In-vitro method for suppression of non-target nucleic acids, kit and implementation thereof

Abstract

A method for enriching pathogen nucleic acid from a sample is in-vitro and includes selectively lysing a sample including pathogens, such that a selectively lysed mixture including intact pathogens is obtained. The method includes adding at least one intercalating dye to the selectively lysed mixture, such that a treated mixture is obtained, and subjecting the treated mixture to photolysis, such that an enriched mixture is obtained. The photolysis allows irreversible binding of the at least one intercalating dye to a nucleic acid present in the treated mixture. The method includes performing an ultrasound lysis with the enriched mixture of step, such that an enriched pathogen nucleic acid is obtained

Description

IN-VITRO METHOD FOR SUPPRESSION OF NON-TARGET NUCLEIC ACIDS, KIT AND IMPLEMENTATION THEREOFFIELD

[0001] The present embodiments relate to the field of healthcare technologies. In particular, the present embodiments relate to an in-vitro method for enriching pathogen nucleic acid in a sample. The present embodiments further relate to an in-vitro method for detecting sepsis causing pathogens in a sample at a low concentration of I’10CFU / mL. Further, the present embodiments also relate to a kit for detecting the presence of Sepsis causing pathogens in the sample.BACKGROUND

[0002] Sepsis is a major cause of morbidity’ and mortality in humans and other animals. Despite the major advances in the treatment of serious infection, the incidence and mortality due to sepsis continues to rise. The physiopathology of sepsis is still poorly understood, even after the development of new diagnostic and therapeutic techniques. The rapid diagnostic methods that are available in the art are unable to detect the sepsis causing pathogens at low levels such as I'10CFU / mL. The current methods such as polymerase chain reaction (PCR) that aim to detect the sepsis causing pathogens fail to amplify sparse copies of pathogens in the abundance of human genomic DNA (human gDNA). It is because the overwhelming quantities of the human gDNA interferes with primer binding to the targets during an amplification reaction. This results in poor limits of detection in the assay. This effect is more pronounced when the target quantities are present at extremely low quantities like I'10CFU / mL.

[0003] Various efforts have been made in the art to develop methods for rapid detection of sepsis causing pathogens. For example, the reference is made to Hasan MR, et al., Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing. J Clin Microbiol.. 2016 Apr, 54(4):919-27, doi: 10.1128 / JCM.03050-15, Epub 2016 Jan 13, PMID: 26763966, PMCID: PMC4809942, which discloses a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.

[0004] Another reference is made to Zhou. L., et al., A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples, BMC Infect Dis 12, 164 (2012), which discloses a method to enrich target bacterial DNA by selective removal of human DNA from blood samples, enhancing the sensitivity of PCR tests.

[0005] However, the methods that are conventionally known in the art aims at depleting the human background and are unable to detect the sepsis causing pathogens effectively. Consequently, the pathogen detection sensitivity is less.

[0006] Accordingly, there is a need in the art to provide a rapid method for enriching the pathogen fraction that does not deplete the human gDNA and allows for a sensitive detection of all sepsis causing pathogens.SUMMARY AND DESCRIPTION

[0007] The scope of the present invention is defined solely by the appended claims and is not affected to any degree by the statements within this summary.

[0008] The present embodiments may obviate one or more of the drawbacks or limitations in the related art. For example, an in-vitro method for enriching pathogen nucleic acid from a sample is provided.

[0009] As another example, an in-vitro method for detecting sepsis causing pathogens in a sample is provided.

[0010] As yet another example, a kit for detecting sepsis causing pathogens in a sample is provided.

[0011] The present embodiments disclose an in-vitro method for enriching pathogen nucleic acid from a sample. The method includes: (a) selectively lysing a sample including pathogens to obtain a selectively lysed mixture including intact pathogens; (b) adding at least one intercalating dye to the selectively lysed mixture of step (a) to obtain a treated mixture; (c) subjecting the treated mixture of step (c) to photolysis to obtain an enriched mixture, where the photolysis allows irreversible binding of the intercalating dye to a nucleic acid present in the treated mixture; and (d) performing an ultrasound lysis with the enriched mixture of step (c) to obtain an enriched pathogen nucleic acid. The present embodiments also include an in-vitro method for detecting sepsis causing pathogens in a sample. The present embodiments further include a kit for detecting the presence of sepsis causing pathogen in a sample. The in-vitro method for enriching pathogen nucleic acid from a sample as described herein helps insuppressing the background effect of nucleic acid that is present in the sample and favors the amplification of the target sequence of the pathogen in the sample. The method of the present embodiments is a rapid method that helps in detecting the target pathogen in the sample at a concentration of T10CFU / mL. in a duration of 15 minutes.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings constitute a part of the description and are used to provide further understanding of the present invention. Such accompanying drawings illustrate the embodiments that are used to describe the principles of the present invention together with the description.

[0013] Figure 1 depicts a schematic diagram of a flowchart depicting steps of a method of enrichment of pathogen fraction by suppression of human genomic DNA using intercalating dye ethidium monoazide (EMA), in accordance with an implementation of the present embodiments.

[0014] Figure 2 depicts stages of EMA upon photoactivation, in accordance with an implementation of the present embodiments.DETAILED DESCRIPTION

[0015] While the invention is susceptible to various modifications and alternative forms, specific embodiment thereof will be described in detail below. It should be understood, how ever, that it is not intended to limit the invention to the particular forms disclosed; 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.

[0016] 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.

[0017] 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.

[0018] Throughout this specification, unless the context requires otherwise, the word “comprise,’’ and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0019] 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.

[0020] The term “detects” or “detection” refers to a detection that has been performed outside of a living patient using a sample from the subject.

[0021] The term “subject” refers to any mammal whose body fluid may be taken for analysis using the in-vitro method of the present invention. The exemplification is based on humans used as subjects.

[0022] The term “selectively lysed mixture” refers to a mixture in which the pathogens remain intact after selectively lysing of cells and nucleic acid (e.g.. DNA) present in the sample from the subject.

[0023] The term “treated mixture” refers to a mixture obtained after treating the selectively lysed mixture including the intact pathogen with the intercalating dye.

[0024] The term “enriched mixture” refers to a mixture obtained after photolysis of the treated mixture in which the intercalating dye binds to nucleic acids covalently, both in solution and in cells that have compromised membranes. The nucleic acid, such as DNA present in the sample from the subject, covalently bound to intercalating dye cannot be amplified. However, the intact pathogens do not allow the intercalating dye to pass through. The enriched mixture includes enriched pathogens and a nucleic acid of the subject covalently bound to the intercalating dye.

[0025] The term “pathogen” refers to prokaryotic microorganisms causing sepsis.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the disclosure, the methods and materials of the present embodiments are now described. All publications mentioned herein are incorporated herein by reference.

[0027] The present disclosure is not to be limited in scope by the specific implementations described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0028] As discussed in the background section of the present embodiments, the method for detecting sepsis causing pathogens is unable to detect the pathogens at low levels such as I'10CFU / mL, and faces the following limitations such as there is maximum carryover of human genomic DNA (human gDNA) and maximum loss of pathogens during enrichment and lysis steps. Further, the overwhelming quantities of the human gDNA may interfere with primer binding to the targets during an amplification reaction. This results in poor limits of detection in the assay. This effect is more pronounced when the target quantities are present at extremely low quantities such as T10CFU / mL. Further, the conventional methods deplete the human background. Hence, the conventional products and methods are not effective for detecting the pathogens at low levels, such as T10CFU / mL. Thus, rapid diagnostic tests that may detect a broad range of sepsis causing pathogens at low levels such as T10CFU / mL are to be provided for the diagnosis / management of sepsis.

[0029] To achieve such low detection levels of T10CFU / mL, it is important to provide a method that handles large starting volumes of whole blood, enriches the pathogen fraction, and lyses the pathogens efficiently in the diagnostic procedure. Not only that, the method may provide 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 qPCR and / or next generation sequencing.

[0030] To circumvent the above-mentioned problems, the present embodiments provide an in- vitro method that suppresses the effect of background human gDNA on the amplification of the target DNA, enriches the pathogen content from the mixed pathogen gDNA. human gDNA. and protein background, and lyses the enriched pathogens. The method of the present embodiments provides the enrichment step allows for a sensitive detection of all sepsis causing pathogens.

[0031] The main difference between the present embodiments and other known solutions / products / methods is that the method of the present embodiments does not deplete the human background, but instead suppresses the human background by binding to a DNA intercalating dye. The method suppresses any remnant human gDNA present in the sample after selective lysis-based enrichment, and this improves the pathogen detection sensitivity.

[0032] In the present embodiments, the samples (e.g., blood samples, respiratory sample) with pathogen targets are selectively lysed, such that only cells from the subject are lysed. After theselective lysis, the intercalating dye is introduced into the selectively lysed mixture to obtain treated mixture or intercalating treated mixture. The intercalating dye used in the present embodiments is cell membrane impermeable. Hence, the intact pathogens do not allow such intercalating dye to pass through. However, the released human genomic DNA may bind to the intercalating dye. Alternatively, selectively lysed mixture may be centrifuged, where the intact pathogens may remain at the bottom, and released human genomic DNA (e.g., subjected to an endonuclease) may be removed by discarding the supernatant from the centrifuged blood sample. The pelleted intact pathogens may then be treated with EMA. This provides that any remnant human gDNA carry-over in the pathogen pellet may be suppressed in PCRs favoring the desired target pathogen amplification. The treated mixture is then subjected to photolysis, which provides that the binding of the intercalating dye to the DNA is irreversible. The crosslinking and photoactivation step deployed in the method of the present embodiments makes the dye binding to DNA irreversibly. Also, the unbound dye is converted into a compound that may no longer bind the DNA. During the photolysis step, the enriched mixture including enriched pathogens and human genomic DNA is bound to the intercalating dye. The enriched mixture enriched with pathogens is then subjected to harsh lysis conditions that break open the pathogens and release the pathogen genomic DNA. The eluate from such extracted sample contains pathogen DNA (e.g., amplifiable) and background human genomic DNA bound to EMA (e.g., non-amplifiable). This way, the background effect of human genomic DNA and the target amplification are favored. This method allows the intercalating dye to bind to the free-floating pathogen DNA in the blood sample, rendering it non-amplifiable, thus only- amplifying DNA from intact bacterial cells.

[0033] Overall, the present embodiments provide a rapid method that allows crosslinking and photo activation of the intercalating dye to the DNA in 15 minutes. The method reduces the human gDNA background effect and hence results in an improved pathogen detection sensitivity. Further, the method is automatable as the photolysis is achieved by PMA-Lite LED Photolysis device.

[0034] In an embodiment, an in-vitro method for enriching pathogen nucleic acid from a sample is provided. The method includes the steps of: (a) selectively lysing a sample including pathogens to obtain a selectively lysed mixture including intact pathogens; (b) adding at least one intercalating dye to the selectively lysed mixture of step (a) to obtain a treated mixture; (c) subjecting the treated mixture to photolysis to obtain an enriched mixture, where the photolysis allows irreversible binding of the intercalating dye to a nucleic acid present in the treatedmixture; and (d) performing an ultrasound lysis with the enriched mixture of step (c) to obtain an enriched pathogen nucleic acid.

[0035] In another embodiment, selectively lysing is done for a period in the range of 2 to 20 minutes.

[0036] In an embodiment, the method includes the steps of: (a) selectively lysing a sample including pathogens to obtain a selectively lysed mixture including intact pathogens; (b) centrifuging the selectively lysed mixture to obtain a pellet including intact pathogens and a supernatant including nucleic acid present in the sample; (c) discarding the supernatant of step (a); (d) adding at least one intercalating dye to the pellet of step (b) to obtain a treated mixture; (e) subjecting the treated mixture of step (d) to photolysis to obtain an enriched mixture, where the photolysis allows irreversible binding of the intercalating dye to a nucleic acid present in the treated mixture; and (f) performing an ultrasound lysis with the enriched mixture of step (e) to obtain an enriched pathogen nucleic acid.

[0037] In an embodiment, the sample is selected from the group consisting of whole blood, swab, bronchoalveolar lavage (BAL), urine, sputum, tissue, cerebrospinal fluid (CSF), and respiratory samples. In another embodiment, the sample is whole blood sample.

[0038] In an embodiment, the method includes the steps of: (a) selectively lysing a sample including pathogens to obtain a selectively lysed mixture including intact pathogens, where the step (a) selectively lyses a mixture of cells and nucleic acids present in the sample leaving the pathogen intact; (b) adding at least one intercalating dye to the selectively lysed mixture of step (a) to obtain a treated mixture; (c) subjecting the treated mixture to photolysis to obtain an enriched mixture, where the photolysis allows irreversible binding of the intercalating dye to a nucleic acid present in the treated mixture; and (d) performing an ultrasound lysis with the enriched mixture of step (c) to obtain an enriched pathogen nucleic acid.

[0039] In an embodiment, the method includes the steps of: (a) selectively lysing a sample including pathogens to obtain a selectively lysed mixture including intact pathogens; (b) adding at least one intercalating dye to the selectively lysed mixture of step (a) to obtain a treated mixture, where the intercalating dye having a concentration in the range of 0. 1 to 20 mM is selected from the group consisting of ethidium monoazide, propidium monoazide, propidium iodide, ethidium monoazide, and thiazole orange; (c) subjecting the treated mixture to photolysis to obtain an enriched mixture, where the photolysis allows irreversible binding of the intercalating dye to a nucleic acid present in the treated mixture; and (d) performing anultrasound lysis with the enriched mixture of step (c) to obtain an enriched pathogen nucleic acid. In another embodiment, the intercalating dye is ethidium monoazide.

[0040] In an embodiment, the method includes the steps of: (a) selectively lysing a sample including pathogens to obtain a selectively lysed mixture comprising intact pathogens; (b) adding at least one intercalating dye to the selectively lysed mixture of step (a) to obtain a treated mixture; (c) subjecting the treated mixture to photolysis to obtain an enriched mixture, where the photolysis allows irreversible binding of the intercalating dye to a nucleic acid present in the treated mixture, and where the photolysis in step (c) includes exposing the treated mixture to an intense visible light for a time period of 5 to 30 minutes using an LED Photolysis device having an output wavelength in the range of 465-475 nm; and (d) performing an ultrasound lysis with the enriched mixture of step (c) to obtain an enriched pathogen nucleic acid.

[0041] In an embodiment, the nucleic acid is DNA .

[0042] In an embodiment, the method includes the steps of: (a) selectively lysing a sample including pathogens to obtain a selectively lysed mixture including intact pathogens; (b) adding at least one intercalating dye to the selectively lysed mixture of step (a) to obtain a treated mixture; (c) subjecting the treated mixture to photolysis to obtain an enriched mixture, where the photolysis allows irreversible binding of the intercalating dye to a nucleic acid present in the treated mixture; and (d) performing an ultrasound lysis with the enriched mixture of step (c) to obtain an enriched pathogen nucleic acid, where the enriched pathogen nucleic acid of step (d) is subjected to an assay selected from the group consisting of nucleic acid amplification methods followed by next generation sequencing.

[0043] In an embodiment, performing an ultrasound lysis ultrasonication is done at 20 to 80% amplitude at energy levels varying from 1000 to 2000 W-Sec.

[0044] In an embodiment, an in-vitro method for detecting sepsis causing pathogens in a sample is provided. The method includes the steps of: (a) performing a method for enriching pathogen nucleic acid from a sample. The method includes the steps of: (i) selectively lysing a sample including pathogens to obtain a selectively lysed mixture including intact pathogens; (ii) adding at least one intercalating dye to the selectively lysed mixture of step (i) to obtain a treated mixture; (iii) subjecting the treated mixture to photolysis to obtain an enriched mixture, where the photolysis allows irreversible binding of the intercalating dye to a nucleic acid present in the treated mixture; and (iv) performing an ultrasound lysis with the enriched mixture of step (iii) to obtain an enriched pathogen nucleic acid; (b) subjecting the enriched pathogennucleic acid to an assay for amplifying target sequence of the pathogen; and (c) detecting for the presence or absence of an amplified target sequence, where the presence of the amplified target sequence indicates the presence of sepsis causing pathogens in the sample.

[0045] In an embodiment, the method detects the sepsis causing pathogens at a concentration of at least 1 to 10 CFU / mL.

[0046] In an embodiment, the assay is selected from the group consisting of nucleic acid amplification methods followed by next generation sequencing. In another embodiment of the present invention, the nucleic acid amplification method is selected from quantitative polymerase chain reaction (qPCR).

[0047] In an embodiment, the pathogen is selected from the group consisting of gram negative, gram positive, fungi, viruses, protozoa, and archae.

[0048] In an embodiment, a kit includes: (a) a lysis buffer; (b) an intercalating dye selected from the group consisting of ethidium monoazide and propidium monoazide; (c) dimethyl sulfoxide (DMSO); and (d) a photolyzer.

[0049] The present embodiments are illustrated hereunder in greater detail in relation to nonlimiting example embodiments as per the following examples.

[0050] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and the description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all and only experiments performed. The methodology' of preparing a few of the example embodiments shall become clearer with working examples provided below.

[0051] In a first example, a method for enriching a pathogen fraction is provided. A workflow of the method of the present embodiments is described as a flowchart in Figure 1.

[0052] In the method of the present embodiments, a sample that was used is a whole blood sample suspected to contain pathogen targets. While the whole blood sample was used in the present embodiments, it may be contemplated that a person skilled in the art may apply the method of the present embodiments to any sample type, including respiratory samples or any other sample, where host DNA is to be depleted / suppressed before targeted pathogen detection / amplification. In the method of the present embodiments, the blood samples with pathogen targets were selectively lysed for a period in the range of 2 to 20 minutes, such that only human cells were lysed. Extremely mild lysis conditions (e.g., including use of mild detergents (2 to 7%) such as Triton X-100, Triton X-l 14, Tween-20, Tween-80, NP-40, Brij 34 and Brij 58, having a pH in the range of 8 to 1 1 and buffer with a high pH such as sodium carbonate buffer having a concentration in the range of 100 to 300 mM) were chosen such that only human cells were lysed leaving the pathogens intact. After the selective lysis, the intercalating dye such as ethidium monoazide (EMA) (e.g., 0. 1 to 10 mM) was introduced into the selectively lysed blood sample. EMA is cell membrane impermeable and a fluorescent photoaffinity label that, after photolysis, binds to nucleic acids covalently, both in solution and in cells that have compromised membranes. DNA covalently bound to EMA cannot be amplified, (e.g., Microbiol. Immunol., 51(8), 763-775, 2007). Hence, intact pathogens do not allow the EMA to pass through. However, the released human gDNA may bind to EMA.

[0053] Alternatively, the selectively lysed blood sample may be centrifuged, where the intact pathogens remain at the bottom, and released human genomic DNA (e.g., subjected to an endonuclease) may be removed by discarding the supernatant from the centrifuged blood sample. The pelleted intact pathogens may then be treated with EMA. This provides that any remnant human gDNA carry-over in the pathogen pellet may be suppressed in PCRs favoring the desired target pathogen amplification.

[0054] After EMA treatment, the photolysis was performed. The photolysis provides that the binding of the dye to the DNA is irreversible. Photolysis involved exposing the EMA treated samples to intense visible light using an LED that has an output wavelength in the range of 465-475 nm. For the purpose of performing the photolysis step, the PMA-Lite LED photolysis device was used for 15 minutes for sufficient photolysis. This photoactivation step made the dye binding to DNA irreversibly. Also, the unbound dye was converted into a compound that was no longer bound to the DNA (Figure 2).

[0055] After photolysis, the EMA treated blood samples enriched with pathogens was subjected to harsh lysis conditions such as enz matic, mechanical, chemical methods that break opened the pathogens and released the pathogen gDNA. In particular, the harsh conditions include enzymes, chaotropes, and detergents that effectively lyse open bacterial, fungi, and viruses. The EMA treated blood samples were subjected to indirect ultrasonication at 20 to 80% amplitude at energy levels varying from 1000 to 2000 W-Sec. The eluate from such extracted sample contained pathogen DNA (e.g., amplifiable) and background human gDNA bound to EMA (e.g.. non-amplifiable). This background effect of human genomic DNA was suppressed, and the target amplification was favored. The method allows EMA to bind to the free-floating pathogen DNA in the blood sample, rendering it non-amplifiable, and thus only amplifying DNA from intact bacterial cells.

[0056] The eluate containing the target pathogen DNA was then subjected to assay such as quantitative polymerase chain reaction (qPCR) and next generation sequencing for the purpose of detecting the sepsis causing pathogen.

[0057] While the method of the present embodiments uses EMA as an intercalating dye, it may be contemplated that a person skilled in the art may use another intercalating dye such as propidium monoazide (PMA), which is highly charged and is known to not penetrate live cells. PMA also has a similar effect on the dead cell chromosomal DNA such as that of EMA (10.1128 / AEM.03962- 13), or any other reagent that does not affect the integrity of a bacterial cell but renders DNA incompatible with PCR may also be used.

[0058] The present embodiments disclose an in-vitro method for enriching pathogen nucleic acid from a sample. The method includes the steps of (a) selectively lysing a sample including pathogens to obtain a selectively lysed mixture including intact pathogens; (b) adding at least one intercalating dye to the selectively lysed mixture of step (a) to obtain a treated mixture; (c) subjecting the treated mixture of step (c) to photolysis to obtain an enriched mixture, where the photolysis allows irreversible binding of the intercalating dye to a nucleic acid present in the treated mixture; and (d) performing an ultrasound lysis with the enriched mixture of step (c) to obtain an enriched pathogen nucleic acid. The present embodiments also include an in-vitro method for detecting sepsis causing pathogens in a sample. The present embodiments further disclose a kit for detecting the presence of sepsis causing pathogen in a sample.

[0059] The present embodiments have the following advantages. (1) The method reduces the human gDNA background effect and hence results in an improved pathogen detection sensitivity. (2) The method of the present embodiments is a rapid method that allows crosslinking and photoactivation of the intercalating dye (e.g.. EMA) to DNA takes roughly 15 minutes of time. (3) The method is automatable; photolysis may be achieved by a PMA-Lite LED Photolysis device. (4) Since this method does not involve removal of any component from the blood sample, and since the selective lysis is done under mild conditions, there are no high microbial losses.

[0060] The elements and features recited in the appended claims may be combined in different ways to produce new claims that likewise fall within the scope of the present invention. Thus, whereas the dependent claims appended below depend from only a single independent or dependent claim, it is to be understood that these dependent claims may, alternatively, be made to depend in the alternative from any preceding or following claim, whether independent or dependent. Such new combinations are to be understood as forming a part of the present specification.

[0061] While the present invention has been described above by reference to various embodiments, it should be understood that many changes and modifications can be made to the described embodiments. It is therefore intended that the foregoing description be regarded as illustrative rather than limiting, and that it be understood that all equivalents and / or combinations of embodiments are intended to be included in this description.

Claims

CLAIMS1. A method for enriching pathogen nucleic acid from a sample, the method being in-vitro and comprising:(a) selectively lysing a sample comprising pathogens, such that a selectively lysed mixture comprising intact pathogens is obtained;(b) adding at least one intercalating dye to the selectively lysed mixture, such that a treated mixture is obtained;(c) subjecting the treated mixture to photolysis, such that an enriched mixture is obtained, wherein the photolysis allows irreversible binding of the at least one intercalating dye to a nucleic acid present in the treated mixture; and(d) performing an ultrasound lysis with the enriched mixture of step, such that an enriched pathogen nucleic acid is obtained.

2. The method of claim 1, wherein the step (a) further comprises:(al) centrifuging the selectively lysed mixture, such that a pellet comprising intact pathogens and a supernatant comprising nucleic acid present in the sample are obtained; and(a2) discarding the supernatant.

3. The method of claim 1, wherein the sample is selected from the group consisting of whole blood, swab, bronchoalveolar lavage (BAL), urine, sputum, tissue, cerebrospinal fluid (CSF), and respiratory samples.

4. The method of claim 1, wherein the step (a) comprises selectively lysing a mixture of cells and nucleic acids present in the sample, leaving the pathogens intact.

5. The method of claim 1. wherein the at least one intercalating dye having a concentration in a range of 0.1 to 20 mM is selected from the group consisting of ethidium monoazide, propidium monoazide, propidium iodide, ethidium monoazide, and thiazole orange.

6. The method of claim 1. wherein the photolysis in step (c) comprises exposing the treated mixture to an intense visible light for a time period of 5-30 minutes using an LED Photolysis device having an output wavelength in a range of 465-475 nm.

7. The method of claim 1, wherein the nucleic acid is DNA.

8. The method of claim 1. wherein the enriched pathogen nucleic acid of step (d) is subjected to an assay selected from the group consisting of nucleic acid amplification methods followed by next generation sequencing.

9. The method of claim 1, wherein selectively lysing is done for a period in a range of 2 to 20 minutes.

10. The method of claim 1, wherein performing an ultrasound lysis ultrasonication is done at 20 to 80% amplitude at energy levels varying from 1000 to 2000 W-Sec.

11. A method for detecting sepsis causing pathogens in a sample, the method being in-vitro and comprising:(a) performing a method for enriching pathogen nucleic acid from a sample as claimed in any one of the claims 1 to 10, such that an enriched pathogen nucleic acid is obtained;(b) subjecting the enriched pathogen nucleic acid to an assay for amplifying a target sequence of a pathogen; and(c) detecting for presence or absence of an amplified target sequence, wherein the presence of the amplified target sequence indicates presence of the sepsis causing pathogens in the sample.

12. The method of claim 1 1, wherein the method detects the sepsis causing pathogens at a concentration of 1 to 10 CFU / mL.

13. The method of claim 11, wherein the assay is selected from the group consisting of nucleic acid amplification methods followed by next generation sequencing.

14. The method of claim 1 or 11 , wherein the pathogen is selected from the group consisting of gram negative, gram positive, fungi, viruses, protozoa, and archae.

15. A kit comprising:(a) lysis buffer;(b) intercalating dye selected from the group consisting of ethidium monoazide and propidium monoazide;(c) dimethyl sulfoxide (DMSO); and(d) photolyzer.

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