Universal microbial lysis method compatible with hybridization based nucleic acid enrichment and extraction techniques

Abstract

The present disclosure provides an in-vitro method of universal microbial lysis compatible with hybridization-based nucleic acid enrichment and extraction methods, comprising the steps of: (a) subjecting a sample to a combination of lysis steps to obtain a lysed sample, wherein the combination of lysis steps is selected from the group consisting of: (i) an ultrasound sonication, (ii) a combination of multiple sonication steps,- (iii) a combination of enzymatic treatment and one or more sonication steps, and (iv) a combination of enzymatic treatment, one or more sonication steps, and thermal step; and (b) treating the lysed sample of step (a) with one or more enzymes, followed by sonication for microbial lysis.

Description

32860HC-004278-WO-POAUNIVERSAL MICROBIAL LYSIS METHOD COMPATIBLE WITH HYBRIDIZATIONBASED NUCLEIC ACID ENRICHMENT AND EXTRACTION TECHNIQUESFIELD

[0001] The present disclosure is in the field of nucleic acid enrichment and extraction and detection of microbes. In particular, the present disclosure relates to a method of universal microbial lysis and enrichment or extraction of nucleic acid from a sample. The present disclosure also relates to an in-vitro method for detecting one or more microbes in the sample.BACKGROUND

[0002] Universal lysis of microbes in various sample types have been extensively studied and explored. The lysis methods generally employ chaotropes and detergents that lyse the microbial targets. While these chemicals and steps employed are compatible with downstream nucleic acid enrichment and extraction methods that are based on charge-based extraction and BOOM chemistry or some form of affinity binding, they are incompatible with hybridization-based nucleic acid extraction methods. Chaotropes and harsh detergents hydrolyze the hydrogen bonds that link the hybridized nucleic acid strands and hence chaotropes and detergents are not compatible in hybridization-based nucleic acid enrichment and extraction methods.

[0003] For instance, reference is made to U.S. Patent No. 10,584,329, which discloses a sonication-based lysing method for hybridization-based nucleic acid extraction. However, the method does not include steps, such as enzyme or chemical or heat treatment during lysis. Also, U.S. Patent No. 10,584,329 does not elaborate on the sonication settings that enable universal lysis of bacteria, fungi, cells, viruses, etc.

[0004] Further reference is made to Masoomi-Aladizgeh F, et al. A universal protocol for high- quality DNA and RNA isolation from diverse plant species. PLoS One. 2023 Dec 14;18(12):e0295852. doi: 10.1371 / joumal.pone.0295852. PMID: 38096235; PMCID: PMC10721051, which discusses a lysing method which combines temperature and other techniques for hybridization-based nucleic acid extraction. However, the lysing method is restricted to plants and not microbes.32860HC-004278-WO-POA

[0005] Accordingly, there is a dire need in the art to provide a universal microbe lysis method that is compatible with hybridization-based nucleic acid extraction and purification methods from multiple sample types.OBJECT

[0006] The principal object of the present disclosure is to provide an in-vitro method of universal microbial lysis specifically compatible with hybridization-based nucleic acid enrichment and extraction of nucleic acid from a sample.SUMMARY

[0007] The present disclosure provides an in-vitro method of universal microbial lysis compatible with hybridization-based nucleic acid enrichment and extraction methods, comprising the steps of: (a) subjecting a sample to a combination of lysis steps to obtain a lysed sample, wherein the combination of lysis steps is selected from the group consisting of: (i) an ultrasound sonication, (ii) a combination of multiple sonication steps, (iii) a combination of enzymatic treatment and one or more sonication steps; and (iv) a combination of enzymatic treatment, one or more sonication steps, and thermal step, and (b) treating the lysed sample of step (a) with one or more enzymes, followed by one or more sonication steps for microbial lysis.

[0008] The lysis method as described herein is compatible with oligo hybridization-based nucleic acid purification methods and can lyse any microbial target in the sample. This makes the described method compatible to be used in an unbiased metagenomic approach-based workflows.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] 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 of the present invention, which are used to describe the principles of the present invention together with the description.

[0010] Figure 1 illustrates one embodiment of the workflow of the universal lysis method described herein.DETAILED DESCRIPTION32860HC-004278-WO-POA

[0011] 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.

[0012] 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.

[0013] 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.

[0014] The term “sample” as used herein relates to a material or mixture of materials derived from a biological and physiological fluid, typically, although not necessarily, in liquid form, containing one or more analytes of interest. In particular, the sample refers to any sample containing bacteria and fungi, virus or any other cells of animal origin.

[0015] The term “microbe” as used herein refers to minute, unicellular organisms that are invisible to the naked eye. The term “microbe” is used interchangeably with “pathogen”, throughout the specification.

[0016] 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.

[0017] The term “universal microbial lysis” refers to a unique method which is capable of breaking down the cell wall of a wide variety of microbes and releasing the intracellular components which includes nucleic acid, proteins, and other cellular components. The said universal microbial lysis method involves a combination of unique ultrasound settings / conditions along with the specific enzymatic treatment conditions that is universal and microbe agnostic.32860HC-004278-WO-POA

[0018] The term “lysed sample” refers to a sample in which the microbial and all other cells are broken down that is caused by the combination of lysis steps as described herein.

[0019] As used herein, the term “hybridization-based nucleic acid extraction method” refers to a method for extracting a nucleic acid from the lysed sample, wherein the method involves binding the target microbial nucleic acids with short complementary nucleic acid strands (probes) at regular sequence intervals and pulling them down using a tether.

[0020] The term “sonication” refers to an act of applying ultrasound energy to agitate particles in a sample, for various purposes such as the extraction of nucleic acid from the lysed sample. The term “ultrasound sonication” refers to sonication method in which an ultrasonic energy is applied to agitate particles in a sample. The term the term “enzymatic treatment” refers to use of lytic enzymes that allows lysis of microbial cells. The term “thermal step” refers to a method that generally utilizes heating of the sample by application of a constant temperature difference between two electrodes.

[0021] The term “amplification method” refers to a process by which a nucleic acid molecule is enzymatically copied to generate a progeny population with the same sequence as the parental one using short nucleic acids called primers and using a DNA synthesizing enzyme called DNA polymerase. The term “sequencing” means to determine the primary structure of an unbranched biopolymer.

[0022] As discussed in the background section, the conventional lysis methods generally employ chaotropes, denaturants, and detergents that lyse the microbial targets. The method deploying these chemicals (chaotropes, denaturants, and detergents) are compatible with silica based nucleic acid extraction methods, however, they are incompatible with hybridization-based nucleic acid extraction methods.

[0023] To circumvent the problems existing in the art, the present invention provides an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method that does not use any chaotropes, denaturants, and detergents that can affect any target capture based nucleic acid extraction and purification methods. The method of the present invention deploys a combination of lysis steps selected from the group consisting of: (i) an ultrasound sonication, (ii) a combination of sonication steps, (iii) a combination of enzymatic treatment and one or more sonication steps, or (iv) a combination of enzymatic treatment, one or more sonication steps, and thermal step.32860HC-004278-WO-POA

[0024] Particularly, in one embodiment the present disclosure provides an in-vitro method of universal microbial lysis which involves a distinct combination of enzymatic and ultrasoundbased methods, which is compatible with hybridization-based nucleic acid extraction methods. The combination of unique ultrasound settings / conditions along with specific enzymatic treatment conditions is compatible with downstream hybridization-based nucleic acid extraction, because the lysis method as described herein does not include reagents like chao tropes, detergents that interfere with nucleic acid hybridization.

[0025] The method may be considered universal and microbe agnostic because it is capable of lysing a wide variety of bacteria, fungi, viruses, and cells of other animal origin. This makes the method compatible to be used in an unbiased, microbe agnostic metagenomic approach-based workflow for targeted and whole genome sequencing of microbes. Further, the method described herein is efficient to release nucleic acids to enable detection between about 1 CFU / mL to about 10000 CFU / mL.

[0026] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization-based nucleic acid enrichment and extraction method, comprising the steps of: (a) subjecting a sample to a combination of lysis steps to obtain a lysed sample, wherein the combination of lysis steps is selected from the group consisting of: (i) an ultrasound sonication, (ii) a combination of multiple sonication steps, (iii) a combination of enzymatic treatment and one or more sonication steps, or (iv) a combination of enzymatic treatment, one or more sonication steps, and thermal step, and (b) treating the lysed sample of step (a) with one or more enzymes, followed by sonication for microbial lysis.

[0027] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method, comprising the steps of: (a) subjecting a sample to a combination of lysis steps to obtain a lysed sample, wherein the combination of lysis steps is selected from the group consisting of: (i) an ultrasound sonication, (ii) a combination of multiple sonication steps, (iii) a combination of enzymatic treatment and one or more sonication steps, or (iv) a combination of enzymatic treatment, one or more sonication steps, and thermal step; (b) treating the lysed sample of step (a) with one or more enzymes, followed by one or more sonication steps for microbial lysis; and (c) subjecting the lysed sample of step (b) to a nucleic acid enrichment and / or extraction based on hybridization method and purification step to extract an enriched nucleic acid from the lysed sample.32860HC-004278-WO-POA

[0028] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method, comprising the steps of: (a) subjecting a sample to ultrasound sonication to obtain a lysed sample, wherein the ultrasound sonication method comprising the steps of placing the sample of step (a) in a sonotrode and agitating the sonotrode at an amplitude of 20 to 80% frequency and at an energy of 1000 to 6000 watt-seconds (Ws); and (b) treating the lysed sample of step (a) with one or more enzymes, followed by sonication for microbial lysis.

[0029] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method, comprising the steps of: (a) subjecting a sample to ultrasound sonication to obtain a lysed sample, wherein the ultrasound sonication method comprising the steps of placing the sample of step (a) in a sonotrode and agitating the sonotrode at an amplitude of 20 to 80% frequency and at an energy of 1000 to 6000 watt-seconds (Ws); (b) treating the lysed sample of step (a) with one or more enzymes, followed by sonication to obtain a treated sample; and (c) subjecting the treated sample of step (b) to a nucleic acid extraction method and a purification step to extract a nucleic acid from the treated sample.

[0030] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method, comprising the steps of: (a) subjecting a sample to ultrasound sonication to obtain a lysed sample, wherein the ultrasound sonication method comprising the steps of placing the sample of step (a) in a sonotrode and agitating the sonotrode at an amplitude of 20 to 80% frequency and at an energy of 1000 to 6000 watt-seconds (Ws), and wherein the sonication comprises sonication in pulse cycles of nON-nOFF-nON cycle, and wherein n is between 0 to 5 seconds; and (b) treating the lysed sample of step (a) with one or more enzymes, followed by sonication for microbial lysis.

[0031] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method, comprising the steps of: (a) subjecting a sample to ultrasound sonication to obtain a lysed sample, wherein the ultrasound sonication method comprising the steps of placing the sample of step (a) in a sonotrode and agitating the sonotrode at an amplitude of 20 to 80% frequency and at an energy of 1000 to 6000 watt-seconds (Ws), and wherein the sonication32860HC-004278-WO-POA comprises nON-nOFF-nON cycle, and wherein n is between 0 to 5 seconds; (b) treating the lysed sample of step (a) with one or more enzymes, followed by one or more sonication steps to obtain a treated sample; and (c) subjecting the treated sample of step (b) to a nucleic acid enrichment and / or extraction method and a purification step to extract an enriched microbial nucleic acid from the treated sample.

[0032] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis specifically compatible with hybridization based nucleic acid enrichment and extraction method, comprising the steps of: (a) subjecting a sample to a combination of sonication steps to obtain a lysed sample, wherein the combination of sonication steps comprises: (i) a first round of sonication having an intensity of 20-80% amplitude and an energy of 1400- 6000 Ws, followed by a second round of sonication having an intensity of 20-80% amplitude and an energy between 1400-6000Ws; or (ii) a first round of sonication having an intensity of 20-50% amplitude and an energy of 500-2500 Ws, followed by a second round of sonication having an intensity of 20-50% amplitude and an energy between 1400-6000Ws; or (iii) a first round of sonication having an intensity of 20-50% amplitude and an energy of 1400-6000 Ws, followed by a second round of sonication having an intensity of 20-50% amplitude and an energy between 1400-2500Ws; and (b) treating the lysed sample of step (a) with one or more enzymes, followed by one or more sonication steps for microbial lysis.

[0033] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method, comprising the steps of: (a) subjecting a sample to a combination of sonication steps to obtain a lysed sample, wherein the combination of sonication steps comprises: (i) a first round of sonication having an intensity of 20-80% amplitude and an energy of 1400-6000 Ws, followed by a second round of sonication having an intensity of 20-80% amplitude and an energy between 1400-6000Ws; or (ii) a first round of sonication having an intensity of 20-50% amplitude and an energy of 500-2500 Ws, followed by a second round of sonication having an intensity of 20-50% amplitude and an energy between 1400-6000Ws; or (iii) a first round of sonication having an intensity of 20-50% amplitude and an energy of 1400-6000 Ws, followed by a second round of sonication having an intensity of 20-50% amplitude and an energy between 1400-2500Ws; (b) treating the lysed sample of step (a) with one or more enzymes, followed by sonication for microbial lysis; and (c) subjecting the treated sample of step (b) to a nucleic acid extraction method and a purification step to extract a nucleic acid from the treated sample.32860HC-004278-WO-POA

[0034] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method, comprising the steps of: (a) subjecting a sample to a combination of enzymatic treatment, one or more sonication steps, and thermal step to obtain a lysed sample, wherein the combination of enzymatic treatment, sonication, and thermal step comprises the steps of (i) incubating the sample of step (a) with one or more enzyme for a time period between 2-40 minutes and at a temperature between 37°C to 70°C to obtain an incubated sample; (ii) sonicating the incubated sample of step (i) at an intensity of 20-80% amplitude and an energy of 1400-6000Ws to obtain a sonicated sample; and (iii) heating the sonicated sample of step (ii) at a temperature between 85°C to 99°C for a time period between 5 to 15 minutes; to obtain the lysed sample; (b) treating the lysed sample of step (a) with one or more enzymes, followed by sonication for microbial lysis.

[0035] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method, comprising the steps of: (a) subjecting a sample to a combination of enzymatic treatment, one or more sonication steps, and thermal step to obtain a lysed sample, wherein the combination of enzymatic treatment, one or more sonication steps, and thermal step comprises the steps of (i) incubating the sample of step (a) with one or more enzyme for a time period between 2-40 minutes and at a temperature between 37°C to 70°C to obtain an incubated sample; (ii) sonicating the incubated sample of step (i) at an intensity of 20-80% amplitude and an energy of 1400-6000Ws to obtain a sonicated sample; and (iii) heating the sonicated sample of step (ii) at a temperature between 85°C to 99°C for a time period between 5 to 15 minutes; to obtain the lysed sample; (b) treating the lysed sample of step (a) with one or more enzymes, followed by sonication to obtain a treated sample; and (c) subjecting the treated sample of step (b) to a nucleic acid extraction method and a purification step to extract a nucleic acid from the treated sample.

[0036] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein treating the lysed sample of step (b) with one or more enzyme, followed by one or more sonication comprises the steps of: incubating the lysed sample of step (b) with one or more enzyme for a time period between 2-40 minutes and at a temperature between 37°C to 70°C to obtain an incubated sample; followed by sonicating the lysed sample at an intensity of 20-80% amplitude and an 1400-6000 Ws.32860HC-004278-WO-POA

[0037] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein one or more enzyme is selected from the group consisting of lysozyme, lyostaphin, chitinase, lyticase, snailase, achromopeptidase, mutanolysin, proteinase K, and combination thereof.

[0038] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein the said sample is selected from the group consisting of a biological sample, an environmental sample, and an artificial contrived sample.

[0039] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein the said biological sample is selected from the group consisting of blood, sputum, sweat, urine, cerebrospinal fluid, broncho respiratory lavage, endotracheal aspirate and any other physiological body fluids.

[0040] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein the said sample is an unprocessed sample, pretreated sample or combination thereof.

[0041] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein the said sample is spiked with nucleic acid controls or free-floating naked DNA.

[0042] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein the said sample has a volume between 0.5 mL to 10.0 mL.

[0043] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein the nucleic acid extraction method is selected from32860HC-004278-WO-POA the group consisting of boom nucleic extraction method, organic solvent-based extraction method, physical adsorption method, charge based separation method, and nucleic acid hybridizationbased extraction method. In another embodiment of the present invention, the nucleic acid extraction method is selected from an organic solvent-based extraction method, wherein the organic solvent-based extraction method is selected from phenol-chloroform (organic solvents), silica beads, guanidine (boom chemistry), and nucleic acid hybridization (complementary DNA sequences). In yet another embodiment of the present invention, the nucleic acid enrichment and extraction method is selected from charge based separation method, wherein the charge based separation method is selected from phenol-chloroform method. In an alternate embodiment of the present invention, the nucleic acid extraction method is selected from nucleic acid hybridizationbased extraction method, wherein the nucleic acid hybridization-based extraction method relies on complementary DNA sequences, and probes.

[0044] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein the nucleic acid is selected from the group consisting of DNA and RNA.

[0045] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method as described herein, wherein the combination of lysis step is carried out in a period of 10-40 minutes.

[0046] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method without any use of chaotropes and detergents in the method.

[0047] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and extraction method, method is compatible with downstream detection techniques selected from the group consisting of a nucleic acid amplification method, a nucleic acid hybridization method, or nucleic acid sequencing.

[0048] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid enrichment and32860HC-004278-WO-POA extraction method as described herein, wherein the amplification method is selected from the group consisting of polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and strand displacement amplification (SDA) and any other isothermal amplification methods.

[0049] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method as described herein, wherein the nucleic acid hybridization method is selected from the group consisting of hybrid capture method, single-strand conformational polymorphism (SSCP), and restriction fragment length polymorphism (RFLP).

[0050] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method as described herein, wherein the sequencing is selected from the group consisting of sanger method, DNA sequencing by synthesis, ion torrent sequencing, and nanopore sequencing based method.

[0051] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method as described herein, wherein the method is efficient to release nucleic acids to enable detection between 1 CFU / mL to 10000 CFU / mL.

[0052] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method as described herein, wherein one or more microbe is selected from the group consisting of one or more bacteria, one or more fungi, and combination thereof.

[0053] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method as described herein, wherein one or more microbe is selected from one or more bacteria, and wherein the bacteria is selected but not limited to the group consisting gram positive bacteria, gram negative bacteria, Bacilli, spirochetes, cocci, vibrio bacteria

[0054] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method as described herein, wherein one or more microbe is selected from one or more fungi, wherein the32860HC-004278-WO-POA fungi is selected but not limited to the group consisting of Ascomycota, Basidiomycota, chytrids, Microspirida, Glomeromycota.

[0055] In an embodiment of the present disclosure, there is provided an in-vitro method of universal microbial lysis compatible with hybridization based nucleic acid extraction method as described herein, wherein the method works on all microbes and can be used to lyse microbe in any microbe agnostic detection method.

[0056] The present invention is illustrated hereunder in greater detail in relation to non-limiting exemplary embodiments as per the following examples:EXAMPLES:

[0057] 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 few of the preferred embodiments shall become clearer with working examples provided below.EXAMPLE 1: Method of universal microbial lysis and extraction of nucleic acid:

[0058] The present example provides a method of universal lysis and extraction of nucleic acids from microbes including but not limited to bacteria and fungi, that is amenable to be used in a molecular diagnostic assay. The universal lysis method is applicable to biological sample, environmental sample, artificial contrived sample such as laboratory test or control sample. Biological samples refer to variety of sample types including but not limited to blood, sputum, sweat, urine, cerebrospinal fluid, broncho respiratory lavage, endotracheal aspirate, and other body fluids. This universal lysis method is compatible with molecular assays which includes unprocessed starting sample. The method is also compatible with pretreated samples.

[0059] The universal lysis method comprises following steps:

[0060] (1) The sample having volume ranging between 0.5 mL to 10.0 mL, was obtained.

[0061] (2) The sample was then subjected to a universal lysis method which is a combination of different steps / techniques as mentioned below-32860HC-004278-WO-POA(a) Ultrasound Sonication - Sonication was done by indirectly sonicating the sample by placing the sample tube in a sonotrode which transmits ultrasound energy to the sample. The sonotrode was agitated at an amplitude ranging from 20 to 80% and at an energy range of 1000 to 6000 Watt-seconds. The sonication was done for a specific amount of time which is referred to as ON cycles. An intermittent cooling off period was given, which is referred to as OFF cycles. ON cycles ranged between 1 to 5 seconds and OFF cycles ranged between 1 to 5 seconds. The ON and OFF cycles were interspersed in multiple formats. 10N-10FF-10N..or 2ON-1OFF-2ON.. or nON-nOFF-nON, wherein the n is anywhere between 0 to 5 seconds.(b) Combination of sonication steps - The sample was subjected to a single round of sonication, combination of 2 or more rounds of sonication at the same or altered settings. i) the combination of sonication having same intensity - 20-80% amplitude 1400-6000Ws round 1 sonication followed by 20-80% amp 1400-6000Ws round 2 sonication, ii) the combination having different intensity - 20-50% amplitude 500-2500Ws round 1 sonication followed by 20- 50% amp 1400-6000Ws round 2 sonication OR 20-50% amp 1400-6000Ws round 1 sonication followed by 20-50% amp 1400-2500Ws round 2 sonication.(c) Combination of enzyme treatment and one or more sonication steps: The sample was subjected to enzyme treatment for 2-40 mins followed by 20-80% amplitude 1400-6000Ws sonication. The method involved treating the sample with multiple enzymes for lysis. The enzymes include but not limited to lysozyme, lysostaphin, chitinase, lyticase, snailase, achromopeptidase, mutanolysin, proteinase K etc. The sample was incubated with these enzymes for a time ranging from 2 minutes to 40 minutes and at temperatures ranging from 37 degrees to 70 degrees followed by sonication.(d) Combination of thermal, enzyme and sonication steps:The sample was subjected to enzyme treatment for 2-40 mins followed by 20-80% amplitude 1400-6000Ws sonication. The method involved treating the sample with multiple enzymes for lysis. The enzymes include but not limited to lysozyme, lysostaphin, chitinase, lyticase, snailase, achromopeptidase, mutanolysin, proteinase K etc. The sample was incubated with these enzymes for a time ranging from 2 minutes to 40 minutes and at temperatures ranging from 37 degrees to 70 degrees followed by sonication. The method was followed by a thermal incubation wherein the sample was heated from 85°C to 99°C for 5 to 15 minutes.

[0062] (3) The lysed sample was then subjected to an enzymatic treatment. The method involved treating the sample with multiple enzymes for lysis. The enzymes include but not limited to lysozyme, lysostaphin, chitinase, lyticase, snailase, achromopeptidase, mutanolysin, proteinase K etc. The sample was incubated with these enzymes for a time ranging from 2 minutes to 40 minutes32860HC-004278-WO-POA and at temperatures ranging from 37 degrees to 70 degrees, which was followed by subjecting the sample with 20-80% amp 1400-6000Ws sonication.

[0063] (4) The lysed sample was then subjected to nucleic acid extraction selected from the group consisting of extraction method, method based on silica beads or filter using BOOM chemistry, organic solvents, or physical adsorption methods, charge based separation-like chitosan beads including nucleic acid hybridization-based extraction and purification techniques, such that it allowed extraction of nucleic acid from the treated sample.

[0064] The universal lysis method as described herein is compatible with any nucleic acid extraction chemistry.Example 2: In-vitro method compatible with downstream detection techniques

[0065] The universal lysis method as described in Example 1 is compatible with downstream detection techniques selected from the group consisting of nucleic acid hybridization, amplification or sequencing which are universal and are microbe agnostic. The amplification methods include but not limited to PCR, isothermal amplification methods like RPA, LAMP, SDA etc. Hybridization techniques include hybrid capture method, SSCP, RFLP etc. Sequencing techniques include but not limited to sanger method, DNA sequencing by synthesis such asMyseq from Illumina, MinlON from Oxford Nanopore Technologies, Ion Genestudio S5 system from Ion torrent, , nanopore sequencing based methods etc. Further, the invention can be used in determining the composition and relative abundance of nucleic acids, for metagenomic analysis and detection of microbes.

[0066] Table 1 shows the universality of the lysis method across all bugs tested.Table 1:32860HC-004278-WO-POA32860HC-004278-WO-POANote: Genus level detection MixedSamples No species level detection within 3 hours

[0067] Referring to Table 1 and Figure 1, it can be inferred that the method of the present invention is efficient to release nucleic acids to enable detection of microbes between 1 CFU / mL to 10000 CFU / mL. In an example, the method was able to detect 10 CFU / mL of various bacteria like Enterococcus faecium, Klebsiella pneumoniae, Enterobacter cloacae, Staphylococcus aureus and various fungi like Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans with the universal lysis conditions described in the present invention.ADVANTAGES OF THE PRESENT INVENTION:

[0068] The advantages of the present invention are as follows:(a) The lysis method is compatible with oligo hybridization based nucleic acid enrichment / extraction and / or purification methods.(b) The method is universal, as it can lyse any target in the sample. This make the method compatible to be used in an unbiased metagenomic approach-based workflows. (c) The lysis method works with multiple sample types- biological samples like blood, sputum, sweat, urine, cerebrospinal fluid, broncho respiratory lavage, endotracheal aspirate, and any other body fluid.32860HC-004278-WO-POA(d) The steps outlined in the universal lysis method -are automation amenable.(e) The method as described here can be used with any metagenomic based workflows with both long read and short read detection methods.(f) The lysis method described herein is devoid of any chaotropes and detergents that can affect any target capture based nucleic acid extraction and purification methods.(g) The lysis method described herein can be completed within 10 to 40 minutes turnaround time.(h) The universal lysis method described above can be used with samples spiked with nucleic acid controls or free floating naked DNA. These molecules are not impacted by the lysis condition.

Claims

32860HC-004278-WO-POAWHAT IS CLAIMED IS:

1. An in-vitro method of universal microbial lysis specifically compatible with hybridizationbased microbial nucleic acid enrichment and extraction, the method comprising:(a) subjecting a sample to a combination of lysis steps to obtain a lysed sample, wherein the combination of lysis steps is selected from the group consisting of: (i) an ultrasound sonication step, (ii) a combination of multiple sonication steps, (iii) a combination of enzymatic treatment and one or more sonication steps, and (iv) a combination of enzymatic treatment, one or more sonication steps, and thermal step; and(b) treating the lysed sample of step (a) with one or more enzymes, followed by one or more sonication steps for microbial lysis.

2. The method as claimed in claim 1, wherein the step (b) is followed by a nucleic acid enrichment and / or extraction based on hybridization method and a purification step to extract an enriched microbial nucleic acid from the lysed sample.

3. The method as claimed in claim 1, wherein the method lyses one or more microbe selected from the group consisting of one or more bacteria, one or more fungi, and a combination thereof.

4. The method as claimed in claim 1, wherein the ultrasound sonication method comprising the steps of placing the sample of step (a) in a sonotrode and agitating the sonotrode at an amplitude of 20 to 80% frequency and at an energy of 1000 to 6000 watt-seconds (Ws).

5. The method as claimed in claim 4, wherein the ultrasound sonication comprises sonication in pulse cycles of nON-nOFF-nON cycle, and wherein n is between zero to 5 seconds.

6. The method as claimed in claim 1, wherein the combination of sonication steps comprises:(i) a first round of sonication performed at an intensity of 20-80% amplitude and an energy of 1400-6000 Ws, followed by a second round of sonication having an intensity of 20-80% amplitude and an energy of 1400-6000Ws; or(ii) a first round of sonication having an intensity of 20-50% amplitude and an energy of 500-2500 Ws, followed by a second round of sonication having an intensity of 20-50% amplitude and an energy between 1400-6000Ws; or(iii) a first round of sonication having an intensity of 20-50% amplitude and an energy of 1400- 6000 Ws, followed by a second round of sonication having an intensity of 20-50% amplitude and32860HC-004278-WO-POA an energy of 1400-2500Ws.

7. The method as claimed in claim 1, wherein the combination of enzymatic treatment and one or more sonication steps comprises the steps of:(i) incubating the sample of step (a) with one or more enzyme for a time period of 2-40 minutes and at a temperature of 37°C to 70°C to obtain an incubated sample; and(ii) sonicating the incubated sample of step (i) at an intensity of 20-80% amplitude and an energy of 1400-6000Ws to obtain the lysed sample, wherein one or more enzyme is selected from the group consisting of lysozyme, lyostaphin, chitinase, lyticase, snailase, achromopeptidase, mutanolysin, proteinase K, and a combination thereof.

8. The method as claimed in claim 1, wherein the combination of enzymatic treatment, sonication, and thermal step comprises the steps of:(i) incubating the sample of step (a) with one or more enzyme for a time period of 2-60 minutes and at a temperature of 37°C to 70°C to obtain an incubated sample;(ii) sonicating the incubated sample of step (i) at an intensity of 20-80% amplitude and an energy of 1400-6000Ws to obtain a sonicated sample, wherein the sonication step is done in two tandem rounds / cycles; and(iii) heating the sonicated sample of step (ii) at a temperature of 85°C to 99°C for a time period of 5 to 15 minutes; or freezing the sonicated sample of step (ii) to obtain the lysed sample9. The method as claimed in claim 1, wherein treating the lysed sample of step (b) with one or more enzyme, followed by sonication to obtain a treated sample, comprising the steps of: incubating the lysed sample of step (b) with one or more enzyme for a time period of 2-40 minutes and at a temperature of 37°C to 70°C to obtain an incubated sample; followed by sonicating the lysed sample at an intensity of 20-80% amplitude and an 1400-6000 Ws.

10. The method as claimed in any one of claims 1, 8 or 9, wherein one or more enzyme is selected from the group consisting of lysozyme, lyostaphin, chitinase, lyticase, snailase, achromopeptidase, mutanolysin, proteinase K, and a combination thereof.

11. The method as claimed in claim 1, wherein the sample is selected from the group consisting of a biological sample, an environmental sample, and an artificial contrived sample.32860HC-004278-WO-POA12. The method as claimed in claim 11, wherein the biological sample comprises blood, sputum, sweat, urine, cerebrospinal fluid, broncho respiratory lavage, or endotracheal aspirate.

13. The method as claimed in claim 11, wherein the sample is an unprocessed sample, pretreated sample, or a combination thereof.

14. The method as claimed in claim 11, wherein the sample is spiked with a nucleic acid control or free-floating naked DNA.

15. The method as claimed in claim 1, wherein the sample has a volume of 0.5 mL to 10.0 mL.

16. The method as claimed in claim 1, wherein the nucleic acid is selected from the group consisting of DNA and RNA.

17. The method as claimed in claim 1, wherein the combination of lysis step is carried out in a period of 10-40 minutes.

18. The method as claimed in claim 1, wherein the universal microbial lysis is compatible with hybridization-based nucleic acid extraction method without any use of chaotropes and detergents in the method.

19. The method as claimed in claim 1, wherein the method is compatible with downstream detection techniques selected from the group consisting of an amplification method, a nucleic acid hybridization method, and sequencing.

20. The method as claimed in claim 19, wherein the amplification method is selected from the group consisting of polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and strand displacement amplification (SDA).

21. The method as claimed in claim 19, wherein the nucleic acid hybridization method is selected from the group consisting of hybrid capture method, single-strand conformational polymorphism (SSCP), and restriction fragment length polymorphism (RFLP).32860HC-004278-WO-POA22. The method as claimed in claim 19, wherein the sequencing is selected from the group consisting of sanger method, DNA sequencing by synthesis, ion torrent sequencing, and nanopore sequencing-based method.

23. The method as claimed in claim 1, wherein the method is efficient to release nucleic acids to enable detection of 1 CFU / mL to 10000 CFU / mL.

24. The method as claimed in claim 3, wherein the one or more bacteria comprises gram positive bacteria, gram negative bacteria, Bacilli, spirochetes, cocci, or vibrio bacteria.

25. The method as claimed in claim 3, wherein the one or more fungi comprises Ascomycota, Basidiomycota, chytrids, Microspirida, or Glomeromycota.

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