Claims:WE CLAIM:
1. A method for extracting DNA or RNA or combination thereof from a test sample in a single container, the said method involving compositions for lysis, wash, and elution steps; wherein,
lysis composition comprises paramagnetic particles;
wash composition comprises alcohol; and
elution composition comprises acidic solution.
2. The method as claimed in claim 1, wherein the test sample is any biological or non-biological sample suspected of containing DNA and/or RNA.
3. The method as claimed in claim 1, wherein the test sample volume is up to 1ml.
4. The method as claimed in claim 1, wherein the use of compositions of the method, as a whole or in part is automated.
5. The method as claimed in claim 1, wherein the compositions of the method, as a whole or in part are lyophilized.
6. A method for extracting DNA or RNA or combination thereof from a test sample in a single container, the said method comprising;
contacting the sample with a lysis composition for simultaneous sample lysis, precipitation, and binding of DNA and/or RNA to paramagnetic particles;
washing the DNA and/or RNA bound paramagnetic particles with wash composition; and

eluting DNA and/or RNA bound to paramagnetic particles with an elution composition.
7. The method as claimed in claim 6, wherein the lysis composition comprises lysis buffer, lysis enhancer buffer, carrier RNA, paramagnetic particles, and isopropanol.
8. The method as claimed in claim 6, wherein the test sample is incubated with the lysis composition at temperature ranging from 50 °C to 60 °C.
9. The method as claimed in claim 7, wherein the lysis buffer is selected from Tris, Tris chloride, Tris hydrochloride, Tris-EDTA, phosphate and carbonate.
10. The method as claimed in claim 7, wherein the lysis buffer comprises sodium salt selected from sodium citrate, sodium chloride, sodium phosphate, sodium carbonate, sodium sulphate and sodium acetate.
11. The method as claimed in claim 7, wherein the lysis buffer comprises detergent selected from sodium dodecyl sulphate, Triton-X and Tween 20.
12. The method as claimed in claim 7, wherein the lysis buffer comprises chaotropic salt selected from guanidinium thiocyanate, guanidine hydrochloride, guanidine isothiocyanate, and cyanoguanidine isosulphate.
13. The method as claimed in claim 7, wherein the lysis enhancer buffer comprises lysis enzyme selected from nuclease, RNase, DNase, protease, and proteinase K.
14. The method as claimed in claim 13, wherein the lysis enzyme is proteinase K.
15. The method as claimed in claim 14, wherein proteinase K is in lyophilized or stabilized liquid form.
16. The method as claimed in claim 15, wherein proteinase K is stabilized using calcium chloride and/or glycerol.
17. The method as claimed in claim 1, wherein the wash composition carries out at least two washes of DNA and/or RNA bound paramagnetic particles; wherein, one wash comprises at least 60% of isopropanol and the other wash comprises at least 80% of ethanol.
18. The method as claimed in claim 17, wherein the wash composition comprises Tris hydrochloride, sodium acetate, and sodium azide.
19. The method as claimed in claim 1, wherein the elution composition comprises acidic solution selected from water having pH less than 7 such as nuclease free water, de-ionized water, DEPC autoclaved water, and acidic buffer.
20. The method as claimed in claim 6, wherein the elution of DNA and/or RNA is carried out at a temperature ranging from 75 °C to 85°C.
21. The method as claimed in claim 1, wherein the lysis composition comprises paramagnetic particles selected from magnetic beads, paramagnetic nanoparticles, microparticles that may be uncoated or coated with polymers, silica, or hydroxyapatite with terminal functionality.
22. A kit for the extraction of DNA or RNA or combination thereof from a test sample in a single container, involving;
Lysis composition comprising paramagnetic particles;
Wash composition comprising alcohol; and
Elution composition comprising acidic solution.
23. The kit as claimed in claim 22, wherein the test sample is any biological or non-biological sample suspected of containing DNA and/or RNA.
24. The kit as claimed in claim 22, wherein the test sample volume is up to 1ml.
25. The kit as claimed in claim 22, wherein the lysis composition comprises lysis buffer, lysis enhancer buffer, paramagnetic particles, carrier RNA, and isopropanol.
26. The kit as claimed in claim 22, wherein the lysis buffer comprises Tris hydrochloride, sodium acetate, sodium dodecylsulphate, ethylenediaminetetraaceticacid, sodium azide, and chaotropic salt.
27. The kit as claimed in claim 26, wherein the chaotropic salt is selected from guanidinium thiocyanate, guanidine hydrochloride, guanidine isothiocyanate, and cyanoguanidine isosulphate.
28. The kit as claimed in claim 25, wherein the lysis enhancer buffer comprises lysis enzyme selected from nuclease, RNase, DNase, protease, and proteinase K.
29. The kit as claimed in claim 28, wherein the lysis enzyme is proteinase K.
30. The kit as claimed in claim 29, wherein proteinase K is in lyophilized or stabilized liquid form.
31. The kit as claimed in claim 30, wherein proteinase K is stabilized using calcium chloride and/or glycerol.
32. The kit as claimed in claim 22, wherein the lysis composition comprises paramagnetic particles selected from magnetic beads, paramagnetic nanoparticles, microparticles that may be uncoated or coated with polymers, silica, or hydroxyapatite with terminal functionality.
33. The kit as claimed in claim 25, wherein the lysis composition comprises isopropanol at a concentration ranging from 50-70%.
34. The kit as claimed in claim 22, wherein the wash composition is used for carrying out at least two washes of the DNA and/or RNA bound paramagnetic particles in a single container; wherein, one wash comprises at least 60% of isopropanol and the other wash comprises at least 80% of ethanol.
35. The kit as claimed in claim 34, wherein the wash composition comprises Tris hydrochloride, sodium acetate, and sodium azide.
36. The kit as claimed in claim 22, wherein the elution composition is used for carrying out elution of DNA and/or RNA bound to paramagnetic particles at an acidic pH ranging from 4-6.
37. The kit as claimed in claim 22, wherein the elution composition comprises acidic solution selected from water having pH less than 7 such as nuclease free water, de-ionized water, DEPC autoclaved water, and acidic buffer.
38. The kit as claimed in claim 36, wherein the elution composition is used at a temperature ranging from 75 °C to 85°C.
39. The method and kit as claimed in claim 22, wherein the kit compositions, as a whole or in part are lyophilized.
40. The kit as claimed in claim 22, wherein the use of kit compositions, as a whole or in part is automated.
41. The kit as claimed in claim 22, wherein lysis composition comprises lysis buffer, lysis enhancer buffer, paramagnetic particles, carrier RNA, and isopropanol; wash composition comprises Tris hydrochloride, sodium acetate, sodium azide, and are reconstituted with up to 60% isopropanol or up to 80% ethanol; and elution composition comprises nuclease free water at an acidic pH ranging from 4-6.
42. The kit as claimed in claim 22, wherein the extracted DNA or RNA or combination thereof is directly applied for further analysis.

Dated this 28th Day of January 2022

Agent for the Applicant
IN/PA-1454
, Description:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
Title of invention:
A METHOD FOR EXTRACTING DNA OR RNA OR COMBINATION THEREOF FROM A TEST SAMPLE

APPLICANT:
MYLAB DISCOVERY SOLUTIONS PRIVATE LIMITED
An Indian entity having address as:
PLOT NO 99-B, LONAVALA INDUSTRIAL CO-OPERATIVE ESTATE LTD,
NANGARGAON, LONAVALA, PUNE – 410401
MAHARASHTRA, INDIA
The following specification describes the invention and the manner in which it is to be performed.
CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY
The present application does not claim priority from any other patent application(s).
TECHNICAL FIELD
The present disclosure relates to the field of nucleic acid extraction. Described subject matter herein, in general relates to a method for extracting DNA or RNA or combination thereof from a test sample.
BACKGROUND
Nucleic acids (NA) such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) are carriers of genetic information that form the material basis for gene expression. They are configured to determine the inherited characteristics of every living thing. According to the central dogma, genetic information stored in the DNA is transcribed into RNA and further translated into proteins that form the functional units of heredity. Thus, the genetic material in all free-living organisms and most viruses is DNA, while RNA is the genetic material of some viruses and is also present in all living cells, where it mediates important processes such as protein synthesis. Since nucleic acids are such vital molecules in all living organisms, they are central to molecular biology research as well as for disease detection or molecular diagnostics. Consequently, nucleic acid extraction (NAE) is an integral step for such analysis and is the subject of constant research.
Commonly employed nucleic acid extraction methods include cesium chloride (CsCl) / ethidium bromide (EtBr), phenol-chloroform, alkaline lysis, sodium dodecylsulphate (SDS) extraction, TriZol extraction, high salt precipitation, and ion exchange.
Extraction of nucleic acids has also been carried out using solid supports such as silica-based membranes or columns or paramagnetic particles as is known to a skilled person in the art. Use of paramagnetic particles eliminates centrifugation steps normally employed in conventional methods. Applying a magnetic field to the sides of a container containing a mixture of such paramagnetic particles and a sample causes them to aggregate without denaturing or damaging the sample or its components. Furthermore, lately, using paramagnetic particles has become popular due to their high potential for automation.
Over a period, advances such as introducing solid phase substrates in conjugation with the conventional methods were made to devise superior extraction protocols. Solid phase methods involve adsorption of nucleic acids on solid substrates or matrices such as silica matrices, glass particles, diatomaceous earth, and paramagnetic particles under suitable conditions of pH, salts, and temperature.
Furthermore, most nucleic acid extraction methods often extract DNA and RNA separately and there are just a few protocols that perform their co-extraction. In general, co-extraction is ideal for obtaining multiple analytes (such as DNA and RNA), along with being timesaving and cost effective. Advancement in polymerase chain reaction (PCR) has led to the advent of numerous techniques employing PCR such as Nucleic Acid Amplification Tests (NAAT) as well as numerous nucleic acid based diagnostic kits that necessitate the extraction of analytes for accelerated analysis. Moreover, many times detection of pathogens with serological tests despite their high sensitivity and specificity and other surrogate markers becomes challenging because of the time gap between getting infected and development of seropositivity (window period). Any serological test undertaken during the window period (WP) leads to a false negative result because the antibodies are normally generated post this period, thus delaying the detection and posing a significant risk to society. Amongst pathogens, DNA or RNA viruses cannot be accurately detected, especially in the window period, increasing the difficulty in terms of detection and cost.
Automated systems are being increasingly utilized to minimize the risk of human errors such as failure to follow the exact protocol, measuring mishaps, laboratorial accidents, contamination of materials due to over-handling, and miscalculations of data. Such risks could be disastrous, especially in routine analytical processes such as extraction of nucleic acids as these may form the basis of health diagnosis and prognosis discussed hereinabove. Additionally, automated extraction methods pose numerous advantages over manual methods such as increased productivity and sample turnover, efficient use of raw materials, standardized protocols assuring high result reproducibility and improved safety. Automations also add versatility and reliability to the processes. In a preferred embodiment, the present disclosure involves paramagnetic particle-based nucleic acid extraction method due to its above advantages as well as a potential for automation and semi-automation. Thus, in a related embodiment, the use of components of the kit, in whole or in part is automated.
Paramagnetic particles are often employed for nucleic acid extraction. This method applies the lysis, wash, and elution buffers to carry out separation of nucleic acids. The lysis buffer or composition helps in cell lysis, precipitation, and binding of the nucleic acids to the paramagnetic particles, while the wash buffer or composition helps in removing the high salt contaminants to improve the purity of the analytes. The elution buffer is usually a low ionic strength composition that not only helps in separating the nucleic acids from the solid phase but also in stabilizing and storing the extracted nucleic acids for their further processing. Most of the protocols for co-extracting DNA and RNA apply an elution buffer with an alkaline pH which helps in facilitating feasible extraction and separation of DNA. But it can denature the RNA, which is co-eluted along with DNA during co-extraction. “Magnetic particles for the separation and purification of nucleic acid”.
Due to the persistent requirement of extracting DNA and RNA in medical and molecular sciences, there is always a need to design protocols which would efficiently and fastidiously extract the target nucleic acids from a wide range of test samples and provide high purity grade analytes, that could directly be applied for further downstream processing. Thus, the present disclosure discloses a method, composition, and kit thereof for efficiently extracting DNA or RNA or combination thereof from a wide range of test samples using a paramagnetic particle-based extraction.
SUMMARY
The instant disclosure relates to a method for extracting DNA or RNA or combination thereof from a test sample in a single container, the said method involving compositions for lysis, wash, and elution steps; wherein, lysis composition comprises paramagnetic particles; wash composition comprises alcohol; and elution composition comprises acidic solution.
Further, the instant disclosure involves a method for extracting DNA or RNA or combination thereof from a test sample in a single container, the said method comprising; contacting the sample with a lysis composition for simultaneous sample lysis, precipitation, and binding of DNA and/or RNA to paramagnetic particles; washing the DNA and/or RNA bound paramagnetic particles with wash composition; and eluting DNA and/or RNA bound to paramagnetic particles with an elution composition.
The present disclosure is also directed to a kit for the extraction of DNA or RNA or combination thereof from a test sample in a single container, involving; lysis composition comprising paramagnetic particles; wash composition comprising alcohol; and elution composition comprising acidic solution.
The instant disclosure also relates to a kit for extracting DNA or RNA or combination thereof from a test sample in a single container comprising lysis, wash, and elution compositions; wherein, lysis composition comprises lysis buffer, lysis enhancer buffer, paramagnetic particles, carrier RNA, and isopropanol; wash composition comprises alcohol, Tris hydrochloride, sodium acetate, and sodium azide; and elution composition comprises acidic solution of pH 4-6.
Further the instant disclosure provides a kit for extracting DNA or RNA or combination thereof from a test sample in a single container using paramagnetic particles, the said kit comprising lysis, wash, and elution compositions; wherein, the lysis composition performs simultaneous sample lysis and precipitation, and binding of DNA and/or RNA to the said paramagnetic particles; the wash composition carries out at least two washes of the DNA and/or RNA bound paramagnetic particles; wherein, one wash comprises at least 60% of isopropanol and the other wash comprises at least 60% of ethanol; and the elution composition carries out elution of DNA and/or RNA at an acidic pH ranging from 4-6.
BRIEF DESCRIPTION OF DRAWINGS
The detailed description is described with reference to the accompanying Figures.
Fig. 1 is a schematic representation of DNA and/or RNA extraction carried out using paramagnetic particles.
Fig. 2 demonstrates PCR efficiency, R^2 and slope for extracted HCV RNA linearity IU/ml wherein the X axis represents the Ct value, and the Y axis represents log IU/ml.
Fig. 3 shows PCR efficiency, R^2 and slope for extracted HBV DNA linearity IU/ml, wherein the X axis represents the Ct value, and the Y axis represents log IU/ml.
Fig. 4 shows PCR efficiency, R^2 and slope for extracted SARS-CoV-2 (N gene), wherein the X axis represents log IU/ml, and the Y axis represents the Ct value.

DETAILED DESCRIPTION
Reference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments,” “in one embodiment,” or “in an embodiment” in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
Before the present method, composition and kit are described, it is to be understood that this disclosure is not limited to the particular method, composition and kit as described, as there can be multiple possible embodiments which are not expressly illustrated in the present disclosure but are still practicable within the scope of the present disclosure.
Further, reference throughout the specification to “composition”, or “compositions” refers to a specific formulation or dosage form of an active/functional element or components either alone or with essential compatible components and carriers for the intended use or a product comprising the specified ingredients in the specified amounts or any product which results, directly or indirectly, from combination of the specified ingredients in the specified amount. Also, the technical solutions offered by the present disclosure are clearly and completely described below. Examples in which specific reagents or conditions may not have been specified, have been conducted under conventional conditions or in a manner recommended by the manufacturer.
The instant disclosure relates to a method for extracting DNA or RNA or combination thereof from a test sample in a single container, the said method involving compositions for lysis, wash, and elution steps; wherein, lysis composition comprises paramagnetic particles; wash composition comprise alcohol; and elution composition comprises acidic solution.
The instant disclosure is enabled as detailed in the following embodiments.
First aspect of the present disclosure relates to a method for extracting DNA or RNA or combination thereof from a test sample in a single container, the said method involving compositions for lysis, wash, and elution steps; wherein, lysis composition comprises paramagnetic particles; wash composition comprises alcohol; and elution composition comprises acidic solution.
Alternatively, the present disclosure relates to a method for co-extracting DNA and RNA from a test sample in a single container, the said method involving compositions for lysis, wash, and elution steps; wherein, lysis composition comprises paramagnetic particles; wash composition comprises alcohol; and elution composition comprises acidic solution.
In an embodiment, the test sample is any biological or non-biological sample suspected of containing DNA and/or RNA. Biological sample includes but is not limited to, K2EDTA blood, K2EDTA plasma, serum, oropharyngeal swab, nasopharyngeal swab, nasal/oral swabs, genital swabs, bronchoalveolar lavage, tracheal aspirate, tissue, sputum, dried blood, and urine.
In a related embodiment, the test sample volume is up to 1ml. The present disclosure may provide flexibility in processing a sample through various volume sizes. Sample size plays an integral role in determining the outcome of extraction method. As known to a skilled person, a large sample size has obvious advantages of providing better and accurate data by reducing sensitivity problems. On the other hand, smaller sample size prevents financial, transportation, storge, and time commitments.
An embodiment involves extraction of DNA or RNA or DNA and RNA together from a test sample in a single container, preferably extraction of DNA and RNA.
In a related embodiment, extraction of DNA or RNA or combination thereof from a test sample involves silica membrane or paramagnetic particles.
It is known to a skilled person that extraction methods using paramagnetic particles have developed into a popular approach due to their high potential for automation. Automated systems have been increasingly utilized to minimize the risk of human errors such as, but are not limited to, failure to follow the exact protocol, measuring mishaps, spillages, laboratorial accidents, contamination of materials due to over-handling, and miscalculations of data. Risk of human error is the combination of the likelihood of occurrence of error and the severity of error, and therefore could be disastrous in a routine analytical process such as extraction of nucleic acids that may form the basis of any health diagnosis and prognosis discussed hereinabove. In addition to the advantages enlisted herein, an automated extraction method highlights numerous advantages over manual method such as higher production rates and increased productivity, efficient use of raw materials, better product quality, assurance of high reproducibility, improved safety, and established protocol. It also encourages versatility and reliability to prepare high yield of pure analytes from a broader variety of samples. Furthermore, the lack of centrifugation steps, normally employed in conventional methods, that can produce shear forces and cause breaking of nucleic acids helps to maintain intact and longer nucleic acid fragments. The application of magnetic field to the side of the vessel containing the sample mixed with the functionalized paramagnetic particles during the extraction is enough to aggregate the target particles without denaturing or damaging them. So, in a preferred embodiment, extraction of DNA or RNA or combination thereof involves paramagnetic particles. It is also known to a skilled person that DNA and/or RNA extraction methods using paramagnetic particles conventionally apply suitable buffers for carrying out lysis, wash, and elution steps depending on the type of extraction process to be devised. In order to limit the risk of cross-contamination, truncate the overall turnaround time, limit multiple pipetting, reduce material wastage, and requirement of equipment and space associated with DNA and/or RNA extraction, in a preferred embodiment, a method for extracting DNA or RNA or combination thereof from a test sample is carried out in a single container. A container is any vessel capable of holding the components or compositions of the method for carrying out the present disclosure. The advantage of using a single container lies with its versatility since it can carry out a sequence of processes without the need to break the containment. This is particularly useful when processing toxic, infectious, or highly potent samples.
One embodiment of the present disclosure is comprised of a method for extracting DNA or RNA or combination thereof from a test sample in a single container, the said method comprising:
contacting the sample with a lysis composition for simultaneous sample lysis, precipitation, and binding of DNA and/or RNA to paramagnetic particles;
washing the DNA and/or RNA bound paramagnetic particles with wash composition; and
eluting DNA and/or RNA bound to paramagnetic particles with an elution composition.

A lysis composition facilitates breaking of proteins and cell walls in order to isolate the nucleic acid/s present within. The type of components and the components present in the lysis composition depends on the cell source (tissue culture, plant, bacteria, fungi, virus, etc.), and whether the cells are in a structure and the type of structure. Cellular proteins are normally bound to the nucleic acids and are required to be separated feasibly during the process. Nucleic acids are generally much more resistant to denaturation than most proteins.
Conventionally, samples are incubated with lysis composition at a temperature ranging from 20°C to 70°C, with the optimum temperature for cell lysis ranging between room temperature and 60°C. The temperature used for lysis composition depends on factors such as the components added, the pH required to be achieved, the type of cell to be lysed, and the type of nucleic acid to be extracted, as known to a skilled person. Generally, the enzymes used for undertaking cell and protein lysis help in determining the optimum lysis temperature.

In a preferred embodiment, the test sample is incubated with the lysis composition at a temperature ranging from 50 °C to 60 °C, preferably from 53°C to 58°C, most preferably 56°C.

For sample lysis, the sample is mixed with the lysis composition and then incubated from about 5 minutes to 45 minutes. The time required for lysis depends on numerous factors such as the composition of lysis buffer, the pH required to be achieved, the type of sample to be lysed, and the type of nucleic acid to be extracted, efficacy and amount of the lysis enzyme used, as known to a skilled person.

In a preferred embodiment, the test sample and lysis composition are incubated for about 15 minutes to 25 minutes.

It is known to a skilled person that alkaline lysis is reliable, fast and relatively clean way to obtain nucleic acids from a sample and is thus the method of choice for isolating DNA and/or RNA, circular plasmid DNA, from human, as well as bacterial cells.

In a preferred embodiment, the pH of the lysis composition ranges from pH 7.0 to pH 8.0.
Lysis composition comprises components that carry out efficient cell lysis during nucleic acid extraction and co-extraction. The type and the concentration of components to be added in the lysis composition depends on factors such as the type of the cell to be lysed, the activity of the enzyme, the pH required for performing efficient lysis, and the temperature at which optimal lysis is undertaken.
In one embodiment, the lysis composition comprises lysis buffer, lysis enhancer buffer, carrier RNA, paramagnetic particles, and isopropanol.
In a preferred embodiment lysis composition comprises lysis buffer, lysis enhancer buffer, carrier RNA, paramagnetic particles and isopropanol that perform simultaneous sample lysis, precipitation, and binding of DNA and/or RNA to paramagnetic particles.
The primary purpose of lysis buffer is isolating the molecules of interest and keeping them in a stable environment. The important factors considered while choosing the buffer are pH, ionic strength, usage of detergent, protease inhibitors to prevent proteolytic processes.
In one embodiment, the lysis buffer may comprise buffers selected from a group of Tris, Tris chloride, Tris hydrochloride, Tris-EDTA, phosphate, carbonate, citrate and others known to a skilled person in the art. In a preferred embodiment, the lysis buffer is selected from Tris, Tris chloride, Tris hydrochloride, Tris-EDTA, phosphate and carbonate. Most preferably, the lysis buffer comprises Tris hydrochloride. Tris is highly soluble in water and hydrochloride prevents overshooting of pH, facilitating achievement of an alkaline pH, that is required for cell lysis and release of nucleic acids. Tris hydrochloride in concentration ranging from 0.5M to 1.5M may be particularly preferred.
In an embodiment, the lysis buffer may further comprise sodium salt selected from sodium citrate, sodium chloride, sodium phosphate, sodium carbonate, sodium sulphate, sodium acetate or combinations thereof that are known to a skilled person in the art. In a preferred embodiment, the lysis buffer comprises sodium salt selected from sodium citrate, sodium chloride, sodium phosphate, sodium carbonate, sodium sulphate and sodium acetate. Most preferably, the lysis buffer comprises sodium acetate. Sodium acetate in concentration ranging from 2.0M to 4.0M may be particularly preferred.
In a further embodiment, the lysis buffer comprises detergent selected from sodium dodecyl sulphate, Triton-X and Tween 20; preferably sodium dodecyl sulphate.
Detergents are organic amphipathic (with hydrophobic tail and a hydrophilic head) surfactants. They are used to separate membrane proteins from membrane because the hydrophobic part of detergent can surround biological membranes and isolate the target molecules. Sodium dodecyl sulphate in up to 5% stock concentration may be particularly preferred.
In another embodiment, the lysis buffer may also comprise additives such as stabilizing/reducing agent selected from dithiothreitol (DTT), dithioerythritol (DTE), glycerol and /or chelating agent such as ethylenediaminetetraacetic acid (EDTA), anti-microbials such as sodium azide or combinations thereof that are known to a skilled person in the art.
In a preferred embodiment, the lysis buffer comprises ethylenediaminetetraaceticacid (EDTA) and more preferably ethylenediaminetetraacetic acid at a concentration ranging from 0.3M to 0.8M.
In a related embodiment, the lysis buffer comprises sodium azide and preferably sodium azide in concentration ranging from 0.5 to 1.5%.
Additionally, the lysis buffer comprises chaotropic salt selected from guanidinium thiocyanate, guanidine hydrochloride, guanidine isothiocyanate, and cyanoguanidine isosulphate. It is known to a skilled person that chaotropic salts increase the solubility of nonpolar substances in water. They denature proteins due to their ability to disrupt hydrophobic interactions. High concentrations of chaotropic salt also facilitates binding of the nucleic acids to membranes or particles. In a preferred embodiment, the lysis buffer comprises chaotropic salt selected from guanidinium thiocyanate, guanidine hydrochloride, guanidine isothiocyanate, and cyanoguanidine isosulphate, most preferably, the lysis buffer comprises guanidine thiocyanate in high concentrations and particularly in concentration ranging from 2M to 6M.
In an embodiment, the lysis enhancer buffer comprises lysis enzyme selected from nuclease, RNase, DNase, protease, and proteinase K.
In a related embodiment, lysis enzyme is in a lyophilized or stabilized liquid form. The presence of lysis enzyme in liquid state exhibits advantages known to a skilled person in the art such as easy transportation, feasible storage, prevention of unnecessary wastage, and easy formulation over the lyophilized enzyme. On the other hand, lyophilization or freeze-drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. A rehydration buffer may be applied in order to rehydrate the constitution and resume their application in the process. Lyophilization ensures feasible storage and transport of the materials as known to a skilled person.
Additionally, the lysis enzyme may be stabilized using additives such as alkaline salts such as sodium, potassium, calcium and/or long-chain alcohols such as glycerol, sorbitol, xylitol.
In a preferred embodiment, lysis enzyme is proteinase K. Proteinase K is a non-specific serine endopeptidase that catalyses the cleavage of peptide bonds at the carboxylic side of aromatic, aliphatic, or hydrophobic amino acid residues. Besides the digestion of unwanted proteins, Proteinase K quickly inactivates the nucleases which might degrade the nucleic acids present in the sample. The proteolytic digestion decreases the level of contaminants in the nucleic acid extract and prevents nucleic acids degradation. Proteinase K in concentrations ranging from 10mg to 30mg may be particularly preferred.
In a related embodiment, proteinase K is in a lyophilized or stabilized liquid form and preferably stabilized using calcium chloride and/or glycerol, and more preferably, stabilized using 1.2mM to 1.6mM of calcium chloride and up to 40% volume fraction of glycerol.
Various modifications to the embodiments will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments.
Further, paramagnetic particles encompassing all its forms such as magnetic beads, paramagnetic nanoparticles, microparticles are constituted of one or several magnetic ores such as magnetite (Fe3O4) or maghemite (gamma Fe2O3), and may be coated with a matrix of polymers, silica, or hydroxyapatite with terminal functionality as is known to a skilled person in the art.
In a preferred embodiment, the lysis composition comprises paramagnetic particles selected from magnetic beads, paramagnetic nanoparticles, microparticles that may be uncoated or coated with polymers, silica, or hydroxyapatite with terminal functionality.
In some embodiments, lysis composition comprises internal control (IC). An internal control is a synthetic fragment that may be endogenously or exogenously added to monitor sample processing during extraction. In a preferred embodiment, the internal control (IC) is exogenously added at the lysis step.
In further embodiments, in addition to the components described hereinabove, lysis composition comprises carrier RNA. As known to a skilled person, the carrier RNA is a seeding agent that is used for qualitative precipitation and purification of DNA and RNA. It facilitates recovery of short fragments (< 200 bp) or low amounts of nucleic acids.
In a preferred embodiment, lysis composition comprises isopropanol as a precipitating agent. Isopropanol when added to the lysis buffer in the presence of salts, facilitates feasible precipitation of nucleic acids isolated from the lysed cell. Isopropanol in concentrations ranging from 50-70% may be particularly preferred.
Wash composition removes contaminants such as protein which also tends to bind non-specifically to the paramagnetic particles. Typically, the wash composition contains a high concentration of ethanol, sodium salts, and a Tris buffer at a slightly alkaline pH. The salt concentration and high pH ensure that the nucleic acids remain bound to the paramagnetic particles, while ethanol promotes the precipitation of nucleic acids.
In a one embodiment, wash composition comprises alcohol such as ethanol, isopropanol, propanol, butanol, isobutanol, and ethylene glycol, preferably comprises isopropanol or ethanol, and more preferably comprises isopropanol and ethanol.
In a preferred embodiment, the wash composition carries out at least two washes of DNA and/or RNA bound paramagnetic particles; wherein, one wash comprises at least 60% of isopropanol and the other wash comprises at least 80% of ethanol.
In a related embodiment, the first wash comprises ethanol and the second wash comprises isopropanol, preferably first wash comprises isopropanol and the second wash comprises ethanol, and more preferably, first wash comprises at least 60% isopropanol and the second wash comprises at least 80% ethanol.
In a preferred embodiment, in addition to isopropanol and ethanol, the wash composition comprises Tris hydrochloride, sodium acetate, and sodium azide.
In further embodiment, wash composition comprises chaotropic salt selected from guanidinium thiocyanate, guanidine hydrochloride, guanidine isothiocyanate, and cyanoguanidine isosulphate.
In a related embodiment, wash composition comprises DNase and/or RNase free deionized buffer and preferably comprises nuclease free water.
One embodiment of the present disclosure comprises elution step after lysis and wash steps; wherein, the elution step involves elution composition.
An elution composition releases the desired nucleic acids from the paramagnetic particles. It is important that elution composition operates without altering the function or the activity of the desired nucleic acids. Elution must be carried out at a pH and a temperature at which nucleic acids bound to the paramagnetic particles are not denatured.
In one embodiment, the elution composition comprises acidic solution selected from water having pH less than 7 such as nuclease free water, de-ionized water, DEPC autoclaved water, and acidic buffer.
In a related embodiment, the elution composition is used at an acidic pH, and preferably at pH ranging from 4-6.
In a further embodiment, the elution of DNA and/or RNA is carried out at a high temperature and preferably at a temperature ranging from 75 °C to 85°C.
In a preferred embodiment, the elution composition comprises nuclease free water at an acidic pH, more preferably at an acidic pH ranging from 4-6, and particularly at a temperature ranging from 75 °C to 85°C.
Thus, the preferred embodiment of the present disclosure comprises a method for extracting DNA or RNA or combination thereof from a test sample in a single container, the said method comprising; contacting the sample with a lysis composition for simultaneous sample lysis, precipitation, and binding of DNA and/or RNA to paramagnetic particles; washing the DNA and/or RNA bound paramagnetic particles with wash composition; and eluting DNA and/or RNA bound to paramagnetic particles with an elution composition.
Alternatively, in one embodiment, the use of compositions of the method described hereinabove, as a whole or in part is automated.
In one embodiment, the compositions of the method described hereinabove, as a whole or in part are lyophilized.
Second aspect of present disclosure relates to lysis, wash, and elution compositions used for carrying out extraction of DNA or RNA or combination thereof.
In one embodiment, extraction of DNA or RNA or combination thereof is carried out using lysis, wash, and elution compositions; wherein, lysis composition comprises lysis buffer, lysis enhancer buffer, paramagnetic particles, carrier RNA, and isopropanol; wash composition comprises alcohol, Tris hydrochloride, sodium acetate, and sodium azide; and elution composition comprises nuclease free water at an acidic pH.

The compositions disclosed herein are applied in accordance with the method that has been previously described.
Third aspect of the present disclosure relates to a kit for the extraction of DNA or RNA or combination thereof from a test sample in a single container, involving;
Lysis composition comprising paramagnetic particles;
Wash composition comprising alcohol; and
Elution composition comprising acidic solution.

In the present disclosure, the term “kit” refers to a consumable or a cartridge comprising a set of articles, components, consumables, cartridges, or compositions in a packet required for a specific purpose.
In the present disclosure, the test sample is any biological or non-biological sample suspected of containing DNA or/and RNA, including but not limited to, K2EDTA blood, K2EDTA plasma, serum, oropharyngeal swab, nasopharyngeal swab, nasal/oral swabs, genital swabs, bronchoalveolar lavage, tracheal aspirate, tissue, sputum, dried blood, and urine.
In a related embodiment, the test sample volume is up to 1ml.
An embodiment involves extraction of DNA or RNA or DNA and RNA together from a test sample, preferably DNA and RNA extraction, and particularly DNA and RNA extraction in a single container.

In another embodiment, extraction of DNA or RNA or combination thereof from the test sample involves paramagnetic particles.
In a related embodiment, extraction of DNA or RNA or combination thereof from a test sample in whole or in part is automated.
A preferred embodiment involves co-extraction of DNA and RNA from a test sample in a single container; more preferably using paramagnetic particle-based co-extraction.
It is also known to a skilled person that DNA or RNA or combination thereof extraction methods using paramagnetic particles conventionally apply suitable buffers for carrying out lysis, wash, and elution steps
In a preferred embodiment, lysis, wash, and elution steps are carried out in a single container. A container is any vessel capable of holding the components or compositions of the kit for carrying out the present disclosure.
Preferred embodiment of the present disclosure is comprised of a kit for extracting DNA and/or RNA from up to 1ml volume of a test sample, the said kit comprising lysis, wash, and elution compositions.
In a related embodiment, the test sample is incubated with the lysis composition at a temperature ranging from 50 °C to 60 °C.

In a related embodiment, the test sample and the lysis composition are incubated for about 15 minutes to 25 minutes.
In a preferred embodiment, the pH of the lysis composition ranges from pH 7.0 to pH 8.0.
In one embodiment, lysis composition comprises lysis buffer, lysis enhancer buffer, carrier RNA, paramagnetic particles, and isopropanol.
In a preferred embodiment, lysis composition comprises lysis buffer, lysis enhancer buffer, carrier RNA, paramagnetic particles and isopropanol that performs simultaneous sample lysis, precipitation, and binding of DNA and/or RNA to paramagnetic particles.
In one embodiment, the lysis buffer comprises Tris hydrochloride, sodium acetate, sodium dodecylsulphate, ethylenediaminetetraaceticacid, sodium azide, and chaotropic salt.
In a preferred embodiment, the lysis buffer comprises Tris hydrochloride and more preferably in concentration ranging from 0.5 to 1.5M.
In a preferred embodiment, the lysis buffer comprises sodium acetate and more preferably in concentration ranging from 2.0M to 4.0M.
In a related embodiment, the lysis buffer comprises sodium dodecyl sulphate and preferably in up to 5% stock concentration.
In a preferred embodiment, the lysis buffer comprises ethylenediaminetetraacetic acid (EDTA) and more preferably in concentration ranging from 0.3M to 0.8M.
In a related embodiment, the lysis buffer comprises sodium azide and preferably in concentration ranging from 0.5 to 1.5%.
In a preferred embodiment, the lysis buffer comprises chaotropic salt selected from guanidinium thiocyanate, guanidine hydrochloride, guanidine isothiocyanate, and cyanoguanidine isosulphate. Most preferably, the lysis buffer comprises guanidine thiocyanate in high concentrations and particularly in concentration ranging from 2M to 6M.
In one embodiment, the lysis enhancer buffer comprises lysis enzyme selected from nuclease, RNase, DNase, protease, and proteinase K, preferably the lysis enzyme is proteinase K, and more preferably proteinase K is in concentrations ranging from 10mg-30mg.
In a related embodiment, proteinase K is in lyophilized or stabilized liquid form.
In further embodiment, proteinase K is stabilized using calcium chloride and/or glycerol, preferably, calcium chloride at a concentration ranging from 1.2mM to 1.6mM and/or up to 40% glycerol.
Additionally, in a related embodiment, the lysis composition comprises paramagnetic particles selected from magnetic beads, paramagnetic nanoparticles, microparticles that may be uncoated or coated with polymers, silica, or hydroxyapatite with terminal functionality.
In a preferred embodiment, lysis composition comprises an internal control (IC) and preferably an internal control exogenously added at the lysis step.
In further embodiments, in addition to the components described hereinabove, lysis composition comprises carrier RNA.
In a preferred embodiment, lysis composition comprises isopropanol at a concentration ranging from 50-70%.
In one embodiment, the wash composition is used for carrying out at least two washes of the DNA and/or RNA bound paramagnetic particles in a single container; wherein, one wash comprises at least 60% of isopropanol and the other wash comprises at least 80% of ethanol.
In a preferred embodiment, in addition to isopropanol and ethanol, the wash composition comprises Tris hydrochloride, sodium acetate, and sodium azide.
In further embodiment, wash composition comprises chaotropic salt selected from guanidinium thiocyanate, guanidine hydrochloride, guanidine isothiocyanate, and cyanoguanidine isosulphate.
In a related embodiment, wash composition comprises DNase and/or RNase free deionized buffer and preferably comprises nuclease free water.
In one embodiment, the elution composition is used for carrying out elution of DNA and/or RNA bound to paramagnetic particles at an acidic pH ranging from 4-6.
In a related embodiment, the elution composition comprises acidic solution selected from water having pH less than 7 such as nuclease free water, de-ionized water, DEPC autoclaved water, and acidic buffer.
In a further embodiment, the elution composition is used at a high temperature and preferably at a temperature ranging from 75 °C to 85°C.
In a preferred embodiment, the elution composition comprises nuclease free water at an acidic pH, more preferably at an acidic pH ranging from 4-6, and particularly at a temperature ranging from 75 °C to 85°C.
Preferred embodiment of the present disclosure comprises a kit for extracting DNA or RNA or combinations thereof from a test sample in a single container using paramagnetic particles, the said kit comprising lysis, wash, and elution compositions; wherein,
the lysis composition performs simultaneous sample lysis and precipitation, and binding of DNA and/or RNA to the said paramagnetic particles;
the wash composition carries out at least two washes of the DNA and/or RNA bound paramagnetic particles; wherein, one wash comprises at least 60% of isopropanol and the other wash comprises at least 80% of ethanol; and
the elution composition carries out elution of DNA and/or RNA at an acidic pH ranging from 4-6.

In a related embodiment, lysis composition comprises lysis buffer, lysis enhancer buffer, paramagnetic particles, carrier RNA, and isopropanol; wash composition comprises Tris hydrochloride, sodium acetate, sodium azide, and are reconstituted with up to 60% isopropanol or up to 80% ethanol; and elution composition comprises nuclease free water at an acidic pH ranging from 4-6.

Alternatively, in one embodiment, the kit compositions, as a whole or in part are lyophilized. Lyophilization or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. A rehydration buffer may be applied in order to rehydrate the constitution and resume their application in the process. It is known to a skilled person that lyophilization makes room temperature storage feasible with desirable stability, and reduces challenges to transport of the materials, thus maximising applications of the kit and ensuring wider reach.
In one embodiment, the use of kit compositions, as a whole or in part is automated. As known to a skilled person, automated systems have been increasingly utilized to minimize the risk of human errors such as failure to follow the exact protocol, measuring mishaps, laboratorial accidents, contamination of materials due to over-handling, and miscalculations of data. Risk of human error is the combination of the likelihood of occurrence of error and the severity of error, and could be disastrous, especially in a routine analytical process such as extraction of nucleic acids that may form the basis of health diagnosis and prognosis discussed hereinabove. In addition to the advantages enlisted herein, an automated system highlights numerous advantages over semi-automated and manual systems such as less labour, higher production rates and increased productivity, efficient use of raw materials, better product quality, assurance of high reproducibility, improved safety, and established protocol. It also encourages versatility and reliability to procure better results.
In a preferred embodiment, the extracted DNA or RNA or combination thereof is directly applied for further analysis.
The compositions of the kit disclosed herein are applied in accordance with the method that has been previously described.

In an embodiment, the kit of the present disclosure may be coupled with any automated or manual extraction setup. Preferably, the kit is used with closed automated system such as Mylab™ Compact series to perform initial steps of extraction during disease diagnosis.

In one aspect, the kit packet comprises containers and other packages for holding the components.
In another aspect, the kit packet also carries a user instructions manual.

Various modifications to the embodiment will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. However, one of ordinary skill in the art will readily recognize that the present disclosure is not intended to be limited to the embodiments illustrated but is to be accorded the widest scope consistent with the principles and features described herein.

The foregoing description shall be interpreted as illustrative and not in any limiting sense. A person of ordinary skill in the art would understand that certain modifications could come within the scope of this disclosure.

The features and properties of the present disclosure are described in further detail below with reference to examples.

Example 1

Extraction of nucleic acids using paramagnetic particles
In this example, viral nucleic acids (DNA and/or RNA) were extracted from up to 1ml of K2EDTA plasma sample obtained from up to 5ml of whole blood using conventional methods known to a skilled person in the art. The extraction process involves sample lysis, wash and elution steps elaborated below. An internal control (IC) was added at the start of the extraction in order to monitor the process. A negative/extraction control was used as a test control.

Sample lysis using lysis composition:
The lysis composition comprises lysis buffer, lysis enhancer buffer, paramagnetic particles, isopropanol, and carrier RNA. The lysis buffer was prepared in a sample tube by mixing the reagents shown in Table 1. Prior to that, a comparative study was carried out to determine the sodium salt to be incorporated in the lysis buffer.

Table 1: Components of lysis buffer
Reagent Stock Concentration pH
Tris HCl 1M 6.0 -6.8
Sodium acetate =3M 9-9.4
Guanidine thiocyanate (freshly prepared) >4 M NA
EDTA 0.5 M 8-8.5
DTT =10% 4-5
SDS =5% 7-8
Sodium azide 1% NA
DEPC treated water for volume make up 5-7
Final pH 7.0-8.0

For this, DNA and RNA extracted separately from plasma samples using commercially available extraction kits (ck) were directly subjected to real time RT-PCR and used as references.
Further, for comparing efficiency of extraction, DNA and RNA extracted separately from plasma using a lysis buffer comprising sodium acetate or sodium chloride was used. Real time RT-PCR data of the extracted samples was analysed. The obtained Ct values demonstrated comparative values with those obtained using commercially available extraction kits (ck).

Table 2: Comparative study between sodium acetate and sodium chloride for RNA extraction
Sample Reference Ct Sodium acetate (=300mM) Sodium chloride (=250mM) ?Ct for Sodium acetate ?Ct for Sodium chloride
Viral RNA IC Viral RNA IC Viral RNA IC Viral RNA IC Viral RNA IC
1 31.45 33.95 31.43 33.52 32.65 34.29 0.02 0.43 -1.2 -0.34
2 31.33 35.03 31.39 35.65 32.34 36.50 -0.06 -0.62 -1.01 -1.47
3 31.65 34.07 31.09 34.89 33.71 35.38 0.56 -0.82 -2.06 -1.31

Table 3: Comparative study between sodium acetate and sodium chloride for DNA extraction
Sample Reference Ct Sodium acetate (=300mM) Sodium chloride (=250mM) ?Ct for Sodium acetate ?Ct for Sodium chloride
Viral DNA IC Viral DNA IC Viral DNA IC Viral DNA IC Viral DNA IC
1 31.40 31.65 31.32 31.69 32.49 32.51 0.08 -0.04 -1.11 -0.86
2 31.32 31.82 31.34 31.78 32.00 33.21 -0.02 0.04 -1.89 -1.39
3 31.76 31.67 31.64 31.83 32.50 32.78 0.12 -0.16 -1.02 -1.11

Thus, Table 2 shows the Ct values for RNA extraction, while Table 3 for DNA extraction performed using sodium acetate and sodium chloride. From Table 2 and Table 3 it was determined that lysis buffer comprising sodium acetate showed comparable results with the reference and thus sodium acetate was incorporated in the lysis buffer.

The lysis enhancer buffer was prepared by dissolving freeze-dried proteinase K powder (up to 20mg/ml) in a stabilizing solution of calcium chloride and glycerol. Optimum concentrations of calcium chloride for stabilizing proteinase K were determined by extracting DNA and RNA separately using lysis enhancer buffer having various concentrations of calcium chloride namely =1 mM, =1.5mM and =2 mM. Ct values from real time RT-PCR of the extracted samples were analysed and compared with reference values obtained using commercial kits (ck). Similarly, 40%, 50%, and 60% (v/v) of glycerol was tested to determine optimal glycerol concentrations.

Lysis enhancer buffer comprising 1.2mM to 1.6mM of calcium chloride and up to 40% volume fraction of glycerol showed comparable results with the reference and were selected. Thus, the lysis enhancer buffer was prepared by dissolving freeze-dried proteinase K powder (up to 20mg/ml), glycerol (up to =40%) and calcium chloride (up to 1.5mM).

The lysis composition further comprises isopropanol and carrier RNA. Addition of isopropanol and carrier RNA enhanced the simultaneous precipitation of the nucleic acids improving their overall yields or recovery. An internal control of synthetic oligonucleotide was used for monitoring the extraction process.

The lysis composition was prepared according to the reagents listed in Table 4 and a K2EDTA plasma sample was mixed with the lysis/binding composition (Table 4) in a sample tube. The mixture was incubated at 56°C for up to 20 minutes with continuous shaking at up to 900 rpm.

Table 4: Lysis/binding composition for 1ml sample volume
Sr. No. Reagents Volume (µl) /Rx
1 Lysis Buffer (see Table 2) 700
2 Lysis enhancer buffer 70
3 Paramagnetic particles 70
4 Internal Control (IC) 2.5
5 Carrier RNA 2.5
6 Isopropanol 1360
Total Volume 2065

Following incubation, magnetic separation of the lysis composition in the sample tube was performed and the supernatant was discarded.

Washing of DNA and/or RNA bound paramagnetic particles:
The nucleic acid bound paramagnetic particles were washed with a wash composition. Comparison of washing with a single wash buffer comprising ethanol (conventional) to washing with two wash buffers, one comprising ethanol and another comprising isopropanol has been shown in Table 5.

Plasma samples with known concentrations of nucleic acid (100ng/µl) were contacted with lysis composition and subjected to one or two washes as mentioned above. An analysis of the resulting washes and the elute after elution demonstrated better quality of eluted nucleic acids as upon performing two washes as shown in Table 5. Thus, two wash buffers, one comprising ethanol and the other comprising isopropanol were selected.

Table 5: Comparative study between nucleic acid recovery using two different wash buffers of Mylab™ and using conventional single wash buffer method (only ethanol)
Sample Two wash buffers method Conventional single wash method
Recovery concentration ng/µl Ratio 260/280 Recovery concentration ng/µl Ratio 260/280
1 90 1.8 62 1.8
2 93 1.7 67 2.0
3 95 1.8 60 1.7

To determine the optimal isopropanol concentration, samples containing known concentration of DNA were used. The binding capacities of DNA to the paramagnetic particles was evaluated using wash buffer 1 comprising 40%, 50%, and 60% of isopropanol. As seen from Table 6, wash composition comprising 60% isopropanol showed better binding and thus was selected. So, for this example, 60% isopropanol was used in the wash composition.

Table 6: Determination of optimal isopropanol concentration in wash buffer 1
Wash buffer 1 with 40% isopropanol Wash buffer 1 with 50% isopropanol Wash buffer 1 with 60% isopropanol
DNA input 1.4ng/µl 1.4ng/µl 1.4ng/µl
Recovery of DNA in supernatant (ng/ul) 0.27 0.14 0.07
0.26 0.15 0.06
0.26 0.13 0.06
0.26 0.14 0.06
In solution % 19.04 10.09 4.63
%Binding capacity to paramagnetic particles 80.95 89.90 95.36

Thus, the nucleic acids bound paramagnetic particles were washed using two wash buffers prepared by mixing the reagents as shown in Tables 7 and Table 8. Wash buffer 1 was reconstituted by adding 60% isopropanol of the final volume and Wash buffer 2 was reconstituted by adding 80% ethanol of the final volume.

About 1200 µl of Wash buffer 1 was added to the sample tube, mixed well with the contents, followed by magnetic separation. The supernatant was discarded.
Then up to 1800 µl of Wash buffer 2 was added to the sample tube, followed by mixing and magnetic separation. The supernatant was again discarded, and the resulting nucleic acids bound paramagnetic particles were dried.

Table 7: Composition of Wash Buffer 1
Reagent Stock Concentration pH
Tris HCl 1M 6-8
Sodium acetate =3M 4-6
Guanidine thiocyanate =1M NA
Sodium azide Upto 1% NA
Nuclease free water for volume make up 5-7
Final pH 4.5-5.5

Table 8: Composition of Wash Buffer 2
Reagent Stock Concentration pH
Tris HCl 1M 6-8
Sodium acetate =3M 8-10
Sodium azide Upto 1%
Nuclease free water for volume make up 5-7
Final pH 7.0-8.0

The washed nucleic acid bound paramagnetic particles were dried and then the bound nucleic acids were eluted using an elution composition.

Elution of DNA and/or RNA from paramagnetic particles:
Several elution compositions were tested to determine optimal recovery of DNA and/or RNA from the paramagnetic particles.
For this, DNA and RNA were separately extracted from plasma samples with known concentrations of DNA and RNA using commercially available extraction kits (ck) which were then used as references for real time RT-PCR.

Elution compositions having acidic, neutral, and alkaline pH were used to elute DNA and RNA from paramagnetic particles and their recoveries were analysed using their real time RT-PCR Ct values compared to those of the reference.
As shown in Table 10 and Table 11, elution at acidic pH showed better recovery and therefore, acidic solutions were used for elution.
Table 9: pH ranges of tested Elution compositions
1 Acidic 5-6
2 Neutral 7-8
3 Alkaline 8-9

Table 10: Elution composition pH for DNA elution
Reference (ck) Elution Buffer pH 5-6 Elution Buffer pH 7-8 Elution Buffer pH 8-9 ?CT for pH 5-6 ?CT for pH 7-8 ?CT for pH 8-9
DNA IC DNA IC DNA IC DNA IC DNA IC DNA IC DNA IC
S1 34.24 26.45 33.43 26.44 33.67 27.42 36.54 29.76 0.81 0.01 0.57 -0.98 -2.3 -2.34
S2 29.43 26.89 27.96 26.86 28.76 27.34 32.65 29.45 1.47 0.03 0.67 -0.48 -3.89 -2.11
S3 24.63 26.73 23.09 26.34 24.98 27.11 27.77 29.12 1.54 0.39 -0.35 -0.77 -2.79 -2.01

Where samples are denoted as S1, S2 and S3

Table 11: Elution composition pH for RNA elution
Reference (ck) Elution Buffer pH 5-6 Elution Buffer pH 7-8 Elution Buffer pH 8-9 ?CT for pH 5-6 ?CT for pH 7-8 ?CT for pH 8-9
RNA IC RNA IC RNA IC RNA IC RNA IC RNA IC RNA IC
S1 33.65 28.33 32.66 28.46 34.56 29.87 35.67 33.12 0.99 -0.13 -0.91 -1.54 -2.02 -1.89
S2 28.31 28.71 26.89 28.68 30.08 29.79 29.99 33.66 1.42 0.03 -1.77 -1.08 -1.68 -2.35
S3 25.13 28.67 23.88 28.19 26.99 29 28.66 33.01 1.25 0.48 -1.86 -0.33 -3.53 -2.25

Where samples are denoted as S1, S2 and S3

Amongst the elution compositions with pH ranging from pH 4-6, DEPC treated autoclaved water was the most promising as there were no additives that may potentially interfere with further processing or analysis.

Thus, up to 120 µl of elution composition was added to the sample tube and the resuspended paramagnetic particles were vortexed and incubated for up to 5 minutes at around 80°C.

Magnetic separation was performed again and the supernatant containing viral nucleic acids was transferred to a new tube for further analysis, or downstream applications or stored at -20°C or -80°C until further use.
Table 12 lists all the components required for the extraction of nucleic acids from 1 mL sample.
Table 12: Compositions of reagents used DNA and/or RNA extraction for 1ml sample volume
Sr. No. Reagents Volume (µl)/ Rx
1 Lysis composition 700
2 Wash buffer 1 1200
3 Wash buffer 2 3600
4 Elution composition 120
5 Lysis enhancer buffer 70
6 Magnetic beads 70
7 Internal control (IC) 2.5
8 Carrier RNA 2.5
9 Isopropanol 1360

Example 2:
Nucleic acid extraction using automated paramagnetic particle-based extraction

Nucleic acids (DNA and/or RNA) were extracted from 200µl or 500µl or 1ml of K2EDTA plasma sample using an automated paramagnetic particle method comprising a thermoshaker and a magnetic assembly with a base plate, a heating block, a single reaction container, a magnetic strip, a nylon plate and temperature sensors. For this, pre-filled extraction cartridge and PCR cartridge, comprising lyophilized compositions were placed on an automated platform (such as Mylab™ Compact series in this example) along with other consumables on the deck in designated place. A skilled person may adjust the compositions depending on the sample size. The cartridges may comprise some or all of the compositions in a lyophilized state. Lyophilization ensures feasible storage and transport of the materials as known to a skilled person. Then, appropriate commands were given to start the pre-uploaded protocol. An automated platform such as from Mylab™ carries out the entire process from viral nucleic acid extraction to real time RT-PCR. Upon nucleic acid extraction, PCR amplification and detection were carried out and an in-built software analysed the data to generate results.
Programmable commands fed within the system help in extracting as well as co-extracting the nucleic acids under automated shaker and temperature conditions, facilitating minimized risk of human errors such as laboratorial accidents, contamination of materials due to over-handling, and miscalculations of data, as known to a skilled person. In addition to the advantages enlisted herein, an automated system highlights numerous advantages over semi-automated and manual systems such as higher production rates and increased productivity, efficient use of raw materials, better product quality, assurance of high reproducibility, improved safety, and established protocol. It also encourages versatility and reliability to procure better results.

In a semi-automated process, the extracted sample and the positive controls may be added to corresponding reaction tubes which may directly be put in a PCR machine and the PCR cycle may be performed as per Table 13.

Table 13: PCR Run conditions
Parameter Stage Temperature Time
Reverse Transcription Hold 50°C Up to 15 minutes
RT inactivation Hold 95°C Up to 20 sec
PCR amplification Cycle
(=50 cycles) 95°C
60°C 05 sec
Up to 30 sec *
(*data collection)

After performing the PCR run, fluorescence data may be collected during the extension step, followed by saving of the data captured by the data analysing software.

Example 3
Comparative study with the commercially available extraction kits
Nucleic acids were extracted from K2EDTA plasma samples using two commercial kits (Ck1 and Ck2) and the kit of the present invention (ML) using an automated process of Example 2. The extracted samples from three different lots were directly subjected to a real-time RT-PCR (total volume up to 50µl) using conditions shown in Table 7.
An analysis of Ct values for samples extracted using commercial kits and extraction kit of the present invention showed comparable results as shown in Table 14.

Table 14: Comparative study with commercially available kits

Reference Ct ML Ck1 Ck2 ?Ct for lot1 ?Ct for lot2 ?Ct for lot3
Viral RNA IC Viral RNA IC Viral RNA IC Viral RNA IC Viral RNA IC Viral RNA IC Viral RNA IC
S1 31.45 33.95 30.43 33.52 30.92 33.63 31.48 34.21 1.02 0.43 0.53 0.32 -0.03 -0.26
S2 31.33 35.03 29.39 33.65 31.37 33.51 31.28 33.53 1.94 1.38 -0.04 1.52 0.05 1.5
S3 31.65 34.07 31.09 33.89 31.7 33.3 31.8 34.37 0.56 0.18 -0.05 0.77 -0.15 -0.3

Where the samples are denoted as S1, S2 and S3

Evaluation based on PCR efficiency, R^2, and slope:
Serial 10 folds dilution of clinical samples and WHO viral standards for HBV and HCV were processed and analyzed for linear range.
As shown in Table 15, Table 16, Figure 2, and Figure 3, the PCR efficiency, R^2 and slope were in recommended range of 90-110%, =0.99 and -3.2 to -3.4 respectively.

Table 15: RNA virus dilutions
HCV linearity
Ct values IU/ml log IU/ml
17.05 1.4E+08 8.15
20.48 14000000 7.15
23.89 1400000 6.15
27.23 140000 5.15
30.39 14000 4.15
33.36 1400 3.15
36.15 140 2.15
38.59 40 1.60
40.01 14 1.15
42 1.4 0.15

Table 16: DNA virus dilutions
HBV Linearity data
Ct value Quantity in IU/ml log IU/ml
13.949 80000000 7.90
17.042 8000000 6.90
20.492 800000 5.90
23.897 80000 4.90
27.434 8000 3.90
30.831 800 2.90
33.936 80 1.90
38.313 8 0.90
39.86 5 0.70

Example 4
Extraction of SARS-CoV-2 RNA using the present extraction kit (ML).
Up to 200µl of respiratory nasal specimen was processed for SARS-CoV-2 viral nucleic acid extraction using the kit of the present invention (ML). The extraction was carried out using an automated process of Example 2. For SARS-Cov-2, each step of the extraction process required less time and thus the overall RNA extraction was accomplished within 30 minutes as described in Table 17.
The total time required for extraction (turnaround time) varies with the type of test sample (respiratory, saliva, blood, plasma and others) and/or specific source organism (HBV, HCV, HIV, or SARS-Cov-2 as in the present example) for the nucleic acids as is known to a skilled person in the art.

Table 17: Extraction procedure for SARS-CoV-2
Steps Time duration
Sample lysis and binding with continuous shaking (at 500C - 600 C) Up to 10 minutes
Washing using wash buffer 1 and/or wash buffer 2 Up to 1 minute each
Drying Up to 3 minutes
Elution (at 750 C - 800 C) Up to 5 minutes

The extracted samples were directly subjected to a real-time RT-PCR (total volume up to 50µl) using conditions shown in Table 7.

Evaluation based on PCR efficiency, R^2, and slope:
Serial 10 folds dilution of Standard/NIBSC code 20/190, winter respiratory panel SARS CoV-2 were processed and analyzed for linear range.
The PCR efficiency, R^2 and slope of SARS-CoV-2 demonstrated in Figure 4 showed broad linear range for all targets indicating efficient extraction of SARS-CoV-2 using the present kit (ML).

The embodiments, examples and alternatives of the preceding paragraphs or the description and drawings, including any of their various aspects or respective individual features, may be taken independently or in any combination. Features described in connection with one embodiment are applicable to all embodiments, unless such features are incompatible.