Abstract:
The present invention is related to a portable apparatus for performing uni-directional convective qPCR or qRT-PCR in a mixing reagent containing a target nucleic acid and a fluorescence dye including denaturation, annealing and extension processes. The apparatus includes at least a temperature controlling unit which comprises at least one heat source and one temperature sensor, a circulation-enabling container, a light source, a photo-detector, a filter, a set of optical elements, and a processor.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of priority of U.S. Provisional Application No. 62/315,660 filed on Mar. 30, 2016, and included herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
       [0002]    This invention is related to a quantitative real-time polymerase chain reaction (hereinafter qPCR) and quantitative reverse transcription real-time PCR (hereinafter qRT-PCR) apparatus by a uni-directional convective circulation. 
       2. Description of the Prior Art 
       [0003]    The real-time quantitative polymerase chain reaction (hereinafter qPCR) and the quantitative reverse transcription real-time PCR (hereinafter qRT-PCR) were developed based on the need for quantifying target at time 0 , during and after a test. Compared to traditional PCR which detects product concentration after the reaction, qPCR and qRT-PCR detect it real-time. Both qPCR and qRT-PCR use fluorescence to detect and quantify the product concentration during the reaction, and thus they are more time effective than traditional PCR. Moreover, both qPCR and qRT-PCR allow for complete reaction and detection within one test zone. Therefore, the advantages of qPCR and qRT-PCR are quantifying products in real-time and minimizing the chance of DNA contamination where PCR products are analyzed by gel electrophoresis. 
         [0004]    For fast and efficient amplification of various targets, lots of apparatuses and methods were developed. The developing principles of these apparatuses and methods are still following the basic PCR rules. In the commercialized PCR kits, methods, and related apparatuses, a sample contained a target DNA, a pair of oligonucleotide primers which are complementary to a specific region of the target DNA, a DNA polymerase which is thermally stable, and deoxynucleotide triphosphates (dNTP). The target DNA is amplified by repeating a designated temperature cycle that sequentially changes the heating temperature of the sample. The temperature cycle includes three different temperature settings, and the temperature settings are set for the following steps. 
         [0005]    The first step is so-called “denaturation” in which the temperature is about 90-95° C. The sample is heated to a relatively high temperature to let a double stranded DNA (hereinafter dsDNA) become a single stranded DNA (hereinafter ssDNA). The second step is so-called “annealing” in which the temperature is decreased to a relative low temperature, that is, about 45 to 65° C., to let the primers bind to the single stranded DNA and form a primer-ssDNA complex. The last step is so-called “extension” in which the temperature is heated or maintained at a suitable temperature, that is 72° C., to let the primer of the primer-ssDNA complex extend by the action of the DNA polymerase to generate a new ssDNA complementary to the template of the target DNA, thus to generate new dsDNA products. Theoretically, the target DNA can be amplified millions or higher number of copies by repeating the three steps for about 20 to 40 times. 
         [0006]    In qPCR or qRT-PCR, the addition of dsDNA fluorescence dyes is prepared as usual. Then the reaction is run in a qPCR instrument, and after each cycle, the intensity of fluorescence is measured with a photo-detector. Two common methods for quantifying the PCR or RT-PCR products in real-time are: (1) non-specific fluorescence dyes that intercalate with any dsDNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence. Both qPCR or qRT-PCR are carried out in a repeating temperature cycle with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore. 
         [0007]    If the non-specific fluorescence dyes added to the PCR bind to the dsDNA, the increase of the products during PCR would lead to an increase in fluorescence intensity measured at each cycle. However, dsDNA dyes such as SYBR® Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as primer dimer). This can potentially interfere with the accuracy of the quantification of the PCR products. If the sequence-specific DNA probes are added to the PCR, the fluorescent reporter probes detect only the DNA containing the sequence complementary to the probe; therefore the use of sequence-specific fluorescence dye significantly increases specificity, and enables performing the technique even in the presence of other dsDNA. The advantage of sequence-specific fluorescence dyes is that it can prevent the interference of measurements caused by primer dimers. 
         [0008]    Typically, the qPCR and qRT-PCR apparatus are large bench top systems that require long reaction time (over 1 hour) because qPCR and qRT-PCR requires a mechanism that cycles through three different temperature ranges allowing for denaturation, annealing and elongation of the target DNA segments. In order to achieve such requirement, qPCR and qRT-PCR apparatus may be of significant weight and size, and also require significant amount of testing time, thus such apparatus eliminates its portability. 
       SUMMARY OF THE INVENTION 
       [0009]    To solve this problem, the present invention discloses a portable uni-directional circulating liquid flow which allows q-PCR and qRT-PCR reaction to take place within a circulation-enabling container by thermal convection. This present invention comprises at least a temperature controlling unit which comprises at least one heat source and one temperature sensor, a circulation-enabling container, alight source, a photo-detector, a filter, a set of optical elements, and a processor. The foresaid components are not limited to any particular arrangement or order. 
         [0010]    The circulation-enabling container comprises at least one opening, a closed-loop system, and a pathway connected from end to end which allows for the mixing reagent to flow through different zones of the circulation-enabling container in one cycle. 
         [0011]    The qPCR and qRT-PCR reagents are poured into the circulation-enabling container when the reaction begins and the circulation-enabling container is placed and contacted to the heat source with a specific region. The circulation-enabling container could be symmetric or asymmetric. When the temperature of the contacted region of the circulation-enabling container increases to the reaction temperature of denaturation, the mixing reagent close to the contacted region would be heated first, and the mixing reagent far from the heat source would be heated thereafter. The density of the mixing reagent closer to the heat source would be lower than the density of the mixing reagent further away from the heat source; with the effect of gravity and buoyancy, a continuous uni-directional circulating flow is created. When the temperature inside the circulation-enabling container reaches the reaction point by aforesaid thermal convection, the PCR reaction is initiated. Without spending time on heating and/or cooling the thermal device for the three different reaction temperatures, the present invention discloses an embodiment of three different temperature zones allowing for denaturation, annealing and elongation of PCR inside the circulation-enabling container with at least one heating source, thus saving reaction time and apparatus size simultaneously. 
         [0012]    This SUMMARY is provided to briefly identify some aspects of the present disclosure that are further described below in the DESCRIPTION. This SUMMARY is not intended to identify key or essential features of the present disclosure nor is it intended to limit the scope of any claims. 
         [0013]    The term “aspects” is to be read as “at least one aspect.” The aspects described above and other aspects of the present disclosure described herein are illustrated by way of example (s) and not limited in the accompanying drawing. 
         [0014]    These and other objectives of the present invention will no doubt become obvious to those of ordinary skill in the art after reading the following detailed description of the preferred embodiment that is illustrated in the various figures and drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0015]    A more complete understanding of the present disclosure may be realized by reference to the accompanying drawings in which: 
           [0016]      FIG. 1  is a schematic diagram illustrating the first configuration of a qPCR and qRT-PCR of the present disclosure; 
           [0017]      FIG. 2  is a schematic diagram illustrating the U-shaped loop of the first configuration; 
           [0018]      FIG. 3  is a schematic diagram illustrating temperature gradient inside the U-shaped loop during the reaction of the first configuration; 
           [0019]      FIG. 4  is a schematic diagram illustrating a second configuration of a qPCR and qRT-PCR of the present disclosure; 
           [0020]      FIG. 5  is a schematic diagram illustrating the U-shaped loop of the second configuration; 
           [0021]      FIG. 6  is a schematic diagram illustrating a third configuration of a qPCR and qRT-PCR of the present disclosure; 
           [0022]      FIG. 7  is a schematic diagram illustrating the U-shaped loop of the third configuration. 
       
    
    
     DETAILED DESCRIPTION 
       [0023]    This present invention comprises at least a temperature controlling unit which comprises at least one heat source and one temperature sensor, a circulation-enabling container, a light source, a photo-detector, a filter, a set of optical elements, and a processor. The foresaid components are not limited to any particular arrangement or order. 
         [0024]    The circulation-enabling container comprises at least one open, a closed-loop system, and a pathway connected from end to end which allows for the mixing reagent to flow through different zones of the asymmetric circulation-enabling container in one cycle. The circulation-enabling container could be asymmetric or symmetric based on the experimental requests. The circulation-enabling container could be a U-shaped loop, a cube, or other structures. 
         [0025]    The temperature controlling unit comprises a heat source for supplying the heat and a temperature sensor for detecting the status of the heat source. The temperature controlling unit is placed and contacted in a specific region in the circulation-enabling container. The temperature controlling unit is configured to adjust the temperature inside of the circulation-enabling container for reaction temperature and flow field distribution. It is possible to use one or more temperature controlling units in different conditions. 
         [0026]    The symmetry of the circulation-enabling container is a key factor to drive and initiate a uni-directional circulation liquid flow by the effect of gravity and buoyancy, and so does the contacted region between the circulation-enabling container and the heat source. The number of heat source (s) is also a key factor. Each of them could cause a uni-directional circulation inside the circulation-enabling container independently or corporately. 
         [0027]    One may further limit the direction of flow of the liquid inside the circulation-enabling container by placing one or more heat sources in the specific regions to control thermal and buoyancy conditions that favor one flow direction over other direction(s). For example, instead of using one heat source on the bottom, the container can be heated at a region off center. In one embodiment described below, using two set of temperature controlling units which are placed in different height from the bottom of the circulation-enabling container can also reach the same outcome. 
         [0028]    The light source is a specific wavelength of light system, such as a LED, a laser diode, or a halogen light. The fluorescence is light emission by a substance that has absorbed the light source. A substance could absorb such light and emit a fluorescent signal which could then be used for monitoring the reaction status. The photo-detector is configured to convert the optical signals to electronic signals. The photo-detector could be a single unit such as a photomultiplier zone (hereinafter PMT), photodiodes, or a photo-detector array, for example a charge-coupled device (hereinafter CCD) or a complementary metal-oxide-semiconductor (hereinafter CMOS). 
         [0029]    The processor is configured to receive and analyze the electronic signals which are transferred from the temperature sensor, photo-detector or the photo-detector array. The filter is configured to filter out these non-predetermined wavelengths of the light sources and let the predetermined wavelengths pass through the filter. One can use one or more optical elements such as a lens or an optical fiber to direct the filtered or unfiltered fluorescent signal to the photo-detector. 
         [0030]    The present invention provides a continuously uni-directional circulating liquid flow to allow the PCR or RT-PCR to react within the circulation-enabling container by thermal convection. Such circulation-enabling container facilitates the uni-directional liquid flow by providing pathway connected from end to end which allows for the reagent to flow through different thermal zones in one cycle and limiting the possible flow paths for predictable reaction efficiency. The thermal convection of the liquid is driven by buoyancy and gravity. When the temperature inside the circulation-enabling container increases to the reaction point by aforesaid thermal convection, the flow of liquid begins and PCR reaction is initiated. And the temperature sensor transfers this status to the processor to initiate the following process. The presence of PCR products will interact with fluorescence dye and fluorescent signal would be emitted and detected by the photo-detector. A programmed algorithm is built into the processor to analyze the fluorescent signal to quantify the PCR or RT-PCR in real-time. 
         [0031]    In one embodiment described below, it is possible to further limit the direction of the flow path by designing an asymmetric pathway connected from end to end of the circulation-enabling container. The angle of one end of the pathway is different from that of the other end, which leads to a different vertical height between these two ends. When the temperature controlling unit starts to provide heat in the contacted region, the density of the mixing reagent closer to the contacted region is lower than the mixing reagent away from the contacted region, thus to lead a buoyancy gap inside the reagent and to initiate a uni-directional circulation liquid flow by the effect of gravity and buoyancy. 
         [0032]    The following merely illustrates the principles of the disclosure. It will thus be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the disclosure and are included within its spirit and scope. 
         [0033]    Furthermore, all examples and conditional language recited herein are principally intended expressly to be only for pedagogical purposes to aid the reader in understanding the principles of the disclosure and the concepts contributed by the inventor(s) to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. 
         [0034]    Moreover, all statements herein reciting principles, aspects, and embodiments of the disclosure, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements later developed that perform the same function, regardless of structure. 
         [0035]    Unless otherwise explicitly specified herein, the drawings are not drawn to scale. 
         [0036]    We now provide some non-limiting, illustrative examples that illustrate operational aspects of a mixing device and associated method preparing materials used in biological or biochemical assays. 
         [0037]    As used herein, directional terms as may be used such as “horizontal,” “vertical,” “proximal,” “distal,” “front”, “rear”, “left,” “right,” “inner,” “outer,” “interior” and “exterior” relate to an orientation of the disclosed apparatus from the perspective of a typical user, and do not specify permanent, intrinsic features or characteristics of the device. 
         [0038]    As used in below three embodiments, the circulation-enabling container is unified by U-shaped loop. 
         [0039]    As used herein, the positional relationship and related terms as may be used such as “before”, “after”, “front”, and “rear” relate to an order of arrival of a reflected light beam traveling among the elements of the disclosed apparatus of each of the embodiments of the invention. For instance, an U-shaped loop is the first element that reflected light beam, and the U-shaped loop is located in the rearmost position of each of the disclosed apparatus. 
       Embodiment 1 
       [0040]    As illustrated in  FIG. 1 , the q-PCR and qRT-PCR device  1  comprises a temperature controlling unit, which is a heat source and a temperature sensor  12  in this embodiment, and a circulation-enabling container, which is a U-shaped loop  11  in this embodiment. A light source  13 , a photo-detector  16 , a filter  15 , a lens  14 , and a processor  17  are also shown in the  FIG. 1 . 
         [0041]    As shown in  FIG. 2 , the asymmetric circulation-enabling container is a U-shaped loop  11  with a pathway at different vertical height between the two ends of the U-shaped loop  11  forming a loop pathway. The U-shaped loop  11  has a left zone  111 , a right zone  112 , a link zone  113 , and a bottom zone  114 . The left and right zone  111 , 112  are perpendicular to the ground, and there is an opening  117  on the top of the left zone  111 . The link zone  113  is connecting the left and right zone  111 ,  112  with a predetermined angle. In this embodiment, the left end of the link zone  113  is higher than the right end thereof. Furthermore, the left zone  111  can be distinguished from a left junction  115  as a left-upper zone  111   a  and a left-lower zone  111   b.  The right zone  112  can be distinguished from a right junction  116  as a right-upper zone  112   a  and a right-lower zone  112   b.  The bottom zone  114  connecting to the left zone  111  and the right zone  112  is a bending zone with symmetrical shape and a predetermined curvature radius. The bottom zone  114  of the U-shaped loop  11  is where the heat source and the temperature sensor  12  contact the surface of the U-shaped loop  11 , and the U-shaped loop  11  is substantially perpendicular to the ground. In this invention, the inner diameter of the left zone  111 , the right zone  112 , the link zone  113 , and the bottom zone  114  of the U-shaped loop  11  is between 0.6 mm to 1.6 mm, and inside the U-shaped loop  11  is a connected space for accommodating the solution. In this embodiment, the inner diameter of the U-shaped loop  11  is 1.6 mm, and the total volume of the solution in the U-shaped loop  11  is 150 μl. When the heat source and a temperature sensor  12  provide heat to the U-shaped loop  11  and make the temperature of the U-shaped loop  11  to a predetermined reaction temperature, the structure of the U-shaped loop  11  is capable of allowing a solution therein to flow in a uni-direction and form a flow field. 
         [0042]    When the heat source and the temperature sensor  12  start to provide heat, the liquid of the bottom zone  114  would be first heated. The left zone  111  and the right zone  112  are both heated from the bottom to the top of the loop. The temperature of the left junction  115  is lower than the temperature of the right junction  116  due to the height of the left junction  115  is shorter than the height of the right junction  116 . Therefore, the density of the left junction  115  is higher than the density of the right junction  116 . The liquid of the left junction  115  flows to the right junction  116  by the effect of buoyancy, and thus the liquid of the right junction  116  pushes downward and back to the left junction  115 , to initiate a clockwise uni-directional circulating flow. The temperature differentiation is as shown in  FIG. 3 , the higher temperature region inside the U-shaped loop  11  is the left-lower zone  111   b,  and the lower temperature region inside the U-shaped loop  11  is the right-lower zone  112   b.  In the embodiment, the heat source and a temperature sensor  12  is set in the range between 90-110° C. The temperature distribution during the reaction is described as below: the temperature of the left-upper zone  111   a  is between 30-40° C., the temperature of the left-lower zone  111   b  is between 90-100° C., the temperature of the right-upper zone  112   a  is between 30-40° C., the temperature of the right-lower zone  112   b  is between 45-60° C., and the temperature of the bottom zone  114  is between 60-90° C. The speed of uni-directional circulating flow is about 1.7-6 mm/s and it takes 10-33 seconds for one cycle. 
         [0043]    The 150 μl solution comprises 30 μl primer (Canine-GAPDH_7-2-F′-GTGGATCTGACCTGCCGCCTGGAGAAAGCT-, 0.5 μM, 15 μl; and Canine-GAPDH_7-2-R′-CCTCAGTGTAGCCCAGGATGCCTTTGAGGG-, 0.5 μM, 15 μl), 75 μl of 2× mastermix (SensiFASTSYBR™ No-ROX, including dNTPs, DNA polymerase, SYBR™ Green), 3 μl plasmid DNA (3*10 3  copies), and 42 μl secondary sterile water. SYBR™ Green is one kind of the fluorescent substances. In this embodiment, the fluorescent substance, which is loaded into the U-shaped loop, will interact with the amplicon and emit fluorescent signal when it is excited by the light source. By measuring this fluorescent signal, we can measure the concentration of amplicon real time. 
         [0044]    The light source  13 , such as LED lights, laser diode lights, or halogen lights, may emit a light beam with predetermined wavelength for a fluorescence excitation. The region where the light source  13  is deployed has a predetermined distance and angle of depression with respect to the U-shaped loop  11 . The left zone  111 , right zone  112 , link zone  113 , and bottom zone  114  have substantially the same irradiation intensity. Besides, the fluorescent substance enters the excited status as receiving the light beam, and exits the excited status with emitting fluorescent. In this embodiment, the light source  13  is a LED light and the wavelength thereof is between 450 to 490 nm, and the SYBR™ Green has max fluorescent value between 510 to 530 nm as excited by the light source with 450 to 490 nm wavelength. 
         [0045]    The lens  14  is disposed in front of the U-shaped loop  11  at a predetermined distance, and at the same side as the light source  13  with respect to the U-shaped loop  11 . The lens  14  is configured to receive the light beam reflected from the U-shaped loop  11 . Besides, each of the distance between the lens  14  and the left zone  111 , the right zone  112 , the link zone  113 , and the bottom zone  114  is substantially the same. The lens  14  is configured to refract a light beam and focus the light beam on the photosensitive unit to form an image of the U-shaped loop  11 . 
         [0046]    The filter  15  is disposed in the front of the lens  14  at a predetermined distance for receiving the light beam from the lens  14 . In other words, the lens  14  is placed between the filter  15  and the U-shaped loop  11 . 
         [0047]    The photo-detector  16  is configured for converting the collected light signal to electrical signal receiving from the filter  15 . The electrical signal is processed by a processor for analysis. In the invention, the photo-detector  16  can be a single element, such as a photomultiplier tube or a photodiode. The photo-detector  16  can also be an array, such as a charge-coupled device or a complementary metal-oxide-semiconductor. In the embodiment, the photo-detector  16  is a CCD. 
       Embodiment 2 
       [0048]    Referring to  FIG. 4  and  FIG. 5 , a q-PCR and qRT-PCR apparatus  2  includes a temperature controlling unit, that is a heat source and a temperature sensor  22  in this embodiment, and a circulation-enabling container, which is U-shaped loop  21 . A light source  23 , a photo-detector  26 , a lens  24 , a filter  25 , and a processor  27  are also shown in the  FIG. 4  and  FIG. 5 . The connection and region of the light source  23 , the photo-detector  26 , the lens  24 , the filter  25 , and the processor  27  are substantially the same as described in  FIG. 1 . However, the connection and the region of the U-shaped loop  21  and the heat source and a temperature sensor  22  are different from  FIG. 1 . 
         [0049]    When the heat source and a temperature sensor  22  provide heat to the U-shaped loop  21 , the asymmetric structure of the U-shaped loop  21  is capable of allowing a solution therein to flow in a uni-directional way and form a flow field. As shown in  FIG. 5 , the U-shaped loop  21  has a left zone  211 , a right zone  212 , a link zone  213 , a bottom zone  214 , and a protruded zone  217  which is connected to the bottom zone  214 . The left and right zones  211 ,  212  are perpendicular to the ground, and there is an opening  218  on the top of the left zone  211 . The link zone  213  is connecting the left and right zones  211 ,  212 . Besides, the angle of both the left junction  215  and the right junction  216  of the link zone  213  is parallel to the ground. In the embodiment, the left end of the link zone  213  is at the same height as the right end thereof. Furthermore, the left zone  211  can be distinguished from a left junction  215  as a left-upper zone  211   a  and a left-lower zone  211   b.  The right zone  212  can be distinguished from a right junction  216  as a right-upper zone  212   a  and a right-lower zone  212   b.  The bottom zone  214  interconnected between the left zone  211  and the right zone  212  is a bending zone with symmetrical shape and a predetermined curvature radius. The protruded zone  217  of the U-shaped loop  21  is where the heat source and a temperature sensor  22  contacts with the U-shaped loop  21 . The protruded zone  217  would transfer the heat from heat source and a temperature sensor  22  to the U-shaped loop  21  during the reaction. In this embodiment, the protruded zone  217  is placed on the right of the bottom zone  214 . The diameter, the solution volume, and the solution content are substantially the same as described in  FIG. 1 . 
         [0050]    When the heat source and a temperature sensor  22  start to provide heat, the protruded zone  217  would be first heated and then the heat is transferred to the left zone  211  and the right zone  212 . The temperature rises faster in the right zone  212  than the left zone  211  since the heat source and a temperature sensor  22  and the protruded zone  217  are closer to the right zone  212 . When the solution nearby the bottom of right zone  212  was heated, the volume of the solution is expanded and the density is decreased. The heated liquid rises up near to the right junction  216  by the effect of buoyancy, and vacated volume would be supplemented by the surrounding liquid. When the supplement liquid is also raised up by the effect of buoyancy, the liquid near to the right junction  216  flows to the left junction  215  and back to the bottom of the right zone  212 , to initiate a counterclockwise uni-directional circulating flow. The higher temperature region inside the U-shaped loop  21  is the left-lower zone  212   b,  the lower temperature region inside the U-shaped loop  21  is  211   b.  In the embodiment, the heat source and a temperature sensor  22  is set in the range between 90-110° C. The temperature distribution during the reaction is described as below: the temperature of the left-upper zone  211   a  is between 30-40° C., the temperature of the left-lower zone  211   b  is between 45-60° C., the temperature of the right-upper zone  212   a  is between 30-40° C., the temperature of the right-lower zone  212   b  is between 90-100° C., and the temperature of the bottom zone  214  is between 60-90° C. The speed of uni-directional circulating flow is about 1.7-6 mm/s and it takes 10-33 seconds for one cycle. 
         [0051]    As nucleic acid amplification takes place, the fluorescent substance, which is loaded into the U-shaped loop, will interact with the amplicon and emit fluorescent signal when it is excited by the light source. The fluorescence is focused by the lens  24  and then passing through the filter  25  to filter out the non-predetermined wavelengths. In this embodiment, the emitted fluorescence signal with a wavelength 510-530 nm is detected by the CCD  26  and then the optical signals are converted to the electronic signals. The processor  27  analyzes the electronic signals. Therefore, this invention could real-time monitor the product concentration. 
       Embodiment 3 
       [0052]    Referring to  FIG. 6  and  FIG. 7 , a q-PCR and qRT-PCR apparatus  3  includes two sets of temperature controlling units, that are heat sources and temperature sensors  32   a  and  32   b  in this embodiment, a circulation-enabling container, that is U-shaped loop  31 , a light source  33 , a photo-detector  36 , a lens  34 , a filter  35 , and a processor  37 . The connection and region of the light source  33 , the photo-detector  36 , the lens  34 , the filter  35 , and the processor  37  are substantially the same as described in  FIG. 1 . However, the connection and the region of the U-shaped loop  31  and the heat sources and temperature sensors  32   a  and  32   b  are different from  FIG. 1 . 
         [0053]    The heat sources and temperature sensors  32   a  and  32   b  could be defined as relatively higher heat source and temperature sensor  32   a  and relatively lower heat source and temperature sensor  32   b.  The preferred placement of both is the height of the relatively higher heat source and temperature sensor  32   a  being lower than the height of the relatively lower heat source and temperature sensor  32   b.  The predetermined temperature of the relatively higher heat source and temperature sensor  32   a  is between 90-120° C. and 5-30° C. for the relatively lower heat source and temperature sensor  32   b.  In this embodiment, the relatively higher heat source and temperature sensor  32   a  are placed in the junction of left zone  311  and bottom zone  314 , while the relatively lower heat source and temperature sensor  32   b  are placed on the right zone  312  and the height of the relative higher heat source and temperature sensor  32   a  is lower than the height of the relatively lower heat source and temperature sensor  32   b.    
         [0054]    When the relatively higher heat source and temperature sensor  32   a  and the relatively lower heat source and temperature sensor  32   b  start to heat, the contact surfaces of the U-shaped loop  31  are first heated. The temperature rises faster in the left zone  311  than the right zone  312 . When the solution nearby the contact surfaces of the U-shaped loop  31  and the relatively higher heat source and temperature sensor  32   a  was heated, the volume of the solution is expanded and the density is decreased. The heated liquid rises up near to the left junction  315  by the effect of buoyancy, and vacated volume would be supplemented by the surrounding liquid which has lower temperature and higher density. When the supplement liquid is also raised up by the effect of buoyancy, the liquid near to the left junction  315  flows to the right junction  316  and back to the bottom of the left zone  311 , to initiate a clockwise uni-directional circulating flow. 
         [0055]    As nucleic acid amplification takes place, the fluorescent substance, which is loaded into the U-shaped loop, will interact with the amplicon and emit a fluorescent signal when it is excited by the light source. The fluorescence is focused by the lens  34  and then passing through the filter  35  to filter out the non-predetermined wavelengths. In this embodiment, the emitted fluorescence signal with a wavelength 510-530 nm is detected by the CCD  36  and then the optical signals are converted to the electronic signals. The processor  37  analyzes the electronic signals. Therefore, this invention could real-time monitor the product concentration. 
         [0056]    Those skilled in the art will readily observe that numerous modifications and alterations of the device and method may be made while retaining the teachings of the invention. Accordingly, the above disclosure should be construed as limited only by the metes and bounds of the appended claims.