WO2020197487A1

WO2020197487A1 - Real-time pcr chip

Info

Publication number: WO2020197487A1
Application number: PCT/SG2019/050172
Authority: WO
Inventors: Lye Hock Sim
Original assignee: Cell Id Pte Ltd
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2020-10-01

Abstract

A real-time PCR chip (10) comprising: a main body (900), the main body comprising a chamber for performing PCR therein and an inlet (101) to allow placement of a test sample and appropriate reagents into the chamber; a bio-sensor formed on the main body and having a sensing portion provided in the chamber to detect amount of PCR product obtained in the chamber after each round of PCR; and a heater formed on the main body to heat the test sample in the chamber according to temperature requirements of the PCR.

Description

REAL-TIME PCR CHIP

FIELD OF THE INVENTION

This invention relates to a polymerase chain reaction (PCR) chip, and in particular, to a real time PCR (RT-PCR) chip.

BACKGROUND OF THE INVENTION

PCR is a commonly used method to amplify or make multiple copies of a DNA sequence for various applications such as DNA cloning for sequencing, diagnosing disease, identifying individuals from DNA samples, and performing functional analyses of genes. In PCR, replication of the DNA sequence takes place in multiple thermal cycles, with each cycle typically having three main steps: denaturation, annealing and extension. In the denaturation step, a double-stranded DNA template is typically heated to about 94-98 °C for 20-30 seconds to yield single-stranded DNA. In the annealing step, primers are typically annealed to the single-stranded DNA by lowering the temperature to about 50-65 °C for 20-40 seconds. In the extension step, using a DNA polymerase (such as Taq), a new double- stranded DNA is synthesized by extending the primer that has been annealed to the single- stranded DNA at an optimum activity temperature of the DNA polymerase (75-80 ^GC for Taq}. Appreciably, replication of the DNA is exponential as the new double-stranded DNA formed in a cycle undergoes denaturation, annealing and extension in the next cycle, such that each cycle effectively doubles the number of DNA sequences obtained. In addition to the three main steps mentioned above, an initialization step may be required if the DNA polymerase used is heat activated, and the final extension step of the last cycle may be held for a longer period of time (e.g 5-15 minutes) to ensure that there are no remaining single- stranded DNA fragment.

Real-time PCR (RT-PCR) is a technique in which amplification of DNA is monitored during performance of the PCR by measuring the accumulation of DNA product, i.e. , double stranded DNA (dsDNA), after each round of PCR amplification. Once the desired amount of DNA has been obtained, the PCR can be halted. In contrast, conventional end-point PCR is typically run for a pre-set number of amplification rounds before measuring DNA amount; in order to ensure that the desired amount of DNA has been obtained, excess rounds of amplification are typically performed so as to avoid under amplification. Appreciably, RT- PCR saves time over convention PCR by eliminating the performance of unnecessary amplification rounds once the desired amount of DNA has been obtained. A current method of RT-PCR uses a fluorescent dye that binds to only dsDNA in the PCR so that the intensity of detected fluorescence is indicative of the amount of replicated DNA. However, dsDNA dyes bind to all dsDNA PCR products, including nonspecific PCR products (such as primer dimers). This can potentially interfere with, or prevent, accurate monitoring of the intended target DNA.

Another method of RT-PCR uses a fluorescent reporter probe that detects only a targeted DNA sequence that is complementary to the probe, thus increasing specificity as presence of other dsDNA would not interfere with detection of the targeted sequence. Using different- coloured labels, fluorescent probes can be used in multiplex assays for monitoring several target sequences in the same tube. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, where the primer dimers compete for DNA polymerase with the targeted DNA, thereby inhibiting its amplification.

In any case, both current RT-PCR methods require fluorescence detection means. Thus, existing RT-PCR devices comprise laboratory bench-top set-ups about the size of at least a desktop document printer. Accordingly, RT-PCR can currently only be performed in laboratory settings. However, PCR is often still needed to diagnose or detect diseases in places where laboratories are not available, such as in the less developed countries and/or remote locations with poor or no infrastructural support. Especially in times of disease outbreak, PCR would be particularly needed in order for molecular diagnoses or detection to be performed as quickly as possible. There is therefore a need to be able to perform PCR without requiring a laboratory set-up to be first established.

SUM MARY OF INVENTION

According to a first aspect, there is provided a real-time PCR chip comprising: a main body, the main body comprising a chamber for performing PCR therein and an inlet to allow placement of a test sample and appropriate reagents into the chamber; a bio-sensor formed on the main body and having a sensing portion provided in the chamber to detect amount of PCR product obtained in the chamber after each round of PCR; and a heater formed on the main body to heat the test sample in the chamber according to temperature requirements of the PCR.

The real-time PCR chip may further comprise a thermo-sensor formed on the main body to obtain temperature of the test sample in the chamber during the PCR.

The main body may comprise a top layer, a middle layer and a bottom layer, a first through hole provided in the top layer to serve as the inlet, and a second through hole provided in the middle layer in fluid communication with the first through hole, wherein the second through hole, part of a lower surface of the top layer, and part of an upper surface of the bottom layer together at least partially define the chamber.

The real-time PCR chip may further comprise a first conductive trace formed on one of: an upper surface of the bottom layer and a lower surface of the top layer, the first conductive trace having a sensing portion located in the chamber to serve as the bio-sensor, the first conductive trace further having terminals configured to be in electrical connection with connection pins of a connector socket.

The heater may comprise a second conductive trace formed on one of: a lower surface of the bottom layer and an upper surface of the top layer, the second conductive trace having an undulating portion disposed at least partially around a discrete heating area located directly adjacent the chamber to heat the chamber, the second conductive trace further having terminals configured to be in electrical connection with connection pins of the connector socket.

The thermo-sensor may comprise a third conductive trace formed on one of: an upper surface of the top layer and a lower surface of the bottom layer, the third conductive trace having a temperature sensing portion located directly adjacent the chamber to sense temperature in the chamber, the third conductive trace further having terminals configured to be in electrical connection with connection pins of the connector socket.

The top layer may have a thickness ranging from 0.15 mm to 0.5 mm, the middle layer may have a thickness ranging from 0.5 mm to 1.2 mm, and the bottom layer may have a thickness ranging from 0.15 mm to 0.5 mm.

The real-time PCR chip may further comprise a sealing sheet layered over the top layer at least over the first through hole to seal the inlet before use of the real-time PCR chip.

The real-time PCR chip may further comprise a cover sheet layered over the sealing sheet at least over the first through hole, the sealing sheet having an integral cover located over the inlet to seal the inlet before use, the cover being attached to an underside of the cover sheet such that at least partially detaching the cover sheet from the sealing sheet detaches the cover from the sealing sheet to expose the inlet.

The real-time PCR chip may further comprise a reagent provider, the reagent provider comprising a reagent container to contain a PCR reagent therein, the reagent container configured to fit into the inlet of the main body, the reagent container having a reagent outlet in fluid communication with the chamber when the reagent container has been fitted into the inlet.

According to a second aspect, there is provided a reagent provider for a PCR chip, the PCR chip having a main body, the main body comprising a chamber for performing PCR therein and an inlet to allow placement of a test sample and appropriate reagents into the chamber, the reagent provider comprising: a reagent container to contain a PCR reagent therein, the reagent container configured to fit into the inlet of the main body, the reagent container having a reagent outlet in fluid communication with the chamber when the reagent container has been fitted into the inlet.

For both aspects, the reagent outlet may open through a side wall and through a bottom of the reagent container.

An inner surface of the bottom of the reagent container may be sloped downwardly towards the reagent outlet.

The reagent container may have a top opening to place the reagent in the reagent container and wherein the reagent provider further comprises a container cap to close the top opening.

The reagent provider may further comprise a strap having a first end connected to the reagent container and a loop provided at a second end of the strap, and wherein the main body comprises an attachment hole for passing the strap therethrough to attach the reagent provider to the main body.

The reagent provider may have a monolithic construction.

The reagent container may contain the PCR reagent in lyophilized form. BRIEF DESCRIPTION OF FIGURES

In order that the invention may be fully understood and readily put into practical effect there shall now be described by way of non-limitative example only exemplary embodiments of the present invention, the description being with reference to the accompanying illustrative drawings.

FIG. 1a is a top view of the exemplary embodiment of a main body of a RT-PCR chip according to the present invention without a sealing sheet and without a cover sheet. FIG. 1 b is a cross-sectional view at A-A of the main body of FIG. 1a.

FIG. 1c is a detailed view at B of the cross-sectional view of FIG. 1b.

FIG. 1d is a detailed view at C of the cross-sectional view of FIG. 1b.

FIG. 2a is a top view of a top layer of the main body of FIG. 1a.

FIG. 2b is a bottom view of the top layer of FIG. 2a.

FIG. 3a is a top view of a middle layer of the main body of FIG. 1a.

FIG. 3b is a bottom view of the middle layer of FIG. 3a.

FIG. 4a is a top view of a bottom layer of the main body of FIG. 1a.

FIG. 4b is a bottom view of the bottom layer of FIG. 3a.

FIG. 5 is a top view of a sealing sheet of the main body.

FIG. 6 is a top view of a cover sheet of the main body.

FIG. 7a is a perspective view of a reagent provider of the RT-PCR chip.

FIG. 7b is a close-up view of an open reagent container of the reagent provider of FIG. 7a. FIG. 7c is a close-up view of the reagent container of FIG. 7b containing reagent therein.

FIG. 7d is a close-up view of the reagent container of FIG. 7b closed by a container cap of the reagent provider.

DETAILED DESCRIPTION

Exemplary embodiments of a real-time PCR (RT-PCR) chip 10 will be described below with reference to FIGS. 1a to 7d. The same reference numerals are used throughout the figures to denote the same or similar parts.

Referring to FIGS. 1a to 4b, the RT-PCR chip 10 comprises a main body 900. The main body 900 comprises chamber 80 for performing PCR therein, as can be seen in particular in FIG. 1d. The chamber 80 has an inlet 101 to allow placement of a test sample and appropriate reagents into the chamber 80. The test sample comprises a liquid that may contain a target nucleic acid to be amplified. The main body 900 comprises a bio-sensor 344 provided in the chamber 80 to detect amount of PCR product obtained in the chamber 80 after each round of PCR. The RT-PCR chip 10 further comprises a heater 355 formed on the main body 900 to heat the test sample in the chamber 80 according to temperature requirements of the PCR. The RT-PCR chip 10 preferably further comprises a thermo sensor 144 formed on the main body 900 to obtain temperature of the liquid in the chamber 80 during the PCR. Overall thickness of the main body 900 may be 1.5 mm.

In an exemplary embodiment, the main body 900 comprises a top layer 100 (FIGS. 2a and b), a middle layer 200 (FIGS. 3a and b), and a bottom layer 300 (FIGS. 4a and b). The top, middle and bottom layers 100, 200, 300 are each preferably made of a biologically inert waterproof material, and may comprise a biologically inert plastics material such as polycarbonate, polyethylene terephthalate, or polypropylene, or paper laminated with a biologically inert plastics material. The top, middle and bottom layers 100, 200, 300 may be made of the same material or may be made of different materials, and are preferably flat and rigid. A lower surface 105 of the top layer 100 is bonded to an upper surface 204 of the middle layer 200. A lower surface 205 of the middle layer 200 is bonded to an upper surface 304 of the bottom layer 300. Bonding between the top and middle layers 100, 200 and between the middle and bottom layers 200, 300 may be achieved using known means such as an adhesive or ultrasonic welding, for example.

As shown in FIGS. 2a and b, the main body 900 comprises a first through hole 101 provided in the top layer 100. The first through hole 101 serves as the inlet 101 of the chamber 80. In the exemplary embodiment, the top layer 100 may be about 0.25 mm thick. In alternative embodiments, the top layer 100 may have a thickness ranging from 0.15 mm to 0.5 mm.

As shown in FIGS. 3a and b, the main body 900 comprises a second through hole 201 provided in the middle layer 200. The second through hole 201 is in fluid communication with the first through hole 101 , and is preferably larger than the first through hole 101. As can be seen in FIG. 1 d, the second through hole 201 , part of the lower surface 105 of the top layer 100, and part of the upper surface 304 of the bottom layer 300 together at least partially define the chamber 80. In the exemplary embodiment, the middle layer 200 may be about 1 mm thick. In alternative embodiments, the middle layer 200 may have a thickness ranging from 0.5 mm to 5 mm. As shown in FIG. 4a, the main body 900 includes a first conductive trace 344 configured to serve as the bio-sensor 344. The first conductive trace 344 is preferably provided on the upper surface 204 of the bottom layer 300. The first conductive trace 344 may comprise a working electrode (WE), a counter Electrode (CE) and a reference electrode (RE). In the exemplary embodiment, the bottom layer 300 may be about 0.25 mm thick. In alternative emodiments, the bottom layer 300 may have a thickness ranging from 0.15 mm to 0.5 mm Overall thickness of the exemplary embodiment of the RT-PCR chip 10 is about 1.5 mm.

A sensing portion 349 of the first conductive trace 344 is located in the chamber 80 to contact the test sample in the chamber 80 during the PCR. The bio-sensor 344 is configured to detect amount of PCR product obtained in the chamber 80 after each round of PCR according to electrical signals obtained the sensing portion 349. The electrical signals obtained by the sensing portion 349 have been calibrated before use of the RT-PCR chip 10 to correspond with actual amount of PCR product in the chamber 80. In this way, the bio sensor 344 is integrated with the main body 900 and real-time PCR can be performed using the presently disclosed RT-PCR chip 10 without requiring fluorescence detecting apparatus. The first conductive trace 344 includes terminals 348 configured to be in electrical connection with connector pins of a connector socket in order to obtain electrical signals obtained by the sensing portion 349. To that end, the terminals 348 are preferably provided on a connector portion 309 that is part of the bottom layer 300, the connector portion 309 of the bottom layer being configured to be inserted into the connector socket. The connector socket is in electrical and signal communication with a computer (not shown) having an application module installed therein to monitor and control PCR being performed in the RT- PCR chip 10. In an alternative embodiment (not shown), the first conductive trace serving as the bio-sensor may instead be provided on the lower surface of the top layer.

The main body 900 further includes a second conductive trace 355 configured to serve as the heater 355, as shown in FIG. 4b. The second conductive trace 355 is preferably formed on a lower surface 305 of the bottom layer 300. The second conductive trace 355 comprises an undulating portion 356 disposed at least partially around a discrete heating area 357 made of a same material as the undulating portion 356. The discrete heating area 357 is located directly adjacent the chamber 80 to heat the test sample in the chamber 80 for the PCR to take place. In this way, the heater 355 is integrated with the main body 900 so that no other heating apparatus is required to perform real-time PCR using the RT-PCR chip 10. The second conductive trace 355 also includes terminals 358 in electrical connection with the undulating portion 356. The terminals 358 are configured to be in electrical connection with connection pins of the connector socket (not shown) in order to power the heater 355, such as a 10P10C connector. To that end, the terminals 348 are preferably also provided on the connector portion 309 of the bottom layer 300. In the alternative embodiment (not shown), the second conductive trace serving as the heater may instead be provided on the upper surface 104 of the top layer 100.

The main body 900 preferably includes a third conductive trace 144 configured to serve as the thermo-sensor 144, the third conductive trace 144 being preferably formed on an upper surface 104 of the top layer 100. The third conductive trace 144 has a temperature sensing portion 149 located directly adjacent the chamber 80 as well as terminals 148 configured to be in electrical connection with connector pins of a connector socket (not shown), such as a 10P10C connector. To that end, the terminals 148 are preferably provided on a connector portion 109 that is part of the top layer 100, the connector portion 109 of the top layer being configured to be inserted into the connector socket. In the alternative embodiment (not shown), the third conductive trace serving as the thermo-sensor may instead be provided on a bottom surface of the bottom layer. The temperature sensing portion 149 may include a resistor 190.

In the exemplary embodiment, the first, second and third conductive traces 344, 355, 144 comprise silver, and may be formed by appropriate printing of a conductive ink on the upper and lower surfaces of the top and bottom layer accordingly as described above. In alternative embodiments, the first, second and third conductive traces 344, 355, 144 may instead by formed by vacuum deposition followed by etching away of unwanted deposited metal, or a combination of both printing and vacuum deposition techniques.

The main body 900 further comprises a sealing sheet 400 (shown in FIG. 5) layered over the top layer 100 at least over the first through hole 101. In this way, the sealing sheet 400 seals the inlet 101 before use of the RT-PCR chip 10. In use, at least partially detaching the sealing sheet 400 exposes the inlet 101.

The main body 900 preferably also comprises a cover sheet 500 (shown in FIG. 6) layered over the sealing sheet 400 at least over the first through hole 101. Where the cover sheet 500 is provided, the sealing sheet 400 may comprise a cover 401 located over the inlet 101 that seals the inlet 101 before use. The cover 401 is integral with the sealing sheet 400 and is also attached to an underside of the cover sheet 500, so that, during use, at least partially detaching the cover sheet 500 from the sealing sheet 400 detaches the cover 401 from the sealing sheet 400 to expose the inlet 101. The cover 401 may be defined in the sealing sheet 400 by a die cut line to facilitate its detachment from the sealing sheet 400 when the cover sheet 500 is at least partially detached from the sealing sheet 400. Thus, in embodiments where the cover sheet 500 is provided, at least partially detaching the cover sheet 500 partially detaches the sealing sheet 400 by detaching the cover 401 from the rest of the sealing sheet 400, thereby exposing the inlet 101.

For all embodiments, the sealing sheet 400 and cover sheet 500 each preferably comprises a moisture barrier polymeric film or laminated paper so that the inlet 101 and chamber 80 are kept well sealed before use of the RT-PCR chip 10 to avoid contamination of the chamber 80 or inlet 101. In this way, the RT-PCR chip 10 requires no additional packaging to keep it free from contamination during storage and transportation of the RT-PCR chip 10 before use. This also greatly reduces the size of the RT-PCR chip 10 and consequently the space it takes up, allowing more units of the RT-PCR chip 10 to be readily transported. Doing away with the need for additional packaging also significantly reduces the cost of providing the RT-PCR chip 10.

The RT-PCR chip 10 may optionally include a reagent provider 800 as shown in FIG. 7. In the exemplary embodiment, as shown in FIG. 7. The reagent provider 800 comprises a reagent container 801 provided to contain a PCR reagent such as DNA polymerase therein. The reagent container 801 is configured to fit into the inlet 101. In the exemplary embodiment, the inlet 101 may be circular in shape and the reagent container 801 may be accordingly generally cylindrical in shape, having a cylindrical wall 806. Preferably, the reagent container 801 fits into the inlet 101 with a sealing fit. A step 803 may be provided in the cylindrical wall 806 of the reagent container 801 to serve as a stop against the upper surface 104 of the top layer 100 for positioning the reagent container 801 relative to the chamber 80 when the reagent container 801 is fitted into the inlet 101.

The reagent container 801 has a reagent outlet 802 that is in fluid communication with the chamber 80 when the reagent container 801 has been fitted into the inlet 101. The reagent outlet 802 is preferably located at a bottom edge of the reagent container 801 such that the reagent outlet 802 opens through the side wall 806 as well as through the bottom 805 of the reagent container 801. An inner surface 855 of the bottom 805 of the reagent container 101 may be sloped downwardly towards the reagent outlet 802 to allow the reagent in the reagent container 801 to flow into the chamber 80 under gravity.

The reagent may be lyophilized in the form of beads 860 that are larger than the reagent outlet 802 so that reagent 860 in the reagent container 801 cannot leave the reagent container 801 before use. Providing the reagent in lyophilized form 860 advantageously eliminates the need for cold storage of the reagent as the lyophilized reagent 860 may be stored for months in room temperature in the reagent provider 800 whereas a liquid form of the reagent typically needs to be kept at refrigerator or freezer temperatures for stable storage. Providing the reagent in lyophilized form 860 thus reduces cost by eliminating the need for cold storage and cold transportation of the reagent.

During use, when the reagent container 801 has been fitted into the inlet 101 , the test sample that has already been placed in the chamber 80 comes into contact with the reagent in the reagent container 801 through the reagent outlet 802 to dissolve the beads of reagent in the container 801. The dissolved reagent then flows out of the reagent outlet 802 into the chamber 80 to allow PCR to take place.

The reagent container 801 preferably has a top opening 804 for placing the reagent in the reagent container 801 before use of the RT-PCR chip 10. The reagent provider 800 may further include a container cap 844 to close the top opening 804 of the reagent container 801 after the reagent has been placed in the reagent container 801. The container cap 844 may be connected to the top opening 804 via a living hinge 830.

The reagent provider 800 may optionally comprise a strap 807 having a first end 891 connected to the reagent container 80. The reagent container 801 and the strap 807 may be integral with each other, and may be made of a bio-inert plastics material. The strap 807 may have a loop 808 provided at its free end 892. The main body 900 may optionally include an attachment hole 17 for attaching the reagent provider 800 to the main body 900. The attachment hole 17 may be provided by providing through holes 107, 207, 307, 407, 507 in each of the top, middle and bottom layers 100, 200, 300 respectively as well as in the sealing sheet 400 and cover sheet 500 (if such is provided), the through holes 107, 207, 307, 407, 507 being aligned with one another. The attachment hole 17 of the main body 900 should be space apart from the inlet 101 without any fluid communication with the chamber 80. To securely attach the reagent provider 800 to the main body 900, the free end 892 of the strap 807 is first passed through the attachment hole 17 without passing the reagent container 801 through the attachment hole 17, followed by passing the reagent container 801 through the loop 808 at the free end 892 of the strap 807.

The reagent provider 800 may optionally include an inlet cap 810 to close the inlet 101 when the reagent container 801 has been removed from the inlet 101 after use of the RT-PCR chip 10. Closing the inlet 101 after use of the RT-PCR chip 10 may be desired to prevent the test sample from leaving the chamber 80 and contaminating surfaces or persons, for instance where the test sample may comprise disease-causing agents such as the human immunodeficiency virus.

The container cap 844 and inlet cap 810 are preferably integral with the reagent container 801 and the strap 807 respectively, as can be seen in FIG. 7. In this way, the reagent provider 800 can have a monolithic construction made of a single material, and may be formed by injection moulding, for example, thereby reducing its manufacturing cost.

Before use, the reagent provider 800 may be provided with the reagent already placed in the container 801 through the top opening 804 and the top opening 804 already closed by the container cap 844, and the entire reagent provider 800 placed in a sealed pouch for safe storage of the reagent.

To use the exemplary embodiment of the RT-PCR chip 10, the cover sheet 500 is at least partially detached from the sealing sheet 400 to expose the inlet 101. The reagent provider 800 is removed from its sealed pouch and attached to the main body 900 at the attachment hole 17 via the strap 807 and loop 808. The test sample is then placed in the chamber 80 via the inlet 101 , followed by fitting the reagent container 801 into the inlet 101. The test sample in the chamber 80 then enters the reagent container 801 via the reagent outlet 802 to dissolve the lyophilized reagent which then flows out of the reagent outlet 802 into the chamber 80 to mix with the test sample. The heater 355 is activated to provide the necessary temperature for PCR to take place in the chamber 80. The bio-sensor 344 is activated to monitor the amount of PCR product obtained after each PCR cycle. The thermo sensor 144 is activated to monitor temperature of the test sample in the chamber 80 during the PCR. When a predetermined amount of PCR product has been obtained as detected by the bio-sensor 344, the PCR in the chamber 80 can be stopped. For all embodiments, the main body 900 is preferably provided with at least one quick response (QR) code 600. The QR code 600 is provided on a visible part of the main body 900, such as on the sealing sheet 400 (as shown in FIG. 5) or, in other embodiments, on the cover sheet 500. The at least one QR code 600 allows information such as manufacturing date, expiry date and source information of the at RT-PCR chip 10 itself to be stored and retrieved, as well as allowing the RT-PCR chip 10 to be associated or tagged with a single specific source of the test sample. The specific source may be a patient or any other test sample supply, depending on the usage application of the RT-PCR chip 10. The QR code 600 should be located on a part of the main body 900 that is never detached so that the RT- PCR chip 10 can always be correctly traced to its specific test sample source. In this way, each RT-PCR chip 10 is indelibly and indubitably associated with only one specific test sample source, thereby minimizing or preventing mix-ups in test results from occurring.

The present invention thus provides a low cost, low bulk, RT-PCR chip 10 that can be easily associated with a specific test sample source and is configured to perform real-time or semi- real-time PCR without requiring a bulky bench-top device in a laboratory setting.

Whilst there has been described in the foregoing description exemplary embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations in details of design, construction and/or operation may be made without departing from the present invention. For example, while the cover sheet shown in FIG. 6 is depicted as being able to be layered only over part of the sealing sheet over at least the cover of the sealing sheet, in other embodiments, the cover sheet may be configured to be layered over all of the sealing sheet. While the inlet and reagent container are circular or cylindrical in the figures, the inlet and reagent container may be of any other desired shape.

Similarly, the chamber may have any appropriate shape other than the generally circular shape shown in the figures.

Claims

1. A real-time PCR chip comprising:

a main body, the main body comprising

a chamber for performing PCR therein and

an inlet to allow placement of a test sample and appropriate reagents into the chamber;

a bio-sensor formed on the main body and having a sensing portion provided in the chamber to detect amount of PCR product obtained in the chamber after each round of PCR; and

a heater formed on the main body to heat the test sample in the chamber according to temperature requirements of the PCR.

2. The real-time PCR chip of claim 1 , further comprising a thermo-sensor formed on the main body to obtain temperature of the test sample in the chamber during the PCR.

3. The real-time PCR chip of claim 1 or claim 2, wherein the main body comprises a top layer, a middle layer and a bottom layer, a first through hole provided in the top layer to serve as the inlet, and a second through hole provided in the middle layer in fluid communication with the first through hole, wherein the second through hole, part of a lower surface of the top layer, and part of an upper surface of the bottom layer together at least partially define the chamber.

4. The real-time PCR chip of any one of claims 1 to 3, wherein the biosensor comprises a first conductive trace formed on one of: an upper surface of the bottom layer and a lower surface of the top layer, the first conductive trace further having terminals configured to be in electrical connection with connection pins of a connector socket.

5. The real-time PCR chip of claim 4, wherein the heater comprises a second conductive trace formed on one of: a lower surface of the bottom layer and an upper surface of the top layer, the second conductive trace having an undulating portion disposed at least partially around a discrete heating area located directly adjacent the chamber to heat the chamber, the second conductive trace further having terminals configured to be in electrical connection with connection pins of the connector socket.

6. The real-time PCR chip of claim 5 when dependent on claim 2, wherein the thermos sensor comprises a third conductive trace formed on one of: an upper surface of the top layer and a lower surface of the bottom layer, the third conductive trace having a temperature sensing portion located directly adjacent the chamber to sense temperature in the chamber, the third conductive trace further having terminals configured to be in electrical connection with connection pins of the connector socket.

7. The real-time PCR chip of claim 3 or any one of claims 4 to 6 when dependent on claim 3, wherein the top layer has a thickness ranging from 0.15 mm to 0.5 mm, the middle layer has a thickness ranging from 0.5 mm to 1.2 mm, and the bottom layer has a thickness ranging from 0.15 mm to 0.5 mm.

8. The real-time PCR chip of any one of the preceding claims, further comprising a sealing sheet layered over the top layer at least over the first through hole to seal the inlet before use of the real-time PCR chip.

9. The real-time PCR chip of claim 8, further comprising a cover sheet layered over the sealing sheet at least over the first through hole, the sealing sheet having an integral cover located over the inlet to seal the inlet before use, the cover being attached to an underside of the cover sheet such that at least partially detaching the cover sheet from the sealing sheet detaches the cover from the sealing sheet to expose the inlet.

10. The real-time PCR chip of any one of the preceding claims, further comprising a reagent provider, the reagent provider comprising a reagent container to contain a PCR reagent therein, the reagent container configured to fit into the inlet of the main body, the reagent container having a reagent outlet in fluid communication with the chamber when the reagent container has been fitted into the inlet.

11. The real-time PCR chip of claim 10, wherein the reagent outlet opens through a side wall and through a bottom of the reagent container.

12. The real-time PCR chip of claim 11 , wherein an inner surface of the bottom of the reagent container is sloped downwardly towards the reagent outlet.

13. The real-time PCR chip of any one of claims 10 to 12, wherein the reagent container has a top opening to place the reagent in the reagent container and wherein the reagent provider further comprises a container cap to close the top opening.

14. The real-time PCR chip of any one of claims 10 to 13, wherein the reagent provider further comprises a strap having a first end connected to the reagent container and a loop provided at a second end of the strap, and wherein the main body comprises an attachment hole for passing the strap therethrough to attach the reagent provider to the main body.

15. The real-time PCR chip of any one of claims 10 to 14, wherein the reagent provider has a monolithic construction.

16. The real-time PCR chip of any one of claims 10 to 15, wherein the reagent container contains the PCR reagent in lyophilized form.

17. A reagent provider for a PCR chip, the PCR chip having a main body, the main body comprising a chamber for performing PCR therein and an inlet to allow placement of a test sample and appropriate reagents into the chamber, the reagent provider comprising: a reagent container to contain a PCR reagent therein, the reagent container configured to fit into the inlet of the main body, the reagent container having a reagent outlet in fluid communication with the chamber when the reagent container has been fitted into the inlet.

18. The reagent provider of claim 17, wherein the reagent outlet opens through a side wall and through a bottom of the reagent container.

19. The reagent provider of claim 18, wherein an inner surface of the bottom of the reagent container is sloped downwardly towards the reagent outlet.

20. The reagent provider of any one of claims 17 to 19, wherein the reagent container has a top opening to place the reagent in the reagent container and wherein the reagent provider further comprises a container cap to close the top opening.

21. The reagent provider of any one of claims 17 to 20, wherein the reagent provider further comprises a strap having a first end connected to the reagent container and a loop provided at a second end of the strap.

22. The reagent provider of any one of claims 17 to 21 , wherein the reagent provider has a monolithic construction.

23. The reagent provider of any one of claims 17 to 22, wherein the reagent container contains the PCR reagent in lyophilized form.