Method for eliminating nucleic acids

Abstract

There is provided a method for eliminating host nucleic acids in a biological sample, the sample having previously been obtained from an animal host, the method comprising the steps of: (a) adding to the sample a cytolysin or an active variant thereof; and (b) performing a process of physically eliminating the nucleic acid released from the host cells within the sample, or making the nucleic acid unrecognizable.

Description

[0001] This application is a divisional application with application number 201780083080.3, application date December 12, 2017, and invention name “Method for Eliminating Nucleic Acids”. Technical Field

[0002] The present invention relates to methods for eliminating host nucleic acids from biological samples. Background Art

[0003] Rapid and comprehensive infectious disease diagnosis is essential for improving patient management and combating antimicrobial resistance. Rapid diagnosis of life-threatening infectious diseases (such as sepsis and pneumonia) is essential. These clinical syndromes have complex etiologies and require identification of pathogens in challenging sample matrices (such as blood, sputum, etc.). Currently, the "gold standard" method for clinical diagnosis is microbial culture, which is labor-intensive, has a long turnaround time and poor clinical sensitivity. Currently available rapid molecular methods (such as PCR) have improved the turnaround time and sensitivity of results, but are limited in scope, and thus rare pathogens and resistance markers may be problematic. The most applicable technology for rapid detection of microbial pathogens is nucleic acid amplification testing (NAAT). NAAT can be used for sepsis diagnosis (such as Septifast (RTM), Roche), but the complexity of use and suboptimal performance prevent their widespread adoption. Most NAATs for respiratory tract infections (RTIs) focus on detecting respiratory viruses (such as Biofire Filmarray Respiratory Panel, SeegeneRV15). The Curetis Unyvero (RTM) test is an exception, which is designed for healthcare-associated pneumonia. However, NAATs are not comprehensive (e.g., the Curetis test covers only 90% of the top pathogens) and only seek a pre-specified target range, which means that less common pathogens will be missed. Therefore, NAAT diagnostics are an adjunct to standard bacteriology, not a replacement, and have limited adoption.

[0004] A paradigm shift in diagnostic technology is urgently needed - a universal diagnostic that can detect any pathogen (e.g., virus, bacteria, fungus) and antibiotic resistance. Agnostic / shotgun metagenomic sequencing has the potential to be the technology of choice to drive this shift. Shotgun metagenomic sequencing can detect and provide the relative proportions of viruses, bacteria, and fungi in a sample without prior knowledge of the microbiome and is increasingly being used to study complex metagenomes in clinical samples.

[0005] So why is shotgun metagenomics not currently widely used for infection diagnostics? One reason is that next-generation sequencing (NGS) has traditionally been expensive, complex to perform, and difficult to analyze. The development of the MinION (RTM) nanopore sequencing technology has transformed the NGS field with an inexpensive, portable sequencer, fast and simple library preparation (15 minutes), and automated real-time analysis tools. Another major obstacle is the large amount of human DNA present in clinical samples, which is often several orders of magnitude higher than the pathogen DNA present. Due to the large amount of human and pathogen nucleic acids (especially DNA) (based on 10 6 WBC / ml, the ratio is usually 10 8 : 1 to 10 9 : 1 [with ~6.6 pg DNA / cell] but as few as 1-10 colony-forming units [CFU] of pathogens per mL [containing ~10 fg DNA / cell]), blood is a particularly challenging matrix for NGS-based pathogen characterization. 5 Elimination of host DNA, potentially resulting in a human:pathogen DNA ratio of 10 3 : 1, is necessary to facilitate NGS-based pathogen characterization, and the level of elimination (causing pathogen nucleic acid enrichment) is not achieved by methods disclosed in the art (such as commercially available pathogen DNA enrichment methods (Looxster (RTM) Enrichment Kit (Analytic Jena); NEBNext (RTM) Microbiome DNA Enrichment Kit (NEB); MolYsis (RTM) Basic 5 Kit (Molzym))).

[0006] One of the objects of the present invention is to solve the above-mentioned problems. Summary of the invention

[0007] Thus, there is provided a method for eliminating host nucleic acids in a biological sample, said sample having been previously obtained from an animal host, said method comprising the steps of:

[0008] (a) adding a cytolysin or an active variant thereof to the sample; and

[0009] (b) performing a process that physically eliminates nucleic acids released from host cells within the sample, or otherwise renders such nucleic acids unrecognizable.

[0010] Preferably, step (b) comprises adding a nuclease to the sample and / or the method further comprises the step of extracting remaining nucleic acid from the sample.

[0011] Preferably, the method further comprises the step of subjecting the extracted nucleic acid to a purification process and / or further comprises the step of amplifying the extracted nucleic acid.

[0012] Preferably, the method further comprises the step of performing a nucleic acid amplification test on the extracted nucleic acid, or preferably, performing a sequencing process on the extracted nucleic acid.

[0013] In a preferred embodiment, the cytolysin is a phospholipase, preferably a phospholipase C (PLC), more preferably a bacterial PLC, more preferably a Group 1 PLC, most preferably a PLC from Clostridium perfringens.

[0014] In a preferred embodiment, the biological sample is a blood sample.

[0015] In a preferred embodiment, the method results in at least 10 times, preferably at least 10 times, the host DNA originally contained in the sample. 2 times, preferably at least 10 3 times, preferably at least 10 4 times, most preferably at least 10 5 times elimination.

[0016] Also provided is a kit comprising i) a cytolysin or an active variant thereof, and ii) means for physically eliminating free nucleic acids in a biological sample or otherwise rendering such nucleic acids unrecognizable. Preferably, the cytolysin is as defined above, and / or wherein the means comprises a nuclease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Amplification curves of human qPCR results after various endonuclease treatments are shown.

[0018] Figure 2 Amplification curves of human qPCR results after endonuclease treatment with various buffer volumes are shown.

[0019] Figure 3 Amplification curves of human qPCR results after HL-SAN DNase and MolDNase treatment with respective buffers are shown.

[0020] Figure 4 Amplification curves showing human qPCR results after cytolysin treatment.

[0021] Figure 5 Shown are amplification curves of human qPCR results showing PLC activity under different sample conditions.

[0022] FIG6 shows amplification curves of qPCR results of blood spiked with bacteria in increasing volumes after PLC and HL-SAN DNase treatment; A: human qPCR; B: Escherichia coli (E. coli) qPCR; C: Staphylococcus aureus (S. aureus) qPCR.

[0023] Figure 7 Amplification curves of human qPCR results showing PLC activity after addition of the active cocktail during host cell lysis.

[0024] FIG8 shows the amplification curves of qPCR results after the inactivation of modified HL-SAN DNase; A: human qPCR; B: Escherichia coli qPCR; C: Staphylococcus aureus qPCR.

[0025] FIG9 shows amplification curves of qPCR results for method comparison; A: human qPCR; B: Escherichia coli qPCR; C: Staphylococcus aureus qPCR.

[0026] Fig.10 Shown is the coverage map of the Candida albicans genome after single-plex MinlON sequencing of Candida albicans (C. albicans). DETAILED DESCRIPTION

[0027] generally

[0028] Provided herein is a method for eliminating host nucleic acids (particularly RNA and / or most preferably DNA) in a biological sample, the sample having been previously obtained from an animal host, the method comprising the steps of:

[0029] (a) adding a cytolysin or an active variant thereof to the sample; and

[0030] (b) performing a process that physically eliminates nucleic acids released from host cells within the sample, or otherwise renders such nucleic acids unrecognizable.

[0031] The animal host may be a vertebrate, such as a bird, fish or, preferably, a mammal, most preferably a human. At the time of sample collection, the host may be alive or dead.

[0032] Biological sample can be any sample containing animal cells (in tissue form or other forms).Special (e.g. clinically) samples of interest include bile, nails, nasal / bronchial lavage fluid, bone marrow, stem cells from the body, bones, non-fetal products of conception, brain, breast milk, organs, pericardial fluid, buffy coat, platelets, cerebrospinal fluid, pleural fluid, cystic fluid, primary cell culture, pus, saliva, skin, fetal tissue, cystic lesion fluid, gastric contents, hair, teeth, tumor tissue, umbilical cord blood, mucus and stem cells.However, particularly preferred samples include joint aspirates, feces, urine, sputum and, especially blood (including plasma).Preferably, the sample is in liquid form.The initial sample may need to be converted to liquid form before carrying out the current method.The volume of the liquid sample can be 10 μl to 100ml, preferably 10 μl to 50ml, such as 10 μl or 100 μl to 20ml (e.g. 0.2ml or 1ml).

[0033] Cytolysin causes (selectively) lysis of host cells, releases host nucleic acid, so that it can be (partially or completely) eliminated. The nucleic acid in non-host cells or particles (e.g., pathogens) remains substantially intact (i.e., not significantly removed or digested from the sample) and identifiable, so that it can be collected and analyzed subsequently, and in particular, identified (by, for example, sequencing or targeted PCR). Nucleic acid is identifiable, for example, if its sequence and / or biological origin can be determined. Therefore, preferably, cytolysin is added to the sample and allowed to act for a period of time so that enough host cell lysis can occur. Steps (a) and (b) ("cytolysin incubation" and "elimination step") can occur simultaneously, or step (b) occurs after step (a).

[0034] Methods for eliminating host nucleic acids include physical elimination (of nucleic acids released from host cells within a sample) and (in the context of the present technology) virtual elimination. Physical elimination can include, for example, digestion of nucleic acids (i.e., breaking down nucleic acid polymers into, for example, basic monomers) or removal of nucleic acids from a sample (e.g., by any nucleic acid capture method known to those skilled in the art, such as deploying nucleic acid-binding magnetic beads in a sample to bind DNA and / or RNA, which can then be removed or harvested from the sample).

[0035] Virtual elimination involves making the (released) nucleic acid (by, in particular, targeted PCR, or most preferably, sequencing) unrecognizable. For DNA, this means making the DNA (e.g., by PCR) unamplifiable and / or (preferably) unsequenceable. For RNA, this means making the RNA unamplifiable, unreversibly transcribed, and / or (preferably) unsequenceable. A preferred method of such representation (in particular of DNA) comprises adding a photoreactive nucleic acid binding dye (such as monopropidium arsenide (PMA) or monoethidium azide (EMA)) to the sample and inducing a photoreaction.

[0036] However, most preferably, the elimination method is by digestion of nucleic acids, most preferably by enzyme digestion. Therefore, preferably step (b) includes adding a nuclease to the sample. Preferably, the nuclease is added to the sample and allowed to act for a period of time so that enough nucleic acid digestion can occur. Therefore, preferably, deoxyribonuclease (DNase) and / or ribonuclease (RNase) are added to the sample (and preferably allowed to act for a period of time so that enough DNA / RNA digestion can occur). Nuclease can have DNase and RNase activity (e.g. HL-SAN DNase). If the analysis of non-host (e.g. pathogen) DNA is to be carried out, the elimination of host DNA is important. If non-host (e.g., pathogen) RNA analysis is carried out, the elimination of host RNA is important, and in fact the optimization of DNA analysis (e.g., DNA sequencing) can be promoted.

[0037] In such embodiments, the method preferably further comprises a subsequent step of neutralizing the nuclease (or each nuclease) (i.e. reducing or substantially eliminating the activity of the nuclease). The skilled person will recognize a range of neutralization options to be identified for each elimination scheme. This may include heat inactivation or preferably buffer exchange (i.e. removing the buffer in which the nuclease is active and / or replacing or adding a buffer in which the nuclease is substantially inactive). Preferably, the temperature of the sample (at any / all stages before / at the time of extracting the remaining nucleic acid from the sample) is maintained at 50°C or less, preferably 45°C or less, preferably 40°C or less, to optimize the subsequent release of nucleic acids from pathogens (particularly bacterial cells).

[0038] Further steps

[0039] In a preferred embodiment, the method further comprises the step of extracting the remaining (preferably non-host) nucleic acids from the sample (or an aliquot thereof). Some or all of the remaining nucleic acids (particularly non-host nucleic acids) will be intact and identifiable.

[0040] Typically, the extraction process will involve a centrifugation step to specifically collect non-host cells / particles (e.g., pathogens) (viral particles and / or particularly bacterial and / or non-animal (e.g., non-mammalian) (e.g., unicellular) eukaryotic cells, such as fungi), from which nucleic acids can be obtained. Centrifugation conditions can be selected so that bacterial cells and non-animal cells are precipitated instead of viral particles, or viral particles are precipitated in addition to bacterial and non-animal cells. In the former case, the supernatant can be subjected to standard virus detection tests. (In fact, before any cytolysin addition, whether or not the present technology is used for enrichment, clinical samples can be centrifuged using standard procedures, the pellet containing cells is retained (for methods of the current technology), and the supernatant is used for virus detection.)

[0041] Nucleic acids can be obtained from pathogens using methods known in the art and may include the addition of lysis buffers, lytic enzymes (degrading or eliminating cell membranes, cell walls and / or viral capsids), and / or proteases such as proteinase K. Preferred lysozymes include lysozymes, mutanolysins, lysostaphins, chitinases and lyticases.

[0042] Alternatively, the extracted nucleic acid (or its aliquot) is subjected to a purification process, such as a purification process known in the art. In DNA purification, RNase may be used to facilitate optimization of subsequent DNA sequencing. However, if non-host (e.g., pathogen) RNA extraction is of interest (e.g., for subsequent RNA sequencing), RNase may be omitted from any purification step (and DNase may be used to assist purification).

[0043] In a preferred embodiment, the extracted nucleic acid (or an aliquot thereof) is subjected to an amplification process (such as whole genome amplification) to increase the copy number of the nucleic acid, particularly where the biological sample is a blood sample. For RNA, this may involve direct amplification, or amplification of the cDNA after conversion of the RNA to cDNA.

[0044] In a preferred embodiment, the method further comprises the step of subjecting the extracted nucleic acid (RNA, DNA or cDNA) (or an aliquot thereof) to a nucleic acid amplification assay (e.g. a targeted PCR amplification process, isothermal amplification, nucleic acid sequence-based amplification (NASBA)) or, preferably, subjecting the extracted nucleic acid (or an aliquot thereof) to a sequencing process, such as (e.g. short or long read) DNA or RNA sequencing using, for example, nanopore or Illumina (RTM) sequencing.

[0045] In the aforementioned embodiments, nucleic acids (particularly host nucleic acids) previously rendered unidentifiable are not amplified by any amplification process and / or (particularly) sequenced by any sequencing process.

[0046] Compared to prior art methods (e.g., MolYsis (RTM) techniques that deploy chaotropes to lyse host cells prior to host nucleic acid digestion), the novel method facilitates highly improved elimination of host nucleic acids (particularly DNA) while leaving non-host (e.g., pathogen, particularly bacterial) nucleic acids intact (and identifiable), resulting in highly improved enrichment of non-host (e.g., pathogen) nucleic acids sufficient for subsequent sequencing-based (e.g., next generation sequencing [NGS]-based) (e.g., pathogen) diagnostics. A key element of this advancement is the ability to achieve, for example, 5×10 of host DNA in a biological sample from a mammalian host. 4 or larger, such as 10 5 or larger (e.g. 10 6 or greater), and these are preferred results characteristic of the present technology (e.g., 10 or greater, 10 2 or greater, 10 3 or larger, 5×10 3 or greater, or 10 4 or greater multiples of elimination). After deployment of the methods of the invention, it is particularly preferred that host nucleic acids (e.g., DNA) are undetectable (e.g., by qPCR). More generally, selective elimination of host nucleic acids can enrich for non-host nucleic acids and thus improve identification of non-host organisms. Thus, the technology is applicable to fields beyond medical microbiology, such as biological research, veterinary medicine / diagnostics, and agriculture / food safety.

[0047] Cytolysin

[0048] Cytolysins (also called cytolytic toxins) are proteins secreted by microorganisms, plants, fungi or animals that are specifically toxic to heterologous cell types, in particular promoting the lysis of target cells. Preferred cytolysins are cytolysins secreted by microorganisms (in particular bacteria) and / or cytolysins that are toxic to animal (e.g. mammalian) cell types.

[0049] The cytolysin may be a cytolysin that has a decontaminating effect on the target cell membrane (e.g., the 26 amino acid delta toxin produced by Staphylococcus) or a cytolysin that forms pores in the target cell membrane (e.g., alpha hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and clostridium perfringens produced by Clostridium perfringens). See, e.g.,

[0050] Alpha hemolysin from Staphylococcus aureus -

[0051] https: / / www.ncbi.nlm.nih.gOv / protein / BBA23710.1(SEQ ID No.2):

[0052] 1 mktrivssvt ttlllgcilm npvanaadsd iniktgttdi gsnttvktgd lVtydkengm

[0053] 6l hkkvfysfid dknhnkkilv irtkgtiagq yrvyseegan ksglawpsaf kvqlqlpdne

[0054] 121 vaqisdyypr nsidtkeyms tltygfngnv tgddsgkigg liganvsigh tlkyvqpdfk

[0055] 181 tilesptdkk vgwkvifnnm vnqnwgpydr dswnpvygnq lfmktmgsm kaadnfldpn

[0056] 241 kassllssgf spdfatvitm drkaskqqtn idviyervrd dyqlywtstn wkgtntkdkw

[0057] 301 tdrsseryki dwekeemtn

[0058] Streptolysin O from Streptococcus pyogenes -

[0059] https: / / www.ncbi.nlm.nih.gov / protein / BAD77794.2(SEQ ID No.3):

[0060] l msnkktflcky srvaglltaa liignlvtan aesnkqntas tettttseqp kpesseltie

[0061] 6l kagqkmddml nsndmiklap kemplesaek eekksedkkk seedhteein dkiyslnyne

[0062] 121 levlaknget ienfvpkegv kkadkfivie rkkkninttp vdisiidsvt drtypaalql

[0063] 181 ankgftenkp davvtkrnpq kihidlpgmg dkatvevndp tyanvstaid nlvnqwhdny

[0064] 24l sggntlpart qytesmvysk sqieaalnvn skildgtlgi dfksiskgek kvmiaaykqi

[0065] 301 fytvsanlpn npadvfdksv tfkdlqrkgv sneapplfvs nvaygrtvfv kletssksnd

[0066] 361 veaafsaalk gtdvktngky sdilenssfi awlggdaae hnkvvtkdfd virnvikdna

[0067] 421 tfsrknpayp isytsvflkn nkiagvnnrt eyvettstey tsgkinlshq gayvaqyeil

[0068] 481 wdeinyddkg kevitkrrwd nnwysktspf stviplgans mirimarec tglawewwrk

[0069] 54l viderdvkls keinvnisgs tlspygsity k

[0070] Preferably, the cytolysin is a cytolysin that digests cell membrane components (e.g. phospholipids, i.e. phospholipase). An example is sphingomyelinase (also known as beta-toxin) from Staphylococcus aureus, see e.g.

[0071] http: / / www.ncbi.nlm.nih.gov / protein / CAA43885.1(SEQ ID No.4):

[0072] 1 mmvkktksns lkkvatlala nlllvgaltd nsakaeskkd dtdlklvshn vymlstvlyp

[0073] 6l nwgqykradl igqssyiknn dvvifneafd ngasdkllsn vkkeypyqtp vlgrsqsgwd

[0074] 121 ktegsysstv aedggvaivs kypikekiqh vfksgcgfdn dsnkgfvytk iekngknvhv

[0075] 181 igthtqseds rcgaghdrki raeqmkeisd fvkkknipkd etvyiggdln vnkgtpefkd

[0076] 241 mlknlnvndv lyaghnstwd pqsnsiakyn ypngkpehld yiftdkdhkq pkqlvnevvt

[0077] 301 ekpkpwdvya fpyyyvyndf sdhypikays k

[0078] The phospholipase may be a phospholipase A, B, C or D, such as PLD from Streptomyces, see e.g. https: / / www.ncbi.nlm.nih.gov / protein / BAL15170.1 (Streptomyces vinaceus) (SEQ ID No. 5):

[0079] 1 mhrhtpslrr psahlpsala vraavpaall alfaavpasa apaagsgadp aphldaveqt

[0080] 61 lrqvspgleg qvwertagnv ldastpggad wllqtpgcwg ddkctarpgt eqllskmtqn

[0081] 121 isqatrtvdi stlapfpnga fqdaivsglk tsaargnklk vrvlvgaapv yhlnvlpsky

[0082] 181 rdelvaklga darnvdlnva smttsktafs wnhskllvvd gqsvitggin dwkddyleta

[0083] 241 hpvadvdlal rgpaaasagr yldelwswtc qnksniasvw fassngaacm pamakdtapa

[0084] 301 apapapgdvp avavgglgvg ikrndpsssf rpalpsapdt kcvvglhdnt nadrdydtvn

[0085] 361 peesalrtli ssanrhieis qqdvnatcpp lprydirvyd alaarmaagv kvrivvsdpa

[0086] 421 nrgavgsggy sqikslseis dtlrdrlalv tgdqgaakat mcsnlqlatf rssqsptwad

[0087] 481 ghpyaqhhkv vsvddsafyi gsknlypawl qdfgyvvesp aaaaqlnarl lapqwqysra

[0088] 541 tatidheral cqs

[0089] Preferably, the phospholipase is a phospholipase C (PLC) (i.e. a phospholipase that cleaves before the phosphate, releasing diacylglycerol and a phosphate-containing head group). Preferably, the PLC is a bacterial PLC selected from any of the following groups:

[0090] Group 1 - Zinc Metallophospholipases

[0091] Group 2 - Sphingomyelinase (eg, sphingomyelinase C)

[0092] Group 3 - Phosphatidylinositol

[0093] Group 4 - Pseudomonas PLC

[0094] PLCs of group 1 are preferred, in particular PLCs from Clostridium perfringens, see for example https: / / www.ncbi.nlm.nih.gov / protein / EDT77687.1 (SEQ ID No. 1):

[0095] 1 mkrkickali caalatslwa gastkvyawd gkidgtgtha mivtqgvsil endmsknepe

[0096] 61 svrknleilk enmhelqlgs typdydknay dlyqdhfwdp dtdnnfskdn swylaysipd

[0097] 121 tgesqirkfs alaryewqrg nykqatfylg eamhyfgdid tpyhpanvta vdsaghvkfe

[0098] 181 tfaeerkeqy kintagcktn edfyadilkn kdfnawskey argfaktgks iyyshasmsh

[0099] 241 swddwdyaak vtlansqkgt agyiyrflhd vsegndpsvg knvkelvayi stsgekdagt

[0100] 301 ddymyfgikt kdgktqewem dnpgndfmtg skdtytfklk denlkiddiq nmwirkrkyt

[0101] 361 afpdaykpen ikviangkvv vdkdinewis gnstynik

[0102] Although the literature reports that purified PLC of C. perfringens is not cytotoxic to leukocytes when used alone, the cytolysin provides efficient lysis of animal host cells in the present technology.

[0103] The cytolysin may be a wild-type cytolysin or an active variant (e.g., produced by recombinant DNA technology). An active variant of a cytolysin is a variant of a cytolysin that retains the ability to lyse target cells, as evidenced by the wild-type protein, for example, in any assay for lytic activity against target cells, showing at least 10%, preferably at least 25%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% of the activity of the wild-type protein.

[0104] "Active variants thereof" include within their scope fragments of wild-type proteins. In a preferred embodiment, the fragment of the wild-type protein is selected to be at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% and most preferably at least 95% of the length of the wild-type protein sequence.

[0105] "Active variants thereof" also include within their scope protein sequences having homology to the wild-type protein sequence, e.g., at least 50% identity, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, and most preferably at least 99% identity, e.g., over the entire wild-type sequence or over a region of contiguous amino acid residues representing 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, and most preferably at least 95% of the length of the wild-type protein sequence. Methods of measuring protein homology are well known in the art and those skilled in the art will appreciate that herein, homology is calculated based on amino acid identity (sometimes referred to as "hard homology").

[0106] Homologous active cytolysin variants typically differ from the wild-type protein sequence by substitutions, insertions or deletions, e.g., 1, 2, 3, 4, 5 to 8 or more substitutions, deletions or insertions. Substitutions are preferably "conservative," that is, amino acids can be substituted with similar amino acids, whereby similar amino acids share one of the following groups: aromatic residues (F / H / W / Y), nonpolar aliphatic residues (G / A / P / I / L / V), polar uncharged aliphatics (C / S / T / M / N / Q), and polar charged aliphatics (D / E / K / R). Preferred subgroups include: G / A / P; I / L / V; C / S / T / M; N / Q; D / E; and K / R.

[0107] Cytolysin or active variant (as described above) can have any number of amino acid residues added to the N-terminus and / or C-terminus, provided that the protein retains lytic activity. Preferably, no more than 300 amino acid residues are added at either or both ends, more preferably no more than 200 amino acid residues, preferably no more than 150 amino acid residues, preferably no more than 100 amino acid residues, preferably no more than 80, 60 or 40 amino acid residues, most preferably no more than 20 or 10 or 5 amino acid residues.

[0108] Preferably, after adding the cytolysin, the sample is mixed.

[0109] Preferably, in order to promote cytolysin activity, specific buffer conditions and / or incubation temperature can be provided for any selected cytolysin. Cytolysin incubation can occur, for example, at 5°C to 50°C, such as at 15°C to 45°C (e.g., 37°C), and lasts for 1 minute to 120 minutes, preferably 1 minute to 60 minutes, more preferably 1 minute to 30 minutes (e.g., 15 minutes or 20 minutes). For partial or all cytolysin incubations, the sample is preferably mixed / shaken, for example, at 1 to 1500rpm, preferably 1 to 1000rpm (e.g., at 500rpm or 1000rpm).

[0110] Preferably, the cytolysin is used in a concentration of at least 0.1 mg / ml in the sample, such as 0.1 mg / ml to 100 mg / ml, preferably 0.1 mg / ml to 100 mg / ml, preferably 1 mg / ml to 100 mg / ml (eg 40 mg / ml).

[0111] DNase

[0112] If a DNase is used in the present methodology, the DNase may be an endonuclease or an exonuclease (or a combination thereof may be provided), preferably an endonuclease.

[0113] Preferred DNases (particularly where the biological sample is a blood sample) include HL-SAN DNase (a heat-labile salt-activated nuclease supplied by Arcticzymes) and MolDNase (endonuclease activity in the presence of chaotropes and / or surfactants supplied by Molzym), and active variants are also contemplated, essentially as discussed above with respect to cytolysins.

[0114] Preferably, the sample is mixed after adding the DNase.

[0115] Preferably, in order to promote DNase activity, specific buffer conditions and / or incubation temperature can be provided for the DNase of any selection. DNase incubation can be, for example, at 5 ℃ to 50 ℃, such as at 15 ℃ to 45 ℃ (for example 37 ℃) and last 1 minute to 120 minutes, preferably 1 minute to 60 minutes, more preferably 1 minute to 30 minutes (for example, 15 minutes). In a particularly preferred embodiment, DNase buffer is added to the sample containing cytolysin, and hatched (for example as mentioned above) before precipitation. Then the precipitation is resuspended in the DNase buffer and DNase itself is added (before further hatching).

[0116] Biological samples

[0117] Preferably, the biological sample is a blood sample.Preferably, when the sample is blood, the cytolysin targets / lyses (eg human) leukocytes.

[0118] Preferably, in particular where the sample is blood and / or the cytolysin is PLC from C. perfringens, the sample comprises a chelating agent (eg EDTA).

[0119] Reagent test kit

[0120] Also provided are kits (according to, for example, any of the above aspects) comprising a cytolysin (preferably a buffer with the cytolysin) and means for physically eliminating free nucleic acids in a biological sample or otherwise rendering such nucleic acids unrecognizable. Free nucleic acids include nucleic acids that are not contained within cells or viral particles (e.g., have been released / liberated from animal cells within the sample due to lysis of these cells).

[0121] According to, for example, for any of the above aspects, the means can be, for example, a means for nucleic acid capture (using, for example, magnetic beads), a means for rendering nucleic acids unrecognizable (such as PMA or EMA), or preferably a means for nucleases (such as DNases) (preferably with a suitable buffer and / or a composition for inactivating nucleases).

[0122] conventional

[0123] Please note that wherever the term "comprising" is used herein, we also contemplate the alternatives where the term "consisting of" or "consisting essentially of" is used. Additionally, please note that the term "protein" as used herein is used interchangeably with the term "polypeptide".

[0124] Example

[0125] In the context of medical microbiology, metagenomic sequencing needs to achieve sufficient genome coverage to identify pathogenic species present, and preferably detect all resistance markers, whether mutated or acquired. To achieve this goal, we estimate that at least 10x genome coverage is required. We directly sequenced (HiSeq) blood spiked with pathogen cells (Escherichia coli), delivering only human reads, highlighting the need for pathogen DNA enrichment (data not shown). Therefore, host DNA elimination is required to reliably and cost-effectively apply metagenomics to infectious disease diagnostics.

[0126] Here, we describe the development of a simple, rapid, and efficient method for depletion of human DNA to enable downstream metagenomic sequencing (and other molecular applications, e.g., PCR) for the detection and identification of pathogens and associated antibiotic resistance markers.

[0127] For efficient and cost-effective metagenomic diagnostics of infections, human DNA depletion or pathogen DNA enrichment is essential. We employed a human DNA depletion approach that focuses on differential lysis of human cells, which has removed human DNA, leaving intact non-human pathogens for further analysis. We used blood as a model sample type because the ratio of human DNA:pathogen DNA is very high (up to 10 9 : 1), blood represents one of the most complex clinical samples to which metagenomic infection diagnostics have been successfully applied.

[0128] We applied cytolysins for differential lysis of human cells and endonucleases (DNases) for digestion of released DNA. We tested many DNases to determine the most effective in blood. We then combined the most effective DNases with various cytolysins to determine if and how effectively these toxins lyse DNA-containing white blood cells in the blood.

[0129] A positive control (PC) was added to each experiment, which was DNA extracted from 200 μl of blood. For the cytolysin experiments, blood was spiked with appropriate pathogen entities (e.g., pathogens causing the most common sepsis (E. coli and S. aureus), Candida albicans, Aspergillus niger (A. niger), HBV, or HIV) to ensure that the pathogens were not lysed during the process. For all qPCR reactions, a no template control (NTC; molecular grade nuclease-free dH2O) was included. A MolDNase control sample (from the MolYsis (RTM) kit, Molzym, Germany) was also appropriately included because it has been shown to work in blood.

[0130] Subsequently, DNA was extracted as follows (unless otherwise stated during the experimental procedure):

[0131] 1. Add bacterial lysis buffer (maximum volume 380 μl) and proteinase K (20 μl) to the treated samples and mix by vortexing. No bacterial lysis buffer was added to the blood samples that were not spiked with bacteria (if necessary, supplement with PBS to a volume of 400 μl).

[0132] 2. Incubate all samples at 65°C for 5 minutes.

[0133] 3. Then purify on MagNAPure (RTM).

[0134] For all experiments, qPCR was used to quantify human and non-human nucleic acids. Specific hydrolysis probe assays were designed or obtained from the literature to detect human, E. coli, and S. aureus DNA (all single-copy gene targets; RNA polymerase II, cyaA, and eap, respectively). In addition, fungal and viral targets included Candida albicans 5.8S rRNA, Aspergillus niger ITS1-2, HBV X gene, and HIV 5′ nuclease assays in LTR genes. All qPCR results are expressed as amplification curves and / or quantitative cycle (Cq) values ​​(this represents the cycle at which the fluorescent signal increases above background, which is directly related to the amount of starting template concentration). The relative concentration of DNA in the sample was calculated using ΔCq (every 3.3 cycles represents a 10-fold difference in concentration; the higher the Cq value, the less starting template DNA is present in the sample).

[0135] Example 1 - Efficacy of endonucleases in digesting DNA in blood

[0136] The initial focus was to identify an endonuclease that would digest DNA released by leukocytes so that the efficacy of the cytolysin could be easily assessed in blood. In this experiment, blood samples were frozen and thawed three times to release human DNA, and endonucleases were added; DS-DNase, HL-SAN DNase (heat-labile, salt-active nuclease), or micrococcal nuclease from Staphylococcus aureus, incubated at 37°C and DNA extracted. Controls included a positive control as described above (PC-DNA from 200 μl of spiked blood without DNase treatment), a MolDNase control (known to work in blood), and a negative control (NTC-nuclease-free water). Human-specific qPCR was performed on all DNA extracts and Cq values ​​were compared to determine if the endonuclease treatment was working.

[0137] Detailed process:

[0138] 1. To lyse blood cells, freeze the sample at -70°C and thaw three times at room temperature (RT)

[0139] 2. Divide the frozen and thawed blood into 5 × 200 μl samples

[0140] 3. For sample 1, add 5 μl of HL-SAN DNase (28.4 U / μl).

[0141] 4. For sample 2, add 5 μl of DS-DNase (2U / μl)

[0142] 5. For sample 3, add 20 μl of micrococcal nuclease (resuspended in 100 μl of nuclease-free water; 0.62 U / μl)

[0143] 6. Mix all samples by vortexing

[0144] 7. Incubate samples 1-3 and PC at 37°C for 30 minutes

[0145] 8. For the MolDNase control sample, add 50 μl of DB1 buffer, then add 5 μl of MolDNase, and then incubate at room temperature for 15 min.

[0146] 9. Terminate all reactions by adding 5 μl DNase inactivation buffer (Ambion (RTM), life technologies (RTM)).

[0147] 10. Extract DNA and quantify by human qPCR (as described above)

[0148] result:

[0149] As shown in Table 1 and Figure 1 As shown, DS-DNase (sample 2) and micrococcal nuclease (sample 3) showed no endonuclease activity on human DNA in blood samples, with a ΔCq of <1 compared to PC. However, HL-SAN DNase (sample 1) with a ΔCq of 2.2 showed endonuclease activity, causing a decrease of about 4 times in human DNA compared to PC. As mentioned above, MolDNase is known to work in blood samples and shows the maximum endonuclease activity with the highest Cq value.

[0150] Table 1: Human qPCR results after various endonuclease treatments

[0151] Sample ID Endonuclease Human qPCR (Cq) 1 HL-SAN DNase 24.77 2 DS-DNase 22.80 3 Micrococcal nuclease 22.35 MolDNase control MolDNase 29.47 PC - 22.58

[0152] in conclusion:

[0153] From all the endonucleases tested in this experiment, HL-SAN DNase was the only one that showed potential to work effectively in blood. HL-SAN DNase was selected as the endonuclease for further testing. Since it is known that HL-SAN DNase is most active under high salt concentrations, our goal was to test high salt buffers to improve activity, and Example 2 details buffer optimization.

[0154] Example 2 - Optimization of HL-SAN buffer conditions

[0155] From Example 1, HL-SAN DNase was selected as the most promising endonuclease to work in blood. Since HL-SAN DNase is a salt-active enzyme, we tested the addition of high salt buffer to optimize the activity of HL-SAN DNase on human DNA in blood samples. High salt buffer was prepared and added to frozen-thawed blood samples with HL-SAN DNase in various volumes, incubated at a known working temperature (37°C), DNase inhibitors were added and samples were further incubated. MolDNase controls, PCs and NTCs were included; all samples were subjected to DNA extraction and human qPCR (as described above).

[0156] HL-SAN buffer composition:

[0157] 10mM Tris HCl, 100mM Mg and 1M NaCl pH 8.5

[0158] Detailed process:

[0159] 1. To lyse blood cells, freeze the sample at -70°C and thaw three times at room temperature (RT)

[0160] 2. Spike the frozen-thawed blood with human DNA and divide into 5 × 200 μl samples

[0161] 3. For sample 1, add 20 μl of HL-SAN buffer and 3 μl of HL-SAN DNase

[0162] 4. For sample 2, add 100 μl of HL-SAN buffer and 3 μl of HL-SAN DNase

[0163] 5. For sample 3, add 180 μl of HL-SAN buffer and 3 μl of HL-SAN DNase

[0164] 6. Incubate the above reaction at 37°C for 15 minutes

[0165] 7. For MolDNase control, add 50 μl of DB1 buffer and 5 μl of MolDNase and incubate at room temperature for 15 minutes

[0166] 8. Terminate all reactions by adding 5 μl DNase inactivation buffer (Ambion (RTM), life technologies (RTM)).

[0167] 9. Extract DNA and quantify by human qPCR (as described above)

[0168] result:

[0169] Table 2 and Figure 2It was shown that the addition of HL-SAN buffer increased the activity of HL-SAN DNase, which was related to the increase in volume. The most effective amount of HL-SAN buffer was 180 μl, which caused similar activity to MolDNase (<1 Cq difference between DNase treatments) compared to no endonuclease treatment (PC), and human DNA levels were reduced by about 32 times (ΔCq5). In the absence of buffer, HL-SAN DNase alone caused a human qPCR Cq value of 24.77 (Table 1), which increased to 27.02 Cq after adding 180 μl of HL-SAN buffer (Table 2), showing that the increase in HL-SAN DNase activity can reduce human DNA by about 4 times (ΔCq2).

[0170] Table 2: Human qPCR results after endonuclease treatment with various volumes of buffer

[0171] Sample ID condition Human qPCR (Cq) 1 20 μl HL-SAN buffer + HL-SAN DNase 22.27 2 100 μl HL-SAN buffer + HL-SAN DNase 24.64 3 1 80 μl HL-SAN buffer + HL-SAN DNase 27.02 MolDNase control 50 μl DB1 buffer + MolDNase 27.63 PC - 22.32 NTC - -

[0172] in conclusion:

[0173] The addition of a high salt buffer (HL-SAN buffer) increased the efficiency of HL-SAN DNase digestion of human DNA present in blood samples after cell lysis by freeze-thaw. The use of this combination (HL-SAN buffer and HL-SAN DNase) resulted in approximately the same level of human DNA elimination as the known control (MolDNase). Therefore, to test the robustness of the optimized HL-SAN DNase method, the experiment was repeated (the volume of HL-SAN buffer needed to be adjusted due to the limitation of the input volume of DNA extraction) compared to MolDNase with the corresponding DB1 buffer (Example 3).

[0174] Example 3 - Comparison of HL-SAN DNase and MolDNase activities

[0175] Here, we tested the robustness of the optimized method selected from Example 2 and compared the activity of HL-SAN DNase and MolDNase with the respective buffers. The volume of HL-SAN buffer that provided the same level of activity between HL-SAN DNase and MolDNase was 180 μl, however, due to volume input limitations of MagNAPure (RTM) used for DNA purification, the volume of HL-SAN buffer was reduced to 150 μl. Blood cells were lysed by freeze-thaw, spiked with human DNA and HL-SAN DNase or MolDNase with the respective buffers, incubated, and then the enzymes were heat inactivated. PCs were also included, and DNA was extracted from all samples and human qPCR was performed.

[0176] Detailed process:

[0177] 1. To lyse blood cells, freeze the sample at -70°C and thaw three times at room temperature (RT)

[0178] 2. Spike frozen-thawed blood with human DNA and divide equally into 4 × 250 μl samples

[0179] 3. For the HL-SAN DNase sample, add 150 μl of HL-SAN buffer (Example 2) and 4 μl of HL-SAN DNase, mix by vortexing and incubate at 37°C for 15 minutes.

[0180] 4. For the MolDNase control sample, add 50 μl of Buffer DB1 and 4 μl of MolDNase, mix by vortexing and incubate at room temperature for 15 minutes.

[0181] 5. For MolDNase control samples and PC, add PBS to increase the sample volume to 400 μl (the input volume required for MagNAPure (RTM))

[0182] 6. Terminate DNase activity by heating the enzyme at 65°C for 10 minutes

[0183] 7. Extract DNA and quantify by human qPCR (as described above)

[0184] result:

[0185] Table 3 and Figure 3 The optimized HL-SAN DNase method was shown to be better than the MolDNase control. There was a difference of approximately ΔCq2, which corresponds to approximately a 4-fold reduction in human DNA.

[0186] Table 3: Human qPCR results for HL-SAN DNase and MolDNase treatment with respective buffers

[0187] Sample ID Human qPCR (Cq) HL-SAN DNase 30.54 MolDNase control 28.23 PC 21.80 NTC -

[0188] in conclusion:

[0189] Under optimized buffer conditions, HL-SAN DNase can work in blood to eliminate human DNA just as well as MolDNase (if not more). At this point, we continued to use HL-SAN DNase as our endonuclease of choice and began the process of selecting an appropriate cytolysin. Example 4 details the different cytolysins we initially selected for evaluation of leukocyte lysis capacity / efficacy.

[0190] Example 4 - Elimination of Host DNA Using Streptolysin O and Alpha Hemolysin

[0191] After identifying HL-SAN DNase as an efficient endonuclease for digesting DNA, we investigated the potential of cytolysins to target and lyse specific cell types. Here, we evaluated the activity of two membrane pore-forming cytolysins, namely streptolysin O (Streptococcus pyogenes) and α-hemolysin (Staphylococcus aureus), in lysing leukocytes. Cytolysins (alone and in combination) were added to blood to lyse host cells. The samples were then incubated and the DNA released from the lysed cells was digested with MolDNase, and a DNase inactivator was added after further incubation. PC and NTC samples were included, and DNA was extracted from all samples and quantified by human qPCR (as described above).

[0192] Cytolysin purchase information:

[0193] Streptolysin O

[0194] ·Article No. S5265-25ku

[0195] Batch No. 025M4059V

[0196] 25000-50000U / vial

[0197] 0.71 mg solid

[0198] ·229577 units / mg solid

[0199] 4794117 units / mg protein

[0200] α-hemolysin

[0201] ·Item No. H9395-5MG

[0202] Batch No. 095M4057V

[0203] ·28840 units / mg solid

[0204] 49647 units / mg protein

[0205] Detailed process:

[0206] 1. Resuspend streptolysin O and α-hemolysin (0.71 mg (163,000 units) and 5 mg (144,200 units), respectively) in 350 μl of nuclease-free water.

[0207] 2. For sample 1, add 50 μl of Streptolysin O to 200 μl of blood

[0208] 3. For sample 2, add 50 μl of α-hemolysin to 200 μl of blood

[0209] 4. For sample 3, add 50 μl of streptolysin O and 50 μl of α-hemolysin to 200 μl of blood.

[0210] 5. Mix all samples by vortexing and incubate at 37°C with shaking at 400 rpm for 30 minutes.

[0211] 6. After incubation, add 150 μl HL-SAN buffer and then add 3 μl HL-SAN DNase

[0212] 7. Incubate the sample at 37°C for a further 15 minutes

[0213] 8. Terminate DNase activity by heating the enzyme at 65°C for 10 minutes

[0214] 9. For samples 1-3, add 100 μl of bacterial lysis buffer, and add 180 μl of bacterial lysis buffer to the PC sample.

[0215] 10. Extract DNA from all samples and quantify human DNA using human qPCR (as described above)

[0216] result:

[0217] When used alone, streptolysin O and α-hemolysin showed approximately the same leukocyte lytic efficacy (Table 3), providing approximately 10 3 Combined use of cytolysins (α-hemolysin and streptolysin O) on the same blood samples resulted in increased efficiency of leukocyte lysis and improved elimination of human DNA, with approximately a further 10-fold reduction in human DNA (ΔCq 3.3).

[0218] Table 4: Human qPCR results after cytolysin treatment

[0219]

[0220]

[0221] in conclusion:

[0222] Here, we show that membrane pore-forming cytolysins can target human cells and eliminate host DNA. Interestingly, it is the combination of these two cytolysins that produces the greatest elimination of human DNA. As we have shown that cytolysins can target human somatic cells and demonstrate that it is possible to cause host DNA elimination in this way, we turn our attention to another member of the cytolysin family, the phospholipase C (PLC) from Clostridium perfringens (C. perfringens), which is a cytolysin that destroys phospholipids in the double membrane of eukaryotic cells (Example 5).

[0223] Example 5 - Study on the activity of PLC on host cell lysis

[0224] As previously described, PLC is a cytolysin produced by Clostridium perfringens that functions by targeting and degrading phospholipids in the bilayer membrane of eukaryotic cells. Therefore, we wanted to test PLC for specific host cell lysis and subsequent host DNA digestion using HL-SAN DNase. PLC is a known zinc metallophospholipase that requires the presence of zinc to function; however, it is unclear whether zinc concentrations in human blood are sufficient for PLC to function. PLC activity also requires calcium and magnesium ions. When performing these experiments using blood collected with EDTA preservative, there was concern that EDTA would chelate calcium and metal ions required for PLC activity. Therefore, we tested PLC in blood without preservative, in blood containing EDTA preservative, and in blood with buffer containing metal ions. PLC was added to each blood sample type and incubated with shaking for host cell lysis. HL-SAN DNase (with HL-SAN buffer) was then added and incubated for host DNA digestion, followed by heat inactivation of the HL-SAN DNase. PC and NTC samples were included, DNA was extracted from all samples, and human qPCR was performed (as described above).

[0225] PLC buffer composition:

[0226] 0.1M ZnCl2 and 0.1M MgCl2

[0227] Detailed process:

[0228] 1. PLC (4 mg) was reconstituted in 100 μl of molecular grade water (40 μg / μl).

[0229] 2. Divide the blood into 4 × 250 μl

[0230] 3. For sample 1 (without EDTA preservative) and sample 2 (with EDTA preservative), add 20 μl PLC and mix thoroughly by vortexing, then incubate at 37°C with shaking at 500 rpm for 15 minutes.

[0231] 4. After incubation, add 150 μl HL-SAN buffer and 4 μl HL-SAN DNase to samples 1 and 2, mix thoroughly by vortexing and incubate at room temperature for 15 minutes.

[0232] 5. For sample 3, add 150 μl of HL-SAN buffer and PLC buffer, then add 4 μl of HL-SAN DNase and 20 μl of PLC, then mix by vortexing and incubate at 37 °C without shaking for 15 min.

[0233] 6. Fill the PC with 150μl PBS (total 400μl)

[0234] 7. Inactivate HL-SAN DNase by incubating all samples at 65°C for 10 minutes

[0235] 8. Extract DNA from all samples and quantify human DNA using human qPCR (see Section 3 for details)

[0236] result:

[0237] When PLC was tested on blood without preservatives or PLC buffer, there was virtually no improvement in human DNA elimination (Tables 5 and Figure 5 ), the absence of EDTA or the addition of PLC buffer reduced the efficacy of elimination. Sample 2 showed the highest level of host DNA elimination, with approximately 100-fold reduction in human DNA compared to PC (ΔCq6).

[0238] Table 5: Human qPCR results of PLC activity under different sample conditions

[0239]

[0240]

[0241] in conclusion:

[0242] Although PLC is known to require calcium, magnesium and zinc ions for activity, the addition of buffers containing these ions appears to reduce the efficiency of PLC in lysing host cells. After concerns that the preservative EDTA would chelate the metal ions required for PLC activity, we observed that PLC worked better in blood samples preserved with EDTA and was not as effective in blood without any preservative. All previous experiments were performed in 200-250 μl of blood to test the efficiency of PLC and HL-SAN DNase in eliminating human DNA. Since the number of bacterial cells present per ml of purulent blood is known to be very low (perhaps only 1 colony forming unit per ml), we next wanted to increase the working volume of blood (Example 6)

[0243] Example 6 - Study of the activity of PLC on host DNA elimination and bacterial DNA recovery in increasing volumes of blood

[0244] The pauci-microbial nature of sepsis means that testing larger volumes of blood will increase diagnostic sensitivity. We therefore wanted to test the activity of PLC in larger volumes of blood (1 ml) and determine if PLC had any undesirable activity against bacterial cells. Blood was spiked with the most common pathogens causing sepsis (E. coli and S. aureus). The spiked blood was incubated with PLC to lyse the host cells, then HL-SAN DNase (with HL-SAN buffer) was added for DNA digestion and the endonuclease was heat inactivated. PC samples were included and DNA was extracted from all samples, and qPCR was performed for human, E. coli, and S. aureus DNA (as described above).

[0245] Detailed procedures:

[0246] 1. Reconstitute PLC (4 mg) in 100 μl of molecular grade water (40 μg / μl).

[0247] 2. Divide the blood with E. coli and Staphylococcus aureus culture into 1x1ml and 1x200μl samples

[0248] 3. For 1 ml of spiked blood, add 100 μl of PLC and incubate at 37°C with shaking at 500 rpm for 20 minutes

[0249] 4. For 200 μl of spiked blood, add 20 μl of PLC and incubate at 37°C with shaking at 500 rpm for 20 minutes

[0250] 5. After incubation, add 500 μl or 150 μl of HL-SAN buffer to 1 ml or 200 μl of sample, respectively, then add 10 μl or 3 μl of HL-SAN DNase to 1 ml or 200 μl, respectively, mix briefly by vortexing, and then incubate at 37°C for 15 minutes.

[0251] 6. Centrifuge the sample at 12000xg for 10 minutes

[0252] 7. Carefully pour off the supernatant and resuspend the pellet in 200 μl PBS

[0253] 8. Inactivate HL-SAN DNase by heating at 68°C for 10 minutes

[0254] 9. Extract DNA from all samples and quantify human, E. coli, and S. aureus using qPCR (as described above)

[0255] result:

[0256] Increasing blood volume resulted in decreased efficiency of human DNA elimination (Table 6 and Fig. 6A). There was approximately 4 times more human DNA remaining in 1 ml of blood compared to 200 μl of blood (ΔCq 2). There was no loss of E. coli between the two volumes, and the 1 ml sample showed an approximately 5-fold increase in E. coli DNA (ΔCq ∼ 2.5) as expected (see Tables 6 and Figure 6B However, there was a loss of S. aureus DNA in both the 200 μl and 1 ml samples, equivalent to a 100-fold reduction (ΔCq ~6 in the 200 μl sample [lower in the 1 ml sample due to a 5-fold increase in the test volume compared to the PC]) (Tables 6 and Figure 6C ).

[0257] Table 6: qPCR results of human, E. coli and S. aureus in increasing volumes of blood spiked with bacteria treated with PLC and HL-SAN DNase

[0258]

[0259]

[0260] in conclusion:

[0261] Increasing the amount of blood results in a decrease in the efficiency of human DNA elimination. The loss of S. aureus DNA was observed, indicating the activity of PLC on the Gram-positive cell wall or a decrease in the efficiency of S. aureus lysis compared to PC (possibly due to the heat inactivation of DNase). There was no loss of E. coli DNA, which confirmed that Gram-negative bacterial cells were not lysed by PLC. We continue to try to improve the efficiency of human DNA elimination in 1 ml of blood by ensuring effective mixing during incubation with PLC (Example 7). The loss of S. aureus was also studied using the hypothesis that heat inactivation of HL-SAN DNase affects the cell wall of S. aureus and reduces the efficiency of cell lysis (Example 8).

[0262] Example 7 - Study of Efficient Mixing During Targeted Lysis of Cells in Increased Volumes of Blood

[0263] First, to investigate the loss of PLC efficiency in host cell lysis in 1 ml of blood, we investigated the effect of effective mixing. After adding PLC to blood spiked with bacteria, the samples were aliquoted into larger volume sample tubes (5 ml) and mixed continuously during incubation to enhance contact of PLC with host cells present in the sample and improve lysis efficiency. HL-SAN DNase (plus HL-SAN buffer) was added to eliminate host DNA and incubated, then heat-inactivated. PC samples were included and DNA was extracted from all samples, and qPCR was performed for human, E. coli, and S. aureus DNA (as described above).

[0264] Detailed process:

[0265] 1. PLC (4 mg) was reconstituted in 100 μl of molecular grade water (40 μg / μl).

[0266] 2. The blood spiked with E. coli and Staphylococcus aureus culture was divided into 1x1ml and 1x200μl samples.

[0267] 3. For 1 ml of spiked blood, add 100 μl of PLC and incubate slowly with a Hulamixer (RTM) at 37°C for 20 minutes.

[0268] 4. For 200 μl of spiked blood, add 20 μl of PLC and incubate at 37°C with shaking at 500 rpm for 20 minutes

[0269] 5. After incubation, add 500 μl or 150 μl of HL-SAN buffer to 1 ml or 200 μl of sample, respectively, then add 10 μl or 3 μl of HL-SAN DNase to 1 ml or 200 μl, respectively, mix briefly by vortexing, and then incubate at 37°C for 15 minutes.

[0270] 6. Centrifuge the sample at 12000xg for 10 minutes

[0271] 7. Carefully pour out the supernatant and resuspend the pellet in 200 μl PBS

[0272] 8. Inactivate HL-SAN DNase by heating at 68°C for 10 minutes

[0273] 9. Extract DNA from all samples and quantify human, E. coli, and S. aureus using qPCR (as described above)

[0274] result:

[0275] Introduction into the larger sample tube and slow mixing after addition of PLC resulted in almost complete elimination of human DNA (approximately 1 cell of human DNA remaining; 1 ml sample eliminated ~2.6 × 10 5 times (Table 7 and Figure 7 ) and complete removal of human DNA from 200 μl sample (at least 10 6 times elimination).

[0276] Table 7: Human qPCR results for PLC activity after addition of the active mix during host cell lysis

[0277] Sample ID Human qPCR (Cq) PLC of 1ml blood 38.04 PLC of 200 μl blood - PC 22.38 NTC -

[0278] in conclusion:

[0279] By ensuring efficient mixing during host cell lysis, the activity of PLC is improved and provides the necessary level of elimination for detection of pathogen sequences in blood by sequencing. However, as described in Example 6, the loss of S. aureus DNA still needs to be investigated (described in detail in Example 8).

[0280] Example 8 - Modified inactivation of HL-SAN DNase to improve DNA recovery from Gram-positive bacteria

[0281] We hypothesized that heat inactivation of HL-SAN DNase was affecting the cell wall of S. aureus, reducing the efficiency of cell lysis and resulting in low levels of DNA recovery. The purpose of this experiment was to try a new method of inactivating HL-SAN DNase to improve the recovery of S. aureus DNA. Rather than heat inactivating HL-SAN, we inactivated the DNase by removing the high salt conditions required for its activity. PLC was added to blood samples spiked with bacteria, incubated and mixed slowly. HL-SAN DNase (+HL-SAN buffer) was added to allow host DNA elimination and incubated. Samples were centrifuged to pellet intact bacterial cells and the supernatant containing the high salt buffer was removed. The PC sample was included and DNA was extracted from all samples, and qPCR was performed for human, E. coli, and S. aureus DNA (as described above).

[0282] Detailed process:

[0283] 1. Reconstitute PLC (4 mg) in 100 μl of molecular grade water (40 μg / μl).

[0284] 2. Divide the blood spiked with E. coli and Staphylococcus aureus culture into 1x1ml (in 5ml tube) and 1x200μl sample

[0285] 3. For 1 ml of spiked blood, add 100 μl of PLC and incubate slowly with a Hulamixer (RTM) at 37°C for 20 minutes.

[0286] 4. For 200 μl of spiked blood, add 20 μl of PLC and incubate at 37°C with shaking at 500 rpm for 20 minutes

[0287] 5. After incubation, add 500 μl or 150 μl of HL-SAN buffer to 1 ml or 200 μl of sample, respectively, then add 10 μl or 3 μl of HL-SAN DNase to 1 ml or 200 μl, respectively, mix briefly by vortexing, and then incubate at 37°C for 15 minutes.

[0288] 6. Centrifuge the sample at 12000xg for 10 minutes

[0289] 7. Carefully pour out the supernatant and resuspend the pellet in 1.5 ml PBS

[0290] 8. Before DNA extraction, pellet the bacterial cells by centrifugation at 12,000 x g for 5 minutes.

[0291] 9. Extract DNA from all samples (including PC) and quantify human, E. coli, and S. aureus using qPCR (as described above)

[0292] result:

[0293] Using buffer exchange rather than heat inactivation on HL-SAN DNase resulted in efficient elimination of human DNA without loss of E. coli or S. aureus DNA (Table 8 and Figure 8). When 1 ml of sample was used, human DNA elimination was effective at ~2.3 × 10 5 times, and (data not shown) when using 200 μl of sample, at least 10 6 times (no human DNA detected).

[0294] Table 8: qPCR results of E. coli and S. aureus after altered HL-SAN DNase inactivation

[0295]

[0296] in conclusion:

[0297] The introduction of buffer exchange to inactivate HL-SAN DNase instead of heat inactivation improved the efficiency of lysis of S. aureus cells (this may also be achieved by using more robust lysis methods such as bead beating or using enzyme cocktails). This method change allowed efficient S. aureus DNA recovery to be successful, without negative impact on E. coli DNA recovery (previously reported in Example 6) or human DNA elimination (previously reported in Example 7). Therefore, an efficient cytolytic human DNA elimination method has been developed that does not result in the loss of microbial components in the sample. To confirm the robustness of the process, we compared it with the commercially available MolYsis (RTM) method and our in-house modified MolYsis (RTM) process (Example 9).

[0298] Example 9 - Cytolysin Human DNA Elimination with MolYsis (RTM) Basic 5 Kit and Modified MolYsis Comparison of (RTM) methods

[0299] To test the robustness of our newly developed human DNA elimination process, we compared it with the commercially available MolYsis (RTM) pathogen DNA isolation protocol and an in-house modified MolYsis (RTM) protocol. Our cytolytic human DNA elimination process was performed using the buffer exchange method of Example 8 instead of heat inactivation of HL-SANDnase. The MolYsis (RTM) pathogen DNA isolation protocol was performed as detailed in the manufacturer's instructions. A modified MolYsis (RTM) protocol (in-house developed) was also tested, which initially removed leukocytes by immunomagnetic separation and then MolYsis (RTM) was performed according to the manufacturer's instructions.

[0300] Method 1 (Cytolysin Human DNA Elimination):

[0301] As described in Example 8.

[0302] Method 2 (MolYsis (RTM)):

[0303] MolYsis (RTM) was used according to the manufacturer's instructions.

[0304] Method 3 (Reformed MolYsis (RTM)):

[0305] 1. Resuspend the anti-CD45 coated magnetic beads by gentle mixing and then aliquot the required volume of beads (250 μl per 1 ml sample).

[0306] 2. Resuspend in 1 ml of separation buffer (25 ml without Ca 2+ Mg 2+ The beads were washed in PBS, 100 μl 0.5 M EDTA and 0.025 g BSA).

[0307] 3. Separate the beads on the magnetic rack and discard the supernatant

[0308] 4. Resuspend the beads in 250 μl of separation buffer

[0309] 5. Deplete leukocytes by adding 250 μl of washed beads to 1 ml of blood and gently mix using a Hulamixer (RTM) at 2-8°C for 30 minutes.

[0310] 6. Separate the beads on a magnetic rack and transfer the supernatant to a new sterile tube.

[0311] 7. Centrifuge at 12,000 × g for 10 minutes to pellet intact bacterial cells and any remaining blood cells, then discard the supernatant.

[0312] 8. Resuspend the pellet in 1 ml PBS

[0313] 9. Further process the samples using the MolYsis (RTM) protocol according to the manufacturer's instructions.

[0314] DNA was extracted from all samples (including PC) and human, E. coli, and S. aureus DNA were quantified using qPCR for all methods (as described above)

[0315] result:

[0316] When comparing our human DNA depletion method to the commercially available MolYsis (RTM), we observed approximately 10 more 4 Our MolYsis(RTM) protocol also showed a 10-fold reduction in human DNA (ΔCq12) compared to MolYsis(RTM). 4 times.

[0317] Table 9: Human, E. coli and S. aureus qPCR results for the compared methods

[0318]

[0319]

[0320] in conclusion:

[0321] Compared with the commercially available MolYsis (RTM) kit, our human DNA depletion method was more efficient in depleting human DNA (showing human DNA depletion of approximately 9.3 × 10 4 Compared to our cytolytic human DNA depletion method, only our modified MolYsis (RTM) protocol showed the same level of efficiency. This suggests that the leading commercially available host depletion kits do not provide sufficient host cell / DNA depletion to achieve effective pathogen DNA detection by sequencing.

[0322] Summary:

[0323] In summary, we developed a rapid pathogen identification method that exploits the properties of a cytolysin (PLC) and an endonuclease (HL-SAN DNase) to specifically target and lyse host cells present in clinical samples (i.e., blood) followed by DNA digestion. This process is a preliminary step to provide adequate pathogen DNA extraction for NGS. As blood represents the most complex clinical sample matrix type with an extremely high ratio of human to bacterial cells, we predicted that clinical sample types would be easily interchangeable without affecting the level of human DNA elimination.

[0324] After several methodological changes, the final process is detailed below.

[0325] Initial Optimization of Human DNA Elimination Method

[0326] PLC solution: 4 mg in 100 μl nuclease-free water

[0327] HL-SAN buffer: 10 mM Tris HCL, 100 mM Mg and 1 M NaCl pH 8.5 in nuclease-free water

[0328] Add 100 μl of PLC solution to 1 ml of blood

[0329] ↓

[0330] Incubate at 37°C with gentle mixing for 20 min

[0331] ↓

[0332] Add 500 μl HL-SAN buffer, 10 μl HL-SAN DNase and mix by vortexing, then incubate at 37 °C for 15 min.

[0333] ↓

[0334] The bacterial cells were pelleted at 12,000 × g for 10 min.

[0335] ↓

[0336] The supernatant was discarded

[0337] ↓

[0338] Resuspend the bacterial cell pellet in 1.5 ml PBS

[0339] ↓

[0340] Pellet bacterial cells at 12,000 x g for 5 min and remove supernatant.

[0341] ↓

[0342] Options for DNA extraction

[0343] [Total time: 50 minutes]

[0344] DNA extraction

[0345] Resuspend the bacterial cell pellet in 350 μl of bacterial lysis buffer and vortex.

[0346] ↓

[0347] Add 30 μl of enzyme mix (lysozyme, mutanolysin and lysostaphin - optional lytic enzyme) and incubate at 37°C and 1000 rpm for 15 minutes.

[0348] ↓

[0349] Add 20 μl of proteinase K.

[0350] ↓

[0351] Mix by vortexing

[0352] ↓

[0353] Incubate at 65 °C for 5 min

[0354] ↓

[0355] MagNAPure (RTM) (Roche) was used to extract DNA

[0356] [Total time: 45 minutes]

[0357] [Therefore, the current solution turnaround time is approximately 90 minutes]

[0358] Example 10 - Validation of fungal enrichment methodology

[0359] 10.1: The above protocol was slightly altered to focus on fungal enrichment, and a final protocol was performed to validate bacterial enrichment. This protocol was tested using ~200 E. coli cells. Blood was spiked with ~200 E. coli cells and processed as detailed in section 10.2.

[0360] 10.2: Revised Protocol (“Enrichment” Procedure):

[0361] 1. Add PLC (0.8 mg / 20 μl) to blood sample (200 μl), vortex and incubate at 37°C in a heating block at 1000 RPM for 15 minutes.

[0362] 2. Add HL-SAN buffer (5 M NaCl and 100 mM MgCl2) and 10 μl HL-SAN DNase in a 1:1 volume ratio (200 μl), vortex and incubate at 37 °C in a heating block at 1000 RPM for 15 minutes.

[0363] 3. Add PBS to a total volume of 2 ml (1.5 ml).

[0364] 4. Pellet the cells by centrifugation at 12,000 x g for 10 minutes and discard the supernatant.

[0365] 5. Resuspend the cell pellet in 1.5 ml PBS.

[0366] 6. Pellet the cells again by centrifugation at 12,000 × g for 10 minutes and discard the supernatant.

[0367] 7. For any test sample; add 350 μl bacterial lysis buffer, 20 μl enzyme mixture (6 μl mutanolysin 25 ku / ml, 5 μl lysozyme 10 mg / ml, 4 μl lytic enzyme 10 ku / ml, 3 μl lysostaphin 4 ku / ml, 2 μl chitinase 50u / ml) and 5 μl RNaseA.

[0368] 8. Incubate all samples in a heating block at 1000 RPM at 37°C for 15 minutes.

[0369] 9. For all samples, add 20 μl of proteinase K and incubate at 65 °C in a heating block for 10 minutes.

[0370] 10. Use the MagnaPure (RTM) Compact automated machine to extract total nucleic acid using the DNA_bacteria_V3_2 protocol.

[0371] 11. Determination of host DNA / RNA depletion and fungal DNA enrichment by qPCR or RT-qPCR.

[0372] result:

[0373] After plate counting, 200 μl of blood was identified to have been spiked with ~110 E. coli cells. This resulted in approximately 10 5 fold elimination of human DNA with no loss of E. coli DNA (Table 10.1a / b).

[0374] Table 10.1a qPCR results of human DNA spiked into blood with -110 E. coli cells with and without fungal / bacterial enrichment.

[0375]

[0376] Table 10.1b qPCR results of E. coli DNA spiked into blood with ~110 E. coli cells with and without fungal / bacterial enrichment.

[0377]

[0378] ~1000 C. albicans cells were added to whole blood and both samples were processed as detailed in Section 10.2. After the enrichment protocol, the elimination of human DNA was ~10 4 to 10 5 albicans DNA was between 2 and 1.5 times that of control (Table 10.2a / b).

[0379] Table 10.2a Duplicate results of human DNA qPCR with ≤1000 Candida albicans cells spiked into blood with and without bacterial / fungal enrichment.

[0380]

[0381] Table 10.2b Results of duplicate qPCR of DNA of C. albicans spiked into blood with ≤1000 C. albicans cells with and without bacterial / fungal enrichment

[0382]

[0383] Whole blood was then spiked with ~200 C. albicans cells and processed as detailed in Section 10.2. After plate counts of C. albicans on Sabouraud agar, 200 μl of blood was identified as spiked with ~60 C. albicans cells. Following the enrichment protocol, this resulted in ~10 5 albicans DNA was eliminated by 1:100% (Table 10.3a / b).

[0384] Table 10.3a Results of qPCR of human DNA from ~60 C. albicans cells with and without bacterial / fungal enrichment.

[0385]

[0386] Table 10.3b Results of C. albicans DNA qPCR of -60 C. albicans cells with and without bacterial / fungal enrichment.

[0387]

[0388] Using a known value of ~10 8 cfu / ml of Aspergillus niger biospheres, and ~10 4 and ~10 3 Serial dilutions were performed. Both samples were processed as described in Section 10.2. After the enrichment protocol, this resulted in ~10 5 fold elimination with no loss of A. niger DNA (Table 10.4ab / 10.5ab).

[0389] Table 10.4a ~200 A. niger cells with and without bacterial / fungal enrichment (10 3 qPCR results of human DNA (diluted).

[0390]

[0391]

[0392] Table 10.4b ~200 A. niger cells with and without bacterial / fungal enrichment (10 3 The results of qPCR of Aspergillus niger DNA (diluted).

[0393]

[0394] Table 10.5a ~2000 A. niger cells with and without bacterial / fungal enrichment (10 4 qPCR results of human DNA (diluted).

[0395]

[0396] Table 10.5b ~2000 A. niger cells with and without bacterial / fungal enrichment (10 4 The results of qPCR of Aspergillus niger DNA (diluted).

[0397]

[0398] *Cq values ​​indicate <10 cells (total input <100 cells)

[0399] Conclusion: Using the protocol detailed in Section 10.2, human DNA was eliminated by ~10 5 times, with no loss of bacterial or fungal DNA.

[0400] Example 11 - Validation of Virus and Phage Enrichment Methodology

[0401] 11.1: Protocols for Virus Enrichment in Plasma

[0402] 1. Spike whole blood with virus particles (up to 200 μl per sample).

[0403] 2. Centrifuge the sample at 20,000 x g for 5 minutes.

[0404] 3. Keep the supernatant and use in the protocol (works effectively in plasma) after aliquoting into equal volumes (maximum 200 μl).

[0405] 4. Add 20 μl of PLC (0.8 mg) to each test sample and incubate at 37°C in a heating block with shaking at 1000 RPM for 15 minutes.

[0406] 5. Add 200 μl HL-SAN buffer (5 M NaCl and 100 mM MgCl2) and 10 μl HL-SAN and incubate at 37°C in a heating block with shaking at 1000 RPM for 15 minutes.

[0407] 6. Add 20 μl of proteinase K to all samples and incubate at 65°C for 10 minutes.

[0408] 7. Use the MagnaPure (RTM) Compact automated machine to extract total nucleic acid using the DNA_bacteria_V3_2 protocol.

[0409] 8. Determine host DNA / RNA depletion and viral DNA / RNA enrichment by qPCR or RT-qPCR.

[0410] 11.2: Protocol for Virus Enrichment in Blood

[0411] 1. Spike whole blood with virus particles (up to 200 μl per sample).

[0412] 2. Add 20 μl of PLC (0.8 mg) to each test sample and incubate at 37°C in a heating block with shaking at 1000 RPM for 15 minutes.

[0413] 3. Add 200 μl HL-SAN buffer (5 M NaCl and 100 mM MgCl2) and 10 μl HL-SAN and incubate at 37°C in a heating block with shaking at 1000 RPM for 15 minutes.

[0414] 4. Centrifuge the test sample at 20,000 x g for 5 minutes and retain the supernatant.

[0415] 5. Add 20 μl of proteinase K to all samples and incubate at 65°C for 10 minutes.

[0416] 6. Use the MagnaPure (RTM) Compact automated machine to extract total nucleic acid using the DNA_Bacteria_V3_2 protocol.

[0417] 7. Determine host DNA / RNA depletion and viral DNA / RNA enrichment by qPCR or RT-qPCR.

[0418] Once the protocol described in sections 11.1 and 11.2 was established, samples were run in triplicate to obtain reproducibility of the protocol (a second blood protocol was also tested at this stage which was identical to section 11.2 except for an additional centrifugation step after step 4).

[0419] result:

[0420] In total, each 200 μl blood sample was spiked with 10,000 IU HIV and 350 IU HBV. For this experiment, all three enrichment protocols were tested in triplicate (as described above). The elimination in human DNA after the virus enrichment protocol in blood was consistently ~10 4 fold, and human DNA was undetectable when enriched in plasma (Table 11.1a / b).

[0421] Although it should be noted that the number of HBV cells in the PCR reaction was ~35 and the Cq value was therefore close to the detection limit of the qPCR assay used, there was no loss of HBV viral DNA targets in blood and plasma (Table 11.2a / b). Regarding RNA viral targets, there was no loss of HIV in blood and plasma (Table 11.3a / b).

[0422] Table 11.1a Human DNA qPCR results in triplicate from spiked blood with and without virus enrichment.

[0423]

[0424] Table 11.1b Human DNA qPCR results in triplicate from spiked plasma with and without virus enrichment.

[0425]

[0426] Table 11.2a HBV DNA qPCR results in triplicate of spiked blood with and without virus enrichment.

[0427]

[0428]

[0429] Table 11.2b HBV DNA qPCR results in triplicate of spiked plasma with and without virus enrichment.

[0430]

[0431] Table 11.3a HIV RNA qPCR results in triplicate from spiked blood with and without virus enrichment.

[0432]

[0433]

[0434] Table 11.3b HIV RNA qPCR results in triplicate of spiked plasma with and without virus enrichment.

[0435]

[0436] Next, phage assays were performed; in summary, each 200 μl blood sample was spiked with 10 4 , 10 5 , 10 6 or 10 7 After the virus enrichment protocol in plasma (Section 11.1), the depletion in human DNA was consistently ~10 3fold with no loss of phage target (Table 11.4a / b).

[0437] Table 11.4a Human DNA qPCR results of spiked blood with and without virus enrichment.

[0438]

[0439]

[0440] Table 11.4b Phage DNA qPCR results of spiked blood with and without virus enrichment.

[0441]

[0442] in conclusion:

[0443] Here, we describe complete protocols for host DNA depletion and enrichment of viruses (DNA and RNA) and bacteriophages (DNA). Two methods have been developed (one that works in plasma; Section 11.1, and one that works in blood; Section 11.2), and both provide human DNA depletion (~10 4 There was no loss of viral and bacteriophage DNA targets or viral HIV RNA targets.

[0444] Example 12 - Alteration of Cytolysin (Blood Sample)

[0445] For all tests using other cytolysins, 200 μl of blood was used following the protocol outlined in section 10.2. The only change was the addition of different volumes / concentrations in place of PLC, i.e. no optimization was performed.

[0446] Phospholipase D (PLD) from Streptomyces

[0447] PLD was purchased from Sigma-Aldrich (RTM) (P0065-25KU), the stock solution was 50KU / ml; different volumes of PLD (2, 5 and 8 μl) were used. 2 fold (Table 12.1a), with no loss of bacterial or fungal targets (Tables 12.1b, c, d).

[0448] Table 12.1a qPCR results of human DNA in blood spiked with and without bacterial / fungal enrichment using PLD.

[0449]

[0450] Table 12.1b qPCR results of E. coli DNA in blood spiked with PLD with and without bacterial / fungal enrichment.

[0451]

[0452] Table 12.1c qPCR results of S. aureus DNA in blood spiked with and without bacterial / fungal enrichment using PLD.

[0453]

[0454] Table 12.1d qPCR results of C. albicans DNA in blood spiked with PLD with and without bacterial / fungal enrichment.

[0455]

[0456] Sphingomyelinase from Staphylococcus aureus

[0457] Sphingomyelinase in solution was purchased from Sigma-Aldrich (RTM) (S8633-25UN) and different volumes (2, 5 and 8 μl) were used. Human DNA elimination <10 2 fold (Table 12.2a), with no loss of bacterial or fungal targets (Tables 12.2b, c, d).

[0458] Table 12.2a qPCR results of human DNA in blood spiked with sphingomyelinase with and without bacterial / fungal enrichment.

[0459]

[0460] Table 12.2b qPCR results of E. coli DNA in blood spiked with sphingomyelinase with and without bacterial / fungal enrichment.

[0461]

[0462] Table 12.2c qPCR results of S. aureus DNA in blood spiked with sphingomyelinase with and without bacterial / fungal enrichment.

[0463]

[0464]

[0465] Table 12.2d qPCR results of Candida albicans DNA in blood spiked with and without bacterial / fungal enrichment using sphingomyelinase.

[0466]

[0467] Alpha-hemolysin from Staphylococcus aureus

[0468] α-hemolysin was purchased from Sigma-Aldrich (RTM) (H9395-5MG) and added to 20 μl of water at 0.01, 0.08 or 0.8 mg. Human DNA elimination <10 2 times (Table 12.3a), with no loss of bacterial or fungal targets (Tables 12.3b, c, d).

[0469] Table 12.3a Human DNA qPCR results of blood spiked with alpha hemolysin with and without bacterial / fungal enrichment.

[0470]

[0471] Table 12.3b E. coli DNA qPCR results of blood spiked with alpha hemolysin with and without bacterial / fungal enrichment.

[0472]

[0473] Table 12.3c S. aureus DNA qPCR results of blood spiked with alpha hemolysin with and without bacterial / fungal enrichment.

[0474]

[0475] Table 12. 3d Candida albicans DNA qPCR results from blood spiked with alpha hemolysin with and without bacterial / fungal enrichment.

[0476]

[0477] Streptolysin O from Streptococcus pyogenes

[0478] Streptolysin O was purchased from Sigma-Aldrich (RTM) (S5265-25KU) and added at 0.08 or 0.8 mg in 20 μl of water. Human DNA was eliminated 10-fold (Table 12.4a) with no loss of bacterial or fungal targets (Table 12.4b, c, d).

[0479] Table 12.4a Human DNA qPCR results using blood spiked with streptolysin O with and without bacterial / fungal enrichment.

[0480]

[0481] Table 12.4b E. coli DNA qPCR results using blood spiked with streptolysin O with and without bacterial / fungal enrichment.

[0482]

[0483] Table 12.4c S. aureus DNA qPCR results using blood spiked with streptolysin O with and without bacterial / fungal enrichment.

[0484]

[0485] Table 12.4d Candida albicans DNA qPCR results using blood spiked with streptolysin O with and without bacterial / fungal enrichment.

[0486]

[0487] in conclusion:

[0488] All cytolysins tested showed efficient elimination of human DNA with no loss of bacterial or fungal DNA.

[0489] Example 13 - Validation of the Methodology for Other Clinical Sample Types

[0490] Using the established protocol detailed in Section 10.2, the initial 200 μl of blood was replaced with 200 μl of sputum, sonicated tissue, or urine to verify that the elimination method works effectively in other clinical sample types.

[0491] Clinical sputum specimens

[0492] In clinical sputum samples, human DNA was eliminated by up to 10 4 times (Table 13.1a) with no bacterial loss (Table 13.1b / c).

[0493] Table 13.1a Human DNA qPCR results for clinical sputum with and without fungal / bacterial enrichment.

[0494]

[0495] Table 13.1b qPCR results of 16S rRNA gene fragments (V3-V4) of clinical sputum with and without fungal / bacterial enrichment.

[0496]

[0497] Table 13.1c S. aureus DNA qPCR results of clinical sputum with and without fungal / bacterial enrichment.

[0498]

[0499] Prosthetic tissue samples

[0500] Staphylococcus epidermidis cells (15TB0821) spiked into biopsies of prosthetic tissue samples, eliminating human DNA <105 times (Table 13.2a), and there was no loss of bacterial targets (Table 13.2b).

[0501] Table 13.2a Human DNA qPCR results for pseudo-spiked tissue samples with and without fungal / bacterial enrichment.

[0502]

[0503] Table 13.2b qPCR results for S. epidermidis DNA from prosthetic spiked tissue samples with and without fungal / bacterial enrichment.

[0504]

[0505] Clinical urine samples

[0506] Elimination of human DNA in clinical sputum samples <10 4 times (Table 13.3a) with no bacterial loss (Table 13.3b / c).

[0507] Table 13.3a Human DNA qPCR results of clinical urine with and without fungal / bacterial enrichment.

[0508]

[0509] Table 13.3b qPCR results of 16S rRNA gene fragments (V3-V4) of clinical urine with and without fungal / bacterial enrichment

[0510]

[0511] Table 13.3c E. coli DNA qPCR results of clinical urine with and without fungal / bacterial enrichment.

[0512]

[0513] Conclusions: All clinical sample types tested showed elimination of host DNA without loss of bacterial DNA.

[0514] Example 14 - Host RNA Elimination (HL-SAN RNase Activity)

[0515] Using the virus blood protocol (Section 11.2 and Table 14.1a), there are >10 2 Using the viral plasma protocol detailed in Section 11.1, host RNA elimination was shown to be >10 2 times (Tables 14.1b and 14.2a), without loss of HIV targets (Table 14.2b).

[0516] Table 14.1a Results of duplicate RT-qPCR of human RNA from unspiked blood with and without virus enrichment (host RNA depletion).

[0517]

[0518] Table 14.1b Results of duplicate human RNA RT-qPCR of unspiked plasma with and without virus enrichment (host RNA depletion).

[0519]

[0520] Table 14.2a Results of duplicate RT-qPCR of human RNA from spiked plasma with and without virus enrichment (host RNA depletion).

[0521]

[0522] Table 14.2b Results of duplicate RT-qPCR of human RNA from spiked plasma with and without virus enrichment (host RNA elimination).

[0523]

[0524] in conclusion:

[0525] Due to the variability of the starting host RNA, it was determined that HL-SAN RNase activity provided maximal host RNA depletion without loss of viral RNA targets and therefore did not require changes to the enrichment protocol (see Section 11.1 for details). Using this method, human RNA was generally undetectable in depleted plasma.

[0526] Example 15 - Removal of human DNA in the absence of nucleases

[0527] Propylmonoarsenide (PMA) for removal of human DNA

[0528] A modified method from that described in section 10.2 is required in order to activate PMA by light. After PLC treatment, the samples were centrifuged at 12,000 × g for 5 min and resuspended in 1.5 ml PBS. PMA was added to a final concentration of 50 μM and incubated in the dark with occasional shaking for 5 min. The samples were then exposed to blue light in a photolysis apparatus for 15 min and the protocol in section 10.2 was followed starting from step 6. Human DNA elimination <10 2 times (Table 15.1), with no loss of bacterial target DNA (Table 15.2).

[0529] Table 15.1 Human DNA qPCR results for spiked blood samples using PMA depleted human DNA with and without fungal / bacterial enrichment.

[0530] Sample ID Human qPCR Assay (Cq) ΔCq relative to PC Unenriched PC blood 22.90 Enriched Blood PMA#1 27.62 4.72 Enriched Blood PMA#2 28.47 5.57

[0531] Table 15.2 E. coli DNA qPCR results from blood samples spiked with PMA-depleted human DNA with and without fungal / bacterial enrichment.

[0532] Sample ID E. coli qPCR assay (Cq) ΔCq relative to PC Unenriched PC blood 20.65 Enriched Blood PMA#1 21.13 0.48 Enriched Blood PMA#2 21.30 0.65

[0533] in conclusion:

[0534] Removal of human DNA using PMA after PLC treatment shows elimination of human DNA and no loss of bacterial target DNA Implementation Example 16 - Improved solution for 1 ml blood sample

[0535] 1. Add PLC (4 mg / 100 μl) to the blood sample (1 ml in a 5 ml bijou tube), vortex and incubate at 37°C in a water bath for 3 minutes, then incubate at 38°C for 20 minutes while mixing at 15 rpm in a hulamixer (RTM).

[0536] 2. Transfer the sample to a 2 ml tube and add 500 μl HL-SAN buffer (5 M NaCl and 100 mM MgCl2) and incubate at 37°C in a heating block at 1000 RPM for 15 minutes.

[0537] 3. Centrifuge at 8000×g for 5 minutes to pellet the cells.

[0538] 4. Resuspend the cell pellet in 200 μl PBS

[0539] 5. Add HL-SAN buffer and 10 μl HL-SAN DNase in a 1:1 volume ratio (200 μl), vortex and incubate at 37°C in a heating block at 1000 RPM for 15 minutes.

[0540] 6. Add PBS to a total volume of 2 ml (1.5 ml).

[0541] 7. Pellet the cells by centrifugation at 12,000 x g for 10 minutes and discard the supernatant.

[0542] 8. Resuspend the cell pellet in 1.5 ml PBS.

[0543] 9. Pellet the cells again by centrifugation at 12,000 × g for 10 minutes and discard the supernatant.

[0544] 10. For any test sample; add 350 μl bacterial lysis buffer, 20 μl enzyme mixture (6 μl mutanolysin 25 ku / ml, 5 μl lysozyme 10 mg / ml, 4 μl lytic enzyme 10 ku / ml, 3 μl lysostaphin 4 ku / ml, 2 μl chitinase 50u / ml) and 5 μl RNaseA.

[0545] 11. Incubate all samples in a heating block at 1000 RPM at 37°C for 15 minutes.

[0546] 12. For all samples, add 20 μl of proteinase K and incubate at 65 °C in a heating block for 10 minutes.

[0547] 13. Use the MagnaPure (RTM) Compact automated machine to extract total nucleic acid using the DNA_bacteria_V3_2 protocol.

[0548] The 200 μl protocol in Section 10.2 was modified to increase the starting volume to 1 ml. This resulted in >10 elimination of human DNA. 6 fold (Table 16a), with no loss of bacterial or fungal target DNA (Tables 16b, c, d).

[0549] Table 16.1a Human DNA qPCR results of 1 ml spiked blood with and without fungal / bacterial enrichment.

[0550]

[0551] Table 16.1b E. coli DNA qPCR results of 1 ml spiked blood with and without fungal / bacterial enrichment.

[0552]

[0553] Table 16.1c S. aureus DNA qPCR results of 1 ml spiked blood with and without fungal / bacterial enrichment.

[0554]

[0555] Table 16. 1d Candida albicans DNA qPCR results of 1 ml spiked blood with and without fungal / bacterial enrichment.

[0556]

[0557] in conclusion:

[0558] A slightly modified method was developed to enrich for fungi when 1 ml of blood was used, and this resulted in ~10 6fold elimination of human DNA without loss of bacterial or fungal target DNA. Larger sample volumes (>1 ml) can also be used.

[0559] The method appears to be applicable to any sample type with host cells having a phospholipid membrane, for example, clinical samples (infectious disease diagnostics) or animal samples (food safety and veterinary / diagnostics).

[0560] Example 17 - NGS after elimination

[0561] Other methodologies

[0562] Following the elimination protocol detailed in Section 10.2, 4 μl of DNA was processed for whole genome amplification (WGA) using the REPLI-g single cell kit (Qiagen 150343). Following the manufacturer's instructions, the amplification time was reduced to 1 hour and 30 minutes. WGA samples (17 μl) were debranched using T7 endonuclease I (NEB M0302S) according to the manufacturer's instructions. The rapid low-input MinlON library preparation used by the PCR barcode kit (ONT SQK-RLB001) was used according to the manufacturer's instructions, with the following changes:

[0563] 2.5 μl FRM with 7.5 μl template DNA (~140 ng)

[0564] 40μμl nuclease-free water, 50μμl LongAmp Taq 2x, 2μl RLB

[0565] ·PCR: [95℃3min]x1, [95℃15s, 56℃15s, 65℃4min]x20, [65℃4min]x20,

[0566] [65℃6min]x1

[0567] A SpotON R9.4MinlON flow cell was prepared and loaded according to the manufacturer's instructions.

[0568] Bioinformatics data analysis: Reads were aligned to the C. albicans reference genome (SC5314NC_003977.2) using minimap2. Genome coverage and aligned reads were identified using samtools and qualimap. The percentage reads are given as reads matching the reference genome out of the total number of reads.

[0569] result

[0570] ~300 cfu / ml C. albicans = 4.5 pg DNA in a ~15 Mb genome

[0571] Average human DNA concentration in 1 ml of blood = 33 μg DNA

[0572] Therefore, before enrichment, the ratio of human: Candida DNA was ∼10 7 :1

[0573] Based on the sequencing data given below, the C. albicans reads are 1% of the total (1.3x genome coverage), so all other reads are assumed to be human = 100:1 (Human:Candida)

[0574] Elimination of pre-human: Candida DNA ratio = 10 7 :1

[0575] After elimination, the ratio of human: Candida DNA = 100:1

[0576] This is equivalent to 10 5 times elimination.

[0577] Table 17 Candida albicans genome alignment from a single-plex MinlON run (input -300 cfu / ml).

[0578]

[0579] The genome coverage of Candida albicans after single-plex MinlON sequencing is shown in Fig.10 shown.

Claims

1. A method for eliminating host nucleic acids in a blood sample, said sample having been previously obtained from a human host, said method comprising the steps of: (a) adding bacterial phospholipase D to the sample; and (b) Add HL-SAN DNase to the sample.

2. The method according to claim 1 further comprises the step of extracting the remaining nucleic acid from the sample.

3. The method according to claim 2, further comprising the step of subjecting the extracted nucleic acid to a purification process.

4. The method according to claim 2, further comprising the step of amplifying the extracted nucleic acid.

5. The method according to claim 2 further comprises the step of performing a nucleic acid amplification test on the extracted nucleic acid.

6. The method according to claim 2 further comprises the step of performing a sequencing process on the extracted nucleic acid.

7. The method according to claim 1, wherein: The bacterial phospholipase D is a phospholipase D from Streptomyces.

8. The method according to claim 7, wherein: The bacterial phospholipase C is a phospholipase D from Streptomyces vinaceus.

9. The method of claim 1, which produces at least a 10-fold elimination of host DNA initially contained within the sample.

10. The method of claim 1, which produces at least 10% of the host DNA initially contained in the sample. 2 times elimination.

11. The method of claim 1, which produces at least 10% of the host DNA initially contained in the sample. 3 times elimination.

12. The method of claim 1, which produces at least 10% of the host DNA initially contained in the sample. 4 times elimination.

13. The method of claim 1, which produces at least 10% of the host DNA initially contained in the sample. 5 times elimination.

Images