CN106442454A - Quick gene amplification detection device and quick gene amplification detection method based on fluorescent quantitation - Google Patents

Abstract

The invention discloses a quick gene amplification detection device and a quick gene amplification detection method based on fluorescent quantitation. The detection device comprises a PCR (polymerase chain reaction) test tube and a temperature control module. The detection device is characterized by further comprising heat-conducting silica gel, a fluorescent detection module, a first transmission mechanism, a first driving mechanism, a connection mechanism, a second transmission mechanism and a second driving mechanism. The problems such as long amplification time, poor temperature uniformity of sample liquor and low fluorescent collection efficiency in the prior art are solved, and temperature control requirements of the gene amplification detection device are reduced. The quick gene amplification detection device and the quick gene amplification detection method based on fluorescent quantitation have the advantages of high sample liquor temperature conversion speed, high uniformity of temperatures among samples, high fluorescent signal collection efficiency, simplicity in temperature control, reliability, low energy consumption and the like.

Description

Fluorescent quantitative gene rapid amplification detection device and amplification detection method

technical field

The invention relates to the fields of biology and medicine, in particular to a fluorescent quantitative gene rapid amplification detection device and an amplification detection method.

Background technique

Polymerase chain reaction (Polymerase Chain Reaction, hereinafter referred to as PCR) is an important in vitro rapid amplification detection technology. PCR technology simulates the DNA replication process in organisms. Under appropriate temperature conditions, the target DNA or RNA fragments undergo three processes of denaturation, annealing, and polymerization extension using templates, primers, and polymerases required for amplification. Cycling to obtain several to several million times amplification of the target DNA or RNA fragment. Fluorescent quantitative PCR technology is to add a specific fluorescent group during the PCR reaction process, and obtain the content of the target DNA or RNA in the sample by collecting the fluorescence signal intensity of the sample during the amplification process. Fluorescence quantitative PCR technology realizes the rapid and large-scale amplification and detection of trace target DNA or RNA fragments in samples, and has important application value in the fields of biology and medicine.

In the rapid amplification detection of genes in vitro, the control of sample temperature and the detection of sample fluorescence signal are two core technologies. The temperature control of the sample is generally realized by the metal block, and the temperature change requirement of the in vitro amplification of the target gene sample is met by changing the temperature of the metal block.

The prior technology "Fluorescence quantitative PCR detection system based on bottom scanning detection" (Chinese invention patent, authorized announcement number: CN101328503A) proposes a fluorescent quantitative PCR detection system based on bottom scanning detection. In this invention, the temperature control device includes a metal block , semiconductor refrigerators and fans, etc. In this invention, the temperature of the sample solution is controlled by a semiconductor refrigerator, and the detection of the fluorescent signal of the sample solution in the test tube is realized by punching holes at the bottom or placing optical fibers. The technical solution of the invention has the following deficiencies:

First, the amplification time is long. The invention only uses one metal block for temperature control. When the temperature of the sample solution needs to be changed, the temperature of the metal block must be changed, and it generally takes a long time to change the temperature of the metal block, so the sample amplification takes a long time .

Second, the temperature uniformity of the sample solution at different locations is poor. In this invention, the temperature of the sample solution is controlled by a semiconductor refrigerator, and the semiconductor refrigerator is generally only placed in the center of the bottom of the metal block, so there will be a problem of strong middle and weak edge when heating or cooling, resulting in the intermediate sample solution and The temperature of the edge sample solution is inconsistent, which will cause the amplification efficiency of the intermediate sample and the edge sample to be inconsistent;

Third, the fluorescence collection efficiency of the sample solution is low. If the invention adopts the method of directly punching holes at the bottom, the sensor for detecting the fluorescent signal cannot approach the sample in the test tube, thereby reducing the excitation and collection efficiency of the fluorescent signal; if the method of optical fiber is used, the fluorescent signal will be transmitted by the optical fiber. The limitation of pore size also reduces the excitation and collection efficiency of fluorescent signals.

Contents of the invention

The purpose of the present invention is to propose a fluorescent quantitative gene rapid amplification detection device and amplification detection method, which not only solves the long time required for amplification, poor temperature uniformity of the sample solution, and low fluorescence collection efficiency in the prior art. problems, and also reduces the requirements for the temperature control algorithm of the gene amplification detection device. The invention has the advantages of fast temperature conversion speed of the sample solution, good temperature uniformity among samples, high fluorescence signal collection efficiency, simple and reliable temperature control, low energy consumption and the like.

Technical solution of the present invention is as follows:

A fluorescent quantitative gene rapid amplification detection device, including a PCR test tube, a temperature control module, and is characterized in that it also has a heat-conducting silica gel, a fluorescence detection module, a first transmission mechanism, a first drive mechanism, a connection mechanism, a second transmission mechanism and a second transmission mechanism. Two driving mechanisms, the temperature control module includes more than one thermostatic block, each two thermostatic blocks are separated by an insulating layer and arranged in sequence, and the heat-conducting silica gel is arranged on the outside of the PCR test tube, so The thermostatic block of the temperature control module is placed around the heat-conducting silica gel; the top of the PCR test tube is fixed on the second transmission mechanism through the connection mechanism, driven by the second drive mechanism The second transmission mechanism drives the PCR test tube to move vertically; the fluorescence detection module is placed on the top, bottom or side of the PCR test tube, and the fluorescence detection module consists of an excitation light source, a detection The optical path and the photoelectric detection unit are formed and fixed on the first transmission mechanism, and the first driving mechanism drives the first transmission mechanism to drive the fluorescence detection module to move.

The first transmission mechanism is a screw rod or a conveyor belt.

The PCR test tube is a single test tube, a single row of test tubes or multiple rows of test tubes.

The sample solution is placed in the PCR test tube, and the same sample solution or different sample solutions with the same temperature cycle conditions required for the amplification of all samples in the same test are placed in the PCR test tubes at different well positions.

The height of the heat-conducting silica gel is greater than the height of the sample solution in the PCR test tube, so as to ensure that the sample can be completely covered by the heat-conducting silica gel.

One PCR test tube, two PCR test tubes, multiple PCR test tubes or all PCR test tubes correspond to one temperature control module.

The fluorescence detection module includes more than one fluorescence detection device, and each fluorescence detection device detects fluorescence of more than one emission wavelength, and the fluorescence detection module can detect the same fluorescence emitted by different sample solutions placed in the PCR test tube. or different fluorescence.

The structure of the fluorescence detection module is:

A point excitation light source, a detection light path, and a non-image photoelectric sensor are used to form and place on the top, bottom or side positions of all PCR test tubes 1. The non-image photoelectric sensor includes a photodiode, a photocell, a photoelectric tube, and a photomultiplier tube. The point excitation light source once irradiates and excites a PCR sample solution, and the non-image photoelectric sensor detects the fluorescent signal of the sample solution in a PCR test tube;

Or use a surface excitation light source, a detection optical path and an area array image sensor to form and place on the top surface, bottom surface or side of all PCR test tubes 1, and the surface excitation light source will irradiate all at one time and excite all PCR sample solutions at the same time. The area array image sensor described above simultaneously detects the fluorescent signals of the sample solutions in all the PCR test tubes.

The above-mentioned fluorescence detection modules are respectively arranged on the top, bottom or side surfaces of all PCR test tubes to detect the fluorescence signals of the sample solutions in the corresponding PCR test tubes.

The method for amplifying and detecting target DNA or RNA by using the above-mentioned fluorescent quantitative gene rapid amplification detection device comprises the following steps:

1) adding the sample solution required for amplification into the PCR test tube;

2) According to the number and value of specific temperature points required for target DNA or RNA amplification, select and set the corresponding temperature control module to determine the number of amplification cycles;

3) The second driving machine drives the second transmission mechanism to drive the PCR test tube to move to the position of the constant temperature block corresponding to the first temperature point required for DNA or RNA amplification, so that the temperature of the sample solution in the PCR test tube reaches the The first temperature value, and then stay for the first amplification time of the gene, and then the second driving mechanism drives the second transmission mechanism to drive the PCR test tube to move to the position of the constant temperature block corresponding to the second temperature point required for DNA or RNA amplification, Make the temperature of the sample solution in the PCR test tube reach the second temperature value, and then stay for the second amplification time of the gene, and then the second driving mechanism drives the second transmission mechanism to drive the PCR test tube to move to DNA or RNA amplification The position of the thermostatic block corresponding to the next temperature point is required, so that the temperature of the sample solution in the PCR test tube reaches the next temperature value, and then stays for the next amplification time of the gene until the target DNA or RNA amplification cycle is completed. All temperature values and corresponding amplification times;

4) The second driving machine drives the second transmission mechanism to drive the PCR test tube to move outside the temperature control module for fluorescent signal detection:

If the detection device adopts the fluorescence detection module that is not an image photoelectric sensor, the fluorescence detection module is fixed on the first transmission mechanism, and the first drive mechanism drives the first transmission mechanism to drive the fluorescence detection module to move, click The excitation light source excites a PCR sample solution, and the non-image photoelectric sensor detects the fluorescent signal of the sample solution in one PCR test tube, and gradually realizes the detection of the fluorescent signal of the sample solution in all PCR test tubes;

If an image sensor is used in the detection device, the surface excitation light source of the fluorescence detection module irradiates all at one time and excites all PCR sample solutions at the same time, and the area array image sensor simultaneously detects the fluorescence signals of the sample solutions in all PCR test tubes. to test;

5) repeat steps 3), 4), and complete another amplification cycle and fluorescence signal detection until the last amplification cycle and fluorescence signal detection are completed;

6) Draw the amplification curve of the fluorescence signal of the sample solution by using the above measurement results, and obtain the target DNA or RNA content of the sample solution according to the existing analysis method.

Compared with the prior art, the present invention has the following technical effects:

First, the amplification speed is fast. In the prior art, when the temperature of the sample solution needs to be changed, the temperature of the metal block must be changed, and it takes a long time to change the temperature of the metal block. For example, it takes about one minute for the temperature of the metal block to rise from 60 degrees to 95 degrees and then drop to 60 degrees, and it usually takes about 40 minutes to complete 40 cycles of amplification. However, in the present invention, the temperature control module includes multiple thermostatic blocks with different temperatures, and the temperature of each thermostatic block is a specific temperature required for gene amplification. The sample solution is moved between the thermostatic blocks of different temperatures to complete the temperature conversion, without repeated heating and cooling of the thermostatic blocks, thus saving most of the time required for heating and cooling, so the present invention has the advantage of fast amplification.

Second, the temperature uniformity of the sample solution at different positions is good. When it is necessary to control all samples at the same amplification temperature, the prior art adopts a semiconductor refrigerator placed at the bottom center of the metal block to control the sample temperature, so there will be problems of strong middle and weak edges when heating or cooling, resulting in The temperature difference between the middle sample solution and the edge sample solution is greater than 0.3 degrees. In the present invention, the temperature of the sample solution in a single PCR test tube can be controlled, and the temperature difference between the sample solutions at different positions can be eliminated. The temperature difference between all samples is less than 0.2 degrees, which improves the uniformity of the sample solution temperature. sex.

Third, the fluorescence detection efficiency is high. Fluorescence detection in the prior art adopts the method of punching holes at the bottom or installing optical fibers. If the hole is directly drilled at the bottom, the sensor for detecting the fluorescent signal cannot approach the sample in the test tube, thereby reducing the excitation and collection efficiency of the fluorescent signal; if the optical fiber is used, the fluorescent signal will be affected by the aperture of the optical fiber. Limitation also reduces the excitation and collection efficiency of fluorescent signals. The present invention uses a constant temperature block and heat-conducting silica gel to control the temperature of the sample solution. When the fluorescence of the sample solution needs to be detected, the PCR test tube is driven by the second driving mechanism to move the sample solution in the PCR to the outside of the temperature control module, which is convenient for the fluorescence detection module. Excite and detect the fluorescence of the sample solution, thus improving the fluorescence detection efficiency of the sample solution.

Fourth, the temperature control is simple. In the prior art, when the temperature of the sample solution needs to be changed, the temperature of the metal block must be changed. In order to meet the two requirements of fast heating and cooling and high temperature control accuracy, the temperature control algorithm needs to consider both dynamic performance indicators and steady-state performance indicators. . However, the temperature control module described in the present invention contains a variety of thermostatic blocks at different temperature points, and the temperature of each thermostatic block is the specific temperature required for gene amplification. The sample solution passes through the thermostatic blocks at different temperatures. Move to complete the temperature conversion, temperature control only needs to consider the steady-state index. Therefore, the present invention has the advantage of simple temperature control.

Fifth, less energy consumption. In the prior art, when the temperature of the sample solution needs to be changed, the temperature of the metal block must be changed, and the rapid and repeated heating and cooling process consumes a lot of energy. However, the temperature control module described in the present invention contains a variety of thermostatic blocks at different temperature points, and the temperature of each thermostatic block is the specific temperature required for gene amplification. To complete the temperature conversion, without repeated heating or cooling, as long as it consumes little energy to maintain the constant temperature of the thermostatic block. Therefore, the temperature control system of the present invention consumes less energy.

Description of drawings

Fig. 1 is a schematic diagram of a single PCR test tube of the fluorescence quantitative gene rapid amplification detection device of the present invention.

Fig. 2 is a schematic diagram of multiple PCR test tubes of the fluorescence quantitative gene rapid amplification detection device of the present invention.

Fig. 3 is a schematic diagram of the sample solution of the present invention when it is in the first constant temperature block.

Fig. 4 is a schematic diagram of the sample solution of the present invention when it is in the second constant temperature block.

Fig. 5 is a schematic diagram of Embodiment 1 of the present invention.

Fig. 6 is a schematic diagram of Embodiment 2 of the present invention.

Fig. 7 is a schematic diagram of Embodiment 3 of the present invention.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention should not be limited thereby. Those skilled in the art will realize that the present invention covers all alternatives, modifications and equivalents as may be included within the scope of the claims.

Please refer to Figures 1-2, Figure 1 is a schematic diagram of a single PCR test tube of the fluorescent quantitative gene rapid amplification detection device of the present invention, and Figure 2 is a schematic diagram of multiple PCR test tubes of the fluorescent quantitative gene rapid amplification detection device of the present invention. Referring to Fig. 1, the fluorescent quantitative gene rapid amplification detection device of the present invention includes a PCR test tube 1, a thermally conductive silica gel 2, a temperature control module 3, a fluorescence detection module 4, a first transmission mechanism 5, a first driving mechanism 6, a connecting mechanism 7, The second transmission mechanism 8 and the second driving mechanism 9 . The temperature control module 3 is composed of a first thermostatic block 301 , an insulating layer 302 and a second thermostatic block 303 . The positional relationship of the above components is as follows:

The sample solution is placed in the PCR test tube 1, and the same sample solution or different sample solutions with the same temperature cycle conditions required for the amplification of all samples in the same detection can be placed in the PCR test tube 1 of different hole positions. The PCR test tube 1 is a single test tube, a single row of test tubes or multiple rows of test tubes, wherein Fig. 1 is a single PCR test tube; Fig. 2 is a single row of PCR test tubes.

The PCR test tube 1 is fixed on the second transmission mechanism 8 through the connection mechanism 7, and the second transmission mechanism 8 is driven by the second driving mechanism 9 to drive the PCR test tube 1 move.

The PCR test tube 1 can be moved to the outside of the temperature control module 3 .

The heat-conducting silica gel 2 is placed on the outside of the PCR test tube 1, and the thermostat block in the temperature control module 3 is placed around the heat-conducting silica gel 2.

The heat-conducting silica gel 2 is attached to the PCR test tube 1. At this time, the heat-conducting silica gel 2 and the PCR test tube 1 can conduct sufficient heat transfer to ensure that the temperature of the sample solution in the test tube is the same as that of the heat-conducting silica gel. . The height of the heat-conducting silica gel 2 is greater than the height of the sample solution in the PCR test tube, so as to ensure that the sample can be completely coated.

The heat-conducting silica gel 2 is located between the PCR test tube 1 and the thermostatic block in the temperature control module 3, the heat-conducting silica gel 2 is attached to the outer wall of the PCR test tube 1, and the thermostatic block Heat transfer is realized through the heat-conducting silica gel 2 with the PCR test tube 1 .

Each of the PCR test tubes 1 corresponds to one temperature control module 3 , or two, more or all PCR test tubes correspond to one temperature control module 3 .

The temperature control module 3 includes more than one thermostatic block, and the thermostatic blocks of different temperatures are separated by an insulating layer to prevent heat exchange between the thermostatic blocks of different temperatures.

The PCR test tube 1 can be moved between thermostatic blocks of different temperatures. The temperature of the thermostatic block is set to one, two or more specific temperature values required for the amplification of the target gene, and the PCR test tube 1 moves back and forth between thermostatic blocks of different temperatures to realize the target gene amplification. Temperature cycling required for rapid amplification.

The number of the constant temperature blocks is consistent with the number of specific temperatures required to complete the gene amplification.

Referring to Fig. 2, the fluorescent detection module 4 is placed on the bottom of the PCR test tube 1, and the photoelectric detection unit of the fluorescent detection module 4 adopts an image sensor or a non-image photoelectric sensor, and the non-image photoelectric sensor includes a photoelectric Diodes, Photocells, Phototubes, Photomultiplier Tubes.

The fluorescence detection module 4 using a non-image photoelectric sensor comprises an excitation light source, a detection optical path, and a non-image photoelectric sensor and is placed on the top, bottom or side positions of all PCR test tubes 1. The point excitation light source irradiates and excites one PCR sample solution, the non-image photoelectric sensor realizes the detection of the fluorescent signal of the sample solution in a PCR test tube. The fluorescence detection module 4 is fixed on the first transmission mechanism 5, and the first driving mechanism 6 drives the first transmission mechanism 5 to drive the fluorescence detection module 4 to move, so as to detect the fluorescence signals of the sample solutions in all PCR test tubes .

The fluorescence detection module 4 using an image photoelectric sensor is composed of a surface excitation light source, a detection optical path and an area array image sensor and is placed on the top surface, bottom surface or side of all PCR test tubes 1. The surface excitation light source is all irradiated at one time and simultaneously All the PCR sample solutions are excited, and the area array image sensor simultaneously detects the fluorescent signals of the sample solutions in all the PCR test tubes.

In the present invention, more than one fluorescence detection module 4 can also be used, respectively arranged on the top, bottom or side surfaces of all PCR test tubes 1, so as to detect the fluorescence signal of the sample solution in the corresponding PCR test tubes.

The method for amplifying and detecting target DNA or RNA using the fluorescent quantitative gene rapid amplification detection device of the present invention comprises the following steps:

1) adding the sample solution required for amplification into the PCR test tube;

2) Select and set the corresponding temperature control module 3 according to the number and value of specific temperature points required for target DNA or RNA amplification;

3) The second driving machine drives the second transmission mechanism to drive the PCR test tube to move to the position of the constant temperature block corresponding to the first temperature point required for DNA or RNA amplification, so that the temperature of the sample solution in the PCR test tube reaches the The first temperature value, and then stay for the first amplification time of the gene, and then the second driving mechanism drives the second transmission mechanism to drive the PCR test tube to move to the position of the constant temperature block corresponding to the second temperature point required for DNA or RNA amplification, Make the temperature of the sample solution in the PCR test tube reach the second temperature value, and then stay for the second amplification time of the gene, and then the second driving mechanism drives the second transmission mechanism to drive the PCR test tube to move to DNA or RNA amplification The position of the thermostatic block corresponding to the next temperature point is required, so that the temperature of the sample solution in the PCR test tube reaches the next temperature value, and then stays for the next amplification time of the gene until the target DNA or RNA amplification cycle is completed. All temperature values and corresponding amplification times.

4) The second driving machine drives the second transmission mechanism to drive the PCR test tube to move outside the temperature control module 3 .

If the detection device adopts the fluorescence detection module that is not an image photoelectric sensor, the fluorescence detection module 4 is fixed on the first transmission mechanism 5, and the first drive mechanism 6 drives the first transmission mechanism 5 to drive the fluorescence detection module. The movement of module 4 realizes the detection of the fluorescent signals of the sample solutions in all the PCR test tubes.

If the fluorescence detection module of the image sensor is adopted in the detection device, the area excitation light source in the fluorescence detection module 4 of the image sensor is all irradiated at one time and all the PCR sample solutions are simultaneously excited, the area array image The sensor simultaneously detects the fluorescent signals of the sample solutions in all PCR test tubes.

5) Steps 3 and 4) are repeated until the last amplification cycle and fluorescence signal detection are completed.

6) Draw the amplification curve of the fluorescence signal of the sample solution by using the above measurement results, and obtain the target DNA or RNA content of the sample solution according to the existing analysis method.

Embodiment one

Please refer to FIGS. 3-5 . FIG. 5 is a schematic diagram of Embodiment 1 of the fluorescence quantitative gene rapid amplification detection device and detection method of the present invention.

This embodiment includes multiple PCR test tubes 1, heat-conducting silica gel 2, temperature control module 3, fluorescence detection module 4, first transmission mechanism 5, first drive mechanism 6, connection mechanism 7, second transmission mechanism 8, and second drive mechanism 9. The temperature control module 3 includes a first thermostatic block 301 , an insulating layer 302 and a second thermostatic block 303 . In this embodiment, the sample solution is placed in the multiple PCR test tubes 1 , and the fluorescence detection module 4 is placed at the bottom of the PCR test tubes 1 . The PCR test tube 1 is surrounded by a temperature control module 3 and a fluorescence detection module 4 . The temperature control module 3 includes a first thermostatic block 301 and a second thermostatic block 303, the first thermostatic block 301 is a high temperature thermostatic block, and the second thermostatic block 303 is a low temperature thermostatic block. The first thermostatic block 301 and the second thermostatic block 303 are separated by the heat insulating layer 302 to prevent heat exchange between the first thermostatic block 301 and the second thermostatic block 303 . The first thermostatic block 301 keeps the temperature constant, and its temperature value is the high temperature required for gene amplification; the second thermostatic block 303 keeps the temperature constant, and its temperature value is required for gene amplification low temperature. Referring to Figures 3-5, the PCR test tube 1 moves between the first thermostatic block 301 and the second thermostatic block 303 to change the temperature of the sample solution, and the PCR test tube 1 and the The heat exchange of the thermostatic block in the temperature control module 3 is accomplished through the heat-conducting silica gel 2 . One fluorescent detection module 4 using a non-image photoelectric sensor is placed on the bottom surface of the PCR test tube 1 . The fluorescence detection module 4 is fixed on the first transmission mechanism 5, and the first driving mechanism 6 drives the first transmission mechanism 5 to drive the fluorescence detection module 4 to move, so as to detect the fluorescence signals of the sample solutions in all PCR test tubes , by analyzing the change curve of the fluorescence signal of each cycle of the sample solution, the content of the target DNA or RNA in the sample is obtained.

Embodiment two

Please refer to FIG. 6 . FIG. 6 is a schematic diagram of Embodiment 2 of the fluorescence quantitative gene rapid amplification detection device of the present invention. This embodiment includes 8 PCR test tubes 1, heat-conducting silica gel 2, temperature control module 3, fluorescence detection module 4, first transmission mechanism 5, the first driving mechanism 6, connection mechanism 7, second transmission mechanism 8, the first Two drive mechanism 9. The temperature control module 3 includes a first thermostatic block 301 , an insulating layer 302 and a second thermostatic block 303 . The fluorescence detection module 4 is placed on the side of the PCR test tube 1 . All PCR test tubes 1 are surrounded by the same temperature control module 3 and equipped with 8 fluorescence detection modules 4 . The temperature control module 3 is provided with a first constant temperature block 301 and a second constant temperature block 303, the first constant temperature block 301 is a high temperature constant temperature block, and the second constant temperature block 303 is a low temperature constant temperature block. The first thermostatic block 301 and the second thermostatic block 303 are separated by the heat insulating layer 302 to prevent heat exchange between the first thermostatic block 301 and the second thermostatic block 303 . The first thermostatic block 301 keeps the temperature constant, and its temperature value is the high temperature required for gene amplification; the second thermostatic block 303 keeps the temperature constant, and its temperature value is the low temperature required for gene amplification. 3-4, the PCR test tube 1 moves between the first thermostatic block 301 and the second thermostatic block 303 to change the temperature of the sample solution, the PCR test tube 1 and the temperature control module 3 The heat exchange of the thermostatic block is accomplished through the heat-conducting silica gel 2 . Eight fluorescence detection modules 4 of non-image photoelectric sensors are respectively arranged on the sides of eight PCR test tubes 1 . The fluorescence detection module 4 is fixed on the first transmission mechanism 5, and the first driving mechanism 6 drives the first transmission mechanism 5 to drive the fluorescence detection module 4 to move, so as to detect the fluorescence signals of the sample solutions in all PCR test tubes , by analyzing the change curve of the fluorescence signal of each cycle of the sample solution, the content of the target DNA or RNA in the sample is obtained.

Embodiment three

Please refer to FIG. 7 . FIG. 7 is a schematic diagram of Embodiment 3 of the fluorescence quantitative gene rapid amplification detection device of the present invention. This embodiment includes a plurality of PCR test tubes 1 , heat-conducting silica gel 2 , temperature control module 3 , a fluorescence detection module 4 , a connection mechanism 7 , a second transmission mechanism 8 , and a second drive mechanism 9 . The temperature control module 3 includes a first thermostatic block 301 , an insulating layer 302 and a second thermostatic block 303 . In this embodiment, the fluorescence detection module 4 is placed on the upper part of the PCR test tube 1 . All PCR test tubes 1 are surrounded by a thermostatic block in the temperature control module 3 and a fluorescence detection module 4 . The temperature control module 3 is provided with the first thermostatic block 301 as a high temperature thermostatic block, and the second thermostatic block 303 as a low temperature thermostatic block. The first thermostatic block 301 and the second thermostatic block 303 are separated by the heat insulating layer 302 . Heat exchange between the first thermostatic block 301 and the second thermostatic block 303 is prevented. The first thermostatic block 301 keeps the temperature constant, and its temperature value is the high temperature required for gene amplification; the second thermostatic block 303 keeps the temperature constant, and its temperature value is the low temperature required for gene amplification. The PCR test tube 1 moves between the first thermostatic block 301 and the second thermostatic block 303 to change the temperature of the sample solution, and the heat exchange between the PCR test tube 1 and the thermostatic block in the temperature control module 3 is It is accomplished by the above-mentioned heat-conducting silica gel 2 .

The fluorescence detection module 4 is composed of a surface excitation light source, a detection optical path and an area array image sensor and is placed on the top surface of all PCR test tubes 1. The surface excitation light source irradiates all at one time and simultaneously excites all PCR sample solutions, The area array image sensor detects the fluorescence signals of the sample solutions in all PCR test tubes at the same time, and obtains the content of the target DNA or RNA in the samples by analyzing the change curve of the fluorescence signals of each cycle of the sample solutions.

Experiments show that the invention not only solves the problems of long amplification time, poor sample solution temperature uniformity, and low fluorescence collection efficiency existing in the prior art, but also reduces the temperature control algorithm requirements of the gene amplification detection device. The invention has the advantages of fast temperature conversion speed of the sample solution, good temperature uniformity among samples, high fluorescence signal collection efficiency, simple and reliable temperature control, low energy consumption and the like.

Claims (10)

1. a kind of fluorescent quantitation gene rapid amplifying detection means, including PCR test tube (1), temperature control modules (3), its feature It is also heat conductive silica gel (2), fluoroscopic examination module (4), the first connecting gear (5), the first drive mechanism (6), bindiny mechanism (7), the second connecting gear (8) and the second drive mechanism (9), described temperature control modules (3) include more than one constant temperature Block, is separated and be arranged in order by heat insulation layer between each two isothermal block and constitute, and described heat conductive silica gel (2) is arranged on described The outside of PCR test tube (1), the isothermal block of described temperature control modules (3) is placed on described heat conductive silica gel (2) around； The top of described PCR test tube (1) is fixed on described the second connecting gear (8) by described bindiny mechanism (7), the Lower described second connecting gear (8) that drives of two drive mechanisms (9) drives described PCR test tube (1) to move in the vertical direction； Described fluoroscopic examination module (4) is placed in the described top surface of PCR test tube (1), bottom surface or side, described fluoroscopic examination module (4) constituted and be fixed on the first connecting gear (5), the first drive mechanism by excitation source, detection light path, photoelectric detection unit (6) the first connecting gear (5) is driven to drive described fluoroscopic examination module (4) motion.

2. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described first biography Mechanism (5) is sent to be screw mandrel or conveyer belt.

3. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described PCR test tube (1) it is single test tube, single platoon test tube or multiple rows of test tube.

4. fluorescent quantitation gene rapid amplifying detection means according to claim 1, its feature sample solution is positioned over institute In the PCR test tube (1) stated, in the PCR test tube (1) of different hole positions, place the temperature required with all samples amplification in one-time detection The consistent identical sample solution of cycling condition or different sample solutions.

5. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described thermal conductive silicon The height of glue (2) is more than sample solution in the described invisible spectro height of PCR it is ensured that sample can be obtained described thermal conductive silicon Glue (2) coats completely.

6. fluorescent quantitation gene rapid amplifying detection means according to claim 1 it is characterised in that PCR test tube, Two PCR test tubes, multiple PCR test tube or whole PCR test tube correspond to a temperature control modules (3).

7. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described fluorescence is examined Survey module (4) and include more than one fluorescence detection device, each fluorescence detection device detects the glimmering of more than one launch wavelength Light, described fluoroscopic examination module can detect be placed on that different sample solution in described PCR test tube (1) sends identical Or different fluorescence.

8. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described fluorescence is examined Survey module (4) structure be：

Top surface, the bottom being placed in all PCR test tubes (1) is constituted using an excitation source, detection light path, non-image light electric transducer Face or lateral location, described non-image light electric transducer includes photodiode, light cell, photocell, photomultiplier tube, institute The point excitation source once irradiating stated simultaneously excites a PCR sample solution, and described non-image light electric transducer is realized to one Sample solution fluorescence signal in PCR test tube is detected；

Or using face excitation source, detection light path dough-making powder array image sensor constitute be placed in all PCR test tubes (1) top surface, Bottom surface or lateral location, the described disposable full illumination of face excitation source simultaneously excites all of PCR sample solution simultaneously, described Array image sensor the sample solution fluorescence signal in all PCR test tubes is detected simultaneously.

9. fluorescent quantitation gene rapid amplifying detection means according to claim 7 is it is characterised in that adopt more than one Described fluoroscopic examination module (4), is arranged respectively at top surface, bottom surface or the side of all PCR test tubes (1), realizes to corresponding The fluorescence signal of the sample solution in PCR test tube is detected.

10. using the fluorescent quantitation gene rapid amplifying detection means described in claim 1, target dna or RNA are expanded With the method detecting it is characterised in that comprising the steps：

1) the required sample solution of amplification is added described PCR test tube；

2) number of specified temp point according to needed for target dna or RNA amplification and numerical value, select and arrange corresponding temperature control Molding block (3), determines amplification cycles number of times；

3) the second driving machine drives the second connecting gear to drive described PCR test tube to move to needed for DNA or RNA amplification first The corresponding isothermal block position of individual temperature spot, makes the temperature of the sample solution in PCR test tube reach the first described temperature value, then stops Stay gene first proliferation time, then the second drive mechanism drives the second connecting gear to drive described PCR test tube to move to DNA Or the corresponding isothermal block position of second temperature point needed for RNA amplification, make described in the temperature arrival of sample solution in PCR test tube Second temperature value, then stop gene second proliferation time, then the second drive mechanism drives described in the second connecting gear drive PCR test tube moves to next temperature spot corresponding isothermal block position needed for DNA or RNA amplification, makes the sample in PCR test tube molten The temperature of liquid reaches next described temperature value, then stops next proliferation time of gene, until completing target dna or RNA amplification All temperature values needed for cycle and corresponding proliferation time；

4) the second driving machine described in drives the second connecting gear to drive described PCR test tube to move to described temperature control mould Carry out fluorescence signal detection outside block (3)：

If adopting the described fluoroscopic examination module of non-image light electric transducer, described fluoroscopic examination module in detection means (4) it is fixed on the first connecting gear (5), the first drive mechanism (6) drives the first connecting gear (5) to drive described fluorescence inspection Survey module (4) motion, point excitation source excites a PCR sample solution, and described non-image light electric transducer is realized to one Sample solution fluorescence signal in PCR test tube is detected, is done step-by-step to the sample solution fluorescence signal in all PCR test tubes Detected；

If adopt imageing sensor in detection means, the face excitation source of described fluoroscopic examination module (4) is disposably whole Irradiate and excite all of PCR sample solution simultaneously, described array image sensor is simultaneously to the sample in all PCR test tubes Solution fluorescence signal is detected；

5) repeat step 3,4), complete another amplification cycles and fluorescence signal detection, until completing last amplification cycles With fluorescence signal detection；

6) utilize above-mentioned measurement result to draw the amplification curve of the fluorescence signal of sample solution, obtain sample by existing analysis method Product solution target dna or the content of RNA.