Method for enriching pathogen DNA and implementation thereof

Abstract

An in-vitro method for enriching pathogen DNA from a sample is described herein. The method comprises the steps of: (a) obtaining a blood sample comprising a mixture of a host nucleic acid and a pathogen DNA; (b) processing the blood sample to obtain a processed blood sample, wherein the processed blood sample is obtained using an anti-CD45 and at least one anti-CD15 antibody coated magnetic beads, sonication, or a combination thereof; and (c) extracting the host RNA / DNA from the processed blood sample and extracting and enriching the pathogen DNA from the processed blood sample to obtain an eluate comprising an enriched pathogen DNA. Also described herein is an in-vitro method for detecting sepsis-causing pathogens in a sample at a low concentration of 1 to 10 CFU / mL.

Description

METHOD FOR ENRICHING PATHOGEN DNA AND IMPLEMENTATION THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Indian Application No. 202311088151, filed on 22 December 2023. The entire disclosure of the application referenced above is incorporated herein by reference.FIELD

[0002] The present disclosure relates to the field of healthcare technologies. In particular, the present disclosure relates to an in-vitro method for enriching pathogen DNA in a sample. The present disclosure further relates to an in-vitro method for detecting sepsis-causing pathogens in a sample at a low concentration of 1 to 10 CFU / mL.BACKGROUND

[0003] Rapid diagnostic tests that can detect a broad range of sepsis-causing pathogens at low levels, such as 1-10 CFU(colony forming units) / mL, are needed for the diagnosis / management of sepsis. To achieve such low detection levels of 1-10 CFU / mL, it is important to have a workflow that handles large starting volumes of whole blood, enriches the pathogen fraction, and lyses the pathogens efficiently in the diagnostic procedure. The workflow should ensure minimum carryover of human genomic DNA, minimum loss of pathogens during enrichment and lysis steps, and complete analysis and detection of the extracted pathogen DNA / RNA using an analytic method, such as quantitative polymerase chain reaction (qPCR) and / or next generation sequencing (NGS).

[0004] The overwhelming quantities of human DNA present in the mixture interferes at multiple steps in the sepsis molecular diagnostic workflow. Human genomic DNA (gDNA) can compete with the target microbial DNA by binding to the solid phase during the sample preparation steps, making the process of extraction inefficient for the microbial targets. Excessive human gDNA can interfere with target microbial DNA by binding to microbe specific primer binding sites in targeted amplification. Human gDNA can also slow the process of sequencing by competing with the microbial DNA and this increases the overall workflow turnaround time. The percentage of target sequences assigned from a nanopore sequencing run is also reduced with increasing quantities ofbackground human DNA. Excessive human gDNA also has to be removed bioinformatically before microbial DNA can be classified and identified. This process makes the workflow computationally intensive. Thus, a human DNA removal step at various stages during a sepsis molecular diagnostic workflow would be very beneficial.

[0005] Accordingly, there is a dire need in the art to provide an efficient and rapid method that would reduce the volume, deplete, or remove human gDNA and protein background, lyse the enriched pathogens, and ensure the enrichment of the pathogen DNA allowing for a sensitive detection of all sepsis-causing pathogens.

[0006] Along with identifying the infection-causing pathogens and characterizing their antimicrobial resistance (AMR) patterns, it is important to also detect the host response biomarkers in the patient’ s blood sample to check if there is an active infection and if that infection is caused by bacteria or fungi. Such an integrated test is beneficial as it will help the physician to do the pathogen identification (ID) and AMR test only if an infection occurrence is detected. Host response biomarkers are generally the human mRNA, miRNA, or protein signatures. These need to be enriched along with the pathogen ID and AMR signatures from the same patient blood sample. The pre-analytical approach for such an integrated workflow is challenging and unique as it has to enable enrichment and extraction of host biomarkers as well as infection-causing pathogen biomarkers.SUMMARY

[0007] This section provides a general summary of the disclosure and is not a comprehensive disclosure of its full scope or all of its features.

[0008] In one embodiment, an in-vitro method for enriching a pathogen DNA from a sample is described herein. The method comprises the steps of: (a) obtaining a blood sample comprising a mixture of a host nucleic acid and a pathogen DNA, wherein the host nucleic acid is selected from the group consisting of a host RNA and a host DNA; (b) processing the blood sample to obtain a processed blood sample, wherein the processed blood sample is obtained by a method selected from the group consisting of a technique using an anti-CD45 and at least one anti-CD15 antibody coated magnetic beads, sonication, or a combination thereof; and (c) extracting the host RNA / DNA from the processed blood sample and extracting and enriching the pathogen DNA from the processed blood sample to obtain an eluate comprising an enriched pathogen DNA, wherein extracting and enriching is done by a technique comprising silica magnetic beadextraction method or oligo-capture approach. The combination of depletion of a host DNA of a subject, at a cellular level before the DNA extraction and at the nucleic acid level at the PCR stage, removes sufficient human genomic DNA to provide high analytical sensitivity. The method described herein is also very rapid and is suitable to be used for a sepsis diagnostic assay that detects both pathogens and host response biomarkers.

[0009] In a particular embodiment, an in-vitro method for enriching a pathogen DNA from a sample is described herein which uses an anti-CD45 and at least one anti-CD15 antibody coated magnetic beads.

[0010] In yet another particular embodiment, an in-vitro method for enriching a pathogen DNA from a sample is described herein which uses an oligo-capture approach.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations and are not intended to limit the scope of the present disclosure.

[0012] Figure 1A-1B depicts pre-analytical steps for a sepsis integrated sequencing assay for host response and pathogen identification using approach 1.

[0013] Figure 2A-2B depicts pre-analytical steps of a sepsis integrated sequencing assay for host response and pathogen identification using approach 2.

[0014] Figure 3 depicts a detailed workflow of an oligo-capture technique to enrich and extract pathogen host DNA / host RNA of interest.

[0015] Figure 4 depicts linearity of calibrated bacterial load estimates for detections shown in Table 1 by using the method of approach 1.

[0016] Figure 5 different depletion volumes of WBCs from fresh whole human blood with anti- CD45 and anti-CD15 coated Dynabeads™ (ThermoFisher), using approach 1.

[0017] Figure 6 depicts depletion of human DNA using biotinylated oligo-capture approach (investigated at multiple probe concentrations), using approach 2.

[0018] Figure 7 depicts Staphylococcus aureus detected within 15 minutes with full panel, in a full assay workflow using approach 2 based pathogen DNA enrichment and extraction.DETAILED DESCRIPTION

[0019] While the invention is susceptible to various modifications and alternative forms, specific embodiment thereof will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the scope of the invention as defined by the appended claims.

[0020] Although one or more features and / or elements may be described herein in the context of only a single embodiment, or alternatively in the context of more than one embodiment, or further alternatively in the context of all embodiments, the features and / or elements may instead be provided separately or in any appropriate combination or not at all. Conversely, any features and / or elements described in the context of separate embodiments may alternatively be realized as existing together in the context of a single embodiment.

[0021] The terminology used herein is for the purpose of describing particular various embodiments only and is not intended to be limiting of various embodiments. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0022] As used herein, the term “host nucleic acid sequence” refers to nucleic acid sequences that are targeted for depletion. Particularly, these sequences are host / non-pathogenic nucleic acid sequences which are targeted for depletion. The nucleic acid sequences are DNA or RNA.

[0023] The term “enriched pathogen DNA” refers to a nucleic acid sequence of interest that is enriched by targeting the sequences that are of interest. As used herein, the “enriched pathogen DNA” is interchangeable with “non-host / microbial / pathogen DNA.”

[0024] As used herein, the term “host” refers to mammals, and the term “non-host” are not mammalian (e.g., bacterial, fungal, viral, protozoan, etc.). In one embodiment, the hosts are human and the non-hosts are bacteria and / or fungi.

[0025] The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in liquid form, containing one or more analytes of interest. In particular, the sample refers to any sample containing nucleic acids that is obtained from a host / mammal. “Nucleic acid sample” refers to genomic DNA obtained from a mammal (e.g. human). In aparticular embodiment, the sample is a blood sample. In a further particular embodiment, the blood sample is a whole blood sample.

[0026] As used herein, the term “processed blood sample” refers to a mixture obtained by subjecting the blood sample to a method comprising a technique using an anti-CD45 and at least one anti-CD15 antibody coated magnetic beads, a sonication technique, or combination thereof.

[0027] The term “depleted blood” refers to a processed blood sample in which about 80-99% of human genomic DNA has been depleted or removed, wherein about 80-99% of human genomic DNA has been depleted or removed by contacting the whole blood sample with anti-CD45 and at least one anti-CD15 antibody coated magnetic beads to allow removal of whole blood fraction.

[0028] The term “lysed blood” refers to a processed blood sample obtained after subjecting the whole blood sample with sonication combined with or without chemical or enzymatic lysis.

[0029] As used herein, the term “in-vitro” refers to a task or method or experiment being performed or taking place in a test tube, culture dish, or elsewhere outside a living organism.

[0030] The term “pathogen” refers to one or more prokaryotic microorganisms causing sepsis.

[0031] The term “silica magnetic bead extraction method” refers to a method of DNA separation that is based on DNA molecules binding to silica surfaces and after binding DNA, an external magnetic field attracts the beads to the outer edge of the container, such as a tube, immobilizing them. While the beads are immobilized, the bead-bound DNA is retained during the washing steps.

[0032] As used herein, the term “oligo-capture approach” refers to a method for enriching pathogen DNA and host mRNA from the background for pathogen ID / AMR and host response assays respectively. Human genomic DNA is the background for enriching pathogen DNA / microbial DNA is the background when enriching the human mRNA. In this approach, biotinylated bait oligos are used to bind to the target pathogen sequences / host mRNA sequences which can be further pulled down using streptavidin coated magnetic beads. The beads can then be further washed, and the bound pathogen / host mRNA sequences can be eluted and used in multiplex PCR reactions that amplify universally conserved pathogen sequences. The capture probes are 18 to 25 bp long oligos that are complimentary to different regions conserved across different bacteria and fungi. These are biotinylated so that they can bind to the streptavidin coated magnetic beads.

[0033] The term “treated mixture” refers to a lysed blood sample contacting the lysed blood sample treated with a preheated lysis buffer and proteinase K. The “preheated lysis buffer” is a lysis buffer with a chaotrope and a detergent that is heated to 60 to 70 degrees.

[0034] As discussed in the background section, the methods for detecting sepsis-causing pathogens are unable to detect pathogens at low levels, such as 1 to 10 CFU / mL, and face the following limitations, such as there is maximum carryover of human genomic DNA (human gDNA) and maximum loss of pathogens during the enrichment and lysis steps. The overwhelming quantities of human DNA present in the mixture interferes at multiple steps in the sepsis molecular diagnostic workflow. Human gDNA can compete with the target microbial DNA to binding to the solid phase during the sample preparation steps, making the process of extraction inefficient for the microbial targets. Excessive human gDNA can interfere with target microbial DNA for binding to microbe specific primer binding sites in targeted amplification. Human gDNA can also slow the process of sequencing by competing with the microbial DNA and this increases the overall workflow turnaround time. The percentage of target sequences assigned from a nanopore sequencing run is also reduced with increasing quantities of background human DNA. Excessive human gDNA also has to be removed bioinformatically before microbial DNA can be classified and identified. All these reasons make it preferable to include a human DNA removal step at various stages during a sepsis molecular diagnostic workflow.

[0035] Thus, in one embodiment, the described invention addresses the problems existing in the art by providing an in-vitro method that allows the removal of human genomic DNA by enrichment at the cellular level before DNA extraction, first via specific binding and removal of the white blood cell (WBC) content of the blood using CD45 and 15 antibody coated beads, and second at the DNA level during the PCR stage (Approach 1). Alternatively, in another embodiment the pathogen DNA can also be enriched from the background human genomic DNA using an oligo-capture approach (Approach 2). In this approach 2, biotinylated bait oligos can be used to bind to the target pathogen sequences which can be further pulled down using streptavidin coated magnetic beads. The beads can then be further washed, and the bound pathogen sequences can be eluted and used in multiplex PCR reactions that amplify universally conserved pathogen sequences. The two stage PCR design used in the pre-analytical workflow also ensures human genomic DNA removal, allowing multiplexed amplification of pathogen DNA and antimicrobial resistance (AMR) targets. Thus, in one embodiment, the described method (e.g., a sepsis pre-analytical workflow) removes the background host / human DNA from the mixture of host / human and non-host / microbial / pathogen DNA.

[0036] Additionally, the method described herein may also extract and enrich the host RNA (i.e., mRNA from a subject) from the depleted whole blood sample using various approaches as described below.

[0037] Multiplexed amplification of host response mRNA targets is performed upon extraction of mRNA from the depleted white blood cell fraction using a commercially available kit. Alternatively, when the pathogen DNA enrichment and extraction is done by approach 2, the same blood sample can be processed substantially in parallel, and RNA can be extracted by a similar oligo capture method. In this embodiment, the lysed whole blood sample was incubated with biotinylated capture probes targeting the host mRNA sequences. These uncapped capture probes are complementary to the selected host mRNA signatures at a specific exon-exon junction towards the 3’ end of the transcripts. The probe design and the denaturation-renaturation conditions are chosen such that the capture probes bind the mRNA molecules of interest and not the host genomic DNA and the microbial DNA which is the background in this case. The buffer background is chosen such that it is inhibitory to RNAse. The capture probes bound to the host mRNA signatures will then be purified by magnetic pull-down using streptavidin coated magnetic beads. The beads are washed, and the bead bound host RNA is subjected to reverse transcription to synthesize the cDNA.

[0038] Thus, the method described herein differs from the known methods in that the method described herein combines the unique steps in the sample preparation and pre-sequencing steps for extracting both human mRNA and pathogen DNA efficiently for an integrated host response and pathogen identification sepsis diagnostic assay.

[0039] Overall, the method described herein is very rapid, (e.g., about 15 minutes of depletion, about 15 minutes of sample DNA extraction and about 60-90 minutes of two- stage amplification followed by rapid library prep), and is suitable to be used for a sepsis diagnostic assay that detects both pathogens and host response biomarkers. Approach 2 is also rapid, in that the target capture method takes about 30-40 minutes and about 60-90 minutes of two-stage amplification followed by an about 10-minute library prep.

[0040] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample, said method comprising the steps of: (a) obtaining a blood sample comprising amixture of a host nucleic acid and a pathogen DNA, wherein the host nucleic acid is selected from the group consisting of a host RNA and a host DNA; (b) processing the blood sample to obtain a processed blood sample, wherein the processed blood sample is obtained by a method selected from the group consisting of a technique using an anti-CD45 and at least one anti-CD15 antibody coated magnetic beads, sonication, and a combination thereof; and (c) extracting the host mRNA from the processed blood sample and extracting and enriching a pathogen DNA from the processed blood sample to obtain an eluate comprising an enriched pathogen DNA, wherein extracting and enriching is done by a technique selected from silica magnetic bead extraction method and oligo-capture approach.

[0041] In one embodiment, there is provided an in-vitro method as described herein, wherein the processed blood sample is a depleted blood, or a lysed blood.

[0042] In one embodiment, there is provided an in-vitro method as described herein, wherein processing the blood sample to obtain the depleted blood is done by the method comprising the steps of: (a) dividing the blood sample into at least two blood subsamples; and (b) contacting the blood subsamples with the anti-CD45 and the at least one anti-CD15 antibody coated magnetic beads to allow the removal of whole blood fraction such that the depleted blood is obtained.

[0043] In one embodiment, there is provided an in-vitro method as described herein, wherein extracting and enriching the pathogen DNA are either done separately or substantially simultaneously (at about the same time).

[0044] In one embodiment, there is provided an in-vitro method as described herein, wherein the processed blood sample is depleted blood and wherein extracting and enriching the pathogen DNA from the depleted blood comprises the steps of: (a) subjecting the depleted blood to sonication combined with or without chemical or enzymatic lysis to obtain a lysed blood sample comprising the pathogen DNA; (b) contacting the lysed blood sample with a preheated lysis buffer and proteinase K to obtain a treated mixture; (c) adding silica coated beads to the treated mixture such that the pathogen DNA binds to the silica coated magnetic beads to form a complex in the treated mixture; and (d) separating the silica coated magnetic beads from the treated mixture to obtain an eluate comprising an enriched pathogen DNA, wherein the preheated lysis buffer is the lysis buffer with a chaotrope and a detergent that is heated to 60 to 70 degrees.

[0045] In one embodiment, there is provided an in-vitro method as described herein, wherein the processed blood is depleted blood, and wherein extracting the host RNA from the depleted bloodis done by subjecting the depleted blood to sonication with or without chemical / enzymatic lysis to obtain a host mRNA eluate.

[0046] In one embodiment, there is provided an in-vitro method as described herein, wherein the processed blood is lysed blood, and wherein processing the lysed blood is done by subjecting the blood to sonication with or without chemical / enzymatic lysis to obtain the lysed blood.

[0047] In one embodiment, there is provided an in-vitro method as described herein, wherein the processed blood is lysed blood and wherein extracting and enriching the pathogen DNA from the lysed blood simultaneously, comprises the steps of: (a) incubating the lysed blood and at least one oligonucleotide bound to a reporter molecule, to obtain a first mixture comprising the complex, wherein the at least one oligonucleotide has a nucleic acid sequence complementary to the sequence of the pathogen DNA present in the lysed blood, and wherein the at least one oligonucleotide binds to the pathogen DNA to form the complex; and (b) separating the oligonucleotide bound to the reporter molecule from the first mixture to obtain an eluate comprising an enriched pathogen DNA.

[0048] In one embodiment, there is provided an in-vitro method as described herein, wherein the processed blood sample is lysed blood and wherein extracting and enriching the pathogen DNA from the lysed blood substantially simultaneously, comprises the steps of: (a) obtaining at least one oligonucleotide bound to a reporter molecule and streptavidin coated beads; and (b) incubating the lysed blood with the at least one oligonucleotide bound to a reporter molecule and streptavidin coated beads to obtain an eluate comprising an enriched pathogen DNA.

[0049] In one embodiment, there is provided an in-vitro method as described herein, wherein the processed blood sample is lysed blood, and wherein extracting the host mRNA from the lysed blood comprises the steps of: (a) incubating the lysed blood and at least one oligonucleotide bound to a reporter molecule, to obtain a second mixture comprising a second complex, wherein the at least one oligonucleotide binds to a host mRNA transcript at the exon-exon junction, present in the lysed blood to form a complex; and (b) separating the oligonucleotide bound to a reporter molecule from the second mixture to obtain an eluate comprising an enriched host mRNA.

[0050] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample, said method comprising the steps of: (a) obtaining a blood sample comprising a mixture of -host nucleic acid and pathogen DNA, wherein the host nucleic acid is selected from the group consisting of a host RNA and host DNA; (b) processing the blood sample to obtain adepleted blood, wherein processing comprises the steps of: (i) dividing the blood sample into at least two blood subsamples; and (ii) contacting the blood subsamples with the anti-CD45 and the at least one anti-CD15 antibody coated magnetic beads to allow the removal of whole blood fraction such that the depleted blood is obtained; and (c) extracting the host mRNA from the processed blood and extracting and enriching a pathogen DNA from the depleted blood to obtain an eluate comprising an enriched pathogen DNA, wherein extracting the host mRNA from the depleted blood is done by subjecting the depleted blood to sonication to obtain a -host mRNA eluate, wherein extracting and enriching the pathogen DNA from the depleted blood comprises the steps of: (i) subjecting the depleted blood to sonication to obtain a lysed blood comprising a pathogen DNA; (ii) contacting the lysed blood with a preheated lysis buffer and proteinase K to obtain a treated mixture; (iii) adding silica coated beads to the treated mixture such that the pathogen DNA binds to the silica coated magnetic beads to form a complex; and (iv) separating the complex from the mixture to obtain an eluate comprising an enriched pathogen DNA.

[0051] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample, said method comprising the steps of: (a) obtaining a blood sample comprising a mixture of host nucleic acid and pathogen DNA, wherein the host nucleic acid is selected from the group consisting of a host RNA and host DNA; (b) processing the blood sample to obtain a lysed blood, wherein processing the lysed blood is done by subjecting the blood sample to sonication with or without the fungal / enzymatic lysis to obtain a lysed blood; and (c) extracting the host RNA from the lysed blood and extracting and enriching a pathogen DNA from the lysed blood to obtain an eluate comprising an enriched pathogen DNA, wherein extracting the host RNA from the lysed blood comprises the steps of: (i) incubating the lysed blood and at least one oligonucleotide bound to a reporter molecule, to obtain a second mixture comprising a second complex, wherein the at least one oligonucleotide binds to a host mRNA transcript at the exonexonjunction, present in the lysed blood to form a complex; and (ii) separating the oligonucleotide bound to a reporter molecule from the second mixture to obtain an eluate comprising an enriched host mRNA, wherein extracting and enriching the pathogen DNA from the lysed blood simultaneously, comprises the steps of: (i) wherein extracting and enriching the pathogen DNA from the lysed blood simultaneously, comprises the steps of: (ii) incubating the lysed blood and at least one oligonucleotide bound to a reporter molecule, to obtain a first mixture comprising the complex, wherein the at least one oligonucleotide has a nucleic acid sequence complementary to the sequence of the pathogen DNA present in the lysed blood, and wherein the at least one oligonucleotide binds to the pathogen DNA to form the complex; and (iii) separating theoligonucleotide bound to the reporter molecule from the first mixture to obtain an eluate comprising an enriched pathogen DNA.

[0052] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample, said method comprising the steps of: (a) obtaining a blood sample comprising a mixture of host nucleic acid and pathogen DNA, wherein the host nucleic acid is selected from the group consisting of a host RNA and host DNA; (b) processing the blood sample to obtain a lysed blood, wherein processing the lysed blood is done by subjecting the blood sample to sonication to obtain a lysed blood; and (c) extracting the host mRNA from the lysed blood and extracting and enriching a pathogen DNA from the lysed blood to obtain an eluate comprising an enriched pathogen DNA, wherein extracting the host RNA from the lysed blood comprises the steps of: (i) incubating the lysed blood and at least one oligonucleotide bound to a reporter molecule, to obtain a second mixture comprising a second complex, wherein the at least one oligonucleotide binds to a host mRNA transcript at the exon-exon junction, present in the lysed blood to form a complex; and (ii) separating the oligonucleotide bound to a reporter molecule from the second mixture to obtain an eluate comprising an enriched host mRNA, wherein extracting and enriching the pathogen DNA from the lysed blood simultaneously, comprises the steps of: (i) wherein extracting and enriching the pathogen DNA from the lysed blood simultaneously, comprises the steps of: (i) obtaining at least one oligonucleotide bound to a reporter molecule and streptavidin coated beads; and (ii) incubating the lysed blood with the at least one oligonucleotide bound to a reporter molecule and streptavidin coated beads to obtain an eluate comprising an enriched pathogen DNA.

[0053] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein contacting the blood sub-samples with the anti-CD45 and fifteen anti-CD15 antibody coated magnetic beads allows the removal of whole blood fraction such that the depleted blood sample is obtained.

[0054] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein contacting the blood sub-samples with the anti-CD45 and the at least one anti-CD15 antibody coated magnetic beads allows the removal of 80-95% of the host DNA.

[0055] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein sonication is done and the sonication is an indirectultrasonication method carried out at an energy ranging between 100 to 3000 W sec and at an amplitude ranging between 20%. This may be accompanied with an enzymatic and / or chemical lysis. Example enzymes include, but are not limited to, lyticase, chitinase, pectinase, lysozyme, lysostaphin, pectinase, and a combination thereof.

[0056] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein adding silica coated beads to the treated mixture comprises the steps of mixing 30 to 50 pl silica coated beads with the treated mixture for a time period in the range of 1 to 10 minutes, at a temperature in the range of 50°C to 70°C, such that the pathogen DNA binds to the silica coated magnetic beads to form the complex.

[0057] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein separating the complex from the mixture to obtain the eluate comprising the enriched pathogen DNA, comprises the steps of: (i) magnetically separating the silica beads from the treated mixture followed by heating the treated mixture at a temperature in the range of 60°C to 90°C to obtain a purified mixture comprising the pathogen DNA; and (ii) adding an elution buffer to the purified mixture, followed by incubating the purified mixture by heating the purified mixture with a combination of orbital shaking and intermittent mixing at a temperature in the range of 30 to 40 °C for a time period in the range of 1 to 20 minutes, to obtain the eluate comprising the enriched pathogen DNA. The elution buffer is an alkaline buffer that helps in reversing the DNA bound to silica.

[0058] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein the elution buffer is tris-EDTA (TE) buffer or plain nuclease-free water.

[0059] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein the reporter molecule is biotin.

[0060] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein incubating the lysed blood, comprises a step of denaturing the first mixture at 95 °C, followed by renaturation of the first mixture at 50°C for a period in the range of 5-20 minutes, such that the at least one oligonucleotide binds to the pathogen DNA to form the complex.

[0061] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein separating the oligonucleotide bound to the reporter molecule from the first mixture is done by adding streptavidin coated beads to the first mixture which pulls out the oligonucleotide bound to the reporter molecule bound to the pathogen DNA, such that the eluate comprising an enriched pathogen DNA is obtained.

[0062] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein the enriched pathogen DNA is subjected to two-stage amplification step done by polymerase chain reaction (PCR).

[0063] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein the enriched pathogen DNA is subjected to an assay selected from next generation sequencing.

[0064] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein the host mRNA eluate is used as a template for a host response multiplex PCR panel.

[0065] In one embodiment, there is provided an in-vitro method for enriching a pathogen DNA from a sample as described herein, wherein the enriched pathogen DNA is used as a template for pathogen identification (ID) multiplex PCR panel and an antimicrobial resistance (AMR) target panel including primers flanking key AMR determinant gene regions.

[0066] In one embodiment, there is provided an in-vitro method for detecting sepsis-causing pathogens in a sample, said method comprises the steps of: (a) performing a method for enriching pathogen nucleic acid from a sample as described herein, to obtain an enriched pathogen DNA; (b) subjecting the enriched pathogen DNA to an assay for amplifying the pathogen DNA; and (c) detecting for the presence or absence of an amplified pathogen DNA, wherein the presence of the amplified pathogen DNA indicates the presence of sepsis-causing pathogens in the sample.

[0067] In one embodiment, there is provided an in-vitro method for detecting sepsis-causing pathogens in a sample as described herein, wherein the method detects the sepsis-causing pathogens at a concentration of at least 1 to 10 CFU / ml.

[0068] In one embodiment, there is provided an in-vitro method for detecting sepsis-causing pathogens in a sample as described herein, wherein the assay is quantitative polymerase chain reaction (qPCR) or next generation sequencing.

[0069] In one embodiment, there is provided an in-vitro method for detecting sepsis-causing pathogens in a sample as described herein, wherein the pathogen is bacteria, virus, protozoa, and / or fungi.

[0070] The invention is illustrated hereunder in greater detail in relation to non-limiting exemplary embodiments as per the following examples.EXAMPLES

[0071] The following examples are merely illustrative, and do not limit this disclosure in any way.Example 1: Method for enriching pathogenic DNA

[0072] The method involves a series of technical steps, which are described below:

[0073] (a) A clinical blood sample was obtained from a human subject (patient).

[0074] (b) The blood sample was divided into, but not limited to, at least two sub-samples.

[0075] (c) (1) Approach 1:

[0076] (i) The sub-samples were subjected to a pre-analytical step where the human genomic WBCs are depleted, thus removing human DNA. This step is called the background depletion step. The blood remaining sub- sample was then mixed with predefined volumes of anti-CD45 and anti-CD15 antibody coated magnetic beads. The anti-CD45 antibody coated magnetic beads are Invitrogen™ Dynabeads™ CD45 (having catalogue number: 11153D) and anti-CD15 antibody coated magnetic beads are Invitrogen™ Dynabeads™ CD 15- Catalog number: 11137D. It can be contemplated that a person skilled in the art can use other antibodies targeted against other CD antigens. The binding conditions were optimized to enable removal of at least 80-95% of human genomic DNA. The depleted blood fractions were then pooled into a single sample for further downstream processing. Sub-sampling resulted in faster enrichment of pathogens due to parallel processing of the samples. Such a depletion step also ensures minimal microbial losses and easy integration with the downstream steps. The anti-CD45 and anti-CD15 coated magnetic beads tethered to WBCs can be retained for extracting RNA from the WBCs. Multiplexed amplification of host response mRNA targets was performed upon extraction of mRNA from the depleted white blood cell fraction using any commercially available kit, such as Versant® Sample preparation1.0 reagents sold by Siemens Healthineers (Approach 1). The pre-analytical workflow using approach 1 is illustrated in Figure 1. The RNA thus extracted (eluate 1) can be used for a host response mRNA expression level test.

[0077] (ii) The depleted blood was then subjected to sample preparation where the target pathogens were lysed and pathogen DNA were extracted and eluted.

[0078] (iii) The depleted blood was subjected to ultrasound lysis of the sample. For this purpose, an indirect ultrasonication method was used to lyse bacteria and fungi in a volume of 0.5 to 5.0 mL blood at an energy ranging between 100 to 3000 W sec and at an amplitude ranging between 20% to 70% Ultrasound lysis using a handheld sonotrode allowed for rapid and universal lysis of the pathogens in the sample. Sonication may be accompanied with enzymatic and / or chemical lysis to improve the lysis efficiency of hard to lyse pathogens like Gram positive and fungal spores. Such a method is especially beneficial in a pathogen agnostic diagnostic workflow. This method also should ensure minimal target losses. Ultrasound based lysis can be automated and easily integrated with the rest of the downstream workflow steps.

[0079] (iii) The lysed sample was then subjected to a nucleic acid binding and extraction using reagents that work on the principle of the Boom Chemistry nucleic acid extraction method. This method involved mixing the lysed sample with preheated lysis buffer (which is the lysis buffer with a chaotrope and a detergent that is heated to 60 to 70 degrees) and proteinase K. To this, 50 pL of silica coated magnetic beads (Versant® sample preparation reagents of Siemens Healthineers). were added that bind to the released nucleic acids. The binding step was facilitated by shaking with heating and intermittent mixing such as vortexing. The binding was done between 1 to 10 minutes at a temperature between 50°C to 70°C.

[0080] (iv) The silica beads were magnetically separated and washed to discard the cellular debris. The purified nucleic acid bound to the beads was then heated to dry the beads to minimize any carryover of the wash buffer components into eluate. This heating was done at a temperature between 60 °C to 90°C.

[0081] (v) The nucleic acids were then eluted (eluate 2) by adding elution buffer (TE buffer or plain nuclease free water) and incubating by heating the mixture with shaking. The elution step was facilitated by shaking which can be a combination of orbital shaking with other intermittent mixing such as vortexing. The eluate was then added as a template to multiplex PCRs. Eluate 1 was used as a template for a host response multiplex PCR panel. This host response multiplexpanel consisted of “n” number of primers that targeted host mRNA targets whose expression levels indicates on the likelihood of the presence and / or severity of infection. Eluate 2 can be used as a template for pathogen identification (ID) multiplex PCR panel. This ID panel has primers that target conserved regions flanking systematically variable regions of ribosomal DNA and protein genes of bacteria and / or fungi to PCR amplify amplicons that, in combination, resolve pathogens to the species level. The eluate is also interrogated with an AMR panel that consists of primers flanking key AMR determinant gene regions.

[0082] (c) (2) Approach 2: The method is explained in detail in Figure 3.

[0083] (i) Alternatively, a simultaneous enrichment and extraction method based on oligonucleotide-capture of pathogenic DNA was used from lysed whole blood. This method ensures both sample enrichment and nucleic acid extraction / purification from the whole blood sample, while compromising neither the extraction of DNA from intact organisms nor potential free-floating DNA in the blood.

[0084] (ii) The blood sample was lysed by a mechanical method like ultrasound, where the nucleic acids released from the cells in the sample was fragmented. For the purposes of lysis, an indirect ultrasonication method was used to lyse bacteria and fungi in a volume of 0.5 to 5.0 mL blood at an energy ranging between 100 to 3000 W sec and at an amplitude ranging between 20% to 70%. This fragmentation further aids in the capture method described below.

[0085] (iii) The method involved incubating a lysed blood sample with multiple biotinylated oligos (capture probes) that are complementary to short genetic regions that are broadly conserved across bacterial and fungal species. The capture probes are 18 to 25 bp long oligos that are complimentary to different regions conserved across different bacteria and fungi. These are biotinylated so that they can bind to the streptavidin coated magnetic beads. The mixture was denatured at 95 °C followed by renaturation at 60°C for few minutes (ranges between 5 mins-20 mins). This denaturation followed by renaturation step ensure binding of biotinylated oligos specifically to pathogen targets. The streptavidin coated paramagnetic beads were used to pull out the biotinylated oligos tethered to the pathogen targets. After washing, the enriched pathogen targets were eluted using pH to denature trapped DNAs from the capture oligos. Alternatively, the biotinylated capture probes pre-bound to the streptavidin coated magnetic beads were used. The beads were directly incubated with the sample to pull the target nucleic acids.

[0086] (iv) At about the same time, the host mRNA markers were extracted from the same lysed blood sample, by using biotinylated oligos targeting the mRNA transcripts at the exon-exon junction. The buffer background was chosen such that it is inhibitory to RNAses. The capture probes bound to host mRNA signatures was then be purified by magnetic pull-down using streptavidin coated magnetic beads. The beads were washed, and the bead bound target RNA was subjected to reverse transcription to synthesize the cDNA. The cDNA is used in the multiplex PCR that amplifies the 10 mRNA host response markers and 3 controls. Approach 2 is illustrated in Figure 2.

[0087] (v) The eluted DNA from steps (ii) and (iii) is then be used in multiplex PCRs for pathogen ID+ AMR and host response PCR respectively.

[0088] (d) The PCR that was used in such a pre-analytical workflow also aids in enrichment of pathogen sequences over remaining background human DNA. Two PCR programs were employed, wherein the first PCR includes broadly targeted PCR using multiple biotinylated broadrange pathogen primer sets containing 5' universal primer site tags that specifically and simultaneously amplified the pathogen sequences. After a limited number of PCR cycles, amplified pathogen sequences were extracted away from background using streptavidin coated magnetic beads. The amplicon bound beads were then washed to remove any human gDNA background.

[0089] (e) The washed amplicons bound to the streptavidin beads were subjected to a second round of PCR which was done using universal PCR primers, allowing relatively even amplification of all targets present. This universal PCR also helps in adding the adapters that are needed for tethering the libraries and allowing the subsequent sequencing runs.

[0090] (f) The amplified products were then be pooled from host response, AMR and pathogen ID panels, library prepared, and subjected to next generation sequencing (NGS).

[0091] (g) This pre-analytical workflow is then made compatible with any downstream NGS method.

[0092] (h) Calibrants (competitive nucleic acid targets containing the same primer target sites as pathogens but differing internal sequence) at known quantities were added directly into the blood sample and served as templates for the primers used in the first round of multiplex PCR. Calibrants were used to estimate the original pathogen DNA load in the blood sample and verify thatextraction was successful by all primers in the multiplex PCRs amplifying the targets as expected. Internal process controls added directly to the PCR at defined quantities, in combination with calibrants spiked into the blood, were used to estimate sample extraction efficiency by measuring the ratio of PCR-amplified products. The ratio of the two known nucleic acid levels (process control and calibrants), where only one is subject to loss during extraction (the calibrant), reveals the proportion of nucleic acid loss during nucleic acid extraction. The control sample as used herein refers are used as quality control to know the efficiency of the process. Here, the control sample is the sample with known amount of RNA markers present at predefined levels. Process controls are added directly to the PCR at predefined quantities and in combination with calibrants spiked in the blood sample, we can use it to verify that extraction was successful by all primers in the multiplex PCRs amplifying the targets as expected.

[0093] (i) The combination of human gDNA depletion, at a cellular level before the DNA extraction and at the nucleic acid level at the PCR stage removed sufficient human genomic DNA to provide high analytical sensitivity.

[0094] Results:

[0095] Table 1. Approach 1 : Results from the NanoSepsID assay utilizing the herein described workflow employing anti-CD45- and anti-CD15-coated beads to deplete WBCs prior to multiplexed PCR amplification of broad bacterial and fungal molecular targets. In this example, nanopore sequencing (Oxford Nanopore Technologies - ONT) using a MinlON device controlled by an external computer with automated sequencing control and analysis software was used for sequencing data collection and analysis. Titered stocks of live organism were used to spike 3mL of whole human blood at 1000, 500, or 100 cfu / mL prior to extraction and analysis. Samples were analyzed using 15, 30, 60 or 180 minutes of total nanopore sequencing data for each sample. All 1000 and 500 cfu / mL samples were sequenced as groups of three barcoded replicates run in a single sequencing flowcell. For 100 cfu / mL samples, six replicates were sequenced in pairs of two barcoded libraries, and three each were sequenced as dedicated runs in their own flowcells.Table 1: Results from the NanoSepsID assay

[0096] Figure 4: Approach 1: Linearity of calibrated bacterial load estimates for detections shown in Table 1. Calibrated estimates of genome counts / mL in the original spiked blood samples were calculated automatically by NanoSepsID software during analysis, giving a rough estimate of the number of bacterial genomes in the blood sample. For a spiked sample, this also allowed a rough estimate of the average number of genomes / CFU present in the original titered stock.

[0097] Figure 5: Approach 1: Depletion of WBCs from fresh whole human blood with anti- CD45 and anti-CD15 coated Dynabeads™ (ThermoFisher). 220uL (for 3.3 mL blood) or 80uL (for 1.2 mL blood) of each anti-CD45 and anti-CD15 were incubated with human blood for the indicated amounts of time either mixed together prior to removal of beads in a magnetic rack. After depletion step, all samples were extracted with Versant extraction chemistry modified for whole human blood and human DNA levels present in each sample were measured with qPCR targeting the human actB gene at an exon: intron junctions to ensure amplification of genomic DNA. There was no substantial difference between 15-minute (B, C and D) vs 30-minute (E) incubation, nor between sequential (B, C) vs mixed (D, E) bead incubation, although there may be a slight advantage to performing depletion in smaller volumes (C, D, E) compared to larger volumes (B).

[0098] Figure 6: Approach 2: Depletion of human DNA using biotinylated oligo capture approach (investigated at multiple probe concentrations) is shown in this figure. Human DNA depletion factor in the sample elutions using approach 2, ranged from 99.928% to 99.999%.

[0099] Figure 7: Staphylococcus aureus detected within 15 minutes with full panel, in a full assay workflow using approach 2 based pathogen DNA enrichment and extraction.

[0100] Thus, overall, it can be inferred that the method described herein combines unique steps in the sample preparation and pre- sequencing steps for extracting both human RNA and pathogen DNA efficiently for an integrated host response and pathogen identification sepsis diagnostic assay.Advantages:

[0101] The described invention has one or more of the following advantages:(i) The method can be tailor made to many sample-target types with minimal upstream changes.(ii) The method can be extremely efficient making it amenable for sensitive detection of targets, when combined with a sensitive amplification step.(iii) The method can be followed by amplification and also sequencing workflows making it agnostic to the downstream application.(iv) The method can be automatable and can be used in a decentralized set-up with minimal user intervention.(v) The method can be suitable to be integrated on to a point of care molecular workflow with minimum hands-on time and user intervention.(vi) The method can be also very rapid (which involves 15 minutes of depletion step, 15 minutes of sample DNA extraction step and 60-90 minutes of 2 stage amplification followed by rapid library preparation).(vii) The method can be suitable to be used for a sepsis diagnostic assay that detects both pathogens and host response biomarkers.(viii) Specifically, the method involving the oligo-capture approach (approach 2) can enrich and extract both free-floating and intact pathogens from a whole blood sample, which is important for detecting low levels of targets present in sepsis samples.(ix) Specifically, the approach 2 pre-analytical method is amenable for testing with frozen blood samples which is a critical requirement during development of a sepsis workflow.

Claims

WHAT IS CLAIMED IS:

1. An in-vitro method for enriching a pathogen DNA from a sample, said method comprising the steps of:(a) obtaining a blood sample comprising a mixture of a host nucleic acid and a pathogen DNA, wherein the host nucleic acid is selected from the group consisting of a host mRNA and a host DNA;(b) processing the blood sample to obtain a processed blood sample, wherein the processed blood sample is obtained by a method selected from the group consisting of a technique using an anti-CD45 and at least one anti-CD15 antibody coated magnetic beads, sonication with or without chemical or enzymatic lysis, or combination thereof; and(c) extracting the host mRNA from the processed blood sample and extracting and enriching a pathogen DNA from the processed blood sample to obtain an eluate comprising an enriched pathogen DNA, wherein extracting and enriching is done by a technique selected from silica magnetic bead extraction method and oligo-capture approach.

2. The method as claimed in claim 1 , wherein the processed blood sample is a depleted blood, or a lysed blood.

3. The method as claimed in claim 1 or 2, wherein processing the blood sample to obtain the depleted blood is done by the method comprising the steps of:(a) dividing the blood sample into at least two blood subsamples; and(b) contacting the blood sub-samples with the anti-CD45 and the at least one antiCD 15 antibody coated magnetic beads to allow the removal of whole blood fraction such that the depleted blood is obtained.

4. The method as claimed in claim 1, wherein extracting and enriching the pathogen DNA are either done separately or simultaneously.

5. The method as claimed in claim 1, wherein the processed blood sample is the depleted blood and wherein extracting and enriching the pathogen DNA from the depleted blood comprises the steps of:(a) subjecting the depleted blood to sonication to obtain a lysed blood sample comprising a pathogen DNA;(b) contacting the lysed blood sample with a preheated lysis buffer and proteinase K to obtain a treated mixture;(c) adding silica coated beads to the treated mixture such that the pathogen DNA binds to the silica coated magnetic beads to form a complex in the treated mixture; and(d) separating the silica coated magnetic beads from the treated mixture to obtain an eluate comprising an enriched pathogen DNA.

6. The method as claimed in claim 1, wherein the processed blood is the depleted blood, and wherein extracting the host mRNA from the depleted blood is done by subjecting the depleted blood to sonication and chemical or enzymatic lysis to obtain a host mRNA eluate.

7. The method as claimed in claim 1, wherein the processed blood is the lysed blood, and wherein processing the lysed blood is done by subjecting the blood to sonication with or without chemical or enzymatic lysis to obtain the lysed blood.

8. The method as claimed in claim 1, wherein the processed blood is the lysed blood and wherein extracting and enriching the pathogen DNA from the lysed blood simultaneously, comprises the steps of:(a) incubating the lysed blood and at least one oligonucleotide bound to a reporter molecule, to obtain a first mixture comprising the complex, wherein the at least one oligonucleotide has a nucleic acid sequence complementary to the sequence of the pathogen DNA present in the lysed blood, and wherein the at least one oligonucleotide binds to the pathogen DNA to form the complex;(b) separating the oligonucleotide bound to the reporter molecule from the first mixture to obtain an eluate comprising an enriched pathogen DNA.

9. The method as claimed in claim 1, wherein the processed blood sample is the lysed blood and wherein extracting and enriching the pathogen DNA from the lysed blood simultaneously, comprises the steps of:(a) obtaining at least one oligonucleotide bound to a reporter molecule and streptavidin coated beads; and(b) incubating the lysed blood with the at least one oligonucleotide bound to a reporter molecule and streptavidin coated beads to obtain an eluate comprising an enriched pathogen DNA.

10. The method as claimed in claim 1, wherein the processed blood sample is the lysed blood, and wherein extracting the host mRNA from the lysed blood comprises the steps of:(a) incubating the lysed blood and at least one oligonucleotide bound to a reporter molecule, to obtain a second mixture comprising a second complex, wherein the at least one oligonucleotide binds to a human mRNA transcript at the exon-exon junction, present in the lysed blood to form a complex; and(b) separating the oligonucleotide bound to a reporter molecule from the second mixture to obtain an eluate comprising an enriched host mRNA.

11. An in-vitro method for enriching a pathogen DNA from a sample, said method comprising the steps of:(a) obtaining a blood sample comprising a mixture of host nucleic acid and pathogen DNA, wherein the host nucleic acid is selected from the group consisting of a host mRNA and host DNA;(b) processing the blood sample to obtain a depleted blood, wherein processing comprises the steps of: (i) dividing the blood sample into at least two blood subsamples; and (ii) contacting the blood subsamples with the anti-CD45 and the at least one anti-CD15 antibody coated magnetic beads to allow the removal of whole blood fraction such that the depleted blood is obtained; and(c) extracting the host mRNA from the processed blood and extracting and enriching a pathogen DNA from the depleted blood to obtain an eluate comprising an enriched pathogen DNA,wherein extracting the host mRNA from the depleted blood is done by subjecting the depleted blood to sonication with or without chemical or enzymatic lysis to obtain a host mRNA eluate, wherein extracting and enriching the pathogen DNA from the depleted blood comprises the steps of: i. subjecting the depleted blood to sonication with or without chemical or enzymatic lysis to obtain a lysed blood comprising a pathogen DNA; ii. contacting the lysed blood with a preheated lysis buffer and proteinase K to obtain a treated mixture; iii. adding silica coated beads to the treated mixture such that the pathogen DNA binds to the silica coated magnetic beads to form a complex; and iv. separating the complex from the mixture to obtain an eluate comprising an enriched pathogen DNA.

12. An in-vitro method for enriching a pathogen DNA from a sample, said method comprising the steps of:(a) obtaining a blood sample comprising a mixture of host nucleic acid and pathogen DNA, wherein the host nucleic acid is selected from the group consisting of a host mRNA and host DNA;(b) processing the blood sample to obtain a lysed blood, wherein processing the lysed blood is done by subjecting the blood sample to sonication with or without chemical or enzymatic lysis to obtain a lysed blood; and(c) extracting the host mRNA from the lysed blood and extracting and enriching a pathogen DNA from the lysed blood to obtain an eluate comprising an enriched pathogen DNA, wherein extracting the host mRNA from the lysed blood comprises the steps of:(i) incubating the lysed blood and at least one oligonucleotide bound to a reporter molecule, to obtain a second mixture comprising a second complex, wherein the at least one oligonucleotide binds to a host mRNA transcript at the exon-exon junction, present in the lysed blood to form a complex; and(ii) separating the oligonucleotide bound to a reporter molecule from the second mixture to obtain an eluate comprising an enriched host mRNA, wherein extracting and enriching the pathogen DNA from the lysed blood simultaneously, comprises the steps of: i. incubating the lysed blood and at least one oligonucleotide bound to a reporter molecule, to obtain a first mixture comprising the complex, wherein the at least one oligonucleotide has a nucleic acid sequence complementary to the sequence of the pathogen DNA present in the lysed blood, and wherein the at least one oligonucleotide binds to the pathogen DNA to form the complex; and ii. separating the oligonucleotide bound to the reporter molecule from the first mixture to obtain an eluate comprising an enriched pathogen DNA.

13. The method as claimed in claim 12, wherein extracting and enriching the pathogen DNA from the lysed blood simultaneously, comprises the steps of: i. obtaining at least one oligonucleotide bound to a reporter molecule and streptavidin coated beads; and ii. incubating the lysed blood with the at least one oligonucleotide bound to a reporter molecule and streptavidin coated beads to obtain an eluate comprising an enriched pathogen DNA.

14. The method as claimed in claim 3 or 11, wherein contacting the blood subsamples with the anti-CD45 and fifteen anti-CD15 antibody coated magnetic beads to allow the removal of whole blood fraction such that the depleted blood sample is obtained.

15. The method as claimed in any one of the claims 3, 11, or 14, wherein contacting the blood subsamples with the anti-CD45 and the at least one anti-CD15 antibody coated magnetic beads allow the removal of 80-95% of the host DNA.

16. The method as claimed in any one of the claims 1, 6, 11, or 12, wherein sonication is an indirect ultrasonication method carried out at an energy ranging between 100 to 3000 W sec and at an amplitude ranging between 20%.

17. The method as claimed in claims 5 or 11, wherein adding silica coated beads to the treated mixture comprises the steps of mixing 30 to 50 pl silica coated beads with the treated mixture for a time period in the range of 1 to 10 minutes, at a temperature in the range of 50°C to 70°C, such that the pathogen DNA binds to the silica coated magnetic beads to form the complex.

18. The method as claimed in claims 5 or 11, wherein separating the complex from the mixture to obtain the eluate comprising the enriched pathogen DNA, comprises the steps of: (i) magnetically separating the silica beads from the treated mixture followed by heating the treated mixture at a temperature in the range of 60°C to 90°C to obtain a purified mixture comprising the pathogen DNA ; and (ii) adding an elution buffer to the purified mixture, followed by incubating the purified mixture by heating the purified mixture with a combination of orbital shaking and intermittent mixing at a temperature in the range of 30 to 40 °C for a time period in the range of 1 to 20 minutes, to obtain the eluate comprising the enriched pathogen DNA.

19. The method as claimed in any one of the claims 8, 9, 10, 12, or 13, wherein the reporter molecule is selected from the group consisting of biotin.

20. The method as claimed in any one of the claims 8, 12, or 13, wherein incubating the lysed blood, comprises a step of denaturing the first mixture at 95 °C, followed by renaturation of the first mixture at 50°C for a period in the range of 5-20 minutes, such that the at least one oligonucleotide binds to the pathogen DNA to form the complex.

21. The method as claimed in claims 8, or 12, wherein separating the oligonucleotide bound to the reporter molecule from the first mixture is done by adding streptavidin coated beads to the first mixture which pulls out the oligonucleotide bound to the reporter molecule, bound to the pathogen DNA such that the eluate comprising an enriched pathogen DNA is obtained.

22. The method as claimed in anyone of the claims 1, 5, 8, 12, or 13, wherein the enriched pathogen DNA is subjected to two-stage amplification step done by polymerase chain reaction (PCR).

23. The method as claimed in claim 22, wherein the enriched pathogen DNA is subjected to an assay selected from next generation sequencing (NGS).

24. The method as claimed in any one of the claims 6 or 11, wherein the host mRNA eluate is used as a template for a host response multiplex PCR panel.

25. The method as claimed in any one of the claims 1, 5, 8, 12, or 13, wherein the enriched pathogen DNA is used as a template for pathogen identification (ID) multiplex PCR panel and an antimicrobial resistance (AMR) target panel that consists of primers flanking key AMR determinant gene regions.

26. An in-vitro method for detecting sepsis-causing pathogens in a sample, said method comprises the steps of:(a) performing a method for enriching pathogen DNA from a sample as claimed in any one of the claims 1 to 25 to obtain an enriched pathogen DNA;(b) subjecting the enriched pathogen DNA to an assay for amplifying the pathogen DNA; and(c) detecting for the presence or absence of an amplified pathogen DNA, wherein the presence of the amplified pathogen DNA indicates the presence of sepsis-causing pathogens in the sample.

27. The in-vitro method as claimed in claim 26, wherein the method detects the sepsis-causing pathogens at a concentration of at least 1 to 10 cfu / ml.

28. The in-vitro method as claimed in claim 26, wherein the assay is selected from the group consisting of quantitative polymerase chain reaction (qPCR) and next generation sequencing (NGS).

29. The in-vitro method as claimed in claim 26 or 27, wherein the pathogen is selected from the group consisting of bacteria, virus, protozoa, and fungi.

30. The in-vitro method as claimed in any one of the claims 1 , 11 , or 12, wherein the enzymatic lysis is carried out in the presence of at least one enzyme selected from the group consisting of lyticase, chitinase, pectinase, lysozyme, lysostaphin, pectinase, and a combination thereof.

31. The in-vitro method as claimed in any one of the claims 1, 11, or 12, wherein the method is a pre-analytical method that works for an integrated workflow for both host response and pathogen identification (ID test.

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