Method and device for isolating and enriching pathogenic nucleic acids from biological samples

Abstract

The present invention relates to a method for isolating and enriching pathogenic nucleic acids from a biological sample thereby reducing host nucleic acid contamination and enrich relative pathogenic DNA in the infectious disease sample and a device thereof. The method involves mixing pathogenic nucleic acid containing biological sample with a lysis buffer at a temperature between 25-35°C for partial or incomplete lysis of the sample; passing the lysed sample through a depth filter wherein the filter paper size ranges from 0.22 microns to 20 microns; passing an elution buffer by reverse plunging action and generating a vibration of 200 to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes for eluting pathogenic nucleic acids into the elution buffer. The invention further provides a device for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample thereby reducing host nucleic acid contamination using said method.

Description

[0001] METHOD AND DEVICE FOR ISOLATING AND ENRICHING PATHOGENIC NUCLEIC ACIDS FROM BIOLOGICAL SAMPLES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a technique and a device for isolating and enriching pathogenic nucleic acids, DNA and / RNA, from biological samples such as blood, saliva, other bodily fluids, solid tissues etc, thereby reducing host nucleic acid contaminants. More specifically, the present invention relates to a device which enables isolation and separation of pathogenic DNA or RNA from nucleic acids of the host DNA in biological samples such as blood, saliva, solid tissues etc.

[0004] BACKGROUND OF THE INVENTION

[0005] Nucleic acid amplification is one of the most reliable and highly sensitive method which is widely used for molecular diagnostic purposes. Said nucleic acid amplification methods including polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), Loop-mediated isothermal amplification (LAMP), and Nucleic acid sequence-based amplification (NASBA), reverse transcriptase LAMP (RT-LAMP) and other such similar techniques are extensively used to detect pathogenic nucleic acids in a host biological sample such as bodily fluids, swab and tissue samples for diagnosis of an infection or a disease. One of the most crucial steps in such a diagnostic method involves isolation of nucleic acids from the diseased or infected host biological sample such as whole blood, saliva, semen, other bodily fluids, or other tissues and tissue swabs

[0006] Isolation of nucleic acid from samples particularly for infectious disease diagnosis faces following challenges (a) presence of host DNA in abundance compared to the pathogen which results in reduced sensitivity and generating false negatives, (b) presence of significant amount of host DNA contributes to change in pH which might affect the downstream applications like sequencing and PCR, and (c) chances of specific primers and probes binding to their respective targets becomes difficult because of the presence of the massive size and amount of host DNA. Several methods are known for reducing host cell nucleic acid contaminants in biological samples for easier detection of pathogenic nucleic acids. One such method is chaotropic salt-based method for isolation along with use of specific restriction enzymes which can reduce the host contamination such as Molzyme but the process requires extensive washing step to reduce the potential inhibitors contributing to the downstream applications like PCR, RT PCR, Genome sequencing etc. Extensive washing steps may severely reduce the amount of pathogenic nucleic acids as well thus increasing the chances of false negatives.

[0007] Another method is use of magnetic based separation of nucleic acids from biological samples which can contribute to size exclusivity but it requires optimization for purifying specific size ranges as well as shearing of the DNA before the process. The process of shearing is dependent on a sonication equipment, further, the signal might get lost during shearing because the binding site of primer might be in the region where the DNA is getting sheared.

[0008] US11148135B2 describes a filtration cartridge system for isolating nucleic acids and a sample preparation method before cartridge filtration. The method involves mechanical force such as ultrasonic frequency for bead beating process which releases nucleic acids from samples. The frequency used for bead beating ranges from 10,000 Hz to 10 MHz which invariably releases all kinds of nucleic acids, both host and pathogenic, from the samples. Further, like other known methods said sample preparation method uses enzymatic degradation of all nucleic acids of the sample in a size range of between about 100 and 10,000 base pairs. Such enzymatic degradation faces the problem of loss of PCR or sequencing primers binding areas in the pathogenic nucleic acids. Such degradation leads to degradation of both pathogenic and host nucleic acids and does not provide an efficient method to enrich pathogenic nucleic acids.

[0009] Therefore, there is a technological gap, which demands an efficient technique to selectively reduce the host nucleic acid from a total nucleic acid extract such that pathogenic nucleic acids relative amounts are higher in the total nucleic acid extract for better sensitivity of any infectious disease diagnostic / detection method based on the pathogenic DNA. This allows for performing better diagnoses and reduce the number of false positive and false negatives. Taking into consideration the drawbacks of the prior art the present invention provides a device and method for isolating and enriching pathogenic nucleic acids from a biological sample thereby reducing host nucleic acid contamination.

[0010] OBJECT(S) OF THE INVENTION

[0011] The main object of the invention is to provide a method for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample thereby reducing host nucleic acid contamination.

[0012] Another object of the invention is to provide a device for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample thereby reducing host nucleic acid contamination.

[0013] Yet another object of the invention is to provide a method for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample for diagnostic techniques such as nucleic acid amplification methods and sequencing methods.

[0014] Yet another object of the invention is to provide a cost-effective method for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample for diagnostic techniques such as nucleic acid amplification methods and sequencing methods.

[0015] Yet another object of the invention is to provide a simple, cost-effective and portable device for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample for diagnostic techniques such as nucleic acid amplification methods and sequencing methods.

[0016] SUMMARY OF THE INVENTION

[0017] The present invention provides a method for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample and a device thereof. Said method and device enable reduction of host nucleic acid contamination, and increase the efficiency of detection of pathogenic nucleic acids in downstream methods for diagnosis and / or sequencing techniques. In the main embodiment the invention provides a method for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample comprising the steps of collecting nucleic acid containing biological sample and mixing with a lysis buffer at a temperature between 25-35°C for partial or incomplete lysis of the sample; passing the lysed sample through a stack of fdters arranged in a specific manner forming a depth filter in cylindrical tube or a syringe wherein the filter paper size ranges from 0.22 microns to 20 microns; discarding the filtrate; passing an elution buffer by reverse plunging action and generating a vibration of 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes for eluting pathogenic nucleic acids into the elution buffer. Said method isolates pathogenic nucleic acids from the sample and reduces the host nucleic acid contamination significantly.

[0018] In another embodiment the invention provides a device for isolating and enriching pathogenic nucleic acids using multiple syringes. The device comprising of a top plate and a base plate mechanical joined together with plurality of support rods, a syringe plunger holding plate, a syringe finger flange holding plate, a syringe barrel holding plate, a tube or container holding plate, plurality of guiding pins, a lead screw to drive syringe plunger holding plate, a lead screw nut attached to the syringe plunger holding plate and a motor with its controller to generates vibrations in the range of 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes and operates the lead screw to perform aspiration and elimination of sample through the syringes.

[0019] In another embodiment the invention provides an alternate device for isolating and enriching pathogenic nucleic acids using multiple syringes. The device comprising of a base unit housing a motor with its controller to generates vibrations in the range of 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes and have a fluid well at top with means to assemble a vail or tube holding plate into the fluid well.

[0020] In an embodiment of the present invention, the operation of device comprises steps of reverse plunging nucleic acid containing biological sample mixed with lysis buffer from first end of the syringe assembly through the depth filter, and pushing the partially lysed nucleic acid containing biological sample using a plunger from the other end of the syringe assembly to remove the filterate. The plunger is further pulled back while the other end is in communication with an elution buffer and by reverse plunging action in synergy with a vibration generated by motor in the range of 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes.

[0021] In another embodiment of the present invention, the present invention provides many unique features and technical advantages such as the frequency range 200 Hz to 350 Hz along with the buffer combinations plays an important role in enrichment without degrading the DNA as well as retaining the pathogenic nucleic acids. Further, post introduction of the sample or the tissue to the buffer and then allowing to pass through filter paper of porosity followed by vibration results in enrichment of pathogenic nucleic acids (which are mostly smaller in size) over the host nucleic acids (which are larger in size or complexed with large biomolecules in partially lysed cells). Furthermore, for calibration of the depth filter specific experiments were devised that checked various combinations of depth filters. This particular range of arrangements specifically helps in reducing host components, and trapping pathogenic nucleic acids on it effectively.

[0022] BRIEF DESCRIPTION OF THE DRAWING(S)

[0023] The object of the invention may be understood in more details and more particularly description of the invention briefly summarized above by reference to certain embodiments thereof which are illustrated in the appended drawings, which drawings form a part of this specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective equivalent embodiments.

[0024] FIG. 1 provides perspective exploded view of syringe assembly which is incorporated in the device for isolating and enriching pathogenic nucleic acids.

[0025] FIG. 2 elucidates a cross-sectional view of the syringe assembly and filter assembly which is incorporated in the device for isolating and enriching pathogenic nucleic acids.

[0026] FIG. 3 shows the perspective view of the device for isolating and enriching pathogenic nucleic acids.

[0027] FIG. 4 shows front view of the device for isolating and enriching pathogenic nucleic acids. FIG. 5 shows sectional view of the device for isolating and enriching pathogenic nucleic acids during aspiration.

[0028] FIG. 6 shows sectional view of the device for isolating and enriching pathogenic nucleic acids during ejection.

[0029] FIG. 7 illustrates perspective view of the syringe plunger holding plate of the device for isolating and enriching pathogenic nucleic acids.

[0030] FIG. 8 illustrates sectional view of the syringe plunger holding plate of the device for isolating and enriching pathogenic nucleic acids.

[0031] FIG. 9 illustrates perspective view of the syringe finger flange holding plate of the device for isolating and enriching pathogenic nucleic acids.

[0032] FIG. 10 illustrates sectional view of the syringe finger flange holding plate of the device for isolating and enriching pathogenic nucleic acids.

[0033] FIG. 11 illustrates perspective view of the syringe barrel holding plate of the device for isolating and enriching pathogenic nucleic acids.

[0034] FIG. 12 illustrates sectional view of the syringe barrel holding plate of the device for isolating and enriching pathogenic nucleic acids.

[0035] FIG. 13 illustrates perspective view of the vial or tube or container holding plate of the device for isolating and enriching pathogenic nucleic acids.

[0036] FIG. 14 illustrates sectional view of the vial or tube or container holding plate of the device for isolating and enriching pathogenic nucleic acids.

[0037] FIG. 15 illustrates perspective view of the base plate of the device for isolating and enriching pathogenic nucleic acids.

[0038] FIG. 16 illustrates sectional view of the base plate of the device for isolating and enriching pathogenic nucleic acids.

[0039] FIG. 17 illustrates perspective view of the top plate of the device for isolating and enriching pathogenic nucleic acids. FIG. 18 illustrates sectional view of the top plate of the device for isolating and enriching pathogenic nucleic acids.

[0040] FIG. 19 illustrates perspective view of alternate embodiment of a device for isolating and enriching pathogenic nucleic acids.

[0041] FIG. 20 illustrates a flow chart showing the steps of method of isolating and enriching pathogenic nucleic acids from biological samples.

[0042] FIG. 21 provides an agarose gel picture of PCR amplification of pathogenic DNA markers extracted from four different infected shrimp samples by spin-column method (commercially available) and the present invention method (D-SWIFT).

[0043] FIG. 22A and 22B provide agarose gel pictures of amplification of pathogenic DNA markers from four different infected shrimp samples by (A) end-point PCR method, (B) real-time PCR method.

[0044] FIG. 22C and 22D provide amplification plots of pathogenic DNA markers from four different infected shrimp samples by (A) end-point PCR method, (B) real-time PCR method.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention will now be described more fully hereinafter with reference to the accompanying drawings in which a preferred embodiment of the invention is shown. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art.

[0047] 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 invention belongs.

[0048] The term "comprising" as used herein will be understood to mean that the list following is non-exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s) and / or ingredient(s) as appropriate. The term "sample" or “biological sample” as used herein will be understood to mean any specimen that potentially contains a substance of interest, in particular a nucleic acid. The term "sample" can encompass a solution, such as an aqueous solution, cell, tissue, biopsy, powder, or population of one or more of the same. The sample can be a biological sample, such as saliva, sputum, buccal swab sample, serum, plasma, blood, buffy coat, pharyngeal, nasal / nasal pharyngeal or sinus swabs or secretions, throat swabs or scrapings, urine, mucous, feces / stool / excrement, rectal swabs, lesion swabs, chyme, vomit, gastric juices, pancreatic juices, gastrointestinal (Gl) tract fluids or solids, semen / sperm, urethral swabs and secretions, cerebral spinal fluid, products of lactation or menstruation, egg yolk, amniotic fluid, aqueous humour, vitreous humour, cervical secretions or swabs, vaginal fluid / secretions / swabs or scrapings, bone marrow samples and aspirates, pleural fluid and effusions, sweat, pus, tears, lymph, bronchial or lung lavage or aspirates, peritoneal effusions, cell cultures and cell suspensions, connective tissue, epithelium, epithelial swabs and smears, mucosal membrane, muscle tissue, placental tissue, biopsies, exudates, organ tissue, nerve tissue, hair, skin, or nails, wherein samples of the foregoing may be obtained from for example, a vertebrate, including a mammal. A mammal can be, for example, a human, a non-human primate, cattle (such as cow, goat, or sheep), as well as a dog, cat, horse, etc.

[0049] The term “nucleic acid” as used herein will be understood to mean deoxyribonucleic acids (DNA), or ribonucleic acids (RNA). The term nucleic acid further encompasses double stranded DNA or RNA, or single stranded DNA or RNA.

[0050] The term “pathogenic nucleic acids” as used herein will be understood to mean DNA or RNA (double or single stranded) derived from pathogens causing infectious diseases in humans, mammals or other animals. Pathogens encompass pathogenic bacteria, fungi, virus, and protozoa which cause infectious diseases in a host.

[0051] The term “host” as used herein will be understood to mean the biological cells harbouring the infectious pathogens. Host encompasses humans, mammals or other animals like aquaculture shrimp, fishes, poultry (birds), etc

[0052] The term “PCR” as used herein will be understood to mean as polymerase chain reaction. The term “qPCR” as used herein will be understood to mean as quantitative polymerase chain reaction.

[0053] The term “PBS” as used herein will be understood to mean as phosphate buffered saline typically comprising of sodium chloride (NaCl), potassium chloride (KC1), disodium hydrogen phosphate (Na2HPC>4), and potassium dihydrogen phosphate (KH2PO4).

[0054] The term “GITC” as used herein will be understood to mean as Guanidinium thiocyanate.

[0055] The term “CTAB” as used herein will be understood to mean as Cetyltrimethylammonium bromide buffer.

[0056] The term “DTT” as used herein will be understood to mean as dithiothreitol.

[0057] The term “DTT” as used herein will be understood to mean as ethylenediaminetetraacetic acid.

[0058] In the main embodiment of the present invention, the invention provides a method for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample. Said method comprises the steps of: i) mixing a pathogenic nucleic acid containing biological sample with a lysis buffer for partial or incomplete lysis of the cells in the biological sample, and homogenizing the mixture if the biological sample is a solid tissue; ii) preparing a stack of fdters forming a depth fdter stacked in a tube or container; iii) filtering the lysed sample obtained from step i) using pressure (aspiration) through the depth filter stack, without the use of any heating or sonication process, to trap the pathogenic nucleic acids in the depth filter stack, and discarding the filtrate produced; iv) placing an elution buffer in a tube or container, and aspirating the elution buffer through the depth filter stack by a reverse plunging action and exposing the depth filter stack to vibration for eluting pathogenic nucleic acids into the elution buffer. In a preferred embodiment, the present invention provides a lysis buffer used for partial lysis of nucleic acid containing biological sample selected from the group comprising of but not limited to -

[0059] (a) PBS with 0.5%- 2% Sodium Lauryl Sulfate (SLS),

[0060] (b) GITC along with Polyvinylpyrrolidone (PVP) (), 10-200 mM Tris-Cl (Tris(hydroxymethyl)aminomethane hydrochloride )(pH 7.5-8.0), 1-10 mM DTT, and

[0061] (c) CTAB 1-10% along with 10-200 mM Tris-Cl (pH 7.5-8.0), 1-50 mM EDTA and NaCl 0.5-3 M NaCl.

[0062] In a preferred embodiment, the present invention provides an elution buffer for solution of nucleic acids from the depth filters is selected from the group comprising of but not limited to -

[0063] (a) Nuclease Free Water,

[0064] (b) Tris buffered at the pH 7.5-8.0, and

[0065] (c) Nuclease Free water with 0.1%SDS

[0066] In a preferred embodiment, the present invention provides a method for isolating and enriching pathogenic nucleic acids from a biological sample, wherein, stack of filters forming a depth filter are utilized. Said filter paper size ranges from 0.22 microns to 20 microns. Further, the stack of filters is arranged in a specific sequence selected from the one of the following arrangements - (a) series of any one type and size of filters having pore size between 1-5 pm; (b) series of any one type and size of filters having pore size between 5-12 pm; (c) series of any one type and size of filters having pore size between size 0.22-0.45 um; (d) combinatorial series of filters between 0.22-20 micron filters. The combinatorial filter stacks can be a single set of combination of filters or repeats of sets of combination ranging from 1-12 repeats. The filters are selected from the group consisting of but not limited to PVDF, nylon, and cellulose.

[0067] In a preferred embodiment, the present invention provides a method for isolating and enriching pathogenic nucleic acids from a biological sample, wherein, the biological sample mixed with lysis buffer is passed through the depth filters at a rate which leads to only partial or incomplete lysis of the sample. The method eliminates the use of any heating or sonication step at the time of lysis to ensure the host cells in the biological sample are not completely lyses. Convention methods mostly use heating or an ultrasonication method which lead to complete lysis of host cells and subsequent release of host nucleic acid contamination in the sample which interferes in the pathogenic nucleic acid identification or detection. In the present invention, the method leads to only partial lysis of host cells whereas the pathogens including bacteria and virus which have simpler and different cell membrane characteristics compared to host cells are mostly completely lysed. The incomplete lysis of host cells enables the retention of the bulky and improperly lyses host cells on the depth filters.

[0068] In a preferred embodiment, the present invention provides a method for isolating and enriching pathogenic nucleic acids from a biological sample, wherein, during the elution step, the depth filters are exposed to vibration of 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes for eluting pathogenic nucleic acids into the elution buffer. Said vibration frequency which is as low as only 200 Hz enables elution of small sized nucleic acids and reduces the elution of larger nucleic acids thus enriching the isolation of pathogenic nucleic acids with less contamination from host nucleic acids. Advantageously, the pathogenic nucleic acids which are almost always smaller in sizes than the host nucleic acids get eluted easily in the elution buffer at such a low frequency vibration, whereas the chances of elution of host nucleic acids is severely reduced as they may be trapped in the partially lysed cells in the biological sample or are bound to large molecular complexes.

[0069] Advantageously, the present invention provides a method to reduce the contamination of host nucleic acids and enriching pathogenic nucleic acids from a biological sample by employing several important techniques and eliminating other traditionally used techniques.

[0070] Said method, unlike traditional methods, eliminates the use of higher frequency shearing forces such as sonication and ultrasonication for cell lysis which is usually higher than 20 kHz. Cell lysis using sonication at higher frequencies tends to completely lyse the host cells in the biological sample which in turn leads to contamination of pathogenic nucleic acids with host nucleic acids. Therefore, the present invention provides a method using a simple lysis buffer containing a detergent or a chelating agent or a reducing agent or a combination thereof which enables cell membrane lysis without use of extensive enzymes or sonication methods. Exemplary detergents include, preferably, sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), sodium taurodeoxycholate (NaTDC), sodium taurocholate (NaTC), sodium glycocholate (NaGC), sodium deoxycholate (NaDC), sodium cholate, sodium alkylbenzene sulfonate (NaABS), N-lauroyl sarcosine (NLS), salts of carboxylic acids (i.e., soaps), salts of sulfonic acids, salts of sulfuric acid, phosphoric and polyphosphoric acid esters, alkylphosphates, monoalkyl phosphate (MAP), and salts of perfluorocarboxylic acids, anionic detergents or a combination thereof.

[0071] Exemplary reducing agents include, preferably, 2-mercaptoethanol (P-ME), tris(2- carboxyethyl) phosphine (TCEP), dithiothreitol (DTT), formamide, dimethylsulfoxide (DMSO), or any combination thereof.

[0072] Exemplary chelators include, preferably, ethylene glycol tetraacetic acid (EGTA), hydroxy ethylethylenediaminetriacetic acid (HEDTA), diethylene triamine pentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (NTA), ethylenediaminetetraacetic (EDTA), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, potassium citrate, magnesium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof.

[0073] In a preferred embodiment, the present invention provides a method for isolating and enriching pathogenic nucleic acids from a biological sample, in which the pathogenic nucleic acid containing biological sample mixed with the lysis buffer is filtered through the depth filter in a manner such that the partially lysed cells are easily separated from the nucleic acids released from pathogens in the biological sample. The higher molecular weight complexes and debris are separated during the filtration process and the filtrate is discarded.

[0074] In a preferred embodiment, the present invention provides a method for isolating and enriching pathogenic nucleic acids from a biological sample which eliminates the use of enzymes or sonication methods to reduce the size of all the nucleic acids in the biological sample. This further advantageously reduces the shearing of host nucleic acids, therefore the chances of host nucleic acids to stay intact or bound to larger molecular complexes increases. Such large sized nucleic acids are not easily eluted when given a low frequency vibration of 200 Hz to 350 Hz. Advantageously said method efficiently reduces contamination of host nucleic acids and enriches the pathogenic nucleic acids when the nucleic acids are extracted from a nucleic acid containing biological sample.

[0075] In another embodiment of the invention, the invention provides a device for isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample thereby reducing host nucleic acid contamination.

[0076] Referring to Fig. 1, provides perspective exploded view of syringe assembly (100) which is incorporated in the device for isolating and enriching pathogenic nucleic acids. The figure clearly elucidates the syringe assembly (100) comprises of a barrel (102) having a narrower tip (104) at first end and broader second end with finger flange (106). The barrel (102) further supports filter assembly (200) and has markings for measurement along its barrel (102) body. A plunger (108) is also provided to be assembled into the barrel (102) to perform suction and ejection of samples.

[0077] Referring to Fig. 2, provides a cross-sectional view of the syringe assembly (100) and filter assembly (200) which is incorporated in the device for isolating and enriching pathogenic nucleic acids. The figure elucidates the mounting of the filter assembly (200) inside the syringe assembly (100). The filter assembly (200) comprises of a top housing (202) having an o-ring (204) and a bottom housing (206). Both the top housing (202) and bottom housing (206) have multiple through holes (208) to let sample pass through it. In between the vacant space of the filter assembly, plurality of filter papers (210) is incorporated. The filter paper (210) size ranges from 0.22 micron to 20 micron. The filter paper (210) stacking has one of the following arrangements (based on the samples used) (a) series of any one type and size of filters having pore size between 1- 5 pm; (b) series of any one type and size of filters having pore size between 5-12 pm; (c) series of any one type and size of filters having pore size between size 0.22-0.45 um; (d) combinatorial series of filters between 0.22-20 micron filters. The combinatorial filter stacks can be a single set of combination of filters or repeats of sets of combination ranging from 1-12 repeats. The filters are selected from the group consisting of but not limited to PVDF, nylon, and cellulose.

[0078] Referring to Fig. 3 and Fig. 4 shows the different views of a device (300) for isolating and enriching pathogenic nucleic acids. The device (300) comprising of a top plate (302) and a base plate (304) mechanical joined together with plurality of support rods (306), a syringe plunger holding plate (308), a syringe finger flange holding plate (310), a syringe barrel holding plate (312), a vial or tube or container holding plate (314), plurality of guiding pins (316), a lead screw (318) to drive syringe plunger holding plate (308), a lead screw nut (320) attached to the syringe plunger holding plate (308) and a motor (322) with its controller to generates vibrations in the range of 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes and operates the lead screw (318) to perform aspiration and elimination of sample through the syringes.

[0079] In preferred embodiment, the syringe plunger holding plate (308) will be derived by the lead screw (318) to perform aspiration and elimination of sample through the syringes. Each of the top plate (302), base plate (304), syringe plunger holding plate (308), syringe finger flange holding plate (310), syringe barrel holding plate (312) and vial or tube or container holding plate (314) are arranged in a concentric manner wherein the centre of each of the plates are in same axis. Furthermore, guiding pins (316) helps in to drive rotations to the syringe finger flange holding plate (310), syringe barrel holding plate (312) and vial or tube or container holding plate (314) when the syringe plunger holding plate (308) derived by the lead screw (318) to perform aspiration and elimination of sample through the syringes.

[0080] In preferred embodiment, syringe finger flange holding plate (310), syringe barrel holding plate (312) and vial or tube or container holding plate (314) are assembled on to the base (304) using bearing means and performs rotation movement only, while the syringe plunger holding plate (308) is assembled on the lead screw (318) and performs rotation and liner movement both.

[0081] In preferred embodiment, on end of the lead screw (318) is attached into the base (302) using bearings and the other end is connected to the motor (322).

[0082] Referring to Fig. 5 shows the sectional view of a device (300) for isolating and enriching pathogenic nucleic acids during aspiration. During aspiration stage the syringe finger flange holding plate (308) is near to the top plate (302). The plunger of the syringe (100) is fully pulled out form the barrel.

[0083] Referring to Fig. 6 shows the sectional view of a device (300) for isolating and enriching pathogenic nucleic acids during ejection. During ejection stage the syringe finger flange holding plate (308) is near to the syringe finger flange holding plate (310). The plunger of the syringe (100) is fully pushed in the barrel. During the ejection stage the discarded buffer sample is collected into the tube or container (400) assembled into the vial or tube or container holding plate (314).

[0084] Referring to Fig. 7 and Fig. 8 illustrates different views of the syringe plunger holding plate (308) of the device (300) for isolating and enriching pathogenic nucleic acids. The syringe plunger holding plate (308) is a solid single molded piece with a surface (308a) and a surface (308b), a centre hole (308c) and through holes (308d) to accommodate guiding pin (316). The surface (308a) is plain and the surface (308b) has plurality of cavities (308e) to hold the plunger head of the syringe (100). The fit and tolerance of each of the cavities (308e) are such that the plunger head will be hold firmly during the operation of the device (300).

[0085] In illustrated figures, syringe plunger holding plate (308) is shown in circular configuration for illustration purpose only and the syringe finger flange holding plate (310) can be made in another geometrical configuration such as but not limited to square, hexagon, pentagon or similar.

[0086] Referring to Fig. 9 and Fig. 10 illustrates different views of the syringe finger flange holding plate (310) of the device (300) for isolating and enriching pathogenic nucleic acids. The syringe finger flange holding plate (310) is a solid single molded piece with a surface (310a) and a surface (310b), a centre hole (310c) and through holes (310d) to accommodate guiding pin (316). Across the surface (310a) and surface (310b), plurality of cavities (310e) are provided to hold the finger flange of the syringe (100). The fit and tolerance of each of the cavities (310e) are such that the finger flange will be hold firmly during the operation of the device (300).

[0087] In illustrated figures, syringe finger flange holding plate (310) is shown in circular configuration for illustration purpose only and the syringe finger flange holding plate (310) can be made in another geometrical configuration such as but not limited to square, hexagon, pentagon or similar.

[0088] Referring to Fig. 11 and Fig. 12 illustrates different views of the syringe barrel holding plate (312) of the device (300) for isolating and enriching pathogenic nucleic acids. The syringe barrel holding plate (312) is a solid single molded piece with a surface (312a) and a surface (312b), a centre hole (312c) and through holes (312d) to accommodate guiding pin (316). Across the surface (312a) and surface (312b), plurality of cavities (312e) are provided to hold the barrel of the syringe (100). The fit and tolerance of each of the cavities (312e) are such that the barrel will be hold firmly during the operation of the device (300).

[0089] In illustrated figures, syringe barrel holding plate (312) is shown in circular configuration for illustration purpose only and the syringe barrel holding plate (312) can be made in another geometrical configuration such as but not limited to square, hexagon, pentagon or similar.

[0090] Referring to Fig. 13 and Fig. 14 illustrates different views of the vial or tube or container holding plate (314) of the device (300) for isolating and enriching pathogenic nucleic acids. The vial or tube or container holding plate (314) is a solid single molded piece with a surface (314a) and a surface (314b), a centre hole (314c) and through holes (314d) to accommodate guiding pin (316). Across the surface (314a) plurality of cavities (314e) are provided to hold the tube or container (400). The fit and tolerance of each of the cavities (314e) are such that the tube or container (400) will be hold firmly during the operation of the device (300).

[0091] In illustrated figures, vial or tube or container holding plate (314) is shown in circular configuration for illustration purpose only and vial or tube or container holding plate (314) can be made in another geometrical configuration such as but not limited to square, hexagon, pentagon or similar.

[0092] Referring to Fig. 15 and Fig. 16 illustrates different views of the base plate (304) of the device (300) for isolating and enriching pathogenic nucleic acids. The base plate (304) is a solid single piece with a flat surface (304a), a centre shaft (304b) for attaching the lead screw (318) and plurality of extrusions (304c) to mount the support rods (306). At the distal end of the centre shaft (304b), a cavity (304d) is provided to hold the lead screw (318) with a bearing. The fit and tolerance of each of the extrusions (304c) are such that the support rods (306) will be hold firmly during the operation of the device (300).

[0093] In illustrated figures, base plate (302) is shown in circular configuration for illustration purpose only and base plate (302) can be made in another geometrical configuration such as but not limited to square, hexagon, pentagon or similar. Referring to Fig. 17 and Fig. 18 illustrates different views of the top plate (302) of the device (300) for isolating and enriching pathogenic nucleic acids. The top plate (302) is a solid single molded piece with a surface (302a) and a surface (302b), a centre hole (302c) for attaching motor (322) to the lead screw (318) and plurality of threaded holes (302d) to mount the motor (322). Across the surface (302b) plurality of extrusions (302e) are provided to hold the support rods (306). The fit and tolerance of each of the extrusions (302e) are such that the support rods (306) will be hold firmly during the operation of the device (300).

[0094] In illustrated figures, top plate (302) is shown in circular configuration for illustration purpose only and top plate (302) can be made in another geometrical configuration such as but not limited to square, hexagon, pentagon or similar.

[0095] Referring to Fig. 19 illustrates perspective view of alternate embodiment of a device (500) for isolating and enriching pathogenic nucleic acids in a semi-automated mode. The device (500) comprising of a base unit (502) housing a motor with its controller to generate vibrations in the range of 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes and has a fluid well (504) at top with extrusions (506) to assemble a vail or tube or container holding plate (508) into the fluid well (504).

[0096] EXAMPLES

[0097] EXAMPLE 1

[0098] Isolating and enriching pathogenic nucleic acids from a nucleic acid containing biological sample

[0099] The present invention relates to a method to isolate and enrich pathogenic nucleic acids from a biological sample (infected or contaminated by a pathogen). The biological sample can be a tissue sample of an animal or human, blood sample, swaps of saliva, genitals or other organs etc.

[0100] FIG. 20 provides a flowchart describing the steps involved in isolating and enriching pathogenic nucleic acids from a biological sample

[0101] Step 1 involves mixing a nucleic acid containing biological sample with a lysis buffer for lysing the cells in the biological sample. For some samples simple addition of lysis buffer is sufficient, while few others like blood sample may require more appropriate lysis buffer. Further some samples such a tissue of small animals (eg. shrimp) or human organs which are slightly tough may require an addition step of crushing the biological sample mixed with lysis buffer using a motor and pestle or pestle alone for few seconds to initiate the lysis process. For some samples homogenization may be required.

[0102] Step 2 involves filtering the nucleic acid containing biological sample mixed with the lysis buffer through a stack of filters forming a depth filter at a rate such that the biological sample is partially lysed and not completely lysed (without the use of any heating or sonication process), and discarding the filtrate produced during the filtration process.

[0103] Step 3 is passing an elution buffer by a reverse plunging action and simultaneously or subsequently exposing the depth filter stack to vibration of 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes for eluting pathogenic nucleic acids into the elution buffer.

[0104] The lysis buffer can vary from biological sample to sample. The lysis buffer is selected from the group comprising of but not limited to - a) PBS with 0.5%- 2% SLS, b) GITC lysis buffer comprising of 1-6 M Guanidinium thiocyanate (GITC), 1-2% (w / v) polyvinylpyrrolidone (PVP), 10-200 mM Tris-Cl (pH 7.5-8.0), 1-10 mM DTT, and c) CTAB lysis buffer comprising of Cetyltrimethylammonium Bromide (CTAB) 1- 10%, 10-200 mM Tris-Cl (pH 7.5-8.0), 1-50 mM EDTA and 0.5-3M NaCl.

[0105] One of the compositions of lysis Buffer for lysis of a biological sample such as blood, sputum, swab, or solid tissue can be GITC lysis buffer, more specifically, lysis buffer comprising of 6 M GITC, 200 mM Tris-Cl (pH 7.8), 10 mM DTT

[0106] The composition of elution buffer for solution of nucleic acids from the depth filters is selected from the group comprising of but not limited to - a) Nuclease Free Water, b) Tris buffered at the pH 7.5 -8.0, and c) Nuclease Free water with 0.1%SDS EXAMPLE 2

[0107] Frequency of Vibration of efficient pathogenic nucleic acid extraction

[0108] Step 4 of the said method requires passing an elution buffer from the opposite side of the filtration process by a reverse plunging action and simultaneously exposing the depth filter stack to vibration of certain frequency with discontinuous pulse of few seconds for certain amount of time for eluting pathogenic nucleic acids into the elution buffer.

[0109] To evaluate the optimum frequency for efficient isolation and enrichment of pathogenic nucleic acid from a biological sample, a range of frequencies were tested for efficiency. For this purpose, each frequency was given as a pulse of 30 seconds for 5 minutes. The range of frequencies tested was 100 Hz - 500 Hz. Table 1 provides the details of the quality of pathogenic nucleic acid, DNA in this case, extracted from biological sample under different frequencies. The biological sample used was sputum samples containing Mycobacterium tuberculosis from infected individuals. The lysis buffer used was GITC along with PVP, 10-200 mM Tris-Cl (pH 7.5-8.0), l-10mM DTT

[0110] Table 1: Nucleic Acid Yield vs Frequency

[0111] Yield is determined by quantifying the amount of DNA, often using spectrophotometry. DNA absorbs light at 260 nm, and the absorbance value is used to calculate DNA concentration. Purity is assessed by examining the ratio of absorbance at different wavelengths (e.g., A260 / A280) to detect contaminants like proteins or salts. For pure DNA, the A260 / A280 ratio is typically around 1.8. A lower ratio suggests protein contamination. Therefore, ratio of 1.8 -2.0 is considered optimal purity. A260 / A230 ratio is used to assess contamination by other compounds like salts and phenol, which absorb at or near 230 nm. A ratio greater than 1.8 is generally considered acceptable.

[0112] It is clearly evident from the results in Table 1 that frequency below 200 Hz does not provide good yield of pathogenic DNA from the sample, and similarly frequency above 350 Hz also does not yield sufficient DNA. The frequency between 200-350 Hz provides good quantities of DNA which have purity levels in the acceptable range (260 / 280 ratio and 260 / 230 ratio between 1.8-2.0).

[0113] Further, the DNA samples were used for conducting qPCR to detect the pathogenic DNA. In qPCR experiments, the Ct (cycle threshold) value is inversely related to the amount of target DNA in a sample. It is evident that samples from frequency below 200 Hz and above 350 Hz did not show amplification and detection of pathogenic DNA. Samples from 200-350 Hz showed amplification suggesting efficient extraction of pathogenic DNA from the biological sample.

[0114] EXAMPLE 3

[0115] Arrangement of filter stacks

[0116] Said method requires stack of filters forming a depth filter wherein the filter paper size ranges from 0.22 microns to 20 microns. Further the stack of filters is arranged in a specific sequence selected from the one of the following arrangements - (a) series of any one type and size of filters having pore size between 1-5 pm; (b) series of any one type and size of filters having pore size between 5-12 pm; (c) series of any one type and size of filters having pore size between size 0.22-0.45 um; (d) combinatorial series of filters between 0.22-20 micron filters. The filters are selected from the group consisting of but not limited to PVDF, nylon, and cellulose.

[0117] Each combination of filter set can be used as a single set or can used up to 12 repetitions for different kinds of biological sample and different pathogenic DNA extraction. To evaluate whether combination of different series of filter stacks efficiently isolates and enriches pathogenic DNA from a biological sample, series of combinations of filter stacks were prepared.

[0118] Filters configuration

[0119] Type A filter-High Pore Size (pm)- 1-5 pm;

[0120] Type B- High Pore size- 5-12 um

[0121] Type C- Low pore size 0.22-0.45 um

[0122] For this experiment blood sample infected with Methicillin-resistant Staphylococcus aureus (MRSA) was used.

[0123] Lysis buffer used was 6 M GITC, 200 mM Tris-Cl (pH 7.8), lOmM DTT

[0124] Vibration frequency - 250 Hz, 30 sec pulse for 5 minutes

[0125] After the pathogenic DNA was extracted, each sample was tested using qPCR to calculate Ct values.

[0126] The Ct value represents the number of PCR cycles required for the fluorescent signal to cross a set threshold, indicating the amplification of a target DNA sequence.

[0127] • Inverse Relationship:

[0128] A lower Ct value means the target DNA was detected earlier in the PCR process, implying a larger initial amount of DNA in the sample. Conversely, a higher Ct value suggests that more PCR cycles were needed to reach the threshold, indicating a smaller initial amount of DNA.

[0129] Table 2: Filter Layer Configurations- All above combinations gave good results- low Ct values that corresponds to high amount of DNA being extracted from sample.

[0130] EXAMPLE 4

[0131] Comparison of present method of pathogenic DNA extraction with available commercial methods

[0132] 1. DNA from Swab sample comparison

[0133] Nisseria gonorrhoea infected genital swabs samples were taken from infected patients and DNA from the swab was extracted using two commercial (conventional) methods and compared to the present invention.

[0134] The two commercial methods used were: a) Boling lysis method - this method involves the following steps: i) resuspending swab sample in 100 pLTE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0); ii) vortexing briefly and incubating at 95°C for 10 minutes to lyse cells; iii) centrifuging at 12,000 x g for 5 minutes to pellet debris; and iv) collecting supernatant containing DNA and storing at -20°C. b) Column extraction method - For this method, commercial kit DNeasy Blood & Tissue Kit (QIAGEN) was used. This method involves the following steps: i) resuspending the swab sample in 180 pl Buffer ATL (proprietor information); ii) adding 20 pl proteinase K and vortexing and incubate at 56°C until completely lysed; iii) adding 200 pl Buffer AL ATL (proprietor information) and vortexing; iv) Adding 200 pl ethanol (96-100%) and vortexing; v) pipeting the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube and centrifuging at >6000 x g (8000 rpm) for 1 min; vi) discarding the flow-through and collection tube; vii) placing the spin column in a new 2 ml collection tube and adding 500 pl Buffer AW1 (proprietor information) and centrifuging for 1 min at >6000 x g; viii) discarding the flow-through and collection tube; ix) placing the spin column in a new 2 ml collection tube, adding 500 pl Buffer AW2 (proprietor information), and centrifuging for 3 min at 20,000 x g (14,000 rpm); x) discarding the flow-through and collection tube; xi) transferring the spin column to a new 1.5 ml or 2 ml microcentrifuge tube, and eluting the DNA by adding 200 pl Buffer AE (proprietor information) to the centre of the spin column membrane; and xii) incubate for 1 min at room temperature (15-25 °C) and centrifuging for 1 min at >6000 x g to extract DNA into the elution buffer.

[0135] The method described in the present invention (D-SWIFT), as described in detail in Example 1, was used to extract DNA from swab samples, briefly, i) the swab sample was resuspended in lysis buffer comprising of 6 M GITC, 200 mM Tris-Cl (pH 7.8), WmM DTT; ii) sample mixed with the lysis buffer was passed through a stack of filters (Stack type 3 of Table 2 in 11 repetitions) forming a depth filter (without the use of any heating or sonication process); iii) the filtrate was discarded; and iv) elution buffer was passed from the opposite side of the filtration process by a reverse plunging action and simultaneously exposed the depth filter stack to vibration of 250 Hz with discontinuous pulse of 30 seconds for 3 minutes for eluting pathogenic nucleic acids into the elution buffer.

[0136] All the three DNA extractions were used to conduct qPCR using pathogen specific primers for specific markers. Table 3 provides a comparison of the Ct values from DNA used of qPCR from all the three types of extractions.

[0137] The results from Table 3 clearly show that the DNA extracted using the present method provides higher yield as compared to commercial and conventional methods (Nanodrop readings). Present method gave a 150-300 % more yield than commercially available spin-column method, and 900-2000% more yield than boiling method. TABLE 3: Comparison of Ct values of pathogen marker

[0138] The formula to calculate Fold Difference to compare Ct values if

[0139] Fold difference = 2(Cl of A Cl of B)

[0140] Difference between Ct value of Boiling method and present method is = 23.17 - 5.94 = 17.23

[0141] Fold difference compared to boiling method = 2(1723)= 157,059 times more efficient or has that much more amplifiable DNA than the boiling method.

[0142] Difference between Ct value of column method and present method is = 19.98 - 5.94 = 14.04

[0143] Fold difference compared to boiling method = 2(1404)= 17,093 times more efficient or has that much more amplifiable DNA than the boiling method

[0144] The results clearly show that the present method of extraction of DNA is several thousand times more efficient than conventional and commercially available methods, making the present invention highly advantageous.

[0145] Table 4 further compares the three methods to elucidate the time involved, equipment required, and other parameters. The methods have been compared to the present invention in manual (semi-automatic) mode and automatic mode.

[0146] Table 4: Process Comparison

[0147] 2. DNA from infected shrimp tissue sample comparison

[0148] White Spot Syndrome Virus (WSSV) is a highly contagious and lethal virus that affects crustaceans, particularly farmed shrimp (but not known to cause disease in humans). Infected shrimps (gill solid tissue) were taken to extract pathogenic DNA using commercially available column method and compared to the present invention (D- SWIFT).

[0149] Briefly for DNA extraction using present method, lysis buffer comprising 6 M GITC, 200 mM Tris-Cl (pH 7.8), lOmM DTT was used. The sample in the lysis buffer was homogenized at 250 Hz for 2 mins only, and the passed though filter stack combination 1 of Table 2 in 12 repetitions. Finally, elution buffer was passed by a reverse plunging action and simultaneously exposed the depth filter stack to vibration of 250 Hz with discontinuous pulse of 30 seconds for 3 minutes for eluting pathogenic nucleic acids into the elution buffer.

[0150] FIG. 21 provides an agarose gel picture of PCR amplification of pathogenic DNA markers extracted from four different infected shrimp samples by both the methods. The results show that pathogenic DNA was more efficiently extracted by the present method (D- SWIFT) compared to negligible extraction by commercial column method. Further, the present method have significantly less contamination of host DNA (shrimp DNA markers used for PCR amplification) compared to column extraction method. Table 5 compares the amount of PCR product amplified for different markers (pathogen markers and host markers), comparison of amount of host DNA contamination in the DNA extracted using both the methods, and comparison of % of pathogen DNA compared to host DNA in the total DNA extracted sample.

[0151] Table 5: Comparison of amount of Pathogen DNA and host DNA in extracted samples

[0152] The results clearly show that the present method provides a purer pathogenic DNA extraction with less host DNA contamination compared to column extraction method. All the four extracted DNA samples contained 70-78.5 % pathogenic DNA in total DNA in present method compared to only 32-42% pathogenic DNA in total DNA by column method. EXAMPLE 5

[0153] Extraction of different types of nucleic acids from different kinds of samples

[0154] 1. Pathogenic nucleic acid - DNA

[0155] Example 2 provides details of extraction of pathogenic DNA from swabs of sputum samples containing Mycobacterium tuberculosis, Example 4 provides details of extraction of pathogenic DNA from swab samples of human genital tissue, and also extraction of pathogenic DNA from solid tissue of shrimp gills.

[0156] Table 6 provides pathogenic DNA extractions from various other kinds of samples to demonstrate that several types of pathogenic DNA can be extracted from different types of host tissue samples. Buffer composition:

[0157] Lysis Buffer used was 6 M GITC, 200 mM Tris-Cl (pH 7.8), lOmM

[0158] Elution buffer was same for all

[0159] Motor frequency for vibration for elution into elution buffer was 250 Hz

[0160] Blood and swab / sputum samples did not require homogenization, whereas, solid tissues had to be homogenized in lysis buffer.

[0161] Table 6: Pathogenic DNA extracted from different infected samples

[0162] Pathogenic DNA was extracted from all types of the samples stated in Table 6 in good quantities and the purity was also within required range. Ct values were also low as desired.

[0163] 2. Pathogenic nucleic acid - RNA The present method can also be used to efficiently extract pathogenic RNA from infected samples. To demonstrate this, Covid 19 virus infected swab samples were collected and subjected to the method as described in Example 2. Lysis Buffer used was 2 M GITC, 100 mM Tris-Cl (pH 7.8), 5mM DTT, and motor frequency for vibration for elution of pathogenic RNA into elution buffer was 250 Hz with discontinuous pulse of 30 seconds for 3 minutes.

[0164] Table 7: Pathogenic RNA extracted from infected swab samples

[0165] The results clearly show the pathogenic nucleic acid including RNA can be efficiently extracted from infected samples using the present method. The Ct values are low in numbers clearly indicating high efficiency in RNA extraction. While certain exemplary embodiments have been described and shown in the accompanying drawings, it is to be understood that such embodiments are merely illustrative of, and not restrictive on, the broad invention, and that this invention not be limited to the specific constructions and arrangements shown and described, since various other changes, combinations, omissions, modifications and substitutions, in addition to those set forth in the above paragraphs, are possible. Those skilled in the art will appreciate that various adaptations and modifications of the just described embodiments can be configured without departing from the scope and spirit of the invention.

Claims

CLAIMSWe claim:

1. A method for isolating and enriching pathogenic nucleic acids from infected biological samples comprising the steps of: i) mixing a pathogenic nucleic acid containing biological sample with a lysis buffer for partial or incomplete lysis of the cells in the biological sample, and homogenizing the mixture if the biological sample is a solid tissue; ii) preparing a stack of fdters forming a depth filter stacked in a tube or container; iii) filtering the lysed sample obtained from step i) using pressure (aspiration) through the depth filter stack, without the use of any heating or sonication process, to trap the pathogenic nucleic acids in the depth filter stack, and discarding the filtrate produced; iv) placing an elution buffer in a tube or container, and aspirating the elution buffer through the depth filter stack by a reverse plunging action and exposing the depth filter stack to vibration for eluting pathogenic nucleic acids into the elution buffer; characterized in that, the vibration frequency used in step iv) is 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes for eluting pathogenic nucleic acids trapped in the depth filter into the elution buffer; the optional homogenization in step i) is achieved by vibrating the biological sample mixed in lysis buffer for 200-350 Hz for 1-2 minutes; the time taken to complete said method for isolating and enriching pathogenic nucleic acids is about 5 minutes; and the temperature at which said method for isolating and enriching pathogenic nucleic acids is carried out is room temperature 25-35°C.

2. The method as claimed in claim 1, wherein, the depth filter stack comprises of filter paper size ranging from 0.22 microns to 20 microns.

3. The method as claimed in claim 1, wherein, the depth filter stack comprises of filter stacks are arranged in a specific sequence selected from one of the following arrangements - (a) series of any one type and size of filters having pore size between 1-5 pm; (b) series of any one type and size of filters having pore size between 5-12 pm; (c) series of any one type and size of filters having pore size between size 0.22-0.45 urn; and (d) combinatorial series of filters between 0.22-20 micron filters.

4. The method as claimed in claim 1, wherein, the depth filter stack comprises of filter stacks are arranged in a specific sequence which can be repeated in the range of 1-12 times.

5. The method as claimed in claim 1, wherein, the lysis buffer is selected from the group consisting of PBS buffer comprising of PBS, and 0.5%- 2% SLS, GITC lysis buffer comprising of 1-6 M Guanidinium thiocyanate (GITC), X% polyvinylpyrrolidone (PVP), 10-200 mM Tris-Cl (pH 7.5-8.0), 1-lOmM DTT, and CTAB lysis buffer comprising of Cetyltrimethylammonium Bromide (CTAB) 1-10%, 10-200 mM Tris-Cl (pH 7.5-8.0), l-50mM EDTA and 0.5-3M NaCl.

6. The method as claimed in claim 1, wherein, the elution buffer is selected from the group consisting of nuclease free water, Tris buffered at the pH 7.5-8.0, and nuclease free water with 0.1% SDS.

7. The method as claimed in claim 1, wherein, pathogenic nucleic acids include DNA and RNA.

8. The method as claimed in claim 1, wherein, pathogenic nucleic acids include that of bacteria, fungi, protozoa, and viruses.

9. The method as claimed in claim 1, wherein, biological samples include blood, sputum, swabs of various infected organs and tissues, urine, and solid tissues of humans and animals.

10. A device (300) for isolating and enriching pathogenic nucleic acids comprising of:atop plate (302) and abase plate (304) mechanical joined together with plurality of support rods (306); a syringe plunger holding plate (308), a syringe finger flange holding plate (310), a syringe barrel holding plate (312) and a vial or tube or container holding plate (314) with plurality of guiding pins (316) assembled through them; a lead screw (318) has on end attached into the base (302) using bearings and the other end is connected to the motor (322) with its controller; and a lead screw nut (320) attached to the syringe plunger holding plate (308); wherein, the motor (322) generates vibrations in the range of 200 Hz to 350 Hz with discontinuous pulse of 20-30 seconds for 2-5 minutes and operates the lead screw (318) to perform aspiration and elimination of sample through the syringes; the top plate (302), base plate (304), syringe plunger holding plate (308), syringe finger flange holding plate (310), syringe barrel holding plate (312) and vial or tube or container holding plate (314) are arranged in a concentric manner wherein the centre of each of the plates are in same axis; the guiding pins (316) helps in to drive rotations to the syringe finger flange holding plate (310), syringe barrel holding plate (312) and vial or tube or container holding plate (314) when the syringe plunger holding plate (308) derived by the lead screw (318); the syringe finger flange holding plate (310), syringe barrel holding plate (312) and vial or tube or container holding plate (314) are assembled on to the base (304) using bearing means and performs rotation movement only; and the syringe plunger holding plate (308) is assembled on the lead screw (318) through lead screw nut (320) and performs rotation and liner movement both.

11. The device (300) for isolating and enriching pathogenic nucleic acids as claimed in claim 10, wherein the syringe plunger holding plate (308) has plurality of cavities (308e) to hold the plunger head of the syringe (100).

12. The device (300) for isolating and enriching pathogenic nucleic acids as claimed in claim 10, wherein the finger flange holding plate (310) has plurality of cavities (310e) to hold the finger flange of the syringe (100).

13. The device (300) for isolating and enriching pathogenic nucleic acids as claimed in claim 10, wherein the syringe barrel holding plate (312) has plurality of cavities (312e) to hold the barrel of the syringe (100).

14. The device (300) for isolating and enriching pathogenic nucleic acids as claimed in claim 10, wherein the vial or tube or container holding plate (314) has plurality of cavities (314e) to hold the tube or container (400).

15. The device (300) for isolating and enriching pathogenic nucleic acids as claimed in claim 10, wherein the base plate (304) has plurality of extrusions (304c) to hold the support rods (306).

16. The device (300) for isolating and enriching pathogenic nucleic acids as claimed in claim 10, wherein the top plate (302) has plurality of extrusions (302e) to hold the support rods (306).

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