DESC:-: Complete Specification :-

“System for extracting nucleic acids and preparing reagents for molecular biology assays”

Cross references to related applications: This complete specification is filed further to application for patent No. 202021027521 dated 29/09/2020 with provisional specification, the contents of which are incorporated herein in their entirety, by reference.

Field of the invention
This invention relates generally to methods and their implementing systems for efficient recovery of nucleic acids from biological samples and furthermore or independently, for reagent preparation required in quantitative polymerase chain reaction assays.

Definitions and interpretations
Before undertaking the detailed description of the invention below, it may be advantageous to set forth definitions of certain words or phrases used throughout this patent document: the terms “include” and “comprise,” as well as derivatives thereof, mean inclusion without limitation; the term “or” is inclusive, meaning and/or; the phrases “associated with” and “associated therewith,” as well as derivatives thereof, may mean to include, be included within, interconnect, with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have, have a property of, or the like; and the term “PCR” refers polymerase chain reaction; “RTPCR” refers real time polymerase chain reaction; “NA” refers Nucleic acid; “DNA” refers deoxyribonucleic acid; “RNA” refers ribonucleic acid; “nucleic acid” refers individual nucleic acids and polymeric chains of nucleic acids, including DNA and RNA, whether naturally occurring or artificially synthesized (including analogs thereof), or modifications thereof, especially those modifications known to occur in nature, having any length – for purpose of this invention, the nucleic acid may be present extracellularly or intracellularly in the biological sample; “quantification” means an accurate and reproducible measurement; “RPM” refers revolutions per minute; “BLDC” refers brushless DC; “DC” refers direct current; “preparator” refers the actor (in this invention, the system proposed herein) which prepares a specimen for scientific processing / examination.
Background of the invention and description of related art
Extraction of nucleic acids, that is DNA or RNA, from complex starting materials like whole blood, tissues, sputum, blood serum, urine or faeces, or any other biological materials is presently undertaken via a variety of methods - traditional organic solvent extraction method, column adsorption method, magnetic bead partition method and the charge method and so on. These processes typically involve lysis of biological material by a solvent or detergent in the presence of protein degrading enzymes, followed by several extractions with organic solvents, for example, phenol and/or chloroform, ethanol precipitation and dialysis of the nucleic acids.

As will be appreciated by the reader, the aforementioned conventional sample preparation and analysis techniques for performing nucleic acid assays are laborious, time-consuming, require trained technicians, and lack precise repeatability. Also, performing these sophisticated techniques require specialized rooms, each for NA extraction, reagent preparation and amplification of the extracted NA. Furthermore, current instruments for performing extraction and PCR through thermal control and cycling are generally exceptionally large (table-top), include manual handling and sometimes take too much time for sample preparation, and amplification.

Additionally in a clinical settling, the relatively large number of steps required to purify NA from such starting materials increases the risk of transmission of NA from sample to sample in the simultaneous processing of several clinical samples, which undesirably results in false positive results. Therefore, the art needs some way of fool-proofing these methods, in order to save efforts and avoid procedural latency as well as error-prone elucidation which are otherwise rampant in state of the art systems.

It shall be readily appreciated by the reader, that efficient isolation and / or purification of nucleic acids from biological samples is critical to research as well as diagnostic applications in molecular biology. As mentioned before, the processes to achieve these have traditionally been manual, and therefore inherently limited by throughput, dependency on level of skills of the operator, and overall low accuracy as well as precision. Hence, there exists a need in art to have some means which are less impeded by, or ideally devoid of, these limitations.

Molecular testing for both screening and analytical purposes has always been in high preferential demand in both research as well as diagnostics industries. The onslaught of diseases the world over, latest being the ongoing COVID-19 pandemic, have heightened this demand manifold, and exponentially increased the sample throughput requirements in laboratories designed to implement such molecular testing protocols.

Instability or denaturation of nucleic acids over time is an issue that plagues molecular testing protocols, especially for sensitive assay protocols when needed to be performed simultaneously on a plurality of biological samples. As nucleic acids tend to degrade rapidly and significantly within hours after sample collection, it is necessary that the molecular testing protocols have to be expedited without compromising precision and / or accuracy. It shall be understood that most molecular testing protocols today have a step of amplification wherein the titer of NA is raised to the minimum resolution of the assay undertaken, but said amplification factor needs to accurately compensate for denaturation of NA or otherwise is susceptible to underestimation or overestimation of actual analyte of interest and hence a failed assay.

Similar considerations exist for reagents which play an exceedingly important part in implementation of the aforementioned molecular testing protocols. Selection of right reagent, correct preparation of reagent, age of reagent, and duration of exposure of the sample to the reagent and many more parameters go in deciding precision and / or accuracy of the molecular testing protocol undertaken. It is needed hence, to some way compensate for these effects while maintaining steady-state precision and / or accuracy of the molecular testing protocol undertaken.

State of art reflects a few solutions directed against the aforementioned wants. For example, there are a few commercial systems that allow RNA isolation completely automatically - MagNa Pure LC Instruments (Roche Diagnostics), AutoGenprep 960 (Autogen), ABI Prism™ 6700 fully automatic nucleic acid workstation (Applied Biosystems), WAVE (registered trademark) nucleic acid analysis system and WAVE (registered trademark) fragment collector FCW200 (Transgenomic) and BioRobot 8000 (Qiagen).

Automation of the reagent preparation and processing steps involved in molecular testing protocols (from extraction through detection) as well as miniaturization of their implementing facilities, has thus been a pressing need of art, and therefore objective of research by the applicant named herein.

However, prior art, to the limited extent presently surveyed, does not list a single effective solution embracing all considerations mentioned hereinabove, thus preserving an acute necessity-to-invent for the present inventors who, as result of their focused research, have come up with novel solutions for resolving all needs of the art once and for all.

A better understanding of the objects, advantages, features, properties and relationships of the present invention will be obtained from the detailed description to follow hereunder which sets forth an illustrative yet-preferred embodiment and other ways in which the invention subject hereof is intended to be performed.

Objectives of the present invention
The present invention is identified in addressing at least all major deficiencies of art discussed in the foregoing section by effectively addressing the objectives stated under, of which:

It is a primary objective to provide a unified platform for automating the upstream processes of nucleic acid extraction and reagent preparation involved in RTPCR based analytics.

It is another objective further to the aforesaid objective(s) that the unified platform so provided has a compact footprint.

It is another objective further to the aforesaid objective(s) that the unified platform so provided is not overly complex and can be operated with none, or minimal if at all, human intervention.

The manner in which the above objectives are achieved, together with other objects and advantages which will become subsequently apparent, reside in the detailed description set forth below in reference to the accompanying drawings and furthermore specifically outlined in the independent claim 1. Other advantageous embodiments of the invention are specified in the dependent claims.

Brief description of drawings
The present invention is explained herein under with reference to the following drawings, in which:
FIG.1 is a side section view of the unified platform system proposed herein.
FIG.2 is a schematic diagram of the deck (4) included in the system proposed herein.
FIG.3 is an illustration of the thermoshaker assembly (5) with block holder (25) for sample processing tubes, with temperature and RPM regulator as per the disclosures hereof.
FIG.4 is a front view of the magnetic assembly as per the disclosures hereof.
FIG.5 is a side view of the magnetic assembly as per the disclosures hereof.
FIG.6 is a side view of the Thermoshaker assembly as per the disclosures hereof.
FIG. 7 shows the structure of the PID controller used in the present invention.
FIG. 8(A to I) are screenshots of the human machine interface (HMI) electronic screen for explaining operations of the system proposed herein.
FIG. 9A is a schematic diagram to illustrate the design of the extraction cartridge included in the present invention.
FIG. 9B is a schematic diagram to illustrate the design of the PCR cartridge included in the present invention.
FIG. 10 is a schematic of the deck shown in FIG. 2 to explain positioning / placement of various components thereupon.

The above drawings are illustrative of particular examples of the present invention but are not intended to limit the scope thereof. The drawings are not to scale (unless so stated) and are intended for use solely in conjunction with their explanations in the following detailed description. In above drawings, wherever possible, the same references and symbols have been used throughout to refer to the same or similar parts, as under-
(1) - Syringe unit
(2) - Assembly for receiving (01)
(3) - Magnetic assembly
(4) - Deck
(5) - Thermoshaker assembly
(6) - Place holder for (5)
(7) - Place holder for (5)
(8) - Footprint area of (5)
(9) - Place holder for (5)
(10) - Place holder for (5)
(11) - Place holder for (5)
(12) - Place holder for (5)
(13) - Place holder for (5)
(14) - BLDC motor
(15) - Base plate of (5)
(16) - Leaf spring assembly
(17) - Heating block
(18) - Wells in (17)
(19) - Up and down movement motor of (3)
(20) - Reverse and forward movement motor of (3)
(21) - Assembly for forward and reverse movement
(22) - Assembly for upward and downward movement
(23) - Magnet holding arm
(24) - Magnets
(25) - Block holder

Though numbering has been introduced to demarcate reference to specific components in relation to such references being made in different sections of this specification, all components are not shown or numbered in each drawing to avoid obscuring the invention proposed.

Attention of the reader is now requested to the detailed description to follow which narrates a preferred embodiment of the present invention and such other ways in which principles of the invention may be employed without parting from the essence of the invention claimed herein.

Summary of the present invention
The present invention is identified in establishment of a system which integrates and automates the extraction of nucleic acids from assay samples, and the preparation of reagents for RTPCR analysis.

Detailed description
The present invention is directed at absorbing all advantages of prior art while overcoming, and not imbibing, any of its shortfalls, to thereby establish an automated system for efficient recovery of nucleic acids from biological samples and furthermore or independently, for reagent preparation required in quantitative polymerase chain reaction assays.

Accordingly, a first aspect of the present application relates to an automated method for eluting nucleic acid (DNA and RNA) from biological samples, particularly those potentially containing genetic material such as blood, tissues, urine, faeces and any other biological fluids. A second aspect of the present application relates to a method for preparation of reagents, particularly those used in RTPCR assays. These and other aspects and advantages will become apparent when the narrative below is read in conjunction with the accompanying drawings which showcase a presently preferred embodiment of construction and operations of the automated sample-to-PCR ready system proposed herein.

The automated sample-to-PCR ready system proposed herein is shown in the accompanying FIG.1 which is identified in comprising a precision plunger type syringe unit (1), a magnetic assembly (3), a deck (4) and a thermoshaker assembly (5). Each of these components (1, 2, 3, and 4) is elaborated in the disclosures to follow.

With reference to the FIG.1, it can be seen that the precision plunger type syringe unit (1) is fixed in an assembly (2) which allows the upward and downward movement of the syringe unit (1). Said upward and downward movement of the syringe unit (1) is facilitated and regulated by a stepper motor. The resultant syringe assembly serves to achieve accurate quantitative suction and / or dispensing of liquid.

With reference to the FIG.1, it can be further seen that the magnetic assembly (3) is positioned behind the aforementioned syringe assembly. This magnetic assembly (3) serves to facilitate and regulate the upward or downward, as well as forward or reverse movement of the syringe assembly for separating a solid magnetic substrate from the liquid contents of the sample processing or the reaction tube. The thermoshaker assembly (5) further includes a block holder (25) for sample processing tubes. Said thermoshaker assembly (5) is provisioned with temperature and RPM regulator. The RPM regulator is software-implemented and controlled by the user / operator via suitable interface chosen among on-screen controls, buttons, sliders and the like as common to art.

As also shown in FIG.2, the deck (4) is provided for housing the consumables required for the entire workflow, and designed for forward and reverse movement, regulated by a stepper motor. The deck (4) has the place holders (6, 7, 9, 10, 11, 12, and 13) being contoured recesses within the deck (4) correspondingly for tips / piercing tips, sample processing tubes, extraction cartridge, elution tube, RTPCR / extraction cartridges, RTPCR tubes and tips being discarded. Area (8) is the footprint of the thermoshaker assembly (5).

In a related aspect, the thermoshaker assembly (5) is an integrated and the most critical unit hereof, which, as shown in FIG. 3, includes a BLDC motor (14) mounted below the base plate (15) of the thermoshaker assembly (5). The thermoshaker assembly (5) is designed, as described elsewhere in this document, for simultaneous heating and mixing for the extraction components with magnetic beads. The speed of the motor (14) ranges from 100 RPM to 1500 RPM and is regulated using the BLDC drive.
A leaf spring assembly (16) made of either Teflon, or a Nylon material is arranged above the base plate (15), which is driven by the BLDC motor (14) being attached below said base plate (15) and driven in an acentric shaft. This acentric shaft is connected with the heating block (17), and is either made up of aluminium or stainless steel. This block is again connected with an electric heater (Peltier unit), which is regulated with a machine controller / PID controller, with which the temperature can be adjusted between 30oC to 150oC.

According to another aspect hereof, the PID is used in tuning for management of temperature, the basic architecture / logic of which is as shown in FIG. 7. The PID controller consists of three terms, namely proportional, integral, and derivative control. The combined operation of these three controllers gives a control strategy for process control. PID controller manipulates the process variables like pressure, speed, temperature, flow, etc. Some of the applications use PID controllers in cascade networks where two or more PID’s are used to achieve control.

As seen in FIG. 7, structure of the PID controller consists of a PID block which gives its output to the process block. Process / plant consists of final control devices like actuators, control valves, and other control devices to control various processes of industry/plant. A feedback signal from the process plant (where the PCR process / protocol is carried out) is compared with a set point or reference signal u(t) and the corresponding error signal e(t) is fed to the PID algorithm. According to the proportional, integral, and derivative control calculations in the algorithm, the controller produces a combined response or controlled output which is applied to plant control devices.

As shown in FIG.3, the block (17) is designed in such a way that the wells (18) are housing at the edge of the block, which facilitates the magnetic beads present in the tube along with the substrate to get attracted to the magnetic assembly (3 shown in FIG.1).

As shown in FIG.4, the magnetic assembly (3) consists of an integrated structure, consisting of two individual assemblies– one for upward and downward movement and another for forward and reverse movement. Each assembly is driven by stepper motors. As shown in FIG.4, the magnetic assembly (3) comprises a motor (19) for up and down movement, a motor (20) for reverse and forward movement, an assembly (21) for forward and reverse movement, an assembly (22) for upward and downward movement, a magnet holding arm (23), and magnets (24).

Nucleic acid extraction in the present invention:
During the process of extraction magnetic beads are used, so that the extracted nucleic acid binds to the magnetic beads and the final nucleic acid is thus obtained for further analysis. Here the Thermoshaker containing the reaction tube is added with the sample and other reagents for extraction of nucleic acid; one of the components added is magnetic beads, the magnetic bead are added so that the extracted nucleic acids bind to the beads, which can later be purified. Once the reaction starts, the magnetic beads which are bound with nucleic acid has to be separated from the rest of the debris, this is where the magnetic arm comes into function. The magnetic assembly lowers down to the reaction tube, contained in the thermoshaker assembly; as soon as it reaches near the Thermoshaker assembly the magnetic beats gets attracted towards the magnet leaving the debris and the other unwanted solution in the tube below. Then the syringe assembly aspirate the unwanted solution and discards them. Once the discarding is completed the magnetic assembly moves back and the magnetic beads containing the extracted nucleic acid falls to the tube. This process is repeated for 3 to 4 times with wash buffer solutions to get rid of any unwanted material, and what is left is the magnetic beads with the nucleic acid. Then again to separate the magnetic beads and the nucleic acids, elution buffer is added and the mixture and is heated at 80o C, at this temperature the nucleic acids detach from the beads and gets suspended in the elution buffer, now again to get rid of the magnetic beads from the elution buffer, the magnetic arm moves towards the thermoshaker and attracts the magnet, leaving behind the elution buffer with the nucleic acid. The elution buffer with the nucleic acid is then aspirated and stored separately in the elution tube. The magnetic arm function ends here and it moves back to its original position behind the syringe assembly.

On basis of construction described above, the workflow of the entire process intended to be implemented therein may be understood by the following sequence of steps-
1) In the first stage, all the plastic wares consisting of tips, piercing tips, sample processing tube, elution tube, RTPCR cartridges and RTPCR tubes are placed onto the deck (4).
2) Once the plastic ware is placed in the system, sample tube consisting of the sample is placed on the deck, and a pre-programmed protocol (NAT/ Extraction 1000 and PCR NAT, Viral Load/ Extraction 200 and PCR Viral Load and Covid-19 and PCR Covid-19 as described later in this document) and steps of lysis, wash and elution- is run;
3) The system will pick the piercing tip and puncture the sealed wells on the cartridges. Next the system will aspirate the reagents required for extraction from the cartridges and dispense the same in the sample processing tube. This sample processing tube is placed on the thermoshaker (5) block for simultaneous heating and mixing of the extraction components with magnetic beads.
4) The extraction components are mixed with the magnetic beads at a pre-set temperature of 52 to 760C at 700-1300 RPM for a definite amount of time, 1300 seconds in particular, after which magnetic assembly attached with magnets moves closer, till about the distance of 12.5mm to the thermoshaker block unit. Once the magnetic assembly is in contact with the block assembly the magnet in the magnetic assembly aggregates the magnetic beads which are present in the sample processing tube along with the extraction reagents. This aggregated magnetic bead is bound with the extracted nucleic acids, leaving the other debris and the other unwanted wastes in the solution, which is discarded, using the syringe assembly.
5) Once the waste is discarded, the magnetic assembly housing the magnets, moves away from the block assembly and the magnetic aggregate falls back to the sample processing tube, which is then washed with buffers or solvents for further purification of the nucleic acids extracted and then housed in the thermoshaker block assembly.

A typical operation run of the automated sample-to-PCR ready system proposed herein is explained now in reference to certain screenshots of the user interface (exhibited on an electronic screen associated with the automated sample-to-PCR ready system proposed herein) as expressed here under in format of a standard operating protocol addressed to the user (you / your etc) as under-
1) Turn ON the Power button on automated sample-to-PCR ready system and Check if the indicators are Red as per FIG. 8A using the (four) tabs of each Station till magnets positions turn into Green indication
2) Once Red indication turn into Green on HMI screen for Magnet, click on Homing tab as seen in FIG. 8B.
3) Loading pipette tips and sample tubes - Once the instrument has completed the Homing position the main screen appears as seen in FIG. 8C. Upload the pipette tips, piercing tips, Sample tubes, elute tubes, extraction cart etc. on Deck (A or B) as per the mentioned positions.
4) Select the Protocol - Select the protocol as seen in FIG. 8D which you want to test like NAT/VIRAL LOAD/ COVID-19 as per your requirement on Deck A or B.
5) Start the Run - Select the appropriate tab of the protocol to be run on the system, observed as a sequence of the following working procedures-
a) At first, Syringe Head assembly will pick the piercing tip and puncture the sealed wells on the cartridges.
b) Next, the instrument will aspirate the reagents required for extraction from the cartridges and dispense the same in the sample processing tube.
c) This sample processing tube is placed on a shaker block for simultaneous heating and mixing of the extraction components with magnetic beads.
d) The extraction components are mixed with the magnetic beads at a pre-set temperature and rpm, for a definite amount of time, after which magnetic assembly attached with magnets moves closer to the shaker block unit.
e) Once the magnetic assembly is in contact with the block assembly the magnet in the magnetic assembly aggregates. The magnetic beads which is present in the sample processing tube along with the extraction reagents. This aggregated magnetic bead is bound with the extracted Nucleic Acid, leaving the other debris and the other unwanted wastes in the solution, which is discarded, using the syringe assembly.
f) Once the waste is discarded in waste box, the magnetic assembly housing the magnets, moves away from the block assembly and the magnetic aggregate falls back to the sample processing tube, which is then washed with buffers or solvents for further purification of the Nucleic Acid, housed in the thermo-shaker block assembly.
g) After completion of run instrument will discard used tips and return to home position and de- highlight protocol.
h) Once the extraction process completed, the consumables are kept for PCR protocol and click on PCR NAT/PCR Viral Load or PCR Covid-19.
i) As the PCR process start, Syringe Head assembly will pick the piercing tip and puncture the sealed wells on the PCR cartridges.
j) Syringe Head assembly will pick up template from elution tube and it will add into 1st well of PCR cartridge. Then it will again aspirate from 1st well and add into 3rd well of PCR cartridge
k) Reagents will transfer from 3rd well of PCR cartridge to PCR tube.
This is the final step in which the user will get the solution in PCR tube and transfer it for further process.
6) After the Run - After completion of RUN, click on Discard button on Monitor screen. On HMI, Warning message will Pop-up as seen in FIG. 8E below once you click the “Discard” button.
In this, Deck will move backward and stop at certain point to get easy access to remove the Waste Box. Discard all consumables from Waste box and fix it again on the deck at its prior position.

All the consumable and extracted nucleic acid are then stored at -20° Celsius and PCR tube is transferred to PCR machine for further use
7) Advance – Protocol Design - There are three types of Protocols that can be Run in the instrument of the present invention as below:
a. NAT/ Extraction 1000 and PCR NAT
b. Viral Load/ Extraction 200 and PCR Viral Load
c. Covid-19 and PCR Covid-19

The three types of protocols are expressed here under in format of a standard operating protocol addressed to the user (you / your etc) as under -
1) NAT/ Extraction 1000 OR PCR NAT:
a. Select NAT/ Extraction 1000 on HMI screen as seen in FIG. 8F on either Station A or B
b. There are three basic steps used in each protocol. They are as below:
i. Lysis - In most purification protocols for nucleic acids, the first step is usually lysis. The type of lysis buffer used depends on the types and source of cells, the desired final molecule or structure, and the level of their functionality. In this step, the cell and the nucleus are broken open to release the DNA inside and there are two ways to do this. This can be done with a tissue homogenizer (like a small blender), by cutting the tissue into small pieces. Second, lysis uses detergents and enzymes such as Proteinase K to free the DNA and dissolve cellular proteins
ii. Wash - The wash steps serve to remove impurities. There are typically two washes, although this can vary depending on the sample type. The first wash will often have a low amount of chaotropic salt to remove the protein and coloured contaminants. This is always followed with an ethanol wash to remove the salts. If the prep is something that didn’t have a lot of protein to start, such as plasmid preps or PCR clean up, then only an ethanol wash is needed
iii. Elution - DNA is soluble in low-ionic-strength solution such as TE buffer or nuclease-free water. When such an aqueous buffer is applied to a silica membrane, the DNA is released from the silica, and the eluate is collected. The purified, high-quality DNA is then ready to use in a wide variety of demanding downstream applications, such as multiplex PCR, coupled in vitro transcription/translation systems, transfection and sequencing reactions.

User control is enabled via selection / configuration of parameters used in the Extraction/PCR process via an HMI screen shown in FIG. 8G and as listed in Table 1 below.

Parameter Value (approximate)
Lysis Temperature 54° Celsius
Tip Drying temperature 74° Celsius
Elution Temperature 76° Celsius
Elution volume 20ul in extraction cartridge
Shaker Temperature actual temperature, which is shown by PID controller while running any step in each protocol
Magnet Height height set for magnet which is used in a bead mixing step
Bead mixing Cycle User-defined, from 0 to upto 99. Normally, 3 no of cycles used for NAT protocol.
Shaking time (prefilled time for Shaking module) As per temperature which is reached to a set value
Shaker magnet time (time for magnet after shaking mode, holds the beads by magnet) As per specifications of the run
Shaker solution mixing cycle (cycle which is used for mixing the solution in Shaker) As per specifications of the run
Tip drying time (time given in Extraction process) 300 seconds
Ex Piercing Selectable between ON and OFF states as per specifications of the run
PCR Piercing Selectable between ON and OFF states as per specifications of the run

Table 1

All the parameters and functional steps are similar as listed in Table 1 above, for all the three protocols but the values are different such as temperature, RPM, mixing time etc. as per the SOP changes

2) Viral load/ Extraction 200 and PCR Viral Load
This protocol is expressed here under in format of a standard operating protocol addressed to the user (instructions to you / your etc) as under- - Select Viral Load/ Extraction 200 on HMI screen show in FIG. 8H on either Station A or B

3) Covid-19 and PCR Covid-19:
This protocol is expressed here under in format of a standard operating protocol addressed to the user (instructions to you / your etc) as under - Covid-19 and PCR Covid-19 - Select Covid-19/ PCR Covid-19 on HMI screen seen in FIG. 8I on either Station A or B.

Reagent preparation in the present invention:
For reagent preparation, cartridges having eight discrete tubes are provided for RTPCR analysis. The Extraction Cartridge, shown in FIG. 9A contains following reagents
• Carrier RNA (poly (A) RNA)
• Internal Control (exogeneous Internal control genes)
• Lysis Enhancer (Consists of Tris HCL, Sodium Acetate, Guanidine Thiocyanate, EDTA, DTT, SDS, Sodium Azide, Proteinase K and DEPC treated water)
• Magnetic Beads (silica coated superparamagnetic particles, 700 nm in diameter and spherical in shape)
• Wash Buffer 1 (consists of Tris HCL, Sodium Acetate, Guanidine Thiocyanate, Sodium azide, DEPC treated water)
• Wash Buffer 2.1 (Consists of Tris HCL, Sodium Acetate, DEPC treated water)
• Wash Buffer 2.2 (Consists of Tris HCL, Sodium Acetate, DEPC treated water)
• Elution Buffer (Consists of Diethyl pyrocarbonate treated HPLC grade water)

The PCR cartridge, shown in FIG. 9B, contains following scheme of reagents in four discrete tubes-
• Reagent-1 (an admixture of Primer, Probes and Nuclease free water)
• Blank (empty / no reagents)
• Reagent-2 (contains PCR enzymes such as Taq polymerase, dNTPs and MgCl2 salts)
• Blank (empty / no reagents)

Once the extraction is completed the extracted nucleic acid is contained in the elution tube as shown in FIG. 2 and FIG. 10.

The elution volume varies according to the different protocol. This nucleic acid is used for further analysis. The syringe assembly aspirates a defined volume of nucleic acid from the elution tube and mixes with the cartridges containing the reagents for RTPCR analysis, once this process is completed, the final mixture is dispensed into a RTPCR tube and the process of reagent preparation is completed.

Thus to summarize, the processing steps involved in reagent preparation and actual assay protocol (from extraction through detection) are efficiently designed to be implemented inside the very system of the present invention, which consists of removable extraction and purification cartridge apparatus for an automated nucleic acid extraction and purification system wherein the nucleic acid extraction and purification system includes a first line component and a second line component for mixing of the RTPCR components with the purified NA.

Industrial applicability
The present invention scores over prior art notably in that there are currently no other systems reported and / or available in the market which can perform two separate functions of nucleic acid extraction and PCR reagent preparation in an automated manner, in a single system. Also, the system proposed herein negates the need for additional pre-processing steps for use, manual intervention (and thus errors due to the same), which are characteristic of prior art systems directed at analogous use-cases. More objectively, the present invention scores above peer technologies in that-

a) Fully automated sample preparation to amplification ready system for sample analysis is ably provisioned which can perform multiple parameters and multiple samples at the same time increasing the sample throughput.
b) all operations from sample handing to PCR tube preparationare fully streamlined and automated within a compact footprint;
c) steps of extraction, reagent preparation and amplification performed conventionally in separate rooms are performed in the same individual system;
d) sample processing burdens are truly reduced;
e) huge infrastructure and capital requirement of molecular testing laboratories are eliminated – specifically, space requirement is reduced from few hundred sq. Ft to Table top;
f) no risk of inter-contamination of samples being processed as the present invention is modelled in a single test single cartridge format.
g) there is flexibility to test multiple parameters with multiple samples at the same time, specifically, high throughput sample processing of 1 to 32 samples is provisioned in single run and 12 runs per day for maximum efficiency, which is applicable across all molecular RT-PCR tests;
h) No manual intervention is required. Complete automated procedure from raw sample to analysis ready sample overcoming manual errors and minimum reagent wastage
i) an able solution is proposed which is not riddled with undue complexities or capital / operative costs.

Once the above process is completed, all the consumables (reagents) and extracted nucleic acid is stored at -20°C or the PCR tube is transferred to a conventional PCR thermocycler / amplifier for further use.

Owing to the advantages recited above, and further ones which will become apparent to the reader, the present invention finds applicability in myriad fields including bench-top research in molecular biology and immunochemistry, scaled screening / diagnostic facilities in clinical laboratories.

Many modifications and other embodiments of the invention will be apparent to those skilled in the art to which the invention pertains in view of the above description and the teachings presented in the accompanying drawings, all of which are intended to be encompassed by spirit of the present invention. The invention disclosed herein shall be limited only by the claims in the complete specification which is intended to be submitted in pursuance of this paper. ,CLAIMS:1] A sample-to-polymerase chain reaction-ready system consisting of:
a) A first sub-system for automated extraction of nucleic acids from test samples, said first sub-assembly consisting of-
? A syringe assembly comprising a precision plunger type syringe unit (1) for aspiration of fluids, said precision plunger type syringe unit (1) being housed in a stepper motor-based assembly (2) to allow regulated upward and downward movement of said precision plunger type syringe unit (1);
? A magnetic assembly (3) positioned behind and connected to the syringe assembly to thereby allow regulated upward or downward movement by means of a motor (19) and coupled assembly (22), as well as forward or reverse movement by means of a motor (20) and coupled assembly (21) of said syringe assembly, magnet holding arm (23) and magnets (24) for separation of solid magnetic substrates from the liquid contents of sample processing tubes; and
? thermoshaker assembly (5) for simultaneous heating and mixing for the extraction components with magnetic beads, said including a base plate (15), a brushless direct current motor (14) mounted below the base plate (15), leaf spring assembly (16) attached below said base plate (15) and driven by said brushless direct current motor (14) about an acentric shaft, a block holder (25) with block (17) having wells (18) in connection with the acentric shaft for holding the sample processing tubes, and regulators for temperature and RPM for performance of the polymerase chain reaction.
b) A second sub-system for automated preparation of reagents needed in performance of polymerase chain reaction, said second sub-assembly consisting of-
? A library of cartridges including an extraction cartridge and a polymerase chain reaction cartridge containing reagents of choice; and
? elution tube for containing the extracted nucleic acids from the sample processing tubes.
c) A deck (4) for housing consumables required in the working of the first and second sub-systems, said deck (4) being provisioned with-
? A stepper motor for regulated forward and reverse movement of the deck (4);
? place holders (6, 7, 9, 10, 11, 12, and 13) being contoured recesses within the deck (4) for receiving piercing tips, sample processing tubes, extraction cartridge, elution tube, RTPCR / extraction cartridges, RTPCR tubes and tips being discarded respectively;
? an area (8) for landing and thus serving as the footprint of the thermoshaker assembly (5).
2] An electronic screen for serving as a human machine interface for presentation of user-selectable operations and parameter values for driving the process to be performed using the sample-to-polymerase chain reaction-ready system.

3] The sample-to-polymerase chain reaction-ready system as claimed in claim 1, wherein speed of the brushless direct current motor (14) ranges between from 100 RPM to 1500 RPM under regulation of a brushless direct current drive.

4] The sample-to-polymerase chain reaction-ready system as claimed in claim 1, wherein the regulator for temperature is a PID controller which combines proportional, integral, and derivative control by comparison of a set point or reference signal with a signal from the process plant, to thereby achieve precise control over the polymerase chain reaction being implemented.

5] The sample-to-polymerase chain reaction-ready system as claimed in claim 1, wherein the PID controller consists of final control devices including actuators, control valves, and other control devices for deployment as per the polymerase chain reaction –based process to be implemented.

6] The sample-to-polymerase chain reaction-ready system as claimed in claim 1, wherein the wells (18) are positioned at the edge of the block (17) to facilitate the magnetic beads present in the tube along with the substrate to get attracted to the magnetic assembly (3).

7] The sample-to-polymerase chain reaction-ready system as claimed in claim 1, wherein the extraction cartridge has eight discrete tubes to contain reagents of choice, respectively being -
a) Carrier RNA, being poly (A) RNA in particular.
b) Internal Control, being exogeneous Internal control genes in particular.
c) Lysis Enhancer consisting of Tris HCL, Sodium Acetate, Guanidine Thiocyanate, EDTA, DTT, SDS, Sodium Azide, Proteinase K and DEPC treated water.
d) Magnetic Beads, being silica coated superparamagnetic particles characterized in having a spherical geometry of 700 nm diameter.
e) Wash Buffer 1, consisting of Tris HCL, Sodium Acetate, Guanidine Thiocyanate, Sodium azide, DEPC treated water
f) Wash Buffer 2.1 consisting of Tris HCL, Sodium Acetate, DEPC treated water
g) Wash Buffer 2.2 consisting of Tris HCL, Sodium Acetate, DEPC treated water
h) Elution Buffer consisting of Diethyl pyrocarbonate treated HPLC grade water

8] The sample-to-polymerase chain reaction-ready system as claimed in claim 1, wherein the polymerase chain reaction cartridge has four discrete tubes to contain reagents of choice, respectively being -
a) Reagent-1, consisting of an admixture of Primer, Probes and Nuclease free water
b) Blank, being empty
c) Reagent-2, consisting of an admixture of PCR enzymes such as Taq polymerase, dNTPs and MgCl2 salts.
d) Blank, being empty

9] The sample-to-polymerase chain reaction-ready system as claimed in claim 1, wherein the process to be performed is selected, at instance of the user, selected among-
a) NAT/ Extraction 1000 and PCR NAT
b) Viral Load/ Extraction 200 and PCR Viral Load
c) Covid-19 and PCR Covid-19