Patent Publication Number: US-2023146570-A1

Title: Pcr rapid detection device and method thereof

Description:
This application claims the benefit of Taiwan application Serial No. 110141503, filed Nov. 8, 2021, the subject matter of which is incorporated herein by reference. 
     TECHNICAL FIELD 
     The disclosure relates in general to a detection device, and more particularly to a rapid polymerase chain reaction (PCR) detection device and a method thereof. 
     BACKGROUND 
     Polymerase chain reaction (PCR) is an enzyme driven process for amplifying the deoxyribonucleic acid (DNA) fragments in vitro. Through denaturation, annealing and elongation, PCR can duplicate millions of DNA fragments. In the denaturation step, double helix DNA is denaturized into single helix DNA at a high temperature (90-95° C.), then the single helix DNA is used as a duplication template. In the annealing step, when the temperature drops to a suitable level, the primers can be annealed to correct positions of the target gene. In the elongation step, the temperature is adjusted to 72° C., and magnesium ions are used as enzyme cofactor, so that DNA polymerase can be synthesized as another strand of DNA fragment according to the code of the duplication template. By repeating the above three steps continuously, a small volume of DNA fragment can be duplicated to a large volume. 
     Although the real-time nucleic acid PCR (RT-PCR) test is simple and has excellent performance in amplification, absolute quantitative result still cannot be obtained. On the other hand, digital nucleic acid PCR (dPCR) test can perform quantitative analysis using direct counting method but requires human intervention at the transition between different stages of the testing process, not only deteriorating testing efficiency but also increasing cost and the operating complexity of device and adding risk to the operator. 
     SUMMARY 
     The disclosure is directed to a rapid PCR detection device whose disposable microfluidic unit, magnetron micro-fluid unit, linear actuator, PCR thermal cycling unit and image recognition unit are integrated in a body, so that integrated rapid testing can be achieved. 
     According to one embodiment of the present disclosure, a rapid PCR detection device is provided. The device includes a body, a disposable microfluidic unit, a magnetron micro-fluid unit, a linear actuator, a PCR thermal cycling unit and an image recognition unit. The microfluidic unit is made of a transparent material, wherein a transparent film is arranged in the middle of the microfluidic channel and has at least one hole for a micro-fluid to flow in the microfluidic channel. The magnetron micro-fluid unit is used to drive the micro-fluid, so that the micro-fluid is divided into a plurality of droplets guided to the lower layer of the microfluidic channel. The linear actuator is used to drive the disposable microfluidic unit to an amplification zone of the body. The PCR thermal cycling unit performs PCR thermal cycling in the amplification zone. The image recognition unit illuminates the droplets with a fluorescent light and determines the number of DNA fragments in the droplets according to the detected fluorescent intensity. 
     According to another embodiment of the present disclosure, a rapid PCR detection method is provided. The method includes the following steps. A micro-fluid is placed in a disposable microfluidic unit, wherein a transparent film is arranged in the middle of the microfluidic channel and has at least one hole, and the micro-fluid flows in an upper layer of the microfluidic channel. A magnet is controlled to move under the microfluidic channel and drive the micro-fluid, so that the micro-fluid is divided into a plurality of droplets guided to the lower layer of the microfluidic channel. The disposable microfluidic unit is driven to an amplification zone of a body. A PCR thermal cycling is performed in the amplification zone. The droplets are illuminated with a fluorescent light and the number of DNA fragments in the droplets is determined according to the detected fluorescent intensity. 
     The above and other aspects of the disclosure will become better understood with regard to the following detailed description of the preferred but non-limiting embodiment(s). The following description is made with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    is a block diagram of a rapid PCR detection device according to an embodiment of the present disclosure; 
         FIGS.  2 A and  2 B  respectively are schematic diagrams of internal configuration of a rapid PCR detection device according to an embodiment of the present disclosure; 
         FIGS.  3 A and  3 B  respectively are an explosion diagram and an assembly diagram of a microfluidic channel according to an embodiment of the present disclosure. 
         FIGS.  4 A- 4 C  respectively are schematic diagrams of droplet division, PCR amplification and image recognition performed in a detection method according to an embodiment of the present disclosure; 
         FIG.  5 A  is a schematic diagram of a magnetron micro-fluid unit according to an embodiment of the present disclosure; 
         FIG.  5 B  is a schematic diagram of droplets moved to a predetermined position along a magnetic induction track according to an embodiment of the present disclosure; 
         FIG.  6    is a schematic diagram of a drive circuit according to an embodiment of the present disclosure; 
         FIG.  7    is a flowchart of a rapid PCR detection method according to an embodiment of the present disclosure. 
     
    
    
     DETAILED DESCRIPTION 
     Technical solutions for the embodiments of the present disclosure are clearly and thoroughly disclosed with accompanying drawings. However, the embodiments disclosed below are only some rather than all of the embodiments of the present disclosure. All embodiments obtained by anyone ordinarily skilled in the technology field of the disclosure according to the disclosed embodiments of the present disclosure are within the scope of protection of the present disclosure if the obtained embodiments lack innovative labor. 
     The disclosed features, structures or characteristics can be combined in one or more embodiments by any suitable way. In the following disclosure, many detailed descriptions are provided for the embodiments of the present disclosure to be better and fully understood. However, anyone ordinarily skilled in the technology field of the disclosure will understand that technical solution for implementing the present disclosure also can dispense with one or more of the details disclosed below or can be implemented using other methods, devices, or steps. In some circumstances, generally known methods, devices, implementations, or operations of the technical solution capable of implementing the present disclosure are not necessarily illustrated or disclosed in detail lest the aspects of the present disclosure might be distracted. 
     Refer to  FIG.  1    and  FIGS.  2 A and  2 B .  FIG.  1    is a block diagram of a rapid PCR detection device  100  according to an embodiment of the present disclosure.  FIGS.  2 A and  2 B  respectively are schematic diagrams of internal configuration of a rapid PCR detection device  100  according to an embodiment of the present disclosure. The rapid PCR detection device  100  of the present embodiment includes a microfluidic unit  120 , a magnetron micro-fluid unit  130 , a linear actuator  140 , a PCR thermal cycling unit  150  and an image recognition unit  160 , which are disposed in a body  110  and form an integrated rapid testing platform. 
     The body  110  is an operating platform on which the user can input setting values, such as thermal cycling temperature value and thermal cycling time, analyze image parameters, detect fluorescent intensity, calculate the number of DNA fragments, and output the detected values. All of the above testing steps are completed in one single body  110  instead of being allocated to different testing devices, hence avoiding human intervention which would occur at the transition between different stages of the testing process. 
     Refer to  FIGS.  2 A and  2 B . The interior of the body  110  is divided into a first area  112  and a second area  114 . The magnetron micro-fluid unit  130  is located in the first area  112 ; the PCR thermal cycling unit  150  and the image recognition unit  160  are located in the second area  114 . The first area  112  of the body  110  for droplet division; the second area  114  is for PCR amplification and fluorescence detection. The linear actuator  140  is movably disposed between the first area  112  and the second area  114 . The disposable microfluidic unit  120  is driven by the linear actuator  140  to reciprocally move between the first area  112  and the second area  114 . The interior of the microfluidic unit  120  has two layers of microfluidic channel  120   a , that is, an upper layer and a lower layer of the microfluidic channel  122   a  and  124   a  as indicated in  FIG.  3 A . 
     In an embodiment, the linear actuator  140  includes a motor  142 , a linear slide  144  and a ball screw  146 . The ball screw  146  is rotated by a torque provided by the motor  142 . The slider  148  on the linear slide  144  and the adapter (not illustrated) on the ball screw  146  are integrally coupled in one piece and move along the linear slide  144 . The disposable microfluidic unit  120  is disposed on the slider  148  and can slide to an amplification zone (the amplification zone is located with the second area  114 ) to perform digital nucleic acid PCR (dPCR) test. 
     Referring to  FIGS.  3 A and  3 B , an explosion diagram and an assembly diagram of a microfluidic channel according to an embodiment of the present disclosure are respectively shown. The microfluidic unit  120  is made of a transparent material (such as acrylic, PMMA or PDMS) and formed of layered plates (such as 5 layers), wherein the plates are such as a cover  121 , an upper plate  122 , an intermediate plate  123 , a lower plate  124  and a bottom plate  125 . The upper plate  122  and the lower plate  124  respectively have microfluidic channels  122   a  and  124   a . The intermediate plate  123  is formed of a transparent film, which has at least one hole  123   a . Each hole  123   a  is interconnected between the microfluidic channel  122   a  of the upper plate  122  and the microfluidic channel  124   a  of the lower plate  124  for a micro-fluid  10  to flow in the microfluidic channel. The micro-fluid  10  moves to the microfluidic channel  124   a  of the lower plate  124  from the microfluidic channel  122   a  of the upper plate  122  through the hole  123   a  arranged in the intermediate plate  123 . 
     In comparison to the conventional microfluidic silicon chip with high cost, the microfluidic unit  120  incurs lower cost and is easier to manufacture. Furthermore, the microfluidic channel can be customized according to customer needs and can be disposed after one time of use, hence reducing the probability of the microfluidic channel being polluted by the residuals of the DNA tester. Besides, the microfluidic unit  120  can completely seal the micro-fluid  10  and divide into the droplets  20  through magnetron. Hence, the transition between different stages of the testing process does not require human intervention, the risk of human contact can be reduced, and the automation of nucleic acid PCR test can be implemented. 
     Refer to  FIGS.  4 A- 4 C , a top view and a side view of droplet division, PCR amplification and image recognition performed in a detection method according to an embodiment of the present disclosure are respectively shown. Refer to  FIGS.  2 A,  3 A and  5 A . As indicated in  FIG.  4 A , when the microfluidic unit  120  is located in the first area  112 , the micro-fluid  10  formed of the to-be-tested DNA tester, ferrofluid, and PCR fluorescent dye is placed in the microfluidic channel  122   a  of the upper plate  122 . Then, the permanent magnet  132  is driven by an electromagnetic force generated by the magnetron micro-fluid unit  130 , and the micro-fluid  10  is driven by the permanent magnet  132  to move in the microfluidic channel  122   a  of the upper plate  122 , and the micro-fluid  10 , after passing through the hole  123   a  of the intermediate plate  123 , is divided to form a droplet  20 . The said droplet division process is repeated to obtain a plurality of droplets  20 . 
     Refer to  FIGS.  2 A,  3 A,  4 B and  5 A . In  FIG.  4 B , after droplet division is completed, the droplet  20  is driven by the permanent magnet  132  and sequentially moved to the microfluidic channel  124   a  of the lower plate  124  for subsequent PCR amplification. Refer to  FIGS.  2 B,  4 B and  5 A . In  FIG.  4 B , the linear actuator  140  drives the microfluidic unit  120  to move to the second area  114  from the first area  112 , and the PCR thermal cycling unit  150  performs PCR thermal cycling in the second area  114 . In the PCR thermal cycling process, double helix DNA is denaturized into single helix DNA at a high temperature. Then, the single helix DNA is used as a duplication template. When the temperature drops to a suitable level, the primers can find the two ends of a target gene fragment and be annealed thereto. Polymerase can correctly add 4 nucleic acid materials (dNTPs:dATP, dGTP, dCTP, dTTP) to the fragment one by one according to the code of the DNA template to form a new strand of DNA fragment. After the PCR thermal cycling process is repeated for a predetermined number of repeats, a large number of duplicated DNA fragments will be obtained. In the present embodiment, the predetermined number of repeats is 30-40 times, but the present disclosure is not limited thereto. 
     Refer to  FIGS.  2 B and  4 C . In  FIG.  4 C , the image recognition unit  160  illuminates the droplets with a fluorescent light,  20 , and determines, according to the detected fluorescent intensity, the number of DNA fragments  12  duplicated in the droplets  20  during the PCR thermal cycling process. Since the number of DNA fragments  12  duplicated in each droplet  20  is not the same, the image recognition unit  160  can calculate the fluorescent intensity of each droplet  20  according to the luminous flux of the image sensor corresponding to each droplet  20  or calculate the fluorescent intensity of each droplet  20  according to the luminous flux of different pixels in one single image sensor. Through image recognition, the number of DNA fragments in each droplet  20  can be determined to complete quantitative analysis. 
     Refer to  FIGS.  5 A,  5 B and  6   .  FIG.  5 A  is a schematic diagram of a magnetron micro-fluid unit  130  according to an embodiment of the present disclosure.  FIG.  6    is a schematic diagram of a drive circuit  133  according to an embodiment of the present disclosure. The magnetron micro-fluid unit  130  includes a magnet  132 , a drive circuit  133  and a magnetic induction track  137 . The drive circuit  133  includes a printed circuit board  134 , a drive element  137  and a plurality of electromagnetic coils  136  disposed on the printed circuit board  134 . The magnet  132  is disposed on the top of the printed circuit board  134 . The electromagnetic coils  136  is disposed under the printed circuit board  134 . The drive element  137  provides a drive current to a portion of electromagnetic coils  136  sequentially, so that the electrified electromagnetic coils  136  can form a magnetic induction track  135  of any shape to generate a magnetic force. The magnetic force generated by the drive circuit  133  drives the magnet  132  to move on the printed circuit board  134 . As indicated in  FIG.  6   , a plurality of electromagnetic coils  136  are arranged as an array on the printed circuit board  134 . Each electromagnetic coil  136  can be powered by an independent power supply switch, so that the electrified electromagnetic coils  136  can be sequentially turned on to form a magnetic induction track  135  according to the predetermined moving path of the magnet  132  and further drives the magnet  132  to move on the printed circuit board  134 . 
     Referring to  FIG.  5 B , a schematic diagram of a droplet  20  moved to a predetermined position along a magnetic induction track  135  according to an embodiment of the present disclosure is shown. The shape of the magnetic induction track  135  is S shape, E shape, F shape, T shape or a combination thereof, and is not subjected to specific restrictions in the present embodiment. The sequential ON/OFF time of the electromagnetic coils  136  can be predetermined, so that the droplet  20  can move to the predetermined position at the terminal of the magnetic induction track  135  and stop there. When the next droplet is desired to move, the predetermined position of the next droplet  20  can be controlled by changing the power-on location of the electromagnetic coils  136  and the said process is repeated until the allocation of all droplets  20  is completed. 
     Referring to  FIG.  7   , a flowchart of a rapid PCR detection method according to an embodiment of the present disclosure is shown. Firstly, in step S 210 , a micro-fluid  10  is placed in a microfluidic unit  120 , and a transparent film is arranged in the middle of the microfluidic channel, wherein the transparent film has at least one hole  123   a  and the micro-fluid  10  can flow in the upper layer of the microfluidic channel. In step S 220 , a magnet  132  is controlled to move under the microfluidic channel, and the micro-fluid  10  is driven by the magnet  132 , so that the micro-fluid  10  is attracted by the magnet  132  and therefore is divided into a plurality of droplets  20  sequentially and each of the droplets  20  is guided to the lower layer of the microfluidic channel. In step S 230 , the microfluidic unit  120  is driven to an amplification zone of the body  110  (the amplification zone is within the second area  114 ). In step S 240 , PCR thermal cycling is performed in the amplification zone, and the droplets  20  are illuminated with a fluorescent light. In step S 250 , the number of DNA fragments in each of the droplets  20  is determined according to the detected fluorescent intensity. In step S 260 , if the number of DNA fragments has reached the target value, the entire testing process is completed. In step S 270 , if the number of DNA fragments has not reached the target value, and the number of repeats of the PCR thermal cycling process does not reach a predetermined number of repeats, the method returns to step S 240  to continue the PCR thermal cycling process. If the number of DNA fragments has not reached the target value, and the PCR thermal cycling process has reached the predetermined number of repeats, the testing method terminates. In the present embodiment, the predetermined number of repeats is 30-40, but the present disclosure is not limited thereto. 
     The above disclosure shows that in actual operation, the rapid PCR detection device and method disclosed in the above embodiments of the present disclosure can be used in the quantitative analysis of digital nucleic acid PCR (dPCR) test and has the advantages of high degree of automation, lower cost and lower pollution risk. Furthermore, since droplet division, PCR thermal cycling and fluorescence detection can be completed in one single body instead of being allocated to different testing devices, human intervention which would occur at the transition between different stages of the testing process can be avoided. Additionally, the PCR testing process of the present embodiment can speed up PCR and reduce the testing time to be within 1.5 hours, so that the testing capacity of each testing center can be increased. 
     While the disclosure has been described by way of example and in terms of the preferred embodiment(s), it is to be understood that the disclosure is not limited thereto. On the contrary, it is intended to cover various modifications and similar arrangements and procedures, and the scope of the appended claims therefore should be accorded the broadest interpretation so as to encompass all such modifications and similar arrangements and procedures.