DESC:TECHNICAL FIELD
The present disclosure relates to filed for molecular biology. The present disclosure particularly relates to simultaneous extraction of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis and virus including but not limited to SARS-CoV2. The present disclosure also relates to simultaneous detection of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium and virus including but not limited to SARS-CoV2. The present disclosure also relates to a kit for simultaneous extraction of nucleic acid or for simultaneous detection of nucleic acid.

BACKGROUND OF THE DISCLOSURE
Nucleic acid isolation is the first step in the sample processing. During this step DNA and/or RNA is released into solution. Biological samples such as sputum requires harsh processing condition for reducing viscosity and for extraction of nucleic acid. Further, nasopharyngeal or oropharyngeal samples in case of respiratory infections, such as SARS CoV-2 can be processed by simple means for isolation of RNA.

It is noted that the respiratory infection such as SARS CoV-2 and the treatment methodology could increase the possibility of infection from Mycobacterium tuberculosis. Also, it is reported that the prognosis for Tuberculosis patients infected with SARS-CoV-2 is poor. Thus, it is essential to arrive at a technique or method that enables simultaneous extraction or detection of nucleic acid belonging to bacteria such as Mycobacterium tuberculosis and virus such as SARS CoV-2.

The present disclosure provides for an improved method that enables simultaneous extraction or simultaneous detection of nucleic acid belonging to bacteria such as Mycobacterium tuberculosis and virus such as SARS CoV-2.

SUMMARY OF THE DISCLOSURE
Accordingly, the present disclosure describes simple, effective and economical method for simultaneous extraction of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis Klebsiella pneumoniae, Streptococcus, Haemophilus Influenza, cornybacterium and Bordetella pertusis; and the virus including but not limited to SARS-CoV-2, Respiratory Syncitial Virus, Influenza and Adenoviruses.

In another embodiment, the present disclosure relates to simple, effective and economical method for simultaneous detection of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis Klebsiella pneumoniae, Streptococcus, Haemophilus Influenza, cornybacterium and Bordetella pertusis; and the virus including but not limited to SARS-CoV-2, Respiratory Syncitial Virus, Influenza and Adenoviruses.

In another embodiment, the present disclosure relates to a kit comprising buffer system, reagent and instructions manual for simultaneous extraction of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis Klebsiella pneumoniae, Streptococcus, Haemophilus Influenza, cornybacterium and Bordetella pertusis; and the virus including but not limited to SARS-CoV-2, Respiratory Syncitial Virus, Influenza and Adenoviruses or for simultaneous detection of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis and virus including but not limited to SARS-CoV-2.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURE
In order that the present disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figure. The figure together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where:

FIGURE 1 provides an exemplification of the method of the present disclosure involved in simultaneous extraction or simultaneous detection of nucleic acid from combination of samples suspected of comprising bacteria and virus.

DETAILED DESCRIPTION OF THE DISCLOSURE
Unless otherwise defined, all terms used in the disclosure, including technical and scientific terms, have meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included for better understanding of the present disclosure.

As used herein, the singular forms ‘a’, ‘an’ and ‘the’ include both singular and plural referents unless the context clearly dictates otherwise.

The term ‘comprising’, ‘comprises’ or ‘comprised of’ as used herein are synonymous with ‘including’, ‘includes’, ‘containing’ or ‘contains’ and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term ‘about’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of ±10% or less, preferably ±5% or less, more preferably ±1% or less and still more preferably ±0.1% or less of and from the specified value, insofar such variations are appropriate to perform the present disclosure. It is to be understood that the value to which the modifier ‘about’ refers is itself also specifically, and preferably disclosed.

As used herein, the term ‘buffer-1’ refers to ‘buffer composition’ comprising chaotropic agent buffering agent and chelating agent. The term ‘buffer-1’ and ‘buffer composition’ may be used interchangeably.

As used herein, the term ‘buffer-2’ refers to ‘lysis buffer’. The term ‘buffer-2’ and ‘lysis buffer’ may be used interchangeably.

The present disclosure relates to method for simultaneous extraction of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis Klebsiella pneumoniae, Streptococcus, Haemophilus Influenza, cornybacterium and Bordetella pertusis; and the virus including but not limited to SARS-CoV-2, Respiratory Syncitial Virus, Influenza and Adenoviruses.

The method of simultaneous extraction of nucleic acid from combination of samples suspected comprising the bacteria and the virus is simple, effective and economical. The inventors have particularly identified that the method of simultaneous extraction of nucleic acid from combination of samples suspected of comprising bacteria and virus, do not damage the nucleic acid to be extracted, thus leading to effective extraction of the nucleic acid.

In some embodiments of the present disclosure, the method involves simultaneous extraction of nucleic acid from combination of samples suspected of comprising the bacteria and the virus. The sample includes but it is not limited to sputum, nasopharyngeal sample, oropharyngeal sample, Tissue, Blood, Serum and stool. Preferably Sputum and Nasopharyngeal sample. In an embodiment, in the method of simultaneous extraction of nucleic acid, at least two different samples are employed. For instance, the combination of samples employed are - combination of sputum and nasopharyngeal sample, combination of sputum and oropharyngeal sample and so on and so forth.

In some embodiments of the present disclosure, the method involves simultaneous extraction of both DNA and RNA from the sample, such as sputum suspected of comprising Mycobacterium tuberculosis and the sample, such as nasopharyngeal and/or oropharyngeal sample suspected of comprising SARS CoV-2.

In some embodiments of the present disclosure, both sputum sample and nasopharyngeal and/or oropharyngeal sample are processed simultaneously for extraction of both DNA and RNA in a single nucleic acid extraction unit.

The inventors have particularly identified that, in the method of simultaneous extraction of both DNA and RNA from the combination of samples, such as sputum sample and nasopharyngeal sample or oropharyngeal sample, the DNA and RNA are extracted without any damage and/or losing stability, thereby leading to efficient extraction of nucleic acid simultaneously.

In some embodiments of the present disclosure, the method of simultaneous extraction of both DNA and RNA from combination of samples suspected of comprising the bacteria and the virus, comprises- adding the sample suspected of comprising bacteria to another sample suspected of comprising the virus, collected in Buffer-1 (buffer composition), followed by mixing to obtain a solution; making both samples ready for nucleic acid extraction. In an embodiment, the sample suspected of comprising containing bacteria can be added directly to the mixture of sample suspected of comprising virus and buffer composition or it can be liquified by employing liquification buffer.

In some embodiments of the present disclosure, the method of simultaneous extraction of both DNA and RNA from combination of samples suspected of comprising the bacteria and the virus, comprises:
i. adding the sample suspected of comprising virus to a buffer composition to obtain a mixture;
ii. adding the sample suspected of comprising bacteria to obtain the mixture of step i), followed by adding lysis buffer (buffer-2); and
iii. subjecting the mixture of step ii) for extraction of the nucleic acid.

In some embodiments of the present disclosure, the method of simultaneous extraction of nucleic acid (both DNA and RNA) from combination of samples suspected of comprising bacteria and the virus comprises:
i. adding the Nasopharyngeal and/or Oropharyngeal sample to buffer composition, followed by mixing to obtain a mixture;
ii. adding liquified sputum sample to the mixture of step i), followed by adding lysis buffer; and
iii. subjecting the mixture of step ii) for extraction of the nucleic acid.

In some embodiments of the present disclosure, the sample including but not limited to sputum, suspected of comprising bacteria is liquified by mixing the sample suspected of comprising bacteria with liquification buffer.

In an embodiment, the liquefication buffer is selected from group comprising Dithiothreitol (DTT), tris(2-carboxyethyl)phosphine, beta mercaptoethanol, and any combinations thereof.

In an embodiment, the liquefication buffer is having concentration ranging from about 0.2 Molar to 1.5 Molar, including all the values in the range, for instance, 0.3 Molar, 0.4 Molar, 0.5 Molar, 0.6 Molar and so on and so forth.

In an embodiment, the liquefication buffer is having pH ranging from about 2 to 9, including all the values in the range, for instance, 2.1, 2.2, 2.3 2.4 and so on and so forth.

The inventors have identified that the combination of the steps employed in the method for simultaneous extraction of nucleic acid from combination of samples suspected of comprising the bacteria and the virus, provides for efficient simultaneous extraction of nucleic acid and thus making the method simple and economical.

The inventors have particularly identified that employing buffer composition (buffer -1) in the described method provides for following advantages-
- inactivates bacteria, such as Mycobacterium tuberculosis and virus such as SARS CoV-2;
- efficiently reduces viscosity of the sample, such as sputum, i.e., liquifies the sample, enabling easy extraction of nucleic acid.
- stabilizes both DNA and RNA from degradation so that there is efficient extraction of both DNA and RNA from combination of samples comprising the bacteria and the virus; and

In some embodiments of the present disclosure, the buffer composition comprises chaotropic agent, buffering agent chelating agent and solvent.

In some embodiments of the present disclosure, the chaotropic agent is in an amount ranging from about 5% to 30%, including all the values in the range, for instance, 5.1%, 5.2%, 5.3%, 5.4% and so on and so forth.

In some embodiments of the present disclosure, the buffering agent is in an amount ranging from about 0.2% to 2%, including all the values in the range, for instance, 0.21%, 0.22%, 0.23%, 0.24% and so on and so forth.

In some embodiments of the present disclosure, the chelating agent is in an amount ranging from about 0.001% to 0.02%, including all the values in the range, for instance, 0.002%, 0.003%, 0.004%, 0.005% and so on and so forth.

In some embodiments of the present disclosure, the solvent is in an amount ranging from about 55% to 68%, including all the values in the range, for instance, 55.1%, 55.2%, 55.3%, 55.4% and so on and so forth.

In some embodiments of the present disclosure, the chaotropic agent includes but it is not limited to n-butanol, ethanol, guanidinium salt, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, protease, RNase inhibitors and any combinations thereof.

In some embodiments of the present disclosure, the buffering agent includes but it is not limited to TAPS, BICINE, Tris(hydroxymethyl)aminomethane, Tricine, MES, Hepes, PIPES, Phosphate, Citrate, Carbonate and any combinations thereof.

In some embodiments of the present disclosure, the chelating agent includes but it is not limited to diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA).

In some embodiments of the present disclosure, the chaotropic agent in the buffer composition is present at a concentration of 0.1Molar to 5 Molar so as to enable the buffer -1 to inactivate the bacteria, such as Mycobacterium tuberculosis and the virus, such as SARS CoV-2 and stabilises nucleic acid, such as DNA and RNA from degradation and liquefies viscous samples.

In an exemplary embodiment of the present disclosure, the chaotropic agent is at a concentration ranging from about 0.1 Molar to 5 Molar, including all the values in the range, for instance, 0.11 Molar, 0.12 Molar, 0.13 Molar, 0.14 Molar and so on and so forth.

In some embodiments of the present disclosure, the buffering agent is present at a concentration ranging from about 0.05 M to 3 Molar.

In an exemplary embodiment, the chaotropic agent, such as Guanidine Thiocyanate in the buffer-1 is present at a concentration ranging from about 0.1 Molar to 5 Molar, including all the values in the range, for instance, 0.11 Molar, 0.12 Molar, 0.13 Molar, 0.14 Molar and so on and so forth.

In an embodiment, the chelating agent is present at a concentration ranging from about 0.01 mM to 1 M.

In some embodiments of the present disclosure, the buffer composition further comprises detergents. In an embodiment, the buffer composition chaotropic agent, buffering agent, chelating agent, solvent and detergent.

In an embodiment, the solvent is selected from a group comprising ethyl alcohol, methyl alcohol, isopropanol, butyl alcohol and any combinations thereof. In an embodiment, the solvent is at a concentration ranging from about 5% to 60%, including all the values in the range, for instance, 5.1%, 5.2%, 5.3%, 5.4% and so on and so forth.

In an embodiment, the detergent is anionic, cationic, non-ionic or zwitterionic. In an embodiment, the detergent includes but not it is not limited to sodium dodecyl sulphate (SDS), other alkali metal alkylsulphate salts, sarkosyl, Triton X-100, Nonidet P-40 and any combinations thereof. In an embodiment, the detergent is at a concentration ranging from about 0.01% to 10%, including all the values in the range, for instance, 0.02%, 0.03%, 0.04%, 0.05% and so on and so forth.

In some embodiments of the present disclosure, the buffer -1 has pH ranging from about 4 to 11, including all the values in the range, for instance, 4.1, 4.2, 4.3, 4.4 and so on and so forth.

In an exemplary embodiment of the present disclosure, the buffer-1 has pH ranging from about 6 to 9, including all the values in the range, for instance, 6.1, 6.2, 6.3, 6.4 and so on and so forth.

In some embodiments of the present disclosure, the buffer-2 is lysis buffer.

In some embodiments of the present disclosure, the buffer-2 is at a concentration ranging from about 0.1 M to 5 Molar

In some embodiments of the present disclosure, the lysis buffer has pH ranging from about 4 to 11, including all the values in the range, for instance, 4.1, 4.2, 4.3, 4.4 and so on and so forth.

In some embodiments of the present disclosure, the method of simultaneous extraction of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis and suspected of comprising virus including but not limited to SARS-CoV-2 employs buffer system comprising combination of liquefication buffer, buffer composition and lysis buffer.

In some embodiments of the present disclosure, the buffer system employed in the method is sufficiently optimized for efficient and simultaneous extraction of both DNA and RNA from combination of samples suspected of comprising the bacteria and the virus.

In some embodiments of the present disclosure, the extraction of nucleic acid upon processing combination samples with the buffer system described above is carried out using Nucleic acid extraction system by transferring the processed sample to a cartridge sample chamber and loading the cartridge into a device for extraction of nucleic acid. In an embodiment, the nucleic acid extraction is carried out using Trueprep® AUTO Universal Cartridge Based Sample Preparation Kit protocol.

In an embodiment, the figure 1 illustrates the process flow involved in simultaneous extraction of nucleic acid from combination of biological samples, such as sputum and nasopharyngeal sample.

The present disclosure further relates to a method of simultaneous detection of nucleic acid from combination of samples suspected of comprising the bacteria including but not limited to Mycobacterium tuberculosis Klebsiella pneumoniae, Streptococcus, Haemophilus Influenza, cornybacterium and Bordetella pertusis; and the virus including but not limited to SARS-CoV-2, Respiratory Syncitial Virus, Influenza and Adenoviruses.

In some embodiments of the present disclosure, in the method of simultaneous detection of nucleic acid of the bacteria and the virus, the combination of samples suspected of comprising the bacteria and the virus are processed together. The inventors have particularly identified that during the simultaneous detection of the nucleic acid from the combination of samples suspected of comprising the bacteria and the virus, the nucleic acid is not damaged, as a result efficient detection is made possible in a simple and economical manner.
In some embodiments of the present disclosure, the method involves simultaneous detection of nucleic acid from combination of samples suspected of comprising the bacteria and the virus. The sample includes but it is not limited to, sputum, nasopharyngeal sample, oropharyngeal sample, Tissue, Blood, Serum and stool. In an embodiment, in the method of simultaneous detection of the nucleic acid, at least two different samples are employed. For instance, the combination of samples employed are- combination of sputum and nasopharyngeal sample, combination of sputum and oropharyngeal sample and so on and so forth.

In some embodiments of the present disclosure, the method of simultaneous detection of nucleic acid of the bacteria and the virus, comprises-
- adding a liquification buffer to a sample suspected of comprising the bacteria, followed by mixing to obtain a solution;
- Adding the solution to a mixture comprising sample suspected of comprising the virus and buffer-1, followed by mixing to obtain further mixture and subjecting to holding time for a predetermined duration;
- Adding the further mixture to buffer -2, followed by mixing and subjecting to holding time for a predetermined duration and subjecting the mixture to extraction of nucleic acid and detecting presence or absence of the nucleic acid of the bacteria and the virus.

In some embodiments of the present disclosure, the method of simultaneous detection of nucleic acid of the bacteria and the virus, comprises-
- Adding a liquefaction buffer to sputum suspected of comprising the bacteria, followed by mixing to obtain a solution;
- Adding the solution to a mixture of nasopharyngeal sample or oropharyngeal sample suspected of comprising the virus and buffer-1, followed by mixing to obtain a further mixture and subjecting to holding time for a predetermined duration; and
- Adding the further mixture to buffer -2, followed by mixing and subjecting to holding time for a determined duration and subjecting the mixture to extraction of nucleic acid and detecting presence or absence of the nucleic acid of the bacteria and the virus.

In some embodiments of the present disclosure, the method of simultaneous detection of nucleic acid of the bacteria and the virus in combination of samples comprises-
- adding the sample suspected of comprising the virus to the buffer composition, followed by mixing to obtain a mixture;
- adding liquified sample suspected of comprising bacteria to the mixture, followed by adding lysis buffer;
- subjecting the mixture to extraction of nucleic acid, followed by detection of the nucleic acid.

In some embodiments of the present disclosure, the sample including but not limited to sputum, suspected of comprising bacteria is liquified by mixing the sample suspected of comprising bacteria with liquification buffer.

In some embodiments of the present disclosure, the extraction of the nucleic acid is carried out using Trueprep® AUTO v2 Universal Cartridge Based Sample Preparation Device.

In some embodiments of the present disclosure, the detection of the nucleic acid is carried out using Truelab® Real Time Quantitative micro PCR Analyzer.

In some embodiments of the present disclosure, the chaotropic agent in the buffer - 1 is present in predetermined amount so as to enable the buffer -1 to inactivate both bacteria, such as Mycobacterium tuberculosis and virus, such as SARS CoV-2, stabilise nucleic acid from degradation and liquefy viscous samples during simultaneous detection of nucleic acid of the bacteria and the virus.

In some embodiments of the present disclosure, in the method of the extraction of the nucleic acid or detection of the nucleic acid, upon processing the combination of samples through the buffer system as per the steps described above, the samples are transferred to cartridge sample chamber, followed by loading the cartridge into a device for extraction of the nucleic acid.

In some embodiments of the present disclosure, in the said method, the detection of presence or absence of the nucleic acid is carried out by techniques including but not limited to PCR, Hybridization, Blotting and UV Spectroscopy.

In some embodiments of the present disclosure, both sputum and nasopharyngeal sample and/or oropharyngeal sample are processed in a single nucleic acid extraction unit and the extracted nucleic acid is used for simultaneous detection of presence or absence of Mycobacterium tuberculosis and SARS CoV-2.

In an embodiment of the present disclosure, during simultaneous detection of nucleic acid belonging to the bacteria and the virus, the extraction of both DNA and RNA is carried out without losing stability of the DNA and the RNA.

In some embodiments of the present disclosure, the method of simultaneous detection of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis and suspected of comprising virus including but not limited to SARS-CoV-2 employs buffer system comprising combination of liquefication buffer, buffer composition and lysis buffer.

In some embodiments of the present disclosure, the buffer system (liquefication buffer, buffer-1 and buffer-2) employed in the method is used for efficient and simultaneous detection of presence or absence of DNA and RNA of the bacteria and the virus.

The method of simultaneous extraction of nucleic acid from combination of samples or the method of simultaneous detection of nucleic acid from combination of samples is an in vitro method.

The present disclosure further relates to a kit comprising buffer system, reagent, and instruction manual.

In some embodiments of the present disclosure, the kit enables simultaneous extraction of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis Klebsiella pneumoniae, Streptococcus, Haemophilus Influenza, cornybacterium and Bordetella pertusis; and the virus including but not limited to SARS-CoV-2, Respiratory Syncitial Virus, Influenza and Adenoviruses.

In some embodiments the present disclosure, the kit enables simultaneous detection of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis and virus including but not limited to SARS-CoV-2.

In some embodiments of the present disclosure, the kit comprises the buffer system comprising liquification buffer, buffer-1 and buffer-2, individually.

In some embodiments of the present disclosure, the liquification buffer is selected from a group comprising Dithiothreitol (DTT), tris(2-carboxyethyl)phosphine, beta mercaptoethanol, and any combinations thereof.

In some embodiments of the present disclosure, the buffer-1comprising chaotropic agent includes but it is not limited to n-butanol, ethanol, guanidinium salt, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, protease and RNase inhibitors.

In some embodiments of the present disclosure, the buffer-1comprising buffer agent includes but it is not limited to TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid), BICINE ([Bis(2-hydroxyethyl)amino]acetic acid), Tris(hydroxymethyl)aminomethane, Tricine, MES (2-(N-morpholino)ethanesulfonic acid), Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Phosphate, Citrate, and Carbonate and any combinations thereof.

In some embodiments of the present disclosure, the buffer-1comprising chelating agent includes but it is not limited to diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA).

The buffer system comprising liquification buffer, buffer-1 (buffer composition) and buffer-2 are produced in the form of kits. Such kits are a packaged to include a container comprising liquification buffer, a container comprising buffer-1, a container comprising buffer-2, respectively and reagents that are essential for simultaneous extraction and/or detection of nucleic acid from combination of samples suspected of comprising bacteria including but not limited to Mycobacterium tuberculosis and virus, including but not limited to SARS-CoV-2. The kit also comprises instructions on how to perform the simultaneous extraction and/or detection of nucleic acid from combination of samples.

The present disclosure further relates to a buffer composition comprising chaotropic agent, buffering agent, chelating agent and solvent.

In some embodiments of the present disclosure, the chaotropic agent is in an amount ranging from about 5% to 30%, including all the values in the range, for instance, 5.1%, 5.2%, 5.3%, 5.4% and so on and so forth.

In some embodiments of the present disclosure, the buffering agent is in an amount ranging from about 0.2% to 2%, including all the values in the range, for instance, 0.21%, 0.22%, 0.23%, 0.24% and so on and so forth.

In some embodiments of the present disclosure, the chelating agent is in an amount ranging from about 0.001% to 0.02%, including all the values in the range, for instance, 0.002%, 0.003%, 0.004%, 0.005% and so on and so forth.

In some embodiments of the present disclosure, the solvent is in an amount ranging from about 55% to 68%, including all the values in the range, for instance, 55.1%, 55.2%, 55.3%, 55.4% and so on and so forth.

In some embodiments of the present disclosure, the chaotropic agent includes but it is not limited to n-butanol, ethanol, guanidinium salt, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, protease and RNase inhibitors.

In some embodiments of the present disclosure, the buffering agent includes but it is not limited to TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid), BICINE ([Bis(2-hydroxyethyl)amino]acetic acid), Tris(hydroxymethyl)aminomethane, Tricine, MES, Hepes, PIPES, Phosphate, Citrate, and Carbonate and any combinations thereof.

In some embodiments of the present disclosure, the chelating agent includes but it is not limited to diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA). According to a preferred embodiment, EDTA is used. As used herein, the term "EDTA" indicates inter alia the EDTA portion of an EDTA compound such as, for example, K2EDTA, K3EDTA or Na2EDTA.

In some embodiments of the present disclosure, the chaotropic agent is present at a concentration ranging from 0.1 M up to the saturation limit, 0.2 to 5M, 0.1 M to 4 M, 0.5M to 3M, 0.75M to 3.5M, most preferred at least 2.75M.

In some embodiments of the present disclosure, the buffering agent is present at a concentration ranging from about 0.05 M to 3M, including all the values in the range, for instance, 0.06 M, 0.07 M, 0.08 M., 0.09 M and so on and so forth.

In some embodiments of the present disclosure, the chelating agent is present at a concentration ranging from about 0.1milli Molar to 1 Molar.

In some embodiments of the present disclosure, the buffer composition further comprises detergents. In an embodiment, the buffer composition chaotropic agent, buffering agent, chelating agent, solvent and detergent.

In an embodiment, the solvent is selected from a group comprising ethyl alcohol, methyl alcohol, isopropanol, butyl alcohol and any combinations thereof. In an embodiment, the solvent is at a concentration ranging from about 5% to 60%, including all the values in the range, for instance, 5.1%, 5.2%, 5.3%, 5.4% and so on and so forth.

In an embodiment, the detergent is anionic, cationic, non-ionic or zwitterionic. In an embodiment, the detergent includes but not it is not limited to sodium dodecyl sulphate (SDS), other alkali metal alkylsulphate salts, sarkosyl, Triton X-100, Nonidet P-40 and any combinations thereof. In an embodiment, the detergent is at a concentration ranging from about 0.01% to 10%, including all the values in the range, for instance, 0.02%, 0.03%, 0.04%, 0.05% and so on and so forth.

In some embodiments of the present disclosure, the buffer-1 has pH ranging from about 6 to 9, including all the values in the range, for instance, 6.1, 6.2, 6.3, 6.4 and so on and so forth.

It is to be understood that the foregoing description is illustrative not a limitation. While considerable emphasis has been placed herein on particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

Descriptions of well-known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for example illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure, certain aspects have been employed. The example used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments may be practiced and to further enable those of skill in the art to practice the embodiments. Accordingly, following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLE

Example 1: Simultaneous detection of nucleic acid from sputum sample and Nasopharyngeal sample/ Oropharyngeal sample
i. Nasopharyngeal sample suspected to have the virus was collected in Buffer I. To the buffer containing the Nasopharyngeal sample, 0.5mL of the liquefied sputum sample suspected of containing Mycobacterium, was added. A portion of the said mixture was added to Buffer 2. Both sputum and nasopharyngeal sample were processed in a single nucleic acid extraction unit, i.e., simultaneous extraction and the extracted nucleic acid is used for simultaneous detection of presence or absence of Mycobacterium tuberculosis and SARS CoV-2.
ii. Oropharyngeal sample suspected to have the virus was collected in Buffer I. To the buffer containing the Oropharyngeal sample, 0.5mL of the liquefied sputum sample suspected of containing Mycobacterium, was added. A portion of the said mixture was added to Buffer 2. Both sputum and Oropharyngeal sample were processed in a single nucleic acid extraction unit, i.e., simultaneous extraction and the extracted nucleic acid is used for simultaneous detection of presence or absence of Mycobacterium tuberculosis and SARS CoV-2.

Table 1 describes the sample processed as per the process described in the Example 1.
Sputum (Comparative Example) Sputum and Nasopharyngeal or Oropharyngeal sample
Sample ID MTB Status SARS Cov 2 Status MTB Status SARS Cov 2 Status
SP 001 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 002 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 003 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 004 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
SP 005 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 006 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 007 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 008 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 009 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 010 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 011 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 012 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
SP 013 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 014 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 015 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
SP 016 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 017 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 018 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 019 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 020 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 021 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 023 MTB Detected SARS Cov 2 Not Detected MTB Detected SARS Cov 2 Not Detected
SP 024 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
SP 025 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
SP 026 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
SP 027 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
SP 028 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
SP 029 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
SP 030 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
PC 1 MTB Detected SARS Cov 2 Detected MTB Detected SARS Cov 2 Detected
PC 2 MTB Detected SARS Cov 2 Detected MTB Detected SARS Cov 2 Detected
NC 1 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected
NC 2 MTB Not Detected SARS Cov 2 Not Detected MTB Not Detected SARS Cov 2 Not Detected

Table 1:

Example 2:
Experiment was conducted for extraction of nucleic acid from sputum and nasopharyngeal sample, individually as per the conventional method (comparative Example).
Further, Experiment was conducted for simultaneous extraction of nucleic acid from combination of samples, i.e., sputum and nasopharyngeal samples employing the process described in Example 1.
Table 2 provides results of the extraction. It can be noted that process described in the Example 1 (method of the present invention) is able to simultaneously extract and simultaneously detect presence of MTB and SARS-CoV2 in combination of samples processed. The method of the present invention is able to provide same result as the result obtained when the samples are processed separately to detect MTB and SARS-CoV2 by conventional method. Thus, making the method of the present invention simple, efficient and economical.

Sample ID Sputum (Comparative Example) Nasopharyngeal Sample (Comparative Example) Sputum + Nasopharyngeal Sample (as per the process of present invention)

MTB Status SARS CoV-2 Status MTB Status SARS CoV2 Status MTB Status SARS CoV2 Status
Sample 1 MTB Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV2 Not Detected MTB Detected SARS CoV2 Not Detected
Sample 2 MTB Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV2 Not Detected MTB Detected SARS CoV2 Not Detected
Sample 3 MTB Not Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV-2 Not Detected
Sample 4 MTB Not Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV-2 Not Detected
Sample 5 MTB Not Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV-2 Detected MTB Not Detected SARS CoV-2 Detected
Sample 6 MTB Not Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV-2 Detected MTB Not Detected SARS CoV-2 Detected
Sample 7 MTB Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV-2 Detected MTB Detected SARS CoV-2 Detected
Sample 8 MTB Detected SARS CoV-2 Not Detected MTB Not Detected SARS CoV-2 Detected MTB Detected SARS CoV-2 Detected
Table 2
Results in Table 2 shows that combining sputum and Nasopharyngeal swab, nucleic acids of both MTB and SARS CoV-2 can be isolated simultaneously.

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.
,CLAIMS:WE CLAIM:
1. A method for simultaneous extraction of nucleic acid from combination of biological samples suspected of comprising bacteria and virus, said method comprises-
i. adding sample suspected of comprising virus to a buffer composition to obtain a mixture;
ii. adding liquified sample suspected of comprising bacteria to the mixture of step i), followed by adding lysis buffer; and
iii. subjecting the mixture of step ii) to simultaneous extraction of the nucleic acid.
2. A method for simultaneous detection of nucleic acid from combination of biological sample suspected comprising bacteria and virus, said method comprises-
i. adding sample suspected of comprising virus to a buffer composition to obtain a mixture;
ii. adding liquified sample suspected of comprising bacteria to the mixture of step i), followed by adding lysis buffer; and
iii. subjecting the mixture of step ii) to simultaneous extraction of the nucleic acid, followed by simultaneous detection of the nucleic acid.
3. The method as claimed in claim 1 or 2, wherein the liquified sample is prepared by mixing the sample suspected of comprising bacteria with liquification buffer.
4. The method as claimed in claim 1 or 2, wherein the buffer composition comprises chaotropic agent, buffering agent chelating agent and solvent.
5. The method as claimed in claim 4, wherein the chaotropic agent is selected from a group comprising n-butanol, ethanol, guanidinium salt, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, protease, RNase inhibitors, and any combinations thereof; the buffering agent is selected from a group comprising TAPS ([tris(hydroxymethyl) methylamino]propanesulfonic acid), BICINE ([Bis(2-hydroxyethyl)amino]acetic acid), Tris(hydroxymethyl)aminomethane, Tricine, MES (2-(N-morpholino)ethanesulfonic acid), Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Phosphate, Citrate, and Carbonate and any combinations thereof; the chelating agent is selected from a group comprising diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA); and the solvent is selected from a group comprising ethyl alcohol, methyl alcohol, isopropanol, butyl alcohol and any combinations thereof
6. The method as claimed in claim 4, wherein the chaotropic agent is at a concentration ranging from about 0.1 M to 5 Molar; the buffering agent is at a concentration ranging from about 0.05 M to 3 M; and the chelating agent is at a concentration ranging from about 0.1 m Molar to 1 Molar.
7. The method as claimed in claim 1 or 2, wherein the bacteria is selected from a group comprising Mycobacterium tuberculosis, Klebsiella pneumoniae, Streptococcus, Haemophilus Influenza, cornybacterium and Bordetella pertusis; and the viruses is selected from a group comprising SARS-CoV-2, Respiratory Syncitial Virus, Influenza and Adenoviruses.
8. The method as claimed in claim 1 or 2, wherein the simultaneous extraction of the nucleic acid is carried out using TruepepAUTO universal cartridge extraction system.
9. The method as claimed in claim 2, wherein the simultaneous detection of the nucleic acid is carried out by technique selected from a group comprising, polymerase chain reaction (PCR), hybridization, blotting and UV spectroscopy.
10. The method as claimed in claim 1 or claim 2, wherein the biological sample is selected from a group comprising sputum, nasopharyngeal sample, oropharyngeal sample, Tissue, Blood, Serum, stool and any combinations thereof.
11. A kit for simultaneous extraction of nucleic acid or simultaneous extraction of nucleic acid from combination of biological samples, the kit comprises-
- buffer system comprising combination of liquification buffer, buffer composition and lysis buffer;
- reagents supporting extraction of the nucleic acid or detection of the nucleic acid; and
- instructions on how to perform simultaneous extraction of the nucleic acid or simultaneous detection of the nucleic acid.
12. The kit as claimed in claim 11, wherein the buffer composition comprises chaotropic agent, buffering agent chelating agent and solvent.
13. The kit as claimed in claim 12, wherein the chaotropic agent is in an amount ranging from about 5% to 30%; the buffering agent is in an amount ranging from about 0.2% to 2%; the chelating agent is in an amount ranging from about 0.001% to 0.02%; and the solvent is in an amount ranging from about 55% to 68%.
14. The kit as claimed in claim 13, wherein the chaotropic agent is selected from a group comprising n-butanol, ethanol, guanidinium salt, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, protease, RNase inhibitors, and any combinations thereof; the buffering agent is selected from a group comprising TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid), BICINE ([Bis(2-hydroxyethyl)amino]acetic acid), Tris(hydroxymethyl)aminomethane, Tricine, MES (2-(N-morpholino)ethanesulfonic acid), Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Phosphate, Citrate, and Carbonate and any combinations thereof; the chelating agent is selected from a group comprising, diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA); and the solvent is selected from a group comprising ethyl alcohol, methyl alcohol, isopropanol, butyl alcohol and any combinations thereof.
15. The kit as claimed in claim 12, wherein the chaotropic agent is at a concentration ranging from about 0.1 M to 5 M; the buffering agent is at a concentration ranging from about 0.05 Molar to 3 Molar; and the chelating agent is at a concentration ranging from about 0.01mM to 1M.
16. The kit as claimed in claim 12, wherein the buffer composition has pH ranging from about 4 to 11.