Rapid sample processing and nucleic acid amplification test on a cartridge

Abstract

The present invention relates to a cartridge (101) for sample pre-treatment. The cartridge (101) includes a sample chamber (103) that is defined at a top portion (101a) of the cartridge (101) and configured to receive the sample and a pre-treatment reagent through an opening that is and are lysed to for a pre-treated sample. The cartridge (101) also includes a plurality of reaction wells (105) that are defined at a bottom portion (101b) of the cartridge (101). Further, at least one valve (104) is fluidly connected to the sample chamber (103) and the plurality of reaction wells (105). The at least one valve (104) is configured to control a flow of the pre-treated sample from the sample chamber (103) to the plurality of reaction wells (105). The cartridge (101) further includes a cap (102) that is externally connectable to the opening defined on the sample chamber (103).

Description

[0001] RAPID SAMPLE PROCESSING AND NUCLEIC ACID AMPLIFICATION TEST ON A CATRIDGE TECHNICAL FIELD

[0002] Present disclosure generally relates to the field of bio-medical engineering. Particularly, but not exclusively, the present disclosure relates to a nucleic acid amplification and detection system. Further, embodiments of the present disclosure disclose a cartridge and an apparatus for rapid sample processing and nucleic acid amplification test and a method thereof.

[0003] BACKGROUND OF INVENTION

[0004] Amplification of nucleic acids is an essential process in biological systems for detecting and sequencing a small amount of nucleic acids such as Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) in molecular diagnostics. Nucleic acid amplification test is a molecular technique to detect a particular sequence of nucleic acid from virus, bacteria or other pathogens in a sample. To amplify the amount of nucleic acid, several techniques of nucleic acid synthesis have been developed accordingly.

[0005] The existing technologies in direct sample-to-result nucleic acid amplification provide foundational capabilities for detecting nucleic acids directly from clinical and environmental samples. However, these methods face limitations in terms of complexity, sensitivity to sample inhibitors, detection challenges, and need for specialized equipment. Conventional processes typically require extensive pre-treatment, including multiple sample purification steps, to remove inhibitors that can interfere with amplification. Additionally, existing isothermal amplification methods, such as Loop-Mediated Isothermal Amplification (LAMP) and Recombinase Polymerase Amplification (RPA), are highly susceptible to contaminants present in raw samples, necessitating additional preparation and equipment that make these technologies less feasible for point-of-care and field applications.

[0006] The detection systems also pose challenges, as real-time or integrated detection is often lacking, leading to multi-step processes that increase the risk of contamination and complexity. This complexity and sensitivity to sample conditions limit the broader applicability of these technologies across different sample types (e.g., sputum, blood) and settings, such as remote areas or field environments where access to laboratory equipment is minimal. Moreover, existing methods are generally sample-specific, requiring tailored protocols for each sample type, which further complicates workflow and restricts usability in varied environments. Thus, by streamlining the pre-treatment and amplification process, enhancing inhibitor tolerance, andintegrating detection into a cohesive, efficient system could result in a rapid, robust, and versatile diagnostic platform that is well-suited for point-of-care, field, and low-resource environments, offering significant advantages over conventional processes.

[0007] The present disclosure is directed to overcome one or more limitations stated above. The background section of the present disclosure should not be considered as a limitation of the present disclosure.

[0008] The drawbacks / difficulties / disadvantages / limitations of the conventional techniques explained in the background section are just for exemplary purpose and the disclosure would never limit its scope only such limitations. A person skilled in the art would understand that this disclosure and below mentioned description may also solve other problems or overcome the other drawbacks / disadvantages of the conventional arts which are not explicitly captured above.

[0009] SUMMARY OF THE DISCLOSURE

[0010] One or more shortcomings of the conventional design are overcome by configuration of a cartridge for sample pre-treatment for an apparatus for sample pre-treatment and amplification of nucleic acid as claimed and additional advantages are provided through the provision of the cartridge as claimed in the present disclosure.

[0011] Additional features and advantages are realized through the configuration of the present disclosure. Other embodiments and aspects of the disclosure are described in detail herein and are considered a part of the claimed disclosure.

[0012] In one non limiting embodiment of the present disclosure, a cartridge for sample pre-treatment is disclosed. The cartridge includes a sample chamber that is defined at a top portion of the cartridge. The sample chamber is configured to receive the sample and a pre-treatment reagent through an opening that is defined on the sample chamber and are lysed to for a pre-treated sample. The cartridge also includes a plurality of reaction wells that are defined at a bottom portion of the cartridge. Further, at least one valve is fluidly connected to the sample chamber and the plurality of reaction wells. The at least one valve is configured to control a flow of the pre-treated sample from eh sample chamber to the plurality of reaction wells. The cartridge further includes a cap that is externally connectable to the opening defined on the sample chamber.In an embodiment of the present disclosure, the cap is configured to store and supply the pretreatment reagent to the sample chamber.

[0013] In an embodiment of the present disclosure, the cartridge includes a plurality of channels extending from each of the plurality of reaction wells and are fluidly connected to the sample chamber.

[0014] In an embodiment of the present disclosure, each of the plurality of reaction wells is configured to contain a predefined concentration of an amplification reaction mixture.

[0015] In an embodiment of the present disclosure, the amplification reaction mixture includes a predefined concentration of primer, enzymes and reagent.

[0016] In an embodiment of the present disclosure, the predefined concentration of primer ranges between 0.2 pM to 2 pM per primer.

[0017] In an embodiment of the present disclosure, the predefined concentration of enzymes between 5 U to 20 U per reaction.

[0018] In one non limiting embodiment of the present disclosure, an apparatus for sample pretreatment and amplification of nucleic acid is disclosed. The apparatus includes a cartridge for sample pre-treatment. The cartridge includes a sample chamber that is defined at a top portion of the cartridge. The sample chamber is configured to receive the sample and a pre-treatment reagent through an opening that is defined on the sample chamber and are lysed to for a pretreated sample. The cartridge also includes a plurality of reaction wells that are defined at a bottom portion of the cartridge. Further, at least one valve is fluidly connected to the sample chamber and the plurality of reaction wells. The at least one valve is configured to control a flow of the pre-treated sample from eh sample chamber to the plurality of reaction wells. The cartridge further includes a cap that is externally connectable to the opening defined on the sample chamber. The apparatus further includes an ultrasonic transducer that is disposed proximal to the sample chamber. The ultrasonic transducer is configured to emit ultrasonic waves to assist lysing the sample and the pre-treatment reagent during pre-treatment. Further, a plurality of heating elements are disposed proximal to the sample chamber and the plurality of reaction wells. The plurality of heating elements are configured to maintain a predefined temperature within the sample chamber and the plurality of reaction wells. The pre-treated sample is amplified in the plurality of reaction wells.In an embodiment of the present disclosure, the apparatus includes an optic module that is disposed proximal to the plurality of reaction wells and opposite to the plurality of heating elements. The optics module is configured to capture and detect a condition of the sample from the plurality of reaction wells.

[0019] In an embodiment of the present disclosure, the cap is configured to store and supply the pretreatment reagent to the sample chamber.

[0020] In an embodiment of the present disclosure, the apparatus includes a plurality of channels that extends from each of the plurality of reaction wells and are connected to the sample chamber.

[0021] In an embodiment of the present disclosure, each of the plurality of reaction wells is configured to contain a predefined concentration of an amplification reaction mixture.

[0022] In an embodiment of the present disclosure, the amplification reaction mixture includes primer, enzymes and reagent.

[0023] In an embodiment of the present disclosure, the predefined concentration of primer ranges between 0.2 pM to 2 pM per primer

[0024] In an embodiment of the present disclosure, the predefined concentration of enzyme ranges between 5 U to 20 U per reaction.

[0025] In an embodiment of the present disclosure, the apparatus includes at least one valve actuator configured to actuate the a least one valve.

[0026] In an embodiment of the present disclosure, the at least one valve actuator is configured to direct infrared radiation towards the at least one valve.

[0027] In an embodiment of the present disclosure, the at least one valve includes a polyethylene black film or infrared spectrum that is configured to block the flow of the pre-treated sample from the sample chamber and thermally deform upon exposure to the infrared radiation. Thereby opening the at least one valve to control the flow of yeh pre-treated sample to the plurality of reaction wells.

[0028] In an embodiment of the present disclosure, actuation of the valve is non-contact type stimulus.In one non limiting embodiment of the present disclosure, a method for sample pre-treatment and nucleic acid amplification is disclosed. The method comprises steps of collecting a predefined quantity of sample from a subject in a sample chamber of a cartridge. Adding a predefined quantity of reagent into the sample chamber and mixing the sample with the pretreatment reagent. The method further includes the step of lysing by delivering ultrasonic wave by an ultrasonic transducer to pre-treat and make the nucleic acids accessible from amplification and form a pre-treated sample. Further steps include actuating a valve to open and supply the pre-treated sample to plurality of reaction wells. The method further includes the step of amplifying the pre-treated at a pre-defined temperature in the plurality of reaction wells and determining through an optic module a condition of the amplified sample form the plurality of reaction wells.

[0029] In an embodiment of the present disclosure, the predefined quantity of sample ranges from 100 pL-400 pL.

[0030] In an embodiment of the present disclosure, the predefined quantity of pre-treatment reagent ranges from 20 pL to 50 pL.

[0031] In an embodiment of the present disclosure, wherein the pre-defined temperature ranges from 37°C - 70°C.

[0032] It is to be understood that the aspects and embodiments of the disclosure described above may be used in combination with each other. Several of the aspects and embodiments may be combined to form a further embodiment of the disclosure.

[0033] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[0034] BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

[0035] The novel features and characteristic of the disclosure are set forth in the appended claims. The disclosure itself, however, as well as a preferred mode of use, further objectives, and advantages thereof, will best be understood by reference to the following detailed descriptionof an illustrative embodiment when read in conjunction with the accompanying figures. One or more embodiments are now described, by way of example only, with reference to the accompanying figures wherein like reference numerals represent like elements and in which:

[0036] Figure 1 illustrates a schematic view of an apparatus for sample pre-treatment and amplification of nucleic acid, in accordance with an embodiment of the disclosure;

[0037] Figure 2 illustrates a schematic view of a cartridge of the apparatus of Figure 1, in accordance with an embodiment of the disclosure;

[0038] Figure 3 illustrates a schematic view of another embodiment of a cartridge of the apparatus of Figure 1, in accordance with an embodiment of the disclosure;

[0039] Figure 4 illustrates a schematic view of a cap of the cartridge of Figure 2, in accordance with an embodiment of the disclosure; and

[0040] Figure 5 illustrates a block diagram of the method of operation of the apparatus of Figure 1, in accordance with an embodiment of the disclosure.

[0041] The figures depict embodiments of the disclosure for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] The foregoing has broadly outlined the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter which form the subject of the claims of the disclosure. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present disclosure. It should also be realized by those skilled in the art that such equivalent processes do not depart from the scope of the disclosure as set forth in the appended claims. The novel features which are believed to be characteristic of the disclosure, both as to its organization and method of operation, together with further objects and advantages will bebetter understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this disclosure.

[0044] The terms “comprises”, “comprising”, or any other variations thereof used in the specification, are intended to cover a non-exclusive inclusion, such that the system comprises a list of features / elements or steps does not include only those features / elements, but may include other features and elements not expressly listed or inherent to such setup or structure. In other words, one or more features / elements in a system proceeded by “comprises... a” does not, without more constraints, preclude the existence of other elements or additional elements in the system thereof. Also, the terms like “at least one” and “one or more” may be used interchangeably or in combination throughout the description.

[0045] Reference will now be made to the exemplary embodiments of the disclosure, as illustrated in the accompanying drawings. Wherever possible, same numerals will be used to refer to the same or like parts. Embodiments of the disclosure are described in the following paragraphs with reference to Figures 1 to 5, the same element or elements which have same functions are indicated by the same reference signs.

[0046] Figure 1 illustrates a schematic view of an apparatus (100) for sample pre-treatment and amplification of nucleic acid. The apparatus (100) may include a housing [not shown]. The housing may be adapted to house plurality of components. The housing may be made of material such as but not limited to polycarbonate, ABS or in combination thereby. The housing is configured to encapsulate the plurality of components housed inside from the outer atmosphere and external factors. The housing may be defined with an opening [not shown]. In an embodiment, the opening may be defined on a top surface of the housing. The opening may be configured to receive a cartridge (101).

[0047] Referring to Figure 2, a schematic view of the cartridge (101) is shown. The cartridge (101) of the present disclosure may be defined with a top portion (101a) and a bottom portion (101b). In an embodiment, the cartridge (101) may be defined with a geometrical shape correspondingto rectangle or square. Such structural configuration of the cartridge (101) should not be considered to limit the scope of the invention. In an embodiment, the cartridge (101) may be defined with a thickness (t). The cartridge (101) may be formed of material such as but not limited to polypropylene, polycarbonate and the like. The cartridge (101) may be manufactured through the processes of heat bonding or ultrasonic welding but not limited to the same. The cartridge (101) includes a sample chamber (103). In an embodiment the sample chamber (103) may be defined on the top portion (101a) of the cartridge (101). The sample chamber (103) may be defined with a thickness (T) defined along an axis A-A [as shown in Figure 1], In an embodiment, the thickness (T) of the sample chamber (103) may be greater that thickness (t) of the cartridge (101). That is, the sample chamber (103) may over hand away from the cartridge (101) to increase volume of the sample chamber (103). Further, the sample chamber (103) may be defined with a first end (103a) and a second end (103b). The sample chamber (103) may be further defined with an opening. In an embodiment, the opening may be defined on the first end (103a) of the sample chamber (103). In an embodiment, the opening may be defined with a threaded portion [not shown]. Further, the sample chamber (103) is configured to receive and hold a predefined quantity of biological sample [herein after alternatively referred as sample] from a subject. The term “subject” in the foregoing description may refer to human or an animal. In an embodiment the predefined quantity of the sample may be ranging from 5 pL to 50 pL. In an embodiment, preferred quantity of the sample may be ranging from 10 pL to 20 pL. Furthermore, the sample chamber (103) is also configured to receive a predefined quantity of pre-treatment reagents. In an embodiment, the pre-defined quantity of the pre-treatment reagent may be stored in the sample chamber (103). The predefined quantity of the pre-treatment reagent may range from 20 pL to 50 pL. The pre-treatment reagent may include one or more buffering agents and chelating agents such as Ethylenediaminetetraacetic Acid (EDTA) and Ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N' -tetraacetic acid (EGTA). Further, the pre-treatment reagent may be defined prerequired pH concentration. The pH concentration of pre-treatment reagent may be ranging between 8-11, preferably between pH 8-9. The pre-treatment reagent may be Tris / Tricine having a concentration range ImM to 200mM and EDTA concentration range between 0.05mM to 250mM. The pre-treatment reagent may contain an antimicrobial agent selected from a list of agents not limited to Triclosan, Azide, and others, preferably sodium azide at a concentration range of 0.005mM to 200mM. The pre-treatment reagent may also contain added resins for example ion exchange resin. The sample may be lysed with the predefined quantity of pre-treatment reagent to form a pre-treated sample. The sample chamber (103) may include plurality of beads. The beadsmay be made of material such as but not limited to glass, Ceramic, silica, zirconia. The beads may be diameter ranging from 0.05-0.4mm. Further, in an embodiment, the beads may be defined with a weight ranging from 20mg-500mg. In an embodiment, the plurality of beads aid in lysing the sample and the pre-treatment reagent to form the pre-treated sample.

[0048] Referring again to Figures 2 and 3, the cartridge (101) includes a plurality of reaction wells (105). In an embodiment the plurality of reaction wells (105) may be defined on the bottom portion (101b) of the cartridge (101). In an illustrated embodiment, the cartridge (101) is defined with eight reaction wells (105) but not limited to the same. In another embodiment, the cartridge (101) is defined with six reaction wells (105) but not limited to the same [as shown in Figure 3], In an embodiment, the cartridge (101) is defined with a conduit (1051) connecting the plurality of reaction wells (105). Furthermore, the cartridge (101) includes at least one valve (104) [hereinafter alternatively referred as valve]. The at least one valve (104) may be fluidly connected to the sample chamber (103) and the plurality of reaction wells (105). In an embodiment, the at least one valve (104) may be between the sample chamber (103) and the plurality of reaction wells (105). That is, one end of the at least one valve (104) may be connected to the second end (103b) of the sample chamber (103), and another end of the at least one valve (104) may be connected to the conduit (1051) connecting the plurality of reaction wells (105). In an alternative embodiment, at least one valve (104) may be fluidly connected to sample chamber (103) and each of the plurality of reaction wells (105). Further, the at least one valve (104) is configured to control the flow of the pre-treated sample from the sample chamber (103) to the plurality of reaction wells (105). In an embodiment, the valve (104) may be a polyethylene black film or infrared spectrum absorbing valve (104). That is, the valve (104) may include a polyethylene black film or an infrared spectrum configured to block the flow of the pre-treated sample from the sample chamber (103) to the plurality of reaction wells (105). In an embodiment, a filter may be provided between the sample chamber (103) and the valve (104). The filter may be configured to prevent the plurality beads flowing along with the pre-treated sample to the plurality of reaction wells (105). Further, the plurality of reaction wells (105) are configured to contain a predefined concentration of an amplification reaction mixture. The predefined concentration of the amplification reaction mixture may include a predefined concentration of primer, enzymes and reagents. The amplification reaction mixture may be lyophilized, desiccated, air dried or glassified. In an embodiment, predefined concentration of primer ranges between 0.2 pM to 2 pM per primer, the predefined concentration of enzymes between 5 U to 20 U per reaction. The pre-treated sample may beamplified in the plurality of reaction wells (105). The cartridge (101) further includes plurality of channels (110) and plurality of vents (111). The plurality of channels (110) may extend from the each of the plurality of reaction wells (105) and are fluidly connected to the sample chamber (103). The plurality of vents (111) may be defined in the sample chamber (103) and proximal to the reaction wells (105). In an embodiment, the plurality of channels (110) and plurality of vents (111) are configured to prevent contamination that may arise due to the vapor coming out of the plurality of reaction wells (105).

[0049] Referring to Figures 2 and 4, the cartridge (101) includes a cap (102). The cap (102) may be externally connectable to the opening defined on the sample chamber (103). In an embodiment, the cap (102) may be defined with an inner surface (102a) and an external surface (102b). The inner surface (102a) may be defined with a threading (1024) complementing the threading defined on the opening of the sample chamber (103) and configured to be threaded to form a seal. In an alternative embodiment, the cap (102) and opening of the sample chamber (103) may be sealed by any means known in the art. In an embodiment, the cap (102) may be configured to store the predefined quantity of pre-treatment reagent. The internal surface of the cap (102) may be defined with a cavity (1021) and configured to store the predefined quantity of pre-treatment reagent. Further, the cap (102) of the present disclosure may include a piercing tool (1022) and a sealing (1023). The sealing (1023) may be aluminium foil sealing (1023) and configured to hold the pre-treatment reagent within the cavity (1021) of the cap (102). The piercing tool (1022) may be configured to puncture into the sample chamber (103) to engage the cap (102) with the cartridge (101) by locking into position upon closure and to break the sealing (1023) to dispense the predefined quantity of pre-treatment reagent into the sample chamber (103). The predefined quantity of pre-treatment may also be added manually into the sample chamber (103).

[0050] Referring back to Figure 1, the apparatus (100) includes an ultrasonic transducer (107). The ultrasonic transducer (107) may be disposed proximal to the sample chamber (103). That is, the ultrasonic transducer (107) is positioned such that the apparatus (100) upon receiving the cartridge (101), the sample chamber (103) of the cartridge (101) may align with the ultrasonic transducer (107). The ultrasonic transducer (107) may be configured to emit ultrasound waves to the sample chamber (103). In an embodiment, the frequency of the ultrasound wave may range between 10 KHz to 100 KHz. The ultrasonic wave may assist in lysing the sample and the pre-treatment reagent during pre-treatment. That is, due to the ultrasonic wave from theultrasonic transducer (107), the beads in the sample chamber (103) may be configured to vibrate and assist in lying the sample and pre-treatment reagent to form the pre-treated sample. In an embodiment, the lysing may be done without the presence of the beads. The lysing configuration of the present disclose facilitates in eliminating the need of manual pre-treatment process and thus reduces errors and contamination during pre-treatment process.

[0051] In an embodiment, the apparatus (100) further includes a plurality of heating elements (106). The plurality of heating elements (106) may be disposed proximal to the sample chamber (103) and the plurality of reaction wells (105). The plurality of heating elements (106) are configured to maintain a predefined temperature within the sample chamber (103) and the plurality of reaction wells (105). That is the amplification of the pre-treated sample in the plurality of reaction wells (105) may be performed at the predefined temperature. The predefined temperature at the plurality of reaction well may range from 37°C to 70°C, preferably 60°C to 65°C. Further, the apparatus (100) includes at least one valve actuator (109) [hereinafter alternatively referred as valve actuator]. The valve actuator (109) may be at least one of infrared spectrum based actuator. The valve actuator (109) may be disposed proximal to the at least one valve (104) defined on the cartridge (101). The at least one valve actuator (109) is configured to actuate the at least one valve (104) to open and control the flow of the pre-treated sample from the sample chamber (103) to the plurality of reaction wells (105). In an embodiment, the valve actuator (109) may direct infrared radiation towards the at least one valve (104) causing the polyethylene black film to deform and open the at least one valve (104). The at least one valve (104) may be non-contact type stimulus, thus does not interfere or contaminate the pretreated sample within the cartridge (101).

[0052] Furthermore, referring to Figure 1, the apparatus (100) of the present disclosure includes an optics module (108). The optics module (108) may be disposed proximal to the plurality of reaction wells (105) and opposite to the plurality of heating elements (106). The optics module (108) may be configured to capture and detect condition of the amplified sample from the plurality of reaction wells (105). The optics module (108) may include a combination of optics fiber, camera and photo diode. Further the apparatus (100) may include a light unit (112). The light unit (112) may be at least one of ultra-violet light, visible light or infrared light. The light unit (112) may facilitate in observing the reaction in cartridge (101). The monitoring of amplification reaction may be real-time, endpoint, melt curve and the like. The apparatus (100) of the present disclosure may further include a control unit [not shown]. The control unit maybe a specialized control unit such as integrated system (bus) controllers, memory management control unit, floating point units, graphics processing units, digital signal processing units, etc. The control unit may include a microprocessor, such as AMD Athlon, Duron or Opteron, ARM’s application, embedded or secure processors, IBM PowerPC, Intel’s Core, Itanium, Xeon, Celeron, or other line of processors, etc. The control unit may be implemented using a mainframe, distributed processor, multi-core, parallel, grid, or other architectures. Some embodiments may utilize embedded technologies like application-specific integrated circuits (ASICs), digital signal processors (DSPs), Field Programmable Gate Arrays (FPGAs), etc. The control unit may be electrically connected to the ultrasonic transducer (107), the plurality of heating element and the at least one valve actuator (109). The control unit may be configured to selectively actuate the ultrasonic transducer (107), the plurality of heating element and the at least one valve actuator (109) upon receiving the cartridge (101) within the housing. In an embodiment, the apparatus (100) includes a battery [not shown]. The battery is configured to supply power to the ultrasonic transducer (107), the plurality of heating element and the at least one valve actuator (109) to work through the control unit.

[0053] Now referring to Figure 5, a method (300) for sample pre-treatment and nucleic acid amplification is illustrated. The method (300) includes a first step (301) of collecting a predefined quantity of sample from the subject. Accordingly, a predefined quantity of pretreatment reagent is added into the sample chamber (103) and mixed roughly with the sample in the second step (302). In an embodiment, the cap (102) is closed and the pre-treatment reagent stored in the cap (102) may be dispensed into the sample chamber (103). In alternate embodiment, the pre-treatment reagent may be manually added. Further, the cartridge (101) may be positioned in the apparatus (100) for further processing the sample and the plurality of heating units may be activated to adjust the temperature at the predefined temperature. In the third step (303), the roughly mixed sample and pre-treatment reagent is lysed by delivering the ultrasonic wave by the ultrasonic transducer (107) to pre-treat and make the nucleic acids accessible for amplification and form a pre-treated sample. The sample may be lysed for a duration ranging from 1 minutes to 5 minutes. Further, in the fourth step (304) the at least one valve (104) may be actuated by the at least one valve actuator (109) to open and supply the pre-treated sample to plurality of reaction wells (105). The pre-treated sample may be amplified at a pre-defined temperature in the plurality of reaction wells (105) at a fifth step (305). The pre-treated sample may be subjected to isothermal amplification reactions for a duration ranging from 10 minutes to 20 minutes in the plurality of reaction wells (105), each containinglyophilized isothermal amplification mixture. As an example, the amplification reactions may be at least one of but not limited to LAMP, RPA, TMA and SDA. The plurality of heating elements (106) are configured to maintain a constant temperature in the sample chamber (103) and plurality of reaction wells (105). In the sixth step (306), the condition of the amplified sample is determined through the optics module (108) from the plurality of reaction wells (105). In other words, the amplified sample is detected in real-time using an embedded detection systems that may be at least one of but not limited to colorimetric, fluorescence and turbidity changes. For instance, LAMP reactions may use fluorescence detection that can be through intercalating dyes or fluorescent probes that fluoresce upon binding to double-stranded DNA, providing an immediate visual or quantitative readout. In an embodiment, the fluorescence detection may be performed with ultra-violet light, visible light or infrared light of wavelength ranging between -350 Nm to 800 Nm supplied from the light unit (112). Further, the sample may be analyzed to indicate the presence or absence of the targeted nucleic acid providing diagnostic results in under 30 minutes.

[0054] The present disclosure offers several advantages, including a significant reduced sample processing time due to the rapid pre-treatment process, which drastically cuts down the time required for nucleic acid preparation, enabling faster diagnostic results. It requires minimal equipment, making the workflow suitable for decentralized or point-of-care testing environments. By effectively removing inhibitors and optimizing the availability of nucleic acids, the present disclosure enhances the sensitivity and specificity of amplification, ensuring more accurate results. Additionally, the streamlined process is user-friendly and minimizes the need for highly trained personnel, making it adaptable for use in a wide range of settings, including resource-limited environments.

[0055] EQUIVALENTS

[0056] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0057] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should typically be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A andB.”In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0058] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.Referral numerals:

[0059]

[0060]

Claims

We claim:

1. A cartridge (101) for sample pre-treatment, the cartridge (101) comprising:a sample chamber (103) defined at a top portion (101a) of the cartridge (101), the sample chamber (103) configured to receive the sample and a pre-treatment reagent through an opening defined on the sample chamber (103) and are lysed to form a pretreated sample;a plurality of reaction wells (105) defined at a bottom portion (101b) of the cartridge (101);at least one valve (104) fluidly connected to the sample chamber (103) and the plurality of reaction wells (105), the at least one valve (104) is configured to control flow of the pre-treated sample from the sample chamber (103) to the plurality of reaction wells (105); anda cap (102) externally connectable to the opening defined on the sample chamber (103).

2. The cartridge (101) as claimed in claim 1, wherein the cap (102) is configured to store and supply the pre-treatment reagent to the sample chamber (103).

3. The cartridge (101) as claimed in claim 1, comprises a plurality of channels (110) extending from each of the plurality of reaction wells (105) and fluidly connected to the sample chamber (103).

4. The cartridge (101) as claimed in claim 1, wherein each of the plurality of reaction wells (105) is configured to contain a predefined concentration of an amplification reaction mixture.

5. The cartridge (101) as claimed in claim 4, wherein the amplification reaction mixture includes a predefined concentration of primer, enzymes and reagent.

6. The cartridge (101) as claimed in claims 4 and 5, wherein the predefined concentration of primer ranges between 0.2 pM to 2 pM per primer.

7. The cartridge (101) as claimed in claims 4 and 5, wherein the predefined concentration of enzymes between 5 U to 20 U per reaction.

8. An apparatus (100) for sample pre-treatment and amplification of nucleic acid, the apparatus (100) comprising:a cartridge (101) for sample pre-treatment, the cartridge (101) comprising:a sample chamber (103) defined at a top portion (101a) of the cartridge (101), the sample chamber (103) configured to receive the sample and a pre-treatment reagent through an opening defined on the sample chamber (103) and are lysed to form a pre-treated sample;a plurality of reaction wells (105) defined at a bottom portion (101b) of the cartridge (101);at least one valve (104) fluidly connected to the sample chamber (103) and the plurality of reaction wells (105), the at least one valve (104) is configured to control a flow of the pre-treated sample from the sample chamber (103) to the plurality of reaction wells (105); anda cap (102) externally connectable to the opening defined on the sample chamber (103);an ultrasonic transducer (107) disposed proximal to the sample chamber (103), the ultrasonic transducer (107) is configured emit ultrasonic waves to assist lysing the sample and the pre-treatment reagent during pre-treatment; anda plurality of heating elements (106) disposed proximal to the sample chamber (103) and the plurality of reaction wells (105), the plurality of heating elements (106) are configured to maintain a predefined temperature within the sample chamber (103) and the plurality of reaction wells (105),wherein the pre-treated sample is amplified in the plurality of reaction wells (105).

9. The apparatus (100) as claimed in claim 8, comprises an optics module (108) disposed proximal to the plurality of reaction wells (105) and opposite to the plurality of heating elements (106), the optics module (108) is configured to capture and detect a condition of the amplified sample from the plurality of reaction wells (105).

10. The apparatus (100) as claimed in claim 8, wherein the cap (102) is configured to store and supply the pre-treatment reagent to the sample chamber (103).

11. The apparatus (100) as claimed in claim 8, comprises a plurality of channels (110) extending from each of the plurality of reaction wells (105) and connected to the sample chamber (103).

12. The apparatus (100) as claimed in claim 8, wherein each of the plurality of reaction wells (105) is configured to contain a predefined concentration of an amplification reaction mixture.

13. The apparatus (100) as claimed in claim 12, wherein the amplification reaction mixture includes primer, enzymes and reagent.

14. The apparatus (100) as claimed in claims 12 and 13, wherein the predefined concentration of primer ranges between 0.2 pM to 2 pM per primer.

15. The apparatus (100) as claimed in claims 12 and 13, wherein the predefined concentration of enzyme ranges between 5 U to 20 U per reaction.

16. The apparatus (100) as claimed in claim 8, comprises at least one valve actuator (109) configured to actuate the a least one valve (104).

17. The apparatus (100) as claimed in claim 16, wherein the at least one valve actuator (109) is configured to direct infrared radiation toward the at least one valve (104).

18. The apparatus (100) as claimed in claim 17, wherein the at least one valve (104) includes a polyethylene black film or infrared spectrum configured to block the flow of the pre-treated sample from the sample chamber (103) and thermally deform upon exposure to the infrared radiation, thereby opening the at least one valve (104) to control the flow of the pre-treated sample to the plurality of reaction wells (105).

19. The apparatus (100) as claimed in claim 17, wherein actuation of the valve (104) is noncontact type stimulus.

20. A method (300) for sample pre-treatment and nucleic acid amplification, the method (300) comprising steps of:collecting, a predefined quantity of sample from a subject in a sample chamber (103) of a cartridge (101);adding, a predefined quantity of pre-treatment reagent into the sample chamber (103) and mixing the sample with the pre-treatment reagent;lysing, by delivering ultrasonic wave by an ultrasonic transducer (107) to pre-treat and make the nucleic acids accessible for amplification and form a pre-treated sample;actuating, at least one valve (104) by the at least one vale actuator to open and supply the pre-treated sample to plurality of reaction wells (105);amplifying, the pre-treated sample at a pre-defined temperature in the plurality of reaction wells (105);determining, through an optics module (108) a condition of the amplified sample from the plurality of reaction wells (105).

21. The method (300) as claimed in claim 20, wherein the predefined quantity of sample ranges from 100 pL-400 pL.

22. The method (300) as claimed in claim 20, wherein the predefined quantity of pretreatment reagent ranges from 20 pL to 50 pL.

23. The method (300) as claimed in claim 20, wherein the pre-defined temperature ranges from 37°C - 70°C.

Images