WO2016012870A1

WO2016012870A1 - System for the characterization of nucleic acid and detection of dna amplification using electrical method

Info

Publication number: WO2016012870A1
Application number: PCT/IB2015/050983
Authority: WO
Inventors: Arun CHANDRESHEKAR; Shashanka DODDAMANI; Sureshkumar Rajendran; Raghu Nanje Gowda; Naveen Vijayan MEKHILERI; Preetham SRIDHAR; Prathap Babu SHETTY; Ravindra Kirana BELEYUR; Raghav ABBOY
Original assignee: Bhat, Shama
Priority date: 2014-07-22
Filing date: 2015-02-10
Publication date: 2016-01-28

Abstract

A system (100) and method (700) for extracting electrical signals from one or more samples and testing the sample for one or more amplicons are provided. The system includes a plurality of vials (102) for receiving samples, a plurality of cover members (106) and electrodes (108) and an electronic circuit (104). Each vial is configured to receive one cover member (106) and a pair of electrodes (108), such that, the pair of electrodes (108) makes contact with the sample. The electronic circuit (104) is connected to the electrodes (108) and is configured to measure electrical properties of the samples present in the vials (102) through the electrodes. The change in the electrical properties of the sample can be used in determining the presence or absence of the amplicons and also in determining which of the amplicons is present in the sample.

Description

SYSTEM FOR THE CHARACTERIZATION OF NUCLEIC ACID AND DETECTION OF DNA AMPLIFICATION USING ELECTRICAL METHOD

The following specification particularly describes the invention and the manner in which it is to be performed.

BACKGROUND

Field

[001] The disclosed subject matter relates to the field of DNA sequence amplification and detection, and more particularly but not exclusively to testing a sample for two or more amplicons.

Discussion of related field

[002] DNA amplification by Polymerase Chain Reaction (PCR) is a common technique used for DNA sequence amplification and detection. DNA detection may also be carried out using isothermal methods.

[003] A sample having a DNA may undergo PCR resulting in a PCR product. Verification of the PCR product is conventionally done using gel electrophoresis method.

[004] Conventional techniques may require sophisticated laboratory, relatively expensive equipment and skilled personnel, which are hard to find in under-developed and developing countries. Moreover, conventional technique may require relatively longer duration to complete the process.

[005] Conventional techniques of PCR are programmed to run a sample through a series of varying temperature cycles. An average of 2 minutes may be consumed by each PCR cycle, and may generally require about 75 minutes to complete 30 PCR cycles, considering time consumed in related activities. PCR may be followed by agarose Gel electrophoresis method, which may take an hour or more to complete. The fluorescent dye method may be used for real time detection, which may require a minimum of 15 cycles and the probe may have to be synthesised priory. [006] Generally a patient might have to be tested for multiple diseases to identify the actual disease the patient is suffering from. To identify the disease a sample might be obtained from the patient and tested. The testing process may include using the sample to conduct amplification and detection processes corresponding to each of the diseases discretely, thereby consuming more time in completing all the tests. In many scenarios, simultaneous testing of samples for detecting amplicon may often be necessary to reduce the time and cost involved in detection.

[007] In light of the foregoing discussion, there is a need for a technique to simultaneously detect two or more amplicons in a sample.

SUMMARY

[008] An embodiment provides a system for extracting electrical signals from one or more samples. The system includes a plurality of vials, a plurality of cover members and electrodes and an electronic circuit. The vials receive samples to be used for carrying out polymerase chain reaction for amplification of target DNA amplicon. Each vial is configured to receive one the cover members and a pair of electrodes at least partially, such that, the pair of electrodes makes contact with the sample. The electronic circuit is configured to measure electrical properties of the samples present in the vials through the electrodes.

[009] Another embodiment provides a method for testing a sample for two or more amplicons. The method includes preparing a reaction mixture, carrying out polymerase chain reaction, determining the presence or absence of the amplicons at least based on the capacitance, determining the presence or absence of the amplicons and determining which of the amplicons is present in the sample. The reaction mixture is prepared by adding primers corresponding to the target template of the sample. The presence or absence of the amplicons in the sample may be determined from the capacitance and which of the amplicons is present in the sample may be determined from the melting temperature.

BRIEF DESCRIPTION OF DRAWINGS

[0010] Embodiments are illustrated by way of example and not limitation in the Figures of the accompanying drawings, in which like references indicate similar elements and in which:

[0011] FIG. 1 illustrates a schematic diagram of the exemplary system 100;

[0012] FIG. 2 is an illustration of the exemplary vials 102, in accordance with an embodiment;

[0013] FIG. 2A is an illustration of an exemplary cover member 106 and the electrodes 108, in accordance with an embodiment;

[0014] FIG. 2B is also an illustration of an exemplary cover member 106 and the electrodes 108, in accordance with an embodiment;

[0015] FIG. 2C is an illustration of the exemplary vial 102 with the cover member 106 and the electrodes 108, in accordance with an embodiment;

[0016] FIG. 2D is also an illustration of the exemplary vial 102 with the cover member 106 and the electrodes 108, in accordance with an embodiment;

[0017] FIG. 3 is an illustration of the electronic circuit 104 used to measure the electrical properties, in accordance with an embodiment;

[0018] FIG. 3A is an illustration of the exemplary engagement members 314, in accordance with an embodiment;

[0019] FIG. 4 is an illustration of the exemplary interconnection board 316, in accordance with an embodiment;

[0020] FIG. 4A is also an illustration of the exemplary interconnection board 316, in accordance with an embodiment;

[0021] FIG. 5 is an illustration of the exemplary first assembly 500, in accordance with an embodiment;

[0022] FIG. 5A is also an illustration of the exemplary first assembly 500, in accordance with an embodiment;

[0023] FIG. 5B is another illustration of the exemplary first assembly 500, in accordance with an embodiment; [0024] FIG. 5C is an illustration of the exemplary lid 503, in accordance with an embodiment;

[0025] FIG. 5D is another illustration of the exemplary lid 503, in accordance with an embodiment;

[0026] FIG. 5E is an illustration of the exemplary user interface unit 510, in accordance with an embodiment;

[0027] FIG. 6 is an illustration of the exemplary second assembly 600, in accordance with an embodiment;

[0028] FIG. 6A is an illustration of the exemplary wells 602, in accordance with an embodiment;

[0029] FIG. 6B is an illustration of the exemplary pressing tool 604 and the pressing device 606, in accordance with an embodiment;

[0030] FIG. 6C is an illustration of the exemplary engagement of the electrodes 108 and cover members 106 with the interconnection board 316, in accordance with an embodiment;

[0031] FIG. 6D is an illustration of the exemplary engagement of the electrodes 108 and cover members 106 with the vials 102, in accordance with an embodiment;

[0032] FIG. 6E is an illustration of the exemplary engagement of the engagement members 314 with the interconnection board 316, in accordance with an embodiment;

[0033] FIG. 7 is a flowchart illustrating the method 700 for testing a sample for two or more amplicons, in accordance with an embodiment;

[0034] FIG. 8 is a graph illustrating the detection curves for positive sample, blank and negative sample plotted using values in Table 2, in accordance with an embodiment;

[0035] FIG. 9 is a graph illustrating the detection plotted using values in Table 3A, in accordance with an embodiment;

[0036] FIG. 10 is a graph plotted using values in Table 4A, illustrating the effect of malachite green on the sensitivity of the detection curve, in accordance with an embodiment; [0037] FIG. 11 is a graph plotted using values in Table 5A, illustrating the effect of different additives that can be used in a PCR to increase the sensitivity of the detection curve, in accordance with an embodiment;

[0038] FIG. 12A is a graph of capacitance against temperature of a sample for 4 PCR cycles, in accordance with an embodiment;

[0039] FIG. 12B is a graph of capacitance against temperatures of a blank for 4 PCR cycles, in accordance with an embodiment;

[0040] FIG. 12C is a graphical representation of the change in capacitance and the corresponding temperature, showing the melting temperature of one amplicon in the positive sample for 4 cycles, in accordance with an embodiment;

[0041] FIG. 12D is a graphical representation of the change in capacitance and the corresponding temperature, showing the melting temperature of blank for 4 cycles, in accordance with an embodiment;

[0042] FIG. 13 is a graphical representation of the temperature and corresponding changes in capacitance of the sample showing a melting temperature for amplicon 246 bp, in accordance with an embodiment;

[0043] FIG. 14 is a graphical representation of the temperature and corresponding changes in capacitance of the sample showing a melting temperature for amplicon (615 bp), in accordance with an embodiment;

[0044] FIG. 15 is a graphical representation of the temperature and corresponding changes in capacitance of the sample under test (multiplexing) showing different melting temperatures/temperature signatures for the sample under test, in accordance with an embodiment; and

[0045] FIG. 16 is a graphical representation of the effect in capacitance with and without adding restriction enzymes, in accordance with an embodiment.

DETAILED DESCRIPTION I. OVERVIEW

II. EXEMPLARY SYSTEM

III. TESTING FOR TWO OR MORE SEQUENCES

IV. CONCLUSION

I. OVERVIEW

[0046] In embodiment provides a system for extracting electrical signals from one or more samples. The system includes a plurality of vials and a plurality of cover members. The vials receive samples for carrying out polymerase chain reaction for amplifying amplicon. Each vial may be configured to at least partially receive a cover member and a corresponding pair of electrodes, such that, the pair of electrodes makes contact with the sample. The system further includes an electronic circuit connected with the electrodes. The electronic circuit is configured to transmit electrical signals to the sample and receive electrical signals from the sample through the electrodes and measure electrical properties of the samples present in the vials. The sample may be tested for two or more amplicons by carrying out polymerase chain reaction. The changes in electrical properties, for example, capacitance, in the sample during the PCR may be used in determining the presence or absence of the amplicons. From the capacitance a melting temperature or a signature temperature may be determined which may provide an identity of a particular amplicon. The melting temperature determines which of the amplicons is present in the sample.

[0047] The following detailed description includes references to the accompanying drawings, which form part of the detailed description. The drawings show illustrations in accordance with example embodiments. These example embodiments are described in enough detail to enable those skilled in the art to practice the present subject matter. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to unnecessarily obscure aspects of the embodiments. The embodiments can be combined, other embodiments can be utilized or structural and logical changes can be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken as a limiting sense.

[0048] In this document, the terms "a" or "an" are used, as is common in patent documents, to include one or more than one. In this document, the term "or" is used to refer to a nonexclusive "or," such that "A or B" includes "A but not B," "B but not A," and "A and B," unless otherwise indicated.

II. EXEMPLARY SYSTEM

[0049] In an embodiment, a system 100 may be provided for extracting electrical signals from one or more samples while they undergo PCR. FIG. 1 illustrates a schematic diagram of the exemplary system 100. The system 100 may include plurality of vials 102 and an electronic circuit 104.

[0050] The vials 102 may be configured to receive a sample, in dry or wet form. The vials 102 may have a cylindrical cross section, which may be conically shaped as it extends inferiorly, and may define an empty space where the sample may be received. The size of the empty space may be such that it can hold the required quantity of the sample along with other ingredients that may be added to the sample.

[0051] FIG. 2 is an illustration of the exemplary vials 102, in accordance with an embodiment. The system 100 may be configured to provide scalability in number of vials 102 used for detection.

[0052] The vials 102 may be configured to receive cover members and electrodes. FIGs. 2A and 2B are illustrations of an exemplary cover member 106 and the electrodes 108, in accordance with an embodiment. Each vial 102 may be configured to receive one cover member

106 and a pair of electrodes 108. The cover member 106 and the pair of electrodes 108 may be configured to be received by the vials 102 such that a portion of the electrodes 108 can be in contact with the PCR sample. The cover members 106 can be inserted into the vials 102 at least during the polymerase chain reaction. The cover members 106 may be impermeable, such that, when the cover member 106 is engaged with the vials 102, the cover member 106 may prevent air to enter into the vial 102 or escape from the vial 102. The cover members 106 may be compressible so as to be snugly fitted with the vials 102.

[0053] In system 100, the cover members 106 engaged with the vials 102 may define enclosures that may prevent vapour from escaping the vials 102, with or without the use of oil. Vapour will be formed while carrying out PCR.

[0054] The cover member 106 may prevent evaporation of the sample which may result in decrease in the volume of PCR mixture, which may alter the electrical parameters thereby introducing error in detection.

[0055] Pair of electrodes 108 may pass through the cover member 106 and may extend out of the cover member 106. The electrodes 108 may extend out of the cover member 106 inferiorly such that when the cover members 106 are inserted into the vial 102, at least a part of the electrodes 108 extending inferiorly make contact the PCR sample. FIGs. 2C and 2D are illustrations of the exemplary vial 102 with the cover member 106 and the electrodes 108, in accordance with an embodiment.

[0056] A part of each of the electrodes 108 may extend out of the cover member 106 such that when the cover member 106 is engaged with the vial 102, the instant part of each of the electrodes 108 may be disposed out of the vial 102. The instant part of the electrodes 108 may be connected to an electronic circuit or interconnection board.

[0057] A cover member 106 and its corresponding pair of electrodes 108 may be configured as a first single unit.

[0058] Alternatively, multiple cover members 106 and their pair of electrodes 108 may be configured as a first single unit.

[0059] In an embodiment, the electronic circuit 104 may be configured to measure electrical properties of the PCR product obtained using the pair of electrodes 108. FIG. 3 is an illustration of the electronic circuit 104 used to measure the electrical properties, in accordance with an embodiment.

[0060] In an embodiment, the electrical properties measured may include impedance and phase among other electrical properties. The electronic circuit 104 may include an impedance and capacitance analyzer 302, which may be configured to measure the magnitude of impedance, relative phase (phase change measured from an initial calibrated value against a known resistance) and capacitance change of the PCR product between the pair of electrodes 108 occurring during the PCR reaction. The impedance and capacitance analyzer 302 determines the impedance and phase of the PCR product as a result of electric signal applied to the PCR sample.

[0061] The electronic circuit 104 may transmit sinusoidal waves in phase voltage and current signals to the PCR sample through one electrode and receive the output signal using the other electrode. Further, the electronic circuit 104 may be configured to compare the amplitude of the transmitted and received signals. The electronic circuit 104 may be configured to detect changes in phase and amplitude introduced by the PCR sample. The electronic circuit 104 may be configured to determine Real (R) and imaginary (I) values of the impedance. The Real (R) and imaginary (I) values may be used to determine the Magnitude and Phase of the sample

[0062] In an embodiment, a single core, extrapolated connection from the electrodes 108 may be connected to the impedance and capacitance analyzer 302. By using impedance and capacitance analyser 302, capacitance, impedance and phase can be derived from applied electric field. Using acquired impedance and phase from impedance and capacitance analyzer 302, capacitance may be calculated at every extension/elongation stage of every PCR cycle, for example at 72 degree Celsius. Alternatively, capacitance may be determined throughout the PCR cycle, for example at predetermined intervals or temperatures.

[0063] A microcontroller 304 may be programmed to enable a PCR machine to choose various modes of PCR operations by sending instructions to the PCR machine. The microcontroller 304 may provide instructions corresponding to the number of cycles to be carried out and/or temperatures variations to be made during each cycle, among others.

[0064] The electronic circuit 104 measures impedance, phase and capacitance after the completion of each PCR cycle. The capacitance calculated at each PCR cycle may be used for plotting the graph between capacitance and number of PCR cycles. The change in capacitance with temperature during PCR cycle may be used to determine melting curve or melting temperature or temperature signature at which change in capacitance may peak.

[0065] The electronic circuit 104 may further include a voltage divider 308, a buffer 310 and an amplifier 312. The electronic circuit 104 may be coupled to a computer 306 through a wired or wireless communication network. The computer 306 may control the operations of the microcontroller 304.

[0066] In an embodiment, the electronic circuit 104 may include engagement members 314. FIGs. 3A and 3B are illustrations of the exemplary engagement members 314, in accordance with an embodiment. The engagement members 314 may extend inferiorly from a board that may have the electronic circuit 104. The engagement members 314 may be operatively or selectively made to interface with the electrodes 108, such that, electrical signals are communicated between the electrodes 108 in contact with the sample and the electronic circuit 104 through the engagement member 314. The engagement members 314 can be spring loaded arrangements such as pogo pins 314. One end of each of the pogo pins 314 may be engaged with the electronic circuit 104 by electrical means and another end of each of the pogo pins 314 may extend from the electronic circuit 104 to interface or establish electrical connection with the electrodes 108.

[0067] In an embodiment, the engagement members 314 and the electronic circuit 314 may be configured as a second single unit.

[0068] In an embodiment, the system 100 may include an interconnection board 316, which may be configured to enable engagement between the engagement members 314 and the electrodes 108. FIGs. 4 and 4A are illustrations of the exemplary interconnection board 316, in accordance with an embodiment.

[0069] The interconnection board 316, multiple cover members 106 and their pair of electrodes 108 may be configured as a first single unit.

[0070] The interconnection board 316 may have a first surface 402 and an opposing second surface 404. The first surface 402 may include conductive pads 406 which may interface with the pogo pins 314. The second surface 404 may be engaged with the electrodes 108 by electrical means. The electrical means may establish connection between the electrodes 108 and the conductive pads 406. The ends of each of the pogo pins 314 that extend from the electronic circuit 104 may be configured to interface with the conductive pads 406. During PCR reaction, the pogo pins 314 make contact with the electrodes 108 through the conductive pads 406 provided in the interconnection board 316.

[0071] In an embodiment, the system 100 may include a first assembly configured to receive electronic circuit 104 and enable connection to be established between electronic circuit 104 and the interconnection board 316. FIGs. 5, 5A and 5B are illustrations of the exemplary first assembly 500, in accordance with an embodiment. [0072] The assembly 500 may include a PCR machine 501 and a lid 503. The lid 503 may be closed to enclose the vial 102 in the PCR machine 501 while carrying out PCR cycles. The lid

503 may include an upper plate 502 and a lower plate 504. The upper plate 502 and the lower plate 504 may define an empty space that may house the electronic circuit 104. The lower plate

504 may be provided with openings or slots 506 such that when the engagement members 314 extend out of the slots 506, they interface with the conductive pads 406. FIG. 5C and 5D are illustrations of the exemplary lid 503, in accordance with an embodiment.

[0073] The upper plate 502 of the assembly 500 may be configured to receive a knob 508. The knob 508 may extend from the upper plate 502 in a direction away from the upper plate 502. The knob 508 may be operated to rotate in a first direction and a second direction. Rotation of the knob 508 may cause the pogo pins 314 to press against the interconnection board 316 thereby establishing electrical connection between electronic circuit 104 and the interconnection board 316. The first assembly 500 can be a lid for the PCR machine 501, such that when the lid is pushed towards the PCR machine 501 to close the PCR machine, the bottom plate 504 of the first assembly 500 faces the surface of the PCR machine 501 on which the vials 102 are disposed.

[0074] The system 100 may include a user interface unit 510 that may be configured to receive input and/or display information. FIG. 5E is an illustration of the exemplary user interface unit 510, in accordance with an embodiment. The user interface unit 510 may be configured to display plots, data or graphs corresponding to different electrical properties or assessments. The user interface unit 510 may also display the status of the PCR cycles and corresponding information.

[0075] In an embodiment, the user interface unit 510 can be a peripheral device connected to the system 100 by means of well known communication protocols. In another embodiment, the user interface unit 510 can be a part of the system 100. The user interface unit 510 may be a touch or non touch enabled device.

[0076] The system 100 may further include a second assembly 600 configured to enable engagement of the interconnection board 316 with the vials 102. FIG. 6 is an illustration of the exemplary second assembly 600, in accordance with an embodiment. The second assembly 600 may receive the plurality of vials 102. The second assembly 600 may include a plurality of wells 602 for receiving the vials 102. FIG. 6A is an illustration of the exemplary wells 602, in accordance with an embodiment.

[0077] The second assembly 600 may be configured to prevent contamination during preparation of PCR samples. The PCR sample may be introduced into the vials 102 inside the second assembly 600. Once the PCR samples and other ingredients are introduced into the vials 102, the interconnection board 316 may be placed on top of the vials 102 such that the electrodes 108 and the cover member 106 engaged with the electrodes 108 are aligned with the vials 102. The interconnection board 316 may be then pressed against the vials 102, such that a portion of the cover member 106 and the electrodes 108 are received by the vials 102.

[0078] In an embodiment, Ultraviolet radiation may be used inside the second assembly 600 during sample preparation which may prevent the sample from contamination. In another embodiment, an equipment for concentrating DNA may be used in preparation of the sample. The UV radiation equipment and the equipment for concentrating DNA may be peripheral devices.

[0079] The second assembly 600 may further include a pressing tool 604 and a pressing device 606. FIG. 6B is an illustration of the exemplary pressing tool 604 and the pressing device 606, in accordance with an embodiment. The pressing tool 604 is configured to apply pressure over the pressing device 606. The pressing device 606 in turn exerts pressure over the interconnection board 316 such that the cover members 106 firmly fit into the vials 102. The pressure applied by the pressing tool 604 on the pressing device 606 is such that the cover members 106 and the electrodes 108 are adequately received by the vials 102 leaving no free space for air to enter into or escape out of the vials 102. FIG. 6C is an illustration of the exemplary engagement of the electrodes 108 and cover members 106 with the interconnection board 316. FIG. 6D is an illustration of the exemplary engagement of the electrodes 108 and cover members 106 with the vials 102. FIG. 6E is an illustration of the exemplary engagement of the engagement members 314 with the interconnection board 316.

TESTING FOR TWO OR MORE SEQUENCES

[0080] In an embodiment a sample may be tested for simultaneous detection of two or more sequences corresponding to multiple organisms or diseases. A reaction mixture may be prepared by introducing the sample into at least one of the vials 102. Additionally a blank (without sample) may be present in one of the vials 102. A part of the reaction mixture may be already provided in the vials 102 as part of a "testing kit". This part of the reaction mixture may include primers (both forward and reverse) corresponding to each of the amplicons for which testing is to be carried out, in addition to other ingredients. The sample may be added to a dry master mix (testing kit) to prepare the reaction mixture. The dry master mix may be prepared and preserved to prevent contamination of sample. The prevention from contamination may be provided by the UV radiation or the equipment for concentrating DNA.

[0081] The dry master mix may include reaction buffer, dNTPs, DNA polymerase enzyme, forward primer and reverse primer. Nuclease-free water may be added to the dry master mix. Each of these components may be present in varied concentration as required for the reaction. For example, to prepare the dry master mix, IX concentration of the reaction buffer may be mixed with 0.2mM dNTPs and 0.2μΜ each of forward primer and reverse primer. The volume of DNA polymerase enzyme and nuclease-free water used in the mixture may be 0.5μΕ and 28μΕ, respectively.

[0082] The enzyme used in the reaction, which may be a part of the dry master mix, may be resistant to inhibitors, thereby eliminating the need for isolation of DNA prior to PCR. Hence pathogen, blood, CSF, sputum, pleural fluid can be added directly to the vials 102 along with water prior to PCR.

[0083] To prepare a positive sample, required volume of double distilled water may be added to the dry master mix. Additionally, required volume of a template of interest may be added to the mixture of dry master mix and distilled water, which gives the positive sample to be used to detect amplicon. The volume of the reaction sample to be used in the PCR is desired to be around 50 L. Examples of different samples include E coli template and MTB Sputum DNA template, among others.

[0084] The sample may be tested against a blank (without sample). The blank may include the dry master mix. The blank may be prepared by adding distilled water to the dry master mix.

However, templates are not added to the mixture of dry master mix and distilled water while preparing the blank. The volume of the blank to be tested is desired to be equal to the volume of the reaction sample, i.e., 50 L. The presence of a template in a positive sample may differentiate the positive sample from the blank.

[0085] The sample may be tested against a negative sample. The negative sample may include the dry master mix, but instead of a template, a negative sample, such as, blood, may be added. The volume of the negative sample to be tested is desired to be equal to the volume of the reaction sample, i.e., 50 L. Negative sample may contain nucleic acid that may not be the target sample.

[0086] The dry master mix may be dispensed in vials 102 with electrical identification code, such as, barcodes. The system 100 or the PCR machine 501 may be configured to read the electrical identification code. The electrical identification code may enable the system 100 or the PCR machine 501 to determine the cycling parameters and the sequences for which the sample is to be tested.

[0087] In an embodiment, to detect whether an amplicon is present in a sample, a set of primers (forward and reverse) may be added to the reaction mixture. In a single PCR reaction, one or more samples can be tested for one or more amplicons. FIG. 7 is a flowchart illustrating a method 700 for testing a sample for two or more amplicons, in accordance with an embodiment. The method 700 includes at step 702, preparing a reaction mixture, comprising at least primers corresponding to the amplicons, whose presence in the sample has to be identified. At step 704, carrying out polymerase chain reaction. At step 706, determining capacitance of the reaction mixture. At step 708, determining the presence or absence of the amplicons at least based on the capacitance. At step 710, determining which of the amplicons is present in the sample at least based on melting curve and restriction enzyme analysis.

DETECTION CURVE (CAPACITANCE VS. PCR CYCLES AT 72 DEGREES)

[0088] The presence of an amplicon may be detected from the change in electrical properties the amplicon may exhibit during amplification. For example, changes in impedance and capacitance may indicate the presence of amplicon in a sample. Capacitance changes due to change in temperature and also due to amplification of the amplicon. Capacitance is a measure of dielectric properties of DNA. [0089] In an embodiment, the capacitance of the positive sample may be measured at every cycle of the PCR. The capacitance can be calculated from the impedance and the phase obtained from the impedance and capacitance analyzer 304 of the electronic circuit 104 during polymerase chain reaction. Data may be acquired continuously right from the first PCR cycle at various temperatures applied during a polymerase chain reaction. Each cycle of a PCR may include temperatures varying from, for example, 50 degree Celsius to 95 degree Celsius. The capacitance measured at 72 degree Celsius in each cycle may be used to determine whether there is change in capacitance.

[0090] The positive sample may be tested against blank and negative sample. Capacitance values for all the three may be obtained and plotted (detection curve) for the number of cycles, the polymerase chain reaction is conducted. Presence of one or more amplicons may be detected from the trend exhibited by the detection curve. It shall be noted that, computer instructions can use the data corresponding to capacitance, for example at 72 degree Celsius, to determine whether there is change in capacitance, and thereby determines whether at least one of the amplicons is present in the sample.

[0091] The positive sample shows an increase in the capacitance with the increase in the number of PCR cycles. An increase in the value of capacitance indicates presence of at least one amplicon in the sample (positive sample).

[0092] The blank and the negative sample are also tested for the presence of amplicon during the polymerase chain reaction. The blank may show a decreasing capacitance trend from an initial value, which may indicate absence of amplicons. Similarly, the negative sample may show a decreasing trend almost similar to the blank, which may indicate the absence of an amplicon. Table 1 below shows typical values of capacitance (in farad) measured at 72 degrees Celsius at each PCR cycle corresponding to the positive sample, blank and negative sample.

PCR Positive sample Negative sample Blank

Cycle

Number

1 0.000000659648800 0.000000574099700 0.000000508092600

2 0.000000660912100 0.000000572638400 0.000000503336600

3 0.000000669289000 0.000000576915100 0.000000503297000

4 0.000000677417800 0.000000577968600 0.000000499496800 5 0.000000680885000 0.000000574775700 0.000000494572400

6 0.000000684817500 0.000000574369700 0.000000490240400

7 0.000000686960800 0.000000571578300 0.000000485730800

8 0.000000689588800 0.000000568606000 0.000000481435800

9 0.000000690258400 0.000000567074900 0.000000477116300

10 0.000000691511000 0.000000565083300 0.000000473388800

11 0.000000687446200 0.000000559369000 0.000000466270300

12 0.000000693653700 0.000000562055700 0.000000467633500

13 0.000000694763300 0.000000560342000 0.000000464380400

14 0.000000696332700 0.000000559321200 0.000000461734200

15 0.000000695843500 0.000000556894100 0.000000458333100

16 0.000000692386000 0.000000550585300 0.000000452183300

Table 1

[0093] Table 2 may be obtained from Table 1 by dividing each value in a column from the first value in the respective column.

Table 2 [0094] FIG. 8 is a graph illustrating the detection curves for positive sample, blank and negative sample plotted using values in Table 2, in accordance with an embodiment.

[0095] In an embodiment, the capacitance may be affected by changing concentrations ("X") or dilution of a sample. Table 3 shows the values of capacitances (in farad) corresponding to different dilutions of the sample and a blank for different PCR cycles.

Table 3

[0096] Table 3A may be obtained from Table 3 by dividing each value in a column from the first value in the respective column.

PCR X X/2 X/3 X/4 Blank

cycle

No.

1 1.000000000 1.000000000 1.000000000 1.000000000 1.000000000

2 1.017689196 1.010265543 1.007498926 1.005167618 0.997577141

3 1.034566264 1.024419859 1.016416870 1.012891785 0.996092022

4 1.042974313 1.025979786 1.020075629 1.011314777 0.992562571

5 1.051853477 1.033286989 1.026028942 1.014106000 0.990623559

6 1.060351300 1.040569147 1.030473371 1.016485713 0.989331689 7 1.067639012 1.046004437 1.035368993 1.018175082 0.987643901

8 1.073479136 1.050959128 1.038795817 1.019901354 0.986119408

9 1.077666678 1.054284277 1.041423918 1.020274097 0.983637366

10 1.083376380 1.059063649 1.046376218 1.022383892 0.983002302

11 1.085051205 1.060805009 1.047668269 1.021630734 0.980280390

12 1.088570801 1.064484741 1.050449813 1.022496876 0.978371090

13 1.091045913 1.066794853 1.052494906 1.022650907 0.976698278

Table 3A

[0097] FIG. 9 is a graph illustrating the detection plotted using values in Table 3A, in accordance with an embodiment. FIG. 9 illustrates changes in capacitance observed by introducing change in dilution with respect to the initial dilution of the sample. From FIG. 9, it can be observed that, at half, one-third and one-fourth of the dilution, the capacitance shows a milder increasing trend with respect to the capacitance at the initial dilution, for 13 PCR cycles.

EFFECT OF ADDITIVES

[0098] In an embodiment, additives can be added to the sample, which may increase the sensitivity of detection. Sensitivity and time taken for detection of the PCR product may be altered using specific additives. Different additives may have different effects on the curve of capacitance against the number of PCR cycles (detection curve). Example of an additive that may be used to increase the sensitivity is malachite green (MG). Table 4 shows the values of capacitance (in farad) of a sample without additive at 72 degree Celsius for 8 PCR cycles, against the capacitance (in farad) measured for the sample after adding malachite green as additive. The concentration of malachite green added in the sample is around 10 mg/mL.

[0099] Mycobacterium tuberculosis (MTB) sample is tested to determine the sensitivity of detection by adding malachite green to the sample. Concentration of the MTB sample used in this reaction is approximately 300 ng/μΕ. Malachite green (around lOmg/mL) is added to the sample to observe its effect on the values of capacitance for each cycle and hence the sensitivity of the detection. FIG. 10 is a graph plotted using values in Table 4A, illustrating the effect of malachite green on the sensitivity of the detection curve, in accordance with an embodiment. PCR Cycle MTB MTB + MG Blank

Number

1 0.0000004959 0.0000004197 0.0000005294

2 0.0000004948 0.0000004231 0.0000005182

3 0.0000004981 0.0000004315 0.0000005065

4 0.0000004983 0.0000004343 0.0000005006

5 0.0000004980 0.0000004370 0.0000004960

6 0.0000005024 0.0000004408 0.0000004940

7 0.0000005019 0.0000004414 0.0000004878

8 0.0000005017 0.0000004438 0.0000004854

Table 4

[00100] Table 4A may be obtained from Table 4 by dividing each value in a column from the first value in the respective column.

Table 4A

[00101] Examples of additives that may be used other than malachite green, include silver, propedium iodide and ethidium bromide. The system 100 may have an inbuilt database of ideal response for pure PCR additives which can be used to identify contaminated PCR additives.

[00102] Table 5 shows the values of capacitance (in farad) for 29 PCR cycles when equal concentration of additives, such as, malachite green, silver, propedium iodide and ethidium bromide are added to the sample chuA. FIG. 11 is a graph plotted using values in Table 5 A, illustrating the effect of different additives that can be used in a PCR to increase the sensitivity of the detection curve, in accordance with an embodiment. Cycles chuA 615bp chuA with chuA with Negative normal silver malachite

green(10mg/ml)

1 0.000000559291 0.000000559605 0.000000497259 0.000000462332

40 00 90 50

2 0.000000565178 0.000000565349 0.000000513881 0.000000455808

80 10 40 60

3 0.000000572208 0.000000573632 0.000000527572 0.000000455412

10 60 10 50

4 0.000000576568 0.000000579244 0.000000537053 0.000000454135

40 70 80 90

5 0.000000582159 0.000000584800 0.000000546089 0.000000452439

50 00 90 60

6 0.000000583377 0.000000587348 0.000000550068 0.000000454024

80 70 80 20

7 0.000000586712 0.000000590355 0.000000555735 0.000000453569

50 40 10 10

8 0.000000588787 0.000000592859 0.000000559880 0.000000452348

80 30 10 80

9 0.000000590085 0.000000594904 0.000000563314 0.000000451199

40 50 00 40

10 0.000000592330 0.000000597527 0.000000567667 0.000000450310

20 80 30 00

11 0.000000591166 0.000000598441 0.000000568761 0.000000449244

30 70 10 00

12 0.000000594669 0.000000600791 0.000000573445 0.000000448498

90 30 10 80

13 0.000000594138 0.000000601665 0.000000575128 0.000000447621

70 00 10 80

14 0.000000593832 0.000000602345 0.000000577065 0.000000446740

20 40 90 00

15 0.000000594302 0.000000603527 0.000000579788 0.000000446034

70 30 90 50

16 0.000000594221 0.000000604250 0.000000582220 0.000000445387

30 40 70 10

17 0.000000593794 0.000000605052 0.000000584530 0.000000444921

50 00 10 10

18 0.000000593629 0.000000605762 0.000000586982 0.000000444278

60 80 50 30

19 0.000000594120 0.000000606737 0.000000590139 0.000000444302

40 50 50 30

20 0.000000593170 0.000000606948 0.000000591682 0.000000444018

60 80 50 60

21 0.000000593110 0.000000607542 0.000000593643 0.000000443564 30 00 00 00

0.000000593355 0.000000608280 0.000000595522 0.000000443324

90 40 70 50

0.000000593468 0.000000608662 0.000000597186 0.000000443268

90 40 50 40

0.000000592008 0.000000608492 0.000000597400 0.000000442867

00 50 20 60

0.000000592877 0.000000609137 0.000000599449 0.000000442807

50 40 60 90

0.000000592857 0.000000609269 0.000000600708 0.000000442753

10 00 90 20

0.000000592664 0.000000609494 0.000000601457 0.000000442702

80 10 90 90

0.000000592266 0.000000609587 0.000000602326 0.000000442476

40 50 80 40

0.000000592930 0.000000609995 0.000000603753 0.000000442511

40 10 00 10

Cycles chuA with chuA with Blank

Propedium iodide Ethidium bromide

1 0.00000055668490 0.00000048890330 0.00000038687300

2 0.00000056511120 0.00000049336700 0.00000038226880

3 0.00000057523690 0.00000049985430 0.00000038054720

4 0.00000058261240 0.00000050459900 0.00000037791970

5 0.00000059055740 0.00000050778280 0.00000037565870

6 0.00000059381800 0.00000050852110 0.00000037236350

7 0.00000059867950 0.00000051072610 0.00000037001430

8 0.00000060155630 0.00000051272690 0.00000036745920

9 0.00000060330380 0.00000051414250 0.00000036507650

10 0.00000060702770 0.00000051613470 0.00000036327520

11 0.00000060582300 0.00000051645660 0.00000036066980

12 0.00000060994390 0.00000051830770 0.00000035937460

13 0.00000060963580 0.00000051864610 0.00000035706530

14 0.00000060945120 0.00000051922750 0.00000035513030

15 0.00000061030180 0.00000051982100 0.00000035341410

16 0.00000061054830 0.00000052017250 0.00000035160770

17 0.00000061048710 0.00000052047580 0.00000034997200

18 0.00000061035380 0.00000052069400 0.00000034844300

19 0.00000061106150 0.00000052133570 0.00000034722420 20 0.00000060985180 0.00000052104540 0.00000034566360

21 0.00000060961590 0.00000052120280 0.00000034435120

22 0.00000060972790 0.00000052135980 0.00000034314160

23 0.00000060975880 0.00000052154100 0.00000034203350

24 0.00000060763790 0.00000052106970 0.00000034057450

25 0.00000060839610 0.00000052134670 0.00000033968470

26 0.00000060791550 0.00000052168500 0.00000033864960

27 0.00000060699520 0.00000052155220 0.00000033759860

28 0.00000060598280 0.00000052133400 0.00000033649580

29 0.00000060620040 0.00000052143040 0.00000033564420

Table 5

[00103] Table 5A may be obtained from Table 5 by dividing each value in a column from the first value in the respective column.

579 202 9145 361 69246 88

12 1.063255 1.073598 1.1532100 1.09567 1.060143 0.9289 0.9700784

934 878 22 1717 591 21377 61

13 1.062306 1.075160 1.1565945 1.09511 1.060835 0.9229 0.9681815

161 158 7 8262 752 52235 58

14 1.061758 1.076376 1.1604915 1.09478 1.062024 0.9179 0.9662742

146 015 26 6656 944 50594 72

15 1.062599 1.078488 1.1659675 1.09631 1.063238 0.9135 0.9647483

389 041 35 463 886 14513 14

16 1.062453 1.079780 1.1708579 1.09675 1.063957 0.9088 0.9633480

848 202 36 743 842 4528 23

17 1.061690 1.081212 1.1755021 1.09664 1.064578 0.9046 0.9623400

739 641 87 7493 21 17278 91

18 1.061395 1.082482 1.1804340 1.09640 1.065024 0.9006 0.9609497

902 823 14 804 515 65076 49

19 1.062273 1.084224 1.1867828 1.09767 1.066337 0.8975 0.9610016

441 587 07 9316 045 14688 6

20 1.060575 1.084602 1.1898858 1.09550 1.065743 0.8934 0.9603880

221 175 12 6273 267 80806 32

21 1.060467 1.085662 1.1938284 1.09508 1.066065 0.8900 0.9594047

406 208 18 2514 212 88479 57

22 1.060906 1.086981 1.1976085 1.09528 1.066386 0.8869 0.9588867

533 71 34 3705 339 61871 32

23 1.061108 1.087664 1.2009544 1.09533 1.066756 0.8840 0.9587653

574 335 71 9213 964 97624 91

24 1.058496 1.087360 1.2013842 1.09152 1.065792 0.8803 0.9578984

519 728 26 9337 97 2636 82

25 1.060051 1.088513 1.2055056 1.09289 1.066359 0.8780 0.9577693

165 148 12 1329 544 26381 54

26 1.060014 1.088748 1.2080380 1.09202 1.067051 0.8753 0.9576510

69 314 9 8004 501 50826 41

27 1.059670 1.089150 1.2095443 1.09037 1.066779 0.8726 0.9575422

862 562 45 4824 872 34172 45

28 1.058958 1.089317 1.2112917 1.08855 1.066333 0.8697 0.9570523

532 465 21 6201 567 83624 38

29 1.060145 1.090045 1.2141598 1.08894 1.066530 0.8675 0.9571273

749 836 39 7087 743 82385 92

Table 5A

[00104] From the table 5A and FIG. 11, it may be interpreted that the effect of malachite green on the detection curve is maximum, in this embodiment. CHARACTERIZATION CURVE

[00105] In addition to detecting whether at least one of the amplicons is present the sample, it might be important to identify which one of the amplicons is present in the sample.

[00106] In an embodiment, the capacitance measured between 72 degree Celsius and 94 degree Celsius in each cycle may be used for determining the melting temperature or temperature signature.

[00107] A temperature signature may be a temperature at which maximum change in capacitance may be observed in a PCR cycle. It may be noted that, each amplicon may have a specific temperature signature. Presence of a particular amplicon in a sample may be identified based on whether temperature at which change in capacitance peaks in the sample matches with expected temperature at which change in capacitance peaks for the amplicon.

[00108] For example, a sample may be tested for amplicons "X" and "Y". Increase in capacitance at 72 degree Celsius may indicate that one of "X" and "Y" is present in the sample.

However, temperature signature may indicate which of "X" and "Y" is/are present in the sample.

The amplicon "X" may have a temperature signature at 80 degree Celsius, whereas amplicon "Y" may have a temperature signature at 84 degree Celsius. The sample may be categorized as having the amplicon "X", if the temperature signature of the sample is 80 degree Celsius. On the other hand, the sample may be categorized as having the amplicon "Y", if the temperature signature of the sample is 84 degree Celsius.

[00109] In an embodiment, temperature signature may be determined after the PCR is concluded.

[00110] In another embodiment, temperature signature may be determined during PCR in each cycle or predefined cycles. A amplicon may be said to be present if temperature signature of the sample determined in each cycle or predefined cycles is consistent and matches with the a pre-identified temperature signature of the amplicon.

[00111] As an example, the capacitance between 72 degree Celsius and 94 degree Celsius may be determined for 10 PCR cycles. Tables 6 and 7 show the capacitances (in farad) corresponding to positive sample and blank respectively, between 72 degree Celsius and 94 degree Celsius for 4 PCR cycles out of 10 cycles. FIG. 12A is a graph of capacitance against temperature of a sample for 4 PCR cycles, in accordance with an embodiment. FIG. 12B is a graph of capacitance against temperatures of a blank for 4 PCR cycles, in accordance with an embodiment.

Table 6

Temperature Cycle 2 Cycle 5 Cycle 8 Cycle 10

72 0.0000004147 0.0000004119 0.0000004059 0.0000004112

74 0.0000004171 0.0000004139 0.0000004071 0.0000004138

76 0.0000004221 0.0000004187 0.0000004114 0.0000004187

78 0.0000004283 0.0000004248 0.0000004172 0.0000004255

80 0.0000004355 0.0000004319 0.0000004242 0.0000004327

82 0.0000004430 0.0000004393 0.0000004313 0.0000004404

84 0.0000004506 0.0000004471 0.0000004390 0.0000004480

86 0.0000004582 0.0000004544 0.0000004460 0.0000004553

88 0.0000004648 0.0000004608 0.0000004519 0.0000004610

90 0.0000004703 0.0000004656 0.0000004564 0.0000004654

92 0.0000004743 0.0000004694 0.0000004601 0.0000004690

94 0.0000004753 0.0000004722 0.0000004626 0.0000004716 Table 7

[00112] Percentage change in capacitance at a temperature may be determined using the below provided formula:

Percentage change in capacitance at X = (((capacitance at X+l) - (capacitance at X))/ (capacitance at X))) * 100

[00113] A graph between change in capacitance against the corresponding value of temperature (characterization curve or melting curve) may determine the melting temperature or temperature signature of a sequence. Table 8 shows the percentage change in capacitance and corresponding temperature for the positive sample, measured for 4 PCR cycles. However, the changes in capacitance and corresponding temperature may be determined up to 10 cycles of a polymerase chain reaction.

[00114] From change in capacitance and the corresponding value of temperature, the temperature at which change in capacitance peaks can be determined. The temperature at which the change in capacitance peaks or deviates maximum from a slope may be referred to as the melting temperature or temperature signature of the sequence present in the sample. It shall be noted that the change in capacitance may be measured continuously from the very first cycle.

MULTIPLEXING

[00115] As recited earlier, a sample may be tested for one or more amplicons. One amplicon may have different primer lengths. The sample may be tested to identify one amplicon with a particular primer length. For example, a sample may have an amplicon with primer length of either 246 bp or 615 bp. To identify the amplicon and which of these primer lengths may be present in the sequence, multiplexing may be conducted.

[00116] Table 8 shows temperature and corresponding capacitance for an amplicon having primer length of 246 bp.

Temperature Capacitance

(°C) (farad)

75 0.000000185908

76 0.000000187572 77 0.000000189346

78 0.000000191272

79 0.000000193238

80 0.000000195062

81 0.000000197108

82 0.000000199002

83 0.000000200910

84 0.000000202886

85 0.000000204806

86 0.000000206678

87 0.000000208584

88 0.000000210450

89 0.000000212224

90 0.000000214104

Table 8

[00117] Table 8A may be determined using the below provided formula:

Change in capacitance at X = (((capacitance at X+l) - (capacitance at X))/ ((temperature at X

+1) - (temperature at X))). FIG. 13 is a graphical representation of the temperature and corresponding change in capacitance of the sample showing a melting temperature for amplicon 246 bp, in accordance with an embodiment.

86 0.000000001906

87 0.000000001866

88 0.000000001774

89 0.000000001880

Table 8A

Table 9 shows temperature and corresponding capacitance for a amplicon having

Table 9

[00119] Table 9A may be determined using the below provided formula:

+1) - (temperature at X))). FIG. 14 is a graphical representation of the temperature and corresponding change in capacitance of the sample showing a melting temperature for amplicon (615 bp), in accordance with an embodiment. Temperature Change in

(°C) Capacitance

(farad)

75 0.000000001902

76 0.000000001750

77 0.000000001902

78 0.000000001874

79 0.000000001512

80 0.000000001646

81 0.000000001568

82 0.000000001472

83 0.000000002460

84 0.000000001598

85 0.000000001592

86 0.000000001570

87 0.000000001536

88 0.000000001410

89 0.000000001588

Table 9A

[00120] A sample may be tested for one or more amplicons, which may be referred to as multiplexing. One of the amplicons may have primer length of 615bp. The melting temperature of the first amplicon may be around 83 degree Celsius. Another amplicon may have a primer length of 246bp. The melting temperature of the second sequence may be around 86 degrees.

[00121] The polymerase chain reaction may be run and capacitances at each temperature value in the cycle may be measured. Table 10 shows temperature and corresponding values of capacitances of the sample under test which may have the two different sequences.

80 0.0000004615

81 0.0000004667

82 0.0000004728

83 0.0000004782

84 0.0000004845

85 0.0000004899

86 0.0000004963

87 0.0000005027

88 0.0000005089

Table 10

[00122] Table 10A is an illustration of the temperature and corresponding changes in capacitance of the sample under test which may have two different sequences. FIG. 15 is a graphical representation of the temperature and corresponding change in capacitance of the sample under test showing different melting temperatures/temperature signatures for the sample under test, in accordance with an embodiment.

Table 10A [00123] From FIG. 15, more than one peak of the change in capacitance can be observed. More than one peak may indicate that the sample under test has more than one melting temperature corresponding to more than one amplicons. A peak may be observed at around 83 degree Celsius, and another peak may be observed at around 86 degree Celsius. It may indicate that amplicons with melting temperatures/temperature signatures 83 degree Celsius and 86 degree Celsius may be present in the sample.

[00124] In an embodiment, the polymerase chain reaction may be carried out in a sequential manner, such that, one cycle of a PCR may be carried out to detect the presence of one amplicon in a sample. In such sequential PCR, the annealing temperature may vary.

[00125] The sequence for carrying out the sequential polymerase chain reaction for each of the amplicons may be in decreasing order of annealing temperatures of the amplicons. For example, a first amplicon may anneal at, say, 65 degree Celsius and a second amplicon may anneal at, say, 55 degree Celsius.

[00126] In another embodiment, a particular annealing temperature for all amplicons may be designed.

RESTRICTION DIGESTION

[00127] After identification of an amplicon in a sample, a further confirmation of presence of the amplicon in the sample may be desired. In an embodiment, the sample used in the PCR may be treated with restriction enzymes. Restriction enzymes are responsible for cutting DNA into fragments if the amplicon is present, which may result in decrease in capacitance from initial capacitance. A decrease in capacitance from initial capacitance may indicate that the amplicon is being digested or divided into smaller fragments by respective enzymes. Table 11 shows the capacitance for samples with different base pair length treated with and without restriction enzyme during incubation.

1 0.0000002262 0.0000002313 0.0000002458 0.0000002339

2 0.0000002275 0.0000002314 0.0000002460 0.0000002331

3 0.0000002286 0.0000002317 0.0000002462 0.0000002330

4 0.0000002293 0.0000002319 0.0000002465 0.0000002329

5 0.0000002297 0.0000002319 0.0000002468 0.0000002328

6 0.0000002301 0.0000002318 0.0000002471 0.0000002328

7 0.0000002303 0.0000002317 0.0000002473 0.0000002327

8 0.0000002304 0.0000002315 0.0000002474 0.0000002327

9 0.0000002305 0.0000002313 0.0000002476 0.0000002327

10 0.0000002305 0.0000002312 0.0000002477 0.0000002327

11 0.0000002304 0.0000002312 0.0000002478 0.0000002327

12 0.0000002303 0.0000002311 0.0000002479 0.0000002327

Table 11

[00128] Table 11 A may be obtained from Table 11 by dividing each value in a column from the first value in the respective column.

Table 11A [00129] FIG. 16 is a graphical representation of the effect in capacitance with and without adding restriction enzymes, in accordance with an embodiment.

[00130] In an embodiment, the presence of at least one amplicon in a sample may be detected by isothermal method of DNA amplification, such as, Loop Mediated Isothermal Amplification (LAMP). It shall be noted that, other isothermal methods for amplification of DNA sequence, well known in the art, may also be used.

[00131] The capacitance may be determined at only one temperature in this method for a predefined duration. An increase in capacitance along with time may indicate the presence of an amplicon in the sample. Further, evaporation may be much less when isothermal methods are used.

[00132] For example, a positive sample (positive HBV blood) may be tested for the presence of an amplicon by the isothermal method. The positive sample may be tested against negative blood sample. The positive sample and the negative sample may be received by two different vials 102. The capacitance for both may be determined by applying a constant temperature to the samples. The samples are heated for about 11 minutes. Table 12 shows the capacitances for both the positive and negative sample against time.

Table 12

[00133] The capacitance of the positive sample increases from an initial value along with the duration for which it is heated. The increase in capacitance over time may indicate the presence of an amplicon. However, capacitance of the negative sample does not exhibit a change over the time.

[00134] The system 100 is configured to determine capacitance sensitively. Hence measurement starts from first cycle, thereby ensuring reduction in time for detection and characterization.

[00135] The system and method can be adapted in detection in processes that release proton, example, Glucose, Cholesterol, NADH or NADPH based reactions.

IV. CONCLUSION

[00136] Embodiments provide a technique that detects multiple sequences in a polymerase chain reaction and identify the amplicon present in the sample responsible for causing a disease.

[00137] Embodiments provide a technique that uses continuous measurement of electrical properties for providing increased sensitivity over other methods.

[00138] Embodiments provide a technique to identify the nature of the amplicon from the melting temperature of the amplicon.

[00139] Embodiments provide a technique that takes lesser time to complete the process with minor human intervention and less sophisticated laboratory and lab equipments.

[00140] Embodiments provide a technique that detects an amplicon of DNA by running the PCR for a relatively lesser number of cycles.

[00141] The example embodiments described herein may be implemented in an operating environment comprising software installed on a computer, in hardware, or in a combination of software and hardware.

[00142] Although embodiments have been described with reference to specific example embodiments, it will be evident that various modifications and changes may be made to these embodiments without departing from the broader spirit and scope of the system and method described herein. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.

[00143] Many alterations and modifications of the present invention will no doubt become apparent to a person of ordinary skill in the art after having read the foregoing description. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. It is to be understood that the description above contains many specifications, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the personally preferred embodiments of this invention. Thus the scope of the invention should be determined by the appended claims and their legal equivalents rather than by the examples given.

We claim:

1. A system for extracting electrical signals from one or more samples, the system comprising: a plurality of vials configured to receive samples for carrying out polymerase chain reaction or isothermal reaction;

a plurality of cover members, wherein each of the cover members comprises a pair of electrodes, wherein each vial is configured to receive one cover member and the corresponding pair of electrodes, wherein the pair of electrodes make contact with the sample; and

an electronic circuit connected to the electrodes, wherein the electronic circuit is configured to measure electrical properties of the samples present in the vials, at least while carrying out polymerase chain reaction or isothermal reaction.

2. The system according to claim 1, wherein the cover members are configured to be disposed at least partially into the vials, wherein the cover members reduce vaporizing of the samples out from the vials upon application of temperature during the polymerase chain reaction.

3. The system according to claim 1, wherein the electrical properties are at least the impedance and phase of polymerase chain reaction products, wherein capacitance is determined using the impedance and phase.

4. The system according to claim 1, further comprising a plurality of engagement members, wherein the engagement members are configured to establish electrical connection between the electronic circuit and the electrodes, wherein the engagement members transfer electrical signals between the samples and the electronic circuit.

Claims

5. The system according to claim 4, further comprising an interconnection board, wherein the interconnection board comprises a first surface and a second surface, wherein the first surface comprises conductive pads, and the second surface is engaged with the electrodes, wherein the electrodes and the corresponding conductive pads are in electrical connection, wherein the engagement members are configured to operatively interface with the conductive pads.

6. The system of claim 5, wherein the interconnection board, the cover members and the electrodes are configured as a first single unit, and the engagement members and the electronic circuit are configured as a second single unit, wherein the second single unit is configured to establish electrical connection with the first single unit.

7. The system according to claim 5, further comprising a first assembly configured to enable connection between the electronic circuit and the interconnection board, wherein the first assembly comprises a plate configured to receive the electronic circuit such that the engagement members extend inferiorly from the plate, wherein the engagement members are configured to translate along its longitudinal axis to facilitate establishing connection between the electronic circuit and the interconnection board.

8. The system according to claim 5, further comprising a second assembly defining a substantially enclosed working area, wherein the second assembly comprises:

a plurality of wells disposed in the working area, wherein the wells are configured to receive the vials; and

a pressing device operable to exert pressure over interconnection board engaged with the vials.

9. A method for testing a sample for two or more target amplicons, the method comprising: preparing a reaction mixture, comprising at least primers corresponding to the amplicons, whose presence in the sample has to be identified;

carrying out polymerase chain reaction;

determining capacitance of the reaction mixture; determining the presence or absence of the amplicons at least based on increase in capacitance; and

determining which of the amplicons is present in the sample at least based on change in capacitance and corresponding temperature.

10. The method of claim 9, wherein determining capacitance of the reaction mixture comprises determining capacitance at varying annealing temperature, wherein each annealing temperature corresponds to the PCR conditions for each amplicon.

11. The method of claim 9, wherein determining which of the amplicons is present in the sample is based on temperature signature at which maximum change in capacitance occurs, wherein the temperature signature is known as melting temperature.

12. The method of claim 9, wherein determining which of the amplicons is present in the sample is based on comparison between predetermined temperature signature corresponding to each of the amplicons and temperature signature determined while testing the sample.

13. The method of claim 9, wherein determining which of the amplicons is present in the sample comprises multiplexing two or more samples and adding restriction enzymes to each sample, wherein multiplexing two or more samples is conducted to obtain the melting temperature for each sample simultaneously.

14. The method of claim 9, wherein the polymerase chain reaction is carried out sequentially for varying annealing temperatures of the amplicons.

15. The method of claim 12, determining which of the amplicons is present in the sample comprises identifying temperatures at which maximum change in capacitance occurs at the end or during the polymerase chain reaction corresponding to each of the amplicons.

16. The method of claim 9, wherein determining the presence or absence of the amplicons at least based on the capacitance comprises determining whether there is increase in capacitance of the reaction mixture at around 72 degree Celsius during multiple cycles of the polymerase chain reaction.

17. The method according to claim 9, wherein preparing the reaction mixture comprises adding the sample to a master mix, wherein the master mix is in dry form.

18. The method according to claim 9, wherein preparing the reaction mixture comprises at least one additive capable of increasing the range of capacitance of the reaction mixture.