CN-115505514-B - Small-sized automatic microfluidic PCR instrument and use method thereof

CN115505514BCN 115505514 BCN115505514 BCN 115505514BCN-115505514-B

Abstract

The invention relates to the technical field of biology, in particular to a small-sized automatic microfluidic PCR instrument and a use method thereof, wherein a temperature control fixing part and a vortex tube fixing part are fixed on the side surface of a substrate, a temperature control body is vertically clamped in the temperature control fixing part, a temperature control reaction cavity is arranged at the axis of the temperature control body, a heating rod and a refrigerating tube are respectively embedded in the peripheral wall of the temperature control body along the axial direction, the reaction tube is sleeved in the temperature control reaction cavity, a sample inlet hole and a sample outlet hole are arranged on the top surface of the reaction tube, the vortex tube is vertically clamped in the inner cavity of the vortex tube fixing part, the middle part of the vortex tube is a compressed air inlet, the top and bottom of the vortex tube are respectively provided with a cold air discharge pipe and a hot air discharge pipe, the top end of the cold air discharge pipe is correspondingly sleeved with the bottom end of the refrigerating tube, a temperature sensor is vertically sleeved in the wall of the temperature control body, the output end of the temperature sensor is respectively and electrically connected with the input end of a controller of a compressed air pump connected with the heating rod and the compressed air inlet, and the problems that the temperature control of the PCR instrument is slow and automatic sample injection is difficult to realize are solved.

Inventors

ZHANG LONGFEI
JIN LIXIN
WANG QIANG
Xiong si
XING YUAN
WANG SONGLING
YAN HAOJIE
WU DEFU
GAO XINGANG

Assignees

青岛佳明测控科技股份有限公司

Dates

Publication Date: 20260505
Application Date: 20220915

Claims (10)

1. A small-sized automatic microfluidic PCR instrument, characterized by comprising the following steps: the device comprises a substrate (1), wherein a temperature control fixing part (11) and a vortex tube fixing part (12) are sequentially fixed on the side surface of the substrate (1) from top to bottom; The temperature control body (2) is vertically clamped in the inner cavity of the temperature control fixing part (11), and a temperature control reaction cavity (21) is arranged at the axis position of the temperature control body (2); The heating rod (3) is embedded in the outer peripheral wall of the temperature control body (2) along the axial direction; the cooling tube (4) is axially embedded in the outer peripheral wall of the temperature control body (2); The reaction tube (5), the reaction tube (5) is sleeved in the temperature control reaction cavity (21), the reaction tube (5) is of a cavity structure, the top surface of the reaction tube is provided with a sample inlet hole (51) and a sample outlet hole (52) for inserting a sample inlet tube and a sample outlet tube respectively, and the sample inlet tube and the sample outlet tube realize the inlet and outlet of solution through a micropump; The vortex tube (6) is vertically clamped in the inner cavity of the vortex tube fixing part (12), the middle part of the vortex tube (6) is a compressed air inlet (61), the top and the bottom of the vortex tube (6) are respectively provided with a cold air discharge pipe (62) and a hot air discharge pipe (63), and the top end of the cold air discharge pipe (62) is correspondingly sleeved with the bottom end of the refrigeration tube (4); The temperature sensor (7), temperature sensor (7) cup joint in along vertical in the wall of the control by temperature change body (2), temperature sensor (7) output with heating rod (3) and the controller input of the compressed air pump that compressed air entry (61) is connected is electric connection respectively.
2. The miniature automatic microfluidic PCR instrument of claim 1, wherein the number of the refrigerating pipes (4) is three, the number of the heating rods (3) is two, the refrigerating pipes (4) and the heating rods (3) are embedded in the peripheral wall of the temperature control body (2) at intervals along the circumferential direction, and the three refrigerating pipes (4) are connected in series.
3. The miniature automatic microfluidic PCR instrument as set forth in claim 2, wherein 5 cambered grooves (22) extending along the axial direction of the outer peripheral wall of the temperature control body (2) are uniformly formed in the circumferential direction, the heating rod (3) and the cooling tube (4) are correspondingly clamped in the cambered grooves (22) respectively, an inserting hole (23) for sleeving the temperature sensor (7) is formed in the position, located on the outer side of the temperature control reaction cavity (21), of the top surface of the temperature control body (2), a tail hole (24) communicated with the bottom end of the temperature control reaction cavity (21) is formed in the axial center of the bottom of the temperature control body (2), and the temperature control body (2) is made of one of metal, semiconductor, glass and plastic.
4. The miniature automatic microfluidic PCR instrument as set forth in claim 1, wherein the inner wall of the reaction tube (5) is provided with a hydrophilic layer, the material of the reaction tube (5) is one of plastic and silicon, and the material of the hydrophilic layer is one of PVA and silicon dioxide.
5. The miniature automatic microfluidic PCR instrument as set forth in claim 3, wherein the temperature control fixing portion (11) comprises a temperature control fixing seat (111) directly fixed with the side surface of the substrate (1), and a temperature control fixing card (112) connected with the temperature control fixing seat (111) through screw stitching, the temperature control body (2) is sleeved in a cavity surrounded by the temperature control fixing seat (111) and the temperature control fixing card (112), and an arc surface clamping groove (113) corresponding to and clamped with the heating rod (3) and the cooling tube (4) respectively is arranged on the inner wall of the cavity.
6. The miniature automatic microfluidic PCR instrument according to claim 1, wherein the vortex tube fixing part (12) comprises a vortex fixing seat (121) directly fixed with the substrate (1) and a vortex fixing clamp (122) connected to the vortex fixing seat (121) through screw stitching, and semicircular column grooves (123) matched with the middle part of the hot gas discharge pipe (63) in a clamping manner are respectively correspondingly arranged on contact surfaces of the vortex fixing seat (121) and the vortex fixing clamp (122).
7. The miniature automated microfluidic PCR instrument according to claim 1, further comprising an upper U-tube (8) and a lower U-tube (9), the upper U-tube (8) and the lower U-tube (9) being adapted to connect three of the refrigeration tubes (4) in series.
8. The miniature automatic microfluidic PCR instrument according to claim 7, wherein a thermal cover (10) is detachably fixed on the top surface of the temperature control fixing portion (11), a through hole III (1003) is formed in the middle of the top surface of the thermal cover (10) and corresponds to the sample inlet hole (51) and the sample outlet hole (52), a through hole I (1001) which is matched with the two ends of the top of the upper U-shaped tube (8) in a sleeved mode is formed in the top surface of the thermal cover (10) in a paired mode, a through hole II (1002) which is matched with the top end of one of the refrigerating tubes (4) in a sleeved mode is further formed in the top surface of the thermal cover (10), a shell (14) is fixedly connected to the periphery of the side surface of the substrate (1) located on the temperature control fixing portion (11), and a heat preservation layer (13) is arranged between the inner wall of the shell (14) and the outer wall of the temperature control fixing portion (11).
9. The method of using a miniature automated microfluidic PCR instrument according to claim 8 comprising the steps of: S1, pretreatment of the reaction tube (5); Firstly, when the reaction tube (5) is used for the first time, 1mL of pure water flows into the sample inlet hole (51), the reaction tube stands for 10min, the pure water is discharged from the sample outlet hole (52) under the action of a micropump, and the process of pure water inflow and discharge is repeated twice; Secondly, reversely evacuating pure water in the flow channel through the sample injection hole (51); Thirdly, blowing air for 5min through the sample injection hole (51), and drying the reaction tube (5) for later use; S2, injecting a PCR solution into the reaction tube (5); sequentially injecting 1uL template, 2uL upstream and downstream mixed primer solution and 47uL enzyme raw material MIX solution into the inner cavity of the reaction tube (5) through a micro pump by a sample injection hole (51); S3, heating the thermal cover (10) to 105 ℃ and then keeping the constant temperature; S4, preheating the reaction tube (5); The heating rod (3) heats the temperature control body (2) to enable the temperature control reaction cavity (21) to rise to 98 ℃ for 2min; S5, performing a first reaction cycle; Compressed air is input into the vortex tube (6) through the compressed air inlet (61), cold air enters the refrigerating tube (4) through the cold air discharge tube (62) so that the temperature of the temperature-controlled reaction cavity (21) is rapidly reduced to be maintained for 10S when the temperature is reduced to 60 ℃, and then the heating rod (3) heats the temperature-controlled reaction cavity (21) to be increased to be 72 ℃ and maintained for 20S so as to complete the first reaction cycle; S6, realizing PCR circulation through multiple times of temperature rise and fall of a refrigerating assembly consisting of the heating rod (3), the vortex tube (6) and the refrigerating tube (4), so that nucleic acid is greatly amplified; s7, after the PCR amplification is finished, the micro pump operates to enable the PCR solution to flow to the next module through the sample outlet (52) for nucleic acid detection; s8, flushing the reaction tube (5); firstly, the heating rod (3) heats the temperature control body (2), and the temperature is controlled at 65 ℃; secondly, 1mL of TE buffer solution with pH of 8 is injected into the reaction tube (5) through the sample injection hole (51) by a micropump, and after standing for 10min, the TE buffer solution is discharged from the sample outlet hole (52) under the control of the micropump and is repeated twice; Thirdly, injecting 1mL of pure water into the reaction tube (5) through the sample injection hole (51) by the micro pump, standing for 10min, and discharging the pure water from the sample outlet hole (52) under the control of the micro pump, wherein the steps are repeated twice; Finally, the micro pump reversely evacuates pure water in the flow channel through the sample injection hole (51), and then blows air for 5min through the sample injection hole (51), so as to dry the reaction tube (5) to facilitate the next reaction cycle.
10. The method for using a miniature automated microfluidic PCR instrument according to claim 9 wherein: when the reaction tube (5) is stored, the reaction tube (5) is washed and dried according to the step S8, and then is placed at normal temperature.

Description

Small-sized automatic microfluidic PCR instrument and use method thereof Technical Field The invention relates to the technical field of biology, in particular to a small-sized automatic microfluidic PCR instrument and a use method thereof. Background The basic principle of PCR (polymerase chain reaction) technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of a target sequence, and PCR basically consists of three basic reaction steps of denaturation-annealing-extension. PCR utilizes the fact that DNA becomes single-stranded at a high temperature of 95 ℃ in vitro, and the primer and the single-stranded are combined according to the base complementary pairing principle at a low temperature (usually about 60 ℃), and then the temperature is regulated to the optimal reaction temperature (about 72 ℃) of DNA polymerase, so that the DNA polymerase synthesizes complementary strands along the direction from phosphoric acid to pentose. The PCR instrument based on polymerase is actually a temperature control device, and can well control the denaturation temperature, the renaturation temperature and the extension temperature. The traditional implementation method is that the sample is fixed in a heating device with variable temperature, the temperature of the device is required to be back and forth to three temperatures, and the sample is continuously heated and cooled to realize the reaction. In such a heating mode, the sample is used in an amount of at least hundreds of microliters, and has a certain thermal inertia, and the device itself also has a thermal inertia, so that the frequency of temperature switching cannot be too fast. Disclosure of Invention The invention aims to provide a small-sized automatic microfluidic PCR instrument and a use method thereof, which are used for solving the problems that the temperature control of the PCR instrument is slow and automatic sample injection is difficult to realize in the prior art. The technical scheme is that a temperature control fixing part and a vortex tube fixing part are sequentially fixed on the side face of a substrate from top to bottom, a temperature control reaction cavity is vertically clamped in an inner cavity of the temperature control fixing part, a temperature control reaction cavity is arranged at the axis position of the temperature control body, a heating rod is axially embedded in the peripheral wall of the temperature control body, a cooling tube is axially embedded in the peripheral wall of the temperature control body, the reaction tube is sleeved in the temperature control reaction cavity, the reaction tube is of a cavity structure, a sample inlet hole and a sample outlet hole for inserting a sample inlet tube and a sample outlet tube are formed in the top face of the reaction tube, the sample inlet tube and the sample outlet tube realize the inlet and the outlet of a solution through a micropump, the vortex tube is vertically clamped in the inner cavity of the temperature control fixing part, a compressed air inlet is formed in the middle of the vortex tube, a cold air outlet tube and a hot air outlet tube are respectively arranged at the top of the vortex tube, the top end of the cold air outlet tube is correspondingly sleeved with the bottom end of the air pump, and the air pump is electrically connected with the air inlet end of the temperature sensor and the air outlet tube in a compressed air sensor in a sleeved mode. Preferably, the number of the cooling pipes is three, the number of the heating rods is two, the cooling pipes and the heating rods are embedded in the peripheral wall of the temperature control body at intervals along the circumferential direction, and the three cooling pipes are connected in series. Preferably, the outer peripheral wall of the temperature control body is uniformly provided with 5 cambered surface grooves which extend along the axial direction of the temperature control body along the circumferential direction, the heating rod and the refrigerating tube are correspondingly clamped in the cambered surface grooves respectively, the position of the top surface of the temperature control body, which is positioned outside the temperature control reaction cavity, is vertically provided with an inserting hole for sleeving the temperature sensor, the axis position of the bottom of the temperature control body is provided with a tail hole communicated with the bottom end of the temperature control reaction cavity, and the temperature control body is made of one of metal, semiconductor, glass and plastic. Preferably, the inner wall of the reaction tube is provided with a hydrophilic layer, the material of the reaction tube is one of plastic and silicon, when the material of the reaction tube is silicon, the material of the hydrophilic layer is silicon dioxide, and when the material of the reaction tube is plastic, the materi