TW201816121A - Convective polymerase chain reaction apparatus and optical detecting method thereof - Google Patents

Abstract

A convective polymerase chain reaction apparatus includes a tube, a temperature control unit, at least one light source and a sensor. The tube includes a cavity used to contain a reaction solution having a liquid level measured from a bottom of the cavity to a top surface of the reaction solution. The temperature control unit is disposed adjacent to the tube used to control the temperature of the reaction solution. The at least one light source provides a light beam passing through an incident portion of the tube to reach the reaction solution used to excite fluorescence in the reaction solution, wherein the incident portion is located at a height greater than 1/2 the liquid level measure from the bottom of the cavity. The sensor is used to detect the excited fluorescence. The light beam has an incident direction forming a non-straight angle with a long axis of the tube.

Description

 Thermal convection polymerase chain reaction device and optical detection method thereof  

The present disclosure discloses a polymerase chain reaction (PCR) device and a detection method thereof. In particular, there is a convective polymerase chain reaction (cPCR) device and an optical detection method thereof.

The polymerase chain reaction is a molecular biology technique used to amplify specific deoxyribonucleic acid (DNA) or Ribonucleic acid (RNA) fragments. A typical polymerase chain reaction device is a repeated thermal cycle using a thermocycler for repeated heating and cooling. Thermal conduction is thermally conducted through a metal block into a reaction tube to allow deoxyribonucleic acid or The ribonucleic acid fragments undergo three different temperature cycles of denaturation (95 ° C), adhesion (45 ° C - 65 ° C) and elongation (72 ° C) at different temperatures. However, the traditional method of using the multi-level indirect heating method to perform the thermal cycle step is not only time-consuming but also bulky, greatly reducing the detection efficiency and its application range.

The thermal convection polymerase chain reaction is a thermal convection principle, which directly heats the reaction vessel containing the reaction liquid, causing a temperature gradient of the reaction liquid, resulting in a thermal convection phenomenon. The reaction liquid can be continuously changed in temperature during the convection process, and the nucleic acid is amplified by looping with the convection of the reaction liquid, shortening the time required for the conventional method to change the temperature, and having the advantages of simple structure and low cost. The thermal convection polymerase chain reaction device currently used is a capillary tube made of plastic or glass as a reaction container, and a fluorescent reagent in the tube is excited by a light source placed under the bottom of the test tube to generate a fluorescent signal. The detector senses the fluorescence signal for the purpose of monitoring the polymerase chain reaction.

However, since the reaction liquid contains a biological sample (for example, whole blood of an organism), its composition is rather complicated. Moreover, during the reaction operation, the contents of the reaction liquid (such as proteins, blood cells, etc.) are mostly deposited on the bottom of the capillary tube, causing interference and hindrance on the excitation light path, so that the light cannot pass through the tube bottom to excite the fluorescent light in the tube. Reagents, fluorescent signals can not be sensed by the detector on the tube side, seriously affecting the quality of control of the polymerase chain reaction.

Therefore, there is a need to provide an advanced thermal convection polymerase chain reaction device and its optical detection method to solve the problems faced by the prior art.

One embodiment of the present specification is to provide a thermal convection polymerase chain reaction device. The thermal convection polymerase chain reaction device comprises: a tube, a temperature control unit, at least one light source, and a sensor. The column includes a cavity for carrying the reaction liquid. The reaction liquid has a liquid height from the liquid level to the bottom of the chamber. The temperature control unit is adjacent to the column for temperature control of the reaction liquid. At least one light source provides light to enter the reaction liquid through the incident portion of the column that is greater than one-half of the liquid height from the bottom to excite the reaction liquid to emit fluorescence. The sensor is adjacent to the column for sensing fluorescence. Wherein an incident direction of the light forms a non-flat angle with the long axis of the column.

Another embodiment of the present specification provides an optical detection method for a thermal convection polymerase chain reaction device. The optical detection method comprises the steps of first providing a column comprising a chamber for carrying a reaction liquid having a liquid level from the liquid level to the bottom of the chamber. Next, light is supplied to the reaction liquid through the incident portion of the column that is greater than one-half of the liquid height from the bottom to excite the reaction liquid to emit fluorescence. Fluorescence is sensed by a sensor adjacent to the column. Wherein an incident direction of the light forms a non-flat angle with the long axis of the column.

In accordance with the above, embodiments of the present specification disclose a thermal convection polymerase chain reaction device and an optical detection method thereof. By flexibly modulating the relative position of the sensor and the incident light to the thermal convection polymerase chain reaction chamber, the reaction fluorescent signal received by the sensor is maximized, and the conventional technique is applied to the light source in the reaction cavity. At the bottom, it causes problems with the sensor's optical path interference and hindrance. At the same time, by adjusting the cavity, the sensor and the incident angle of the light, the space application of the thermal convection polymerase chain reaction device can be more efficient, and the volume miniaturization is achieved.

100,200‧‧‧Hot convection polymerase chain reaction device

101‧‧‧ column

101a‧‧‧ cavity

101b‧‧‧Bottom of the cavity

101c‧‧‧上盖

101d‧‧‧ long axis of the column

101e‧‧‧ scale

102‧‧‧temperature control unit

102a‧‧‧ Groove

103, 107, 108, 109‧‧‧ light source

103a, 107a, 108a, 109a‧‧‧ rays

104, 204‧‧‧ sensor

104a, 204a‧‧‧Light receiving direction

105‧‧‧Reactive liquid

105a‧‧‧ liquid level

106A, 106B, 106C‧‧‧ incident section

110‧‧‧Filter

301, 302, 303, 304, 304, 305, 306, 307, 308‧‧‧ curves

U‧‧‧Liquid height

Θ1, θ2, θ3, θ4, θ5‧‧‧ angle

The above-described embodiments and other objects, features and advantages of the present invention will become more apparent from the embodiments of the invention. A partial structural perspective view of a thermal convection polymerase chain reaction device is shown; FIG. 1B is a partial side view of the thermal convection polymerase chain reaction device according to FIG. 1A; FIG. 2 is a Another embodiment is a side view of a partial structure of a thermal convection polymerase chain reaction device.

3A to 3D are diagrams showing the fluorescence measured by the sensor after the thermal convection polymerase chain reaction of the two reaction liquids containing the whole blood sample and the pure water content, respectively, using light sources of different angles. strength.

The embodiments disclosed in the present specification relate to a thermal convection polymerase chain reaction device and an optical detection method thereof, which can solve the problem that the sensor optical path of the conventional thermal convection polymerase chain reaction device is disturbed and hindered, and The purpose of miniaturizing the volume of the thermal convection polymerase chain reaction device. The above described objects, features and advantages of the present invention will become more apparent from the description of the appended claims.

It should be noted, however, that the specific embodiments and methods are merely illustrative and are not intended to limit the invention. The invention may be practiced with other features, elements, methods and parameters. The embodiments are merely intended to illustrate the technical features of the present invention and are not intended to limit the scope of the invention. Equivalent modifications and variations will be made without departing from the spirit and scope of the invention. In the different embodiments and the drawings, the same elements will be denoted by the same reference numerals.

Referring to FIGS. 1A and 1B, FIG. 1A is a partial perspective view of a thermal convection polymerase chain reaction device 100 according to an embodiment of the present specification. Fig. 1B is a side view showing a partial structure of the thermal convection polymerase chain reaction device 100 according to Fig. 1A. The thermal convection polymerase chain reaction device 100 includes at least one column 101, one temperature control unit 102, one light source 103, and one sensor 104.

In some embodiments of the present specification, the column 101 may be a material such as plastic or glass, and has a long-transmissive tube, such as a capillary. Among them, the column 101 includes a cavity 101a for carrying the reaction liquid 105. The reaction liquid 105 carried in the cavity 101a has a liquid height U from the liquid surface 105a to the bottom portion 101b of the cavity 101a.

The temperature control unit 102 is adjacent to the column 101 for temperature control of the reaction liquid 105. In some embodiments of the present specification, the temperature control unit 102 is designed to have a heating block capable of accommodating the column 101, which is shaped as a groove 102a. The recess 102a has a shape and size similar to that of the stem 101 to enable the temperature control unit 102 to be in close proximity to the stem 101 to efficiently transfer thermal energy and reduce energy loss.

In this embodiment, the temperature control unit 102 includes a metal heating block that tightly encloses the bottom of the column 101 composed of a plastic capillary tube, and can transfer heat to the reaction liquid 105 in the plastic capillary tube, and the column 101 is The bottom is maintained at 90 degrees Celsius. The upper cover 101c of the tubular string 101 can be open, and the top of the tubular string 101 can be maintained at 50 degrees Celsius. Thus, a temperature gradient of the lower heat and cold is formed, so that the deoxyribonucleic acid or ribonucleic acid in the reaction liquid 105 in the column 101 is amplified by continuous temperature cycling.

The light source 103 can be a light emitting diode (LED) die, a halogen lamp, a xenon lamp, a xenon lamp, a laser source, or any combination thereof, for providing light 103a having a special color light, passing through The column 101 is incident on the reaction liquid 105 from the cavity bottom portion 101b of the cavity 101a by more than one-half of the liquid height (1/2 U) to excite the reaction liquid 105 to emit fluorescence. It is to be noted that since the reaction liquid 105 generates different degrees of sediment depending on the type of the biological sample, in order to avoid interference of the sediment at the bottom portion 101b of the cavity, it is recommended that the incident portion 106A of the light source 103 is disposed at the bottom of the cavity. 101b is calculated to be greater than one-half of the liquid height (1/2U), that is, where the fluorescent product is distributed more, a good detection effect can be obtained.

In an embodiment of the present specification, the light source 103 may be a single color light emitting diode die for providing red light having a wavelength range substantially between 600 nm and 750 nm, and the wavelength range is substantially Green light from 500 nm to 570 nm or blue light with a wavelength range from 420 nm to 500 nm. In another embodiment of the present specification, the light source 103 may be a white light emitting diode die, and a filter 110 is disposed between the light source 103 and the column 101 to filter out white light emitted by the light source 103. Part of the wavelength allows light of a particular wavelength to pass through the filter 110 and the column 101 into the reaction liquid 105. The purpose of providing light rays 103a of different color lights by a single light source 103 can be achieved by selecting (replacement) different filters 110.

In some embodiments of the present specification, the position of the light source 103 relative to the cavity 101a of the column 101 may be adjusted in accordance with the liquid level 105a of the reaction liquid 105. However, in the present embodiment, the reaction liquid 105 placed in the cavity 101a of the column 101 is a fixed volume, and the height of the liquid surface 105a and the scale 101e are taken as an example. Therefore, the liquid surface 105a of the reaction liquid 105 and the position of the light source 103 with respect to the cavity 101a of the column 101 can be relatively fixed to a specific scale 101e of the column 101.

The sensor 104 is adjacent to the column 101 for detecting and sensing the fluorescent light emitted by the reaction liquid 105 by the light 103a. In an embodiment of the present specification, the sensor 104 may include a photodiode for converting the received fluorescence intensity into a current or voltage signal. In another embodiment of the present specification, the sensor 104 may include an optical fiber for transmitting the received fluorescent signal to the photoelectric conversion device inside or outside the convective polymerase chain reaction device 100, and then performing subsequent signals. deal with.

The sensor 104 is disposed relative to the light source 103 and the column 101. In an embodiment of the present specification, the sensor 104 has a light receiving direction 104a and an incident direction of the light ray 103a (shown by an arrow) having an angle substantially between 60° and 180°, and further, a sense The angle between the light receiving direction 104a of the detector 104 and the long axis 101d of the column 101 is not a flat angle. The angle θ3 formed by the incident direction of the light ray 103a provided by the light source 103 and the long axis 101d of the column 101 is not a flat angle.

For example, in the present embodiment, the angle θ1 formed by the light receiving direction 104a of the sensor 104 and the incident direction of the light ray 103a is substantially 120°; the light receiving direction 104a of the sensor 104 and the long axis of the column 101 The angle θ2 formed by 101d is substantially 90°; the angle θ3 formed by the incident direction of the light ray 103a and the long axis 101d of the column 101 is substantially 30°; and the incident direction of the light ray 103a, the light receiving direction 104a of the sensor 104 And the long axis 101d of the column 101 is located on the same plane.

In some embodiments of the present specification, the thermal convection polymerase chain reaction device 100 may further include other light sources, such as light sources 107 and 108, which may provide light rays 107a and 108a of the same or different wavelengths as the light rays 103a, respectively. In the present embodiment, the light 107a provided by the light source 107 passes through the column 101 from the cavity bottom 101b of the cavity 101a, and the incident portion 106B which is greater than one-half of the liquid height (1/2 U) enters the reaction liquid 105. . The light 108a supplied from the light source 108 enters the reaction liquid 105 through the incident portion 106C of the column 101 from the cavity bottom portion 101b of the cavity 101a by more than one-half of the liquid height (1/2 U). It should be noted that in the present embodiment, the incident direction of the light ray 108a, the light receiving direction 104a of the sensor 104, and the long axis 101d of the column 101 are perpendicular to each other and not in the same plane. In detail, the light receiving direction 104a of the sensor 104 is substantially perpendicular to the incident direction of the light ray 108a; and the incident direction of the light ray 108a is substantially perpendicular to the long axis 101d of the stem 101.

In one embodiment, the light receiving direction 104a of the sensor 104, the long axis 101d of the column 101, and the incident directions of the light rays 103a and 107a are in the same plane. The angle θ4 formed by the light receiving direction 104a of the sensor 104 and the incident direction of the light ray 107a is substantially 60°; the angle formed by the incident direction of the light ray 107a and the long axis 101d of the stem 101 is substantially 30°.

It should be noted that the relative positions of the sensor 104, the column 101, and the light sources 103, 107, and 108 are not limited thereto. For example, in other embodiments of the present specification, the light receiving direction 104a of the sensor 204 and the long axis 101d of the stem 101 may be clipped at a non-right angle. Please refer to FIG. 2, which is a partial structural side view of a thermal convection polymerase chain reaction device 200 according to an embodiment of the present specification. Wherein, the structure of the thermal convection polymerase chain reaction device 200 is as large as that of the thermal convection polymerase chain reaction device 100, with the difference that the thermal convection polymerase chain reaction device 200 includes the light source 109, and the light receiving direction 204a of the sensor 204 and The long axis 101d of the column 101 forms an included angle θ5 of substantially 60°. In this embodiment, the light receiving direction 204a of the sensor 204, the long axis 101d of the column 101, and the incident direction of the light ray 109a are located on the same plane; and the light receiving direction 204a of the sensor 204 and the incident direction of the light ray 109a The direction is vertical.

Subsequently, the light sources 103, 107, 108, and 109 of different angles shown in FIG. The convective polymerase chain reaction was measured and its fluorescence intensity was measured by a sensor 104 to verify the optical detection effect of the thermoconvergence polymerase chain reaction device 100.

Please refer to Figures 3A to 3D. The curves 301 and 302 depicted in FIG. 3A respectively represent the thermal convection polymerase chain reaction of the two reaction liquids 105 containing the whole blood sample and the content of pure water by the light source 103, and the sensor 104 measures the measured result. Fluorescence intensity. The curves 303 and 304 depicted in FIG. 3B respectively represent the thermal convection polymerase chain reaction of the two reaction liquids 105 containing the whole blood sample and the content of pure water by the light source 107, and the sensor 104 measures the measured result. Fluorescence intensity. The curves 305 and 306 depicted in FIG. 3C respectively represent the thermal convection polymerase chain reaction of the two reaction liquids 105 containing the whole blood sample and the pure water content by the light source 108, and the sensor 104 measures the measured result. Fluorescence intensity. The curves 307 and 308 shown in Fig. 3D represent the results of the sensor 104 measurement after the thermal convection polymerase chain reaction of the two reaction liquids 105 containing the whole blood sample and the pure water content by the light source 109, respectively. Fluorescence intensity.

It can be seen from the detection results of FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D that the light sources 103, 107, 108 and 109 having different incident directions are used to contain the whole blood sample and the content is pure water. After the two reaction liquids 105 are subjected to the thermal convection polymerase chain reaction, the sensor 104 measures the fluorescence signal (as shown by the curves 301, 303, 305, and 307) obtained by the reaction liquid 105 containing the whole blood sample. The fluorescent signal obtained by measuring the pure water reaction liquid 105 (as depicted by curves 302, 304, 306, 308) clearly produces a segmentation. Therefore, after deducting the background value of the fluorescent signal of pure water, the fluorescence signal of the actual polymerase chain reaction can still be clearly obtained, showing the adjustment of the relative position of the chain reaction chamber by the sensor and the light incident to the thermal convection polymerase. It can solve the problem of sensing optical path interference and hindrance caused by the deposition of biological samples in the reaction liquid by the prior art.

In accordance with the above, embodiments of the present specification disclose a thermal convection polymerase chain reaction device and an optical detection method thereof. By flexibly modulating the relative position of the sensor and the incident light to the thermal convection polymerase chain reaction chamber, the reaction fluorescent signal received by the sensor is maximized, and the conventional technique is applied to the light source in the reaction cavity. At the bottom, it causes problems with the sensor's optical path interference and hindrance. At the same time, by adjusting the cavity, the sensor and the incident angle of the light, the space application of the thermal convection polymerase chain reaction device can be more efficient, and the volume miniaturization is achieved.

Although the present specification has been disclosed above in the preferred embodiments, it is not intended to limit the invention. A person skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the scope of the appended claims.

Claims (10)

 A convective polymerase chain reaction (cPCR) device comprising: a tube comprising a cavity for carrying a reaction liquid, the reaction liquid having a liquid level from the liquid surface to the cavity a liquid height at the bottom; a temperature control unit adjacent to the column for temperature control of the reaction liquid; at least one light source providing a light passing through the column from the bottom by more than two centimeters An incident portion of the liquid level enters the reaction liquid to excite the reaction liquid to emit a fluorescent light; and a sensor adjacent to the column for sensing the fluorescent light; wherein, the light The incident direction forms an angle with a long axis of the column, and the included angle is not a flat angle.    The thermal convection polymerase chain reaction device according to claim 1, further comprising a filter disposed between the at least one light source and the column to filter a part of the wavelength of the light to allow a specific wavelength The light passes through the incident portion into the reaction liquid.    The thermal convection polymerase chain reaction device of claim 1, wherein the sensor has a light receiving direction and the incident direction of the light is substantially at an angle of between 60° and 180°.    The thermal convection polymerase chain reaction device of claim 3, wherein the long axis of the column, the light receiving direction, and the incident direction of the light ray are in the same plane.    The thermal convection polymerase chain reaction device of claim 3, wherein the long axis of the column, the light receiving direction, and the incident direction of the light are not coplanar.    The thermal convection polymerase chain reaction device of claim 5, wherein the long axis of the column, the light receiving direction, and the incident direction of the light are perpendicular to each other.    The thermal convection polymerase chain reaction device of claim 3, wherein an angle between the long axis of the column and the light receiving direction is not a flat angle.    The thermal convection polymerase chain reaction device of claim 7, wherein the light receiving direction is perpendicular to the incident direction of the light.    An optical detection method for a thermal convection polymerase chain reaction device, comprising: providing a column comprising a cavity for carrying a reaction liquid having a liquid level from a liquid level to a bottom of the cavity a liquid height; providing a light passing through the column from the bottom portion to an incident portion greater than one-half of the liquid height to enter the reaction liquid to excite the reaction liquid to emit a fluorescent light; and providing a sensor Adjacent to the column for receiving the fluorescent light; wherein an incident direction of the light forms an angle with a long axis of the column, the angle being not a flat angle.    The optical detection method of the thermal convection polymerase chain reaction device according to claim 9, wherein the sensor has a light receiving direction and the incident direction of the light is substantially between 60° and 180°. An angle.