • the contact time with the DNase is between 1 minute and 90 minutes (for example between 5 minutes and 60 minutes, for example between 5 minutes and 45 minutes, for example for 10 minutes to 40 minutes, for example for about 30 minutes.
  • the contact time with the one or more cholesterol-targeting surfactant (which may run concurrently with the contact time for the DNase) is between 1 minute and 10 minutes (for example between 2 minutes and 8 minutes, for example between 3 minutes and 7 minutes, for example for 4 minutes to 6 minutes, for example for about 5 minutes.
  • step d may be carried out in the presence of one or more cyclodextrins. Further details of cyclodextrins suitable for use in this step are provided below.
  • the DNase is preferably an endonuclease. It may for example be DNase I or a derivative thereof.
  • Suitable nucleases include DNase I derivates such as DNase I-XT (New England Biolabs) and salt Active Nuclease (SAN) (or HL-SAN), DNase II or derivatives, Benzonase, Micrococcal endonuclease, Turbonuclease (Serratia marcescens), TurboDNase (ThermoFisher), Serratia marcescens nuclease and derivates such as Benzonase (Sigma), Salt Active Nuclease (Sigma), Heat-Labile Salt Active Nuclease 5 (Arctizymes), Vibrio salmonicida Endonuclease I and other salt tolerant nucleases from halophilic organisms, Exonuclease V (RecBCD), Micrococcal Nuclease, SI
  • the one or more cholesterol-targeting surfactant is selected from the one or more cholesterol-targeting surfactant.
  • a cholesterol-targeting surfactant is a chemical compound (or mixture thereof) which is able to disrupt cell membranes of which are relatively rich in cholesterol relative to cell membranes which are relatively poor in cholesterol.
  • mammalian cells membranes are relatively high in cholesterol compared to prokaryotic pathogens.
  • a “cholesterol-targeting surfactant” is simply a surfactant which differentially disrupts mammalian cell membranes versus prokaryotic cell membranes.
  • non-ionic surfactants may be regarded as cholesterol-targeting surfactants.
  • the one or more cholesterol-targeting surfactant is one or more non-ionic surfactants.
  • the one or more non-ionic surfactants may comprise Triton-X 100 ( 2-[4-(2,4,4- trimethylpentan-2-yl)phenoxy]ethanol), Ecosurf , for example Ecosurf EH (2-Ethyl hexanol EO-PO nonionic surfactant) and/or an alternative alcohol ethoxylate and/or a polysorbate surfactant such as polysorbate 80.
  • suitable alcohol ethoxylate include C9 to Cl l alcohol ethoxylates, C12 to C15 alcohol ethoxylates, 2- ethyl hexanol, 2-propylheptanol and isodecyl alcohol.
  • the one or more cholesterol-targeting surfactants comprise one or more artificial cholesterol- based detergents such as CHOBIMALT (Cholesterol a-F)-Glucopyranosyl-( l ⁇ 4)-p- D-Glucopyranosyl-(1— >6)-a-D-Glucopyranosyl-(l— >4)-P-D-Glucyopyranoside) or CHAP STEROL (3 -[(3 - ⁇ [3 -hydroxy- 19-oxocholest-5 -en- 19- yl]amino ⁇ propyl)(dimethyl)-ammonio]propan-sulfonate)
  • the one or more cholesterol-targeting surfactant is one or more saponins. According to certain embodiments, the one or more cholesterol-targeting surfactant is one or more glycoalkaloid.
  • Saponins are a family of steroid glycosides and triterpene glycosides. They exist as extracts from plants.
  • the saponins of embodiments of the invention may be a single saponin or a mixture of one or more saponins, including tomatine and/or solanine.
  • the one or more cholesterol-targeting surfactants (for example one or more saponins, glycoalkaloids or non-ionic surfactants) will be selected for their ability to differentially lyse mammalian cells versus bacterial or fungal cells. This selectivity of lysis is due to the different composition of mammalian cells versus fungal/bacterial cells. Mammalian membranes are cholesterol rich whereas this is not the case for bacterial cells.
  • the invention uses one or more of the following saponins: Digitalis purpurea saponins, Quillaja saponaria saponins, tomatine, a-chaconine, a-hederin, glycyrrhizic acid, 20S-glycyrrhizic acid, dioscin, a-solanine, timosaponin A-III, ginsenoside Rb3, 10 astragaloside I, K-strophanthoside, ginsenoside Rgl, ginsenoside Rc, lanatoside B, lanatoside C, gitoxin, hederacoside C, protodioscine, Chrysanthellin A, Chrysanthellin B, hederagenin diglycosides, platycodigenin-type saponins, and Astragalus membranaceus saponins.
  • Digitalis purpurea saponins Digitalis purpur
  • Glycoalkaloids are chemical compounds consisting of an alkaloid moiety attached to sugar groups.
  • the one or more cholesterol-targeting surfactants comprise one or more saponins.
  • the one or more cholesterol-targeting surfactants are substantially (for example at least 90%) one or more saponins.
  • the lysis of mammalian cell membranes in step d of methods of the invention is carried out in the presence of one or more cyclodextrins.
  • the one or more cyclodextrins may optionally be as described herein.
  • the one or more cholesterol-targeting surfactants are present at individual and combined concentrations sufficient to cause lysis of cell membranes, such as eukaryotic cell membranes, containing cholesterol, whilst causing no or minimal lysis of cell membranes, such as bacterial cell membranes, containing no cholesterol.
  • the combined concentration, in use, of the one or more cholesterol-targeting surfactants is between 0.005% and 0.5%, for example between 0.01% and 0.2%, for example between 0.02% and 0.2%, for example between 0.02% and 0.1%, for example between 0.02% and 0.08%, for example between 0.03% and 0.07%, for example about 0.05%.
  • the combined concentration, in use, of the one or more cholesterol- targeting surfactants, especially if the one or more cholesterol targeting surfactants are alcohol ethoxylates is between 0.5% and 50%, for example between 1% and 50%, for example between 2% and 50%, for example between 5% and 50%, for example between 5% and 25%, for example between 5% and 20%, for example about 10%, for example between 0.5% and 2.5%, for example between 0.5% and 2%, for example about 1%.
  • the combined concentration is between 0.01 pM and 20 pM, for example between 0.02 pM and 15 pM, for example between 0.05 pM and 10 pM, for example between 0.1 pM and 10 pM, for example between 0.2 pM and 10 pM, for example between 0.5 pM and 10 pM, for example between 0.5 pM and 8 pM, for example between 1 pM and 5 pM, for example between 1 pM and 3 pM, for example about 2 pM.
  • Step e of both the first and second aspects of the invention is a second filtration step. It has the purpose of removing products of the lysis of mammalian cells from any bacterial cells. It may be carried out in the presence of a second protease in order to reduce membrane clogging. In certain embodiments it uses the same filtration membrane as the first filtration step. In other embodiments it uses a second filtration membrane. A second filtration membrane may optionally be provided in a second filtration device.
  • the second protease is a broad-spectrum serine protease, for example proteinase K or a derivative thereof.
  • suitable proteases include trypsin and trypsin-like proteases, chymotrypsin and chymotrypsin-like proteases, thrombin and thrombin-like proteases, elastase, and elastase-like proteases, subtilisin and subtilisin- like proteases.
  • the protease is used as part of a mixture of more than one protease, for example two or more of the proteases disclosed above.
  • the second protease may be as described above in respect of the first protease.
  • the contacting of the depleted blood sample with the second protease to produce the protease-treated blood sample is optionally for sufficient time and under sufficient conditions for at least 50% of the protein present in the pathogen-enriched sample to be digested to peptides of 20 or fewer residues.
  • it may be at a temperature of between 10°C and 50°C (for example between 25°C and 45°C, for example 33°C to 42°C, for example between 35°C and 39°C, for example about 37°C) for between 1 minute and 20 minutes (for example for between 2 minutes and 10 minutes, for example between 1 minute and 5 minutes, for example for about 5 minutes).
  • the incubation time may be between 5 minutes and 120 minutes, for example between 5 minutes and 90 minutes, for example between 5 minutes and 60 minutes, for example, between 10 minutes and 90 minutes, for examples, between 20 minutes and 90 minutes.
  • the contacting of the depleted blood sample with the second protease to produce the protease-treated blood sample may be carried out in the presence of one or more cyclodextrins.
  • the one or more cyclodextrins may optionally be as described herein.
  • the purpose of the second filtration step is to retain any pathogen cells, whilst allowing smaller substances including the lysed remains of the mammalian cells to pass through the filter.
  • This filtration step may be carried out using the first filtration membrane or using a separate second filtration membrane
  • the pore size of the filtration membrane used for the second filtration step may be at less than about 0.5 pm.
  • the pore size can be considerably smaller than that.
  • smaller pore sizes tend to produce slower filtration because they have a greater tendency to clog and so a compromise pore size may be selected in preferred embodiment of the invention.
  • the pore size is less than 0.5 pm, less than 0.4pm, less than 0.3 pm, or less than 0.2 pm.
  • the pore size may be larger, for example less than 0.7 pm, less than 1pm, less than 2 pm or less than 5 pm.
  • the minimum pore size is a factor in filtration speed and so in preferably 0.01pm, 0.02pm, 0.05pm.
  • Any suitable filtration membrane may be used, for example a nylon PES, MES, PVDF or PCTE membrane.
  • the pore size is less than 1 pm, less than 0.8pm, less than 0.6 pm, or less than 0.5 pm.
  • the pore size may be between 0.1 pm, and 1.0 pm, between 0.2 pm, and 0.8 pm, or between 0.3 pm and 0.6 pm.
  • Any suitable filtration membrane may be used, for example a nylon, PES, MES, PVDF or PCTE membrane.
  • a filtration device in any aspect, contains a filtration membrane.
  • the first filtration membrane may optionally be provided in a first filtration device.
  • a second filtration membrane may optionally be provided in a second filtration device.
  • the first and/or second filtration devices may optionally additionally comprise further elements, for example filtration membrane support elements.
  • the filtration device may contain a single filtration membrane.
  • the invention is based, in part, on an appreciation that whilst cholesterol-targeting surfactants are useful in the differential lysis of cell types, cholesterol-targeting surfactants and/or the products of cholesterol-targeting surfactants -lysis are deleterious to downstream steps and particular to downstream DNA amplification steps. These deleterious compounds could be removed by various methods, but further handling steps have the potential to reduce pathogen DNA yield and thus reduce the sensitivity of any pathogen detection or identification method which uses a method of the invention.
  • the present inventors have found that one or more cyclodextrins may be used to effectivity neutralise inhibition of DNA amplification.
  • One or more cyclodextrins may also be used in steps of methods of the invention prior to DNA amplification and/or DNA sequencing.
  • a suitable concentration of one or more cyclodextrins may be used, for example, to effectively neutralise inhibition of DNA amplification, for example from 1 mM to 500 mM, for example from 2 mM to 200 mM, for example from 5 mM to 100 mM, for example from 10 mM to lOOmM, for example from 20 mM to 100 mM, for example from 25 mM to 75mM, for example about 50 mM.
  • Cyclodextrins are a family of cyclic oligosaccharides, consisting of a macrocyclic ring of glucose subunits joined by a-1,4 glycosidic bonds. They may optionally be alphacyclodextrins comprising o 6 glucose subunits, beta cyclodextrins consisting of 7 glucose subunits, or gamma cyclodextrins consisting of 8 glucose subunits, or a mixture of any thereof.
  • one or more cyclodextrins are contacted with the second retentate, or where step e is absent the pathogen-enriched sample, in step f of methods of the invention. This may be achieved by adding one or more cyclodextrins at step f and/or at a prior step. According to certain embodiments one or more cyclodextrins is added at step f, but one or more cyclodextrins may have additionally been added prior to step f of a method of the invention.
  • cyclodextrins are present during multiple method steps, it may be preferred to add additional cyclodextrin at (or immediately prior to) each of said multiple steps rather than rely on the presence of cyclodextrin carried forwards from an earlier step of the method. That is because the cyclodextrins of a previous step may already have their binding ability wholly or partly “used up”.
  • the cyclodextrin may comprise DIMEB (2,6-Dimethyl beta cyclodextrin) and or RAMEB (Methyl-beta- cyclodextrin (DS-12)), or a mixture of DIMEB (2,6-Dimethyl beta cyclodextrin) and or RAMEB (Methyl-beta-cyclodextrin (DS-12).
  • Suitable cyclodextrins include TRIMEB (Heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin) and 2HPPCD (2- hydroxypropyl-P-cyclodextrin) as well as alpha cyclodextrin and gamma cyclodextrin.
  • steps b, d and e are carried out in the presence of one or more cyclodextrins in addition to the cyclodextrins used in step f. Additionally or alternatively, step h may also be carried out in the presence of one or more cyclodextrins.
  • Amplification is the presence of amplification inhibitors.
  • amplification takes place in the presence of one or more inhibitors of nucleic acid amplification selected from the groups consisting of: saponin, products of protease digestion, detergents, solvents, surfactants and glycoalkaloids.
  • said one or more inhibitors of nucleic acid amplification are present at a concentration which is sufficient, in the absence of the one or more cyclodextrins, to inhibit nucleic acid amplification at least 2-fold, 5-fold, 10-fold or 20-fold.
  • the molar concentration of the one or more inhibitors or nucleic acid amplification is between 10 and 120% or the molar concentration of the one or more cyclodextrins present.
  • Step h of methods of the invention entails lysis of the pathogen cells to release and thus obtain the pathogen DNA.
  • this lysis may be carried out by subjecting the cells to hydroxide ions, for example by the addition of potassium or sodium hydroxide (for example at between 10 mM and lOOmM followed by incubation of at least 1 minute (for example for between 1 minute and 10 minutes, for example for about 5 minutes) at between 5 °C and 50 °C, for example between 10 °C and 45 °C, for example between 20 °C and 45 °C, for example between 30 °C and 45 °C, for example between 32 °C and 43 °C for example at about 37 °C.
  • hydroxide ions for example by the addition of potassium or sodium hydroxide (for example at between 10 mM and lOOmM followed by incubation of at least 1 minute (for example for between 1 minute and 10 minutes, for example for about 5 minutes) at between 5 °C and 50 °C, for example between
  • Lysis is preferably carried out in the presence of a reducing agent, for example dithiothreitol (DTT) or di thioerythritol (DTE) or Tris(3-hydroxypropyl)phosphine (THP) or beta-mercaptoethanol or Tris(2-carboxyethyl)phosphine (TCEP) at a concentration between 5 mM and 500 mM, such as between 10 mM and 200 mM, such as between 10 mM and 100 mM, such at between 10 mM and 50 mM, such as about 25 mM DTT or DTE.
  • a reducing agent such as DTT or DTE causes reduction of protein disulfide bridges to dithiols which assists in release of DNA from protein.
  • the alkali is preferably neutralised or buffered after the hydrolysis period to achieve an approximately neutral pH (for example a pH of between 5.5 and 8.5, for example between 6 and 8). This may optionally be carried out by the addition of an acid or the addition of a buffer.
  • step h is carried out in the presence of one or more cyclodextrins.
  • the one or more cyclodextrins may be as described herein.
  • amplification of pathogen DNA optionally takes place after step h.
  • amplification is preferably whole genome amplification.
  • it may be selective amplification of known target sequence by PCR or LAMP or a similar method which uses specific primers.
  • it is an amplification method which does not use selective primers and which instead uses primers designed to anneal at essential random places on the target DNA, for example random oligonucleotide primers.
  • the amplification reaction may be a hot-start amplification reaction.
  • it is non-hot-start amplification reaction which does not require thermal cycling and instead is carried out isothermally.
  • whole genome amplification uses a highly progressive strand displacement DNA polymerase, such as phi-29, to obtain amplified DNA of substantially the whole genome.
  • Amplification methods may optionally include one or more additional features to favour amplification of pathogen DNA versus mammalian DNA, for example reagents capable of selectively binding to mammalian DNA and blocking amplification may be employed.
  • DNA amplification is carried out in the presence of one or more cyclodextrins.
  • the one or more cyclodextrins may be as described herein.
  • Said sequencing step may employ any suitable sequencing technology.
  • Preferably at least 80% of the genome of at least one pathogen is sequenced.
  • Suitable sequencing technologies include fourth generation sequencing technologies for example nanopore sequencing.
  • the sequencing may be followed by analysis (for example in silled) of the sequences in order to identify pathogen species and optionally identify pathogen susceptibility to antibiotics or other antipathogen therapeutics.
  • this analysis may be followed by the selection, and optional subsequent administration of a suitable antipathogen therapeutic agent.
  • sequencing is carried out in the presence of one or more cyclodextrins.
  • the one or more cyclodextrins may be as described herein.
  • the sequencing method employs a strand-displacement highly progressive DNA polymerase (for example Phi-29).
  • the DNA polymerase employs primers which are not highly specific for target pathogen DNA versus mammalian DNA.
  • the primers may comprise mixtures of essentially random oligonucleotide sequences (for example random hexamers) or otherwise be primers capable of annealing and thus initiating amplification at multiple sites of the DNA sequence of the pathogen target and also on the mammalian DNA.
  • a method of obtaining a DNA sequence of a bacterial or fungal pathogen from a mammalian blood sample comprising the steps of: a, bulk removal of mammalian cells from the mammalian blood sample to produce a depleted blood sample, b, optionally, contacting the depleted blood sample with a first protease to produce a protease-treated sample, c, optionally, reducing the volume of the protease-treated sample, by filtration to produce a first filtrate and a first retentate, d, contacting the first retentate, or where step c is absent the protease-treated sample, or where steps b and c are absent the depleted blood sample, with DNase and one or more cholesterol-targeting surfactant, in order to differentially lyse mammalian cells and differentially degrade mammalian DNA to produce a pathogen-enriched sample, e, optionally contacting the pathogen-enriched
  • Steps a to h of embodiments of methods of the second aspect of the invention may optionally include features as described above primarily in reference to the first aspect of the invention.
  • a method which is a method (or part of a method) for the diagnosis of suspected sepsis in a human subject, or which is a method (or part of a method) for the identification of the pathogen causing sepsis in a human subject, or which is a method (or part of a method) for the identification of suitable anti-pathogen therapies for treating sepsis in a human subject.
  • the mammalian blood sample may be a blood sample previously obtained from the human subject.
  • first or second aspect of the invention carrying out steps a to h of the method takes less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours or less than 1 hour. This is considerably more rapid than isolating a pathogen from a blood sample by pathogen culture methods.
  • Optional steps of method of the invention takes less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours or less than 1 hour. This is considerably more rapid than isolating a pathogen from a blood sample by pathogen culture methods.
  • Methods of the invention in the first and second aspect comprise multiple method steps. Two of those steps - steps e and g are optional steps. According to some embodiments, steps e and g are both absent from the method. According to some embodiments steps e and g and both present from the method. According to some embodiments, step e is present, and step g is absent. According to certain embodiments, step g is present, and step e is absent.
  • Step e consists of contacting the pathogen-enriched sample with a second protease, and then optionally filtering it to produce a second filtrate and a second retentate.
  • step e may consist of contacting the pathogen-enriched sample with a second protease and then filtering it to produce a second filtrate and a second retentate.
  • step e may consist of contacting the pathogen-enriched sample with a second protease, without then filtering it to produce a second filtrate and a second retentate.
  • step g may or may not be present.
  • step g when step e is absent, or when step e consists only of contacting the pathogen- enriched sample with a second protease, without then filtering it to produce a second filtrate and a second retentate, that step g is present.
  • step g when step e is present and where it comprises both contacting the pathogen-enriched sample with a second protease and then filtering it to produce a second filtrate and a second retentate, step g may be absent. This may because the rection in sample volume provided by a concentration step may be unnecessary if the volume has been sufficiently reduced by the filtration of step e.
  • steps b and c are absent.
  • steps b and c are absent steps e and g may also both absent from the method.
  • steps b and c are absent steps e and g and both present from the method.
  • step e is present, and step g is absent.
  • steps b and c are absent, step g is present, and step e is absent.
  • Step e consists of contacting the pathogen-enriched sample with a second protease, and then optionally filtering it to produce a second filtrate and a second retentate.
  • step e may consist of contacting the pathogen-enriched sample with a second protease and then filtering it to produce a second filtrate and a second retentate.
  • step e may consist of contacting the pathogen- enriched sample with a second protease, without then filtering it to produce a second filtrate and a second retentate.
  • step g may or may not be present. However, it is preferred, that where steps b and c are both absent, and when step e is absent, or when step e consists only of contacting the pathogen-enriched sample with a second protease, without then filtering it to produce a second filtrate and a second retentate, that step g is present. According to certain embodiments, where steps b and c are both absent and where step e is present and where it comprises both contacting the pathogen-enriched sample with a second protease and then filtering it to produce a second filtrate and a second retentate, step g may be absent. This may because the rection in sample volume provided by a concentration step may be unnecessary if the volume has been sufficiently reduced by the filtration of step e.
  • the invention provides in a third aspect a kit for carrying out a method according to the first aspect of the invention or the second aspect of the invention, said kit comprising, a cholesterol-targeting surfactant, a protease, one or more cyclodextrins, a DNase, one or more filtration devices.
  • Components of such a kit may comprise features described above in reference to the first or second aspects of the invention.
  • Such a kit may optionally additionally comprise a highly progressive DNA polymerase (optionally having further features as described in reference to the first or second aspect of the invention).
  • Such a kit may optionally additionally comprise reagents and or equipment for sequencing pathogen DNA (optionally having further features as described above in reference to the first or second aspect of the invention).
  • kit may optionally additionally comprise instructions, for example in written form such as in a leaflet or booklet or in electronic form for carrying out a method according to the first aspect of the invention or a method according to the second aspect of the invention.
  • a kit of the invention may optionally additionally comprise computer encoded instructions for a robotic system to carry out one or more steps of a method of the invention automatically.
  • the invention provides a method of amplifying a target nucleic acid derived from biological material comprising carrying out a nucleic acid amplification reaction in the presence of one or more cyclodextrins.
  • the invention in accordance with this further aspect may include further features as described in reference to the other aspects of the invention.
  • the amplification reaction, nucleic acid, its source, polymerase, amplification reaction, primers, cyclodextrins, genome coverage etc may optionally be in this further aspect as described above in reference to the first and/or second aspect.
  • Step 1 Bulk removal of human cells via antibody-bound magnetic beads This method step depletes the bulk of human cells present. Various methods for carrying this step out are known in the art. Step 2 - Filtration of human-cell-depleted blood
  • Step 3 Differential lysis of remaining human cells
  • Step 4 Filtration of saponin-treated-depleted blood
  • Step 5 Removal of remaining inhibitors using cyclodextrins
  • a cyclodextrin for example DIMEB, RAMEB or any other cyclodextrin
  • Step 6 Lysis and whole-genome-amplification
  • Example 1 Proteinase K treatment of plasma prevents clogging of filtration membranes.
  • Example 3 Absence of saponin increases human DNA and decreases target bacterial DNA post whole genome amplification
  • This experiment uses qPCR to detect the amount of each of human and bacterial DNA amplified. It demonstrates that when the full method of the invention is carried out as described in steps 1 to 6, the inhibitors of whole genome amplification illustrated in figure 2 can be sufficiently removed to permit DNA amplification. It also demonstrates that using saponin versus carrying out the method without saponin, leads to an increase in bacterial DNA amplification versus human DNA amplification.
  • Whole blood was spiked with bacteria and processed as described in the general method, with a comparator method used which lacked saponin. The final DNA amplified was tested via qPCR to detect the amount of human vs bacterial DNA present in the sample.
  • Example 4 A second proteinase K treatment following saponin-lysis of human cells enhances DNA amplification by phi29 in sample preparation system.
  • This method demonstrates the value of using proteinase K a second time in step 4 of the general method described above. Briefly whole blood was depleted, followed by treatment with proteinase K at 37 °C for 5 min, flow through a 0.1 pM filter, withdrawal, and treatment with saponin and DNase. Samples were then treated with or without (+/-) an additional proteinase K treatment in step 4 of bulk human cell depleted the general method before samples were filtered again and washed with PBS before bacterial lysis and phi29 amplification. DNA concentrations were quantified using QubitTM IX dsDNA High Sensitivity kits.
  • Example 5 Cyclodextrin treatment of sample increases whole genome amplification.
  • the general method as described above was carried out from step 1 to step 6. Briefly, blood spiked with bacteria was filtered post proteinase K treatment and then diluted in PBS plus saponin and DNase. This was incubated for 5 minutes at RT before being treated with proteinase K for an additional 5 minutes and filtered to recover the final sample.
  • this sample was either treated with PBS or PBS+DIMEB (2,6-Dimethyl beta cyclodextrin) for 10 minutes at RT before being concentrated by centrifugation, lysed and amplified with Phi29 for 3 hours. The DNA concentration was then assayed by Qubit reagent and fluorescence detection.
  • ESKAPE is an acronym comprising the scientific names of six highly virulent and antibiotic-resistant bacterial pathogens including: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.
  • Staphylococcus aureus (2x), Acinetobacter baumannii (2x) and the RAMEB treated samples were used with Pseudomonas aeruginosa (2x) and Enterococcus faecalis (2x) and treated with PBS+DIMEB or PBS+RAMEB for 10 minutes at RT before being concentrated by centrifugation, lysed, and amplified with Phi29 for 3 hours. The DNA concentration was then assayed by Qubit reagent and fluorescence detection.
  • Example 7 Demonstration of detection of pathogen DNA at very low CFUs
  • Samples of 10 ml of human blood were spiked with various strains of E. colt or S. aureus at defined CFU levels (CFU/ml).
  • Samples were processed according to the general method of the invention with steps b, d, and e in addition to step g carried out in the presence of cyclodextrin.
  • DNA was amplified using phi-29 and random hexamer primers and sequenced. The sequence was aligned in silico with the appropriate reference sequence to calculate a percentage of total genome coverage. Results are presented below and in figure 7.
  • Figure 8 shows reduced amplification in the -cyclodextrin conditions. This likely demonstrates the capacity of cyclodextrins to remove a variety of enzymatic inhibitors that can stymie DNA amplification. It appears that at least some of those inhibitors arises during earlier stages of DNA preparation and are best mitigated against by including cyclodextrin in those earlier stages.
  • FIG. 9 shows that higher pressure was needed on the filter to maintain a consistent flow rate in the “control” samples in comparison with the “protease” samples. This suggests that protease treatment mitigates sample-induced blocking of the filter.
  • Figure 9A shows the first run of the experiment using a new filter.
  • Figures 9B and 9C show, respectively, repeats using the same filter used in the previous experimental run.

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Abstract

L'invention concerne des procédés d'obtention d'ADN d'un pathogène bactérien ou fongique issu d'un échantillon de sang de mammifère, qui comprennent les étapes suivantes : extraction de la majorité des cellules de mammifère de l'échantillon de sang de mammifère, traitement à la protéase, filtrage pour réduire le volume, utilisation d'un ou plusieurs tensioactifs de ciblage de cholestérol et de DNase pour lyser de manière différentielle des cellules de mammifère et dégrader de manière différentielle l'ADN de mammifère afin de produire un échantillon enrichi en agents pathogènes, qui est lysé pour obtenir l'ADN de pathogène. L'invention concerne également des procédés de séquençage de l'ADN de pathogène ainsi que des kits associés pour mettre en œuvre les procédés.
PCT/GB2025/051582 2024-07-18 2025-07-17 Procédé de purification d'adn de pathogène bactérien ou fongique issu d'un échantillon sanguin Pending WO2026018002A1 (fr)

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