CN107557356B - Small multichannel library construction instrument and library construction method - Google Patents
Small multichannel library construction instrument and library construction method Download PDFInfo
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Abstract
The invention provides a small multichannel library construction instrument and a library construction method, and particularly relates to the technical field of gene sequencing. The invention thoroughly solves the problems of time consumption and low flux of manually preparing samples, and the whole flow of the system is automatically completed by a one-stop workstation, thereby avoiding errors and being more suitable for laboratory standardization.
Description
Technical Field
The invention belongs to the technical field of gene sequencing, and particularly relates to a small multichannel library construction instrument and a library construction method.
Background
In recent years, the market of gene sequencing is rapidly developed, and the advantages of high throughput, high accuracy, low cost and the like of the second generation sequencing technology are particularly realized, so that the method is widely applied. The industrial chain of second generation sequencing can be divided into an upstream instrument equipment end, a midstream sequencing service and a downstream sequencing data analysis. Foreign second generation sequencer manufacturers are mainly Illumina, life technologies, rogowski, etc. Global sequencing instrument market research results from genome web pairs 2013 showed that Illumina occupied 71% of the global sequencing instrument market, life technologies ranked the second 16% market share, roche and pacbi were ranked third and fourth by 10% and 3%.
The procedure for second generation sequencing is sample preparation (DNA purification and library construction), sequencing and data analysis. Where sequencing is automated by a sequencer, sample preparation requires a large number of manual operations, which is time consuming and low throughput, and can only be done for 4 samples per day. Furthermore, manual preparation of samples is relatively demanding for the skilled person, and errors and wastage are not allowed due to the expensive sequencing reagents.
There is thus an urgent need for a small multi-channel library construction instrument and method of constructing a library that solves the existing problems.
Disclosure of Invention
The invention aims to provide a small multichannel library construction instrument and a library construction method, which thoroughly solve the problems of time consumption and low flux of manually preparing samples, and the whole process of the system is automatically completed by a one-stop workstation, so that errors are avoided, and the system is more suitable for laboratory standardization.
The invention provides the following technical scheme:
the utility model provides a small-size multichannel library constructs appearance, includes the frame, be equipped with first backup pad and controller in the frame, be equipped with test-tube rack and the application of sample device that are used for depositing reagent strip and rifle head in the first backup pad, test-tube rack one side is equipped with the waste liquid box, the activity is equipped with heating device and magnetism separator respectively in test-tube rack bottom, heating device with magnetism separator is connected with first drive mechanism and second drive mechanism respectively, the top of test-tube rack is equipped with the application of sample device, the third drive mechanism is connected to the application of sample device, first drive mechanism the second drive mechanism with third drive mechanism all electrically links the controller. The number of the first supporting plates is 1, 2 or more, that means that a plurality of reaction working stations can be arranged in the same machine, the heating device is used for controlling the temperature of the system, the magnetic separation device is convenient for controlling the magnetic field, when the magnetic beads and the supernatant are required to be separated, the magnetic beads and the supernatant are enabled to be separated, when the magnetic beads and the solution are required to be uniformly mixed, the magnetic separation device is removed, the magnetic beads can be uniformly distributed in the solution, and the sample adding device is mainly used for adding reagents, uniformly mixing the reagents and the magnetic beads and sucking the supernatant when the magnetic beads and the supernatant are separated. The first transmission mechanism, the second transmission mechanism and the third transmission mechanism respectively move the heating device, the magnetic separation device and the sample adding device to required positions, and the controller controls the actions of the first transmission mechanism, the second transmission mechanism and the third transmission mechanism. The reagent strips and the gun heads can be alternately arranged on the test tube rack, and the test tube rack can be divided into a reagent strip placing area and a gun head placing area.
Preferably, a bar code reader for scanning the information of the reagent kit is movably arranged on the test tube rack, the bar code reader is connected with a fourth transmission mechanism, and the fourth transmission mechanism is electrically connected with the controller. The bar code reader is used for scanning the information of the kit, so that whether the used kit is correct or not can be conveniently determined.
Preferably, the fourth transmission mechanism comprises a second driving motor, a second motor gear, a second synchronous belt, a second driven gear and a third linear guide rail which is parallel to the second synchronous belt and arranged along the length direction of the frame, and the bar code reader is connected with the second synchronous belt and moves on the third linear guide rail.
Preferably, the sample adding device comprises a sample adding pump, a sample adding needle assembly and a lifting mechanism fixing plate, wherein the lifting mechanism fixing plate is arranged on the first supporting plate, the sample adding pump is connected with the sample adding needle assembly through a hose, the third transmission mechanism is a third linear motor which is connected with the sample adding needle assembly and drives the sample adding needle assembly to move up and down on the lifting mechanism fixing plate, and the bottom of the test tube rack is provided with a Y-direction transmission mechanism which drives the test tube rack to move back and forth along the width direction of the rack and an X-direction transmission mechanism which drives the test tube rack to move left and right along the length direction of the rack. The sample adding pump drives the sample adding needle component to suck and discharge liquid to finish the function of sample adding or waste liquid discharging. The sample adding needle component is arranged to move up and down, and the test tube rack can move left and right or Y forwards and backwards to cooperate with the sample adding device to work. The number of the sample adding needles and the sample adding pumps can be 8, and 8 samples can be prepared at one time. The sample adding pumps are arranged on two sides of the frame every four.
Preferably, a second support plate is arranged between the test tube rack and the first support plate, a Y-direction transmission mechanism and an X-direction transmission mechanism are respectively arranged on the first support plate and the second support plate, the Y-direction transmission mechanism and the X-direction transmission mechanism are respectively connected with the test tube rack and the second support plate, and the Y-direction transmission mechanism and the X-direction transmission mechanism are electrically connected with the controller. And the X-direction transmission mechanism drives the test tube rack to move by driving the second supporting plate to move. Because the X-direction movement of the test tube rack is smaller than the Y-direction movement frequency of the test tube rack in the whole operation process, the Y-direction transmission mechanism is arranged above the X-direction transmission mechanism. The X-direction transmission mechanism only needs to be used when the gun head is replaced in the whole operation process.
Preferably, the Y-direction transmission mechanism comprises a first synchronous belt, a first motor gear and a first driven gear which are arranged at two ends of the first synchronous belt, a first driving motor and two first guide rails which are parallel to each other, the first guide rails are parallel to the first synchronous belt, a first linear bearing which is used for crossing the first guide rails and a synchronous belt pressing sheet which is connected with the first synchronous belt are arranged on the bottom surface of the test tube rack, the first motor gear, the first driven gear and the first guide rails are arranged on the second support plate, a second linear bearing is arranged at the bottom of the second support plate, the X-direction transmission mechanism comprises a fourth linear motor and two second guide rails which are parallel to each other, the second guide rails are crossing the second linear bearing, the fourth linear motor is connected with the second support plate, a strip through hole is formed in the first support plate, and the first driving motor passes through the strip through hole and is connected with the first motor gear. The first driving motor is arranged below the first supporting plate, so that space is saved, and meanwhile materials for fixing the first driven gear are saved.
Preferably, the heating device comprises a plurality of incubation grooves for inserting test tubes, a Wen Yocao fixing plate for fixing the incubation grooves and heating resistors or heating pads for heating the incubation grooves, the first transmission mechanism comprises a plurality of first linear guide rails, a plurality of first sliding blocks movably intersected with the first linear guide rails and a first linear motor for driving the heating device to move up and down, the first sliding blocks and the first linear motor are all arranged on the Wen Yocao fixing plate, the first linear motor is connected with the controller, and the first linear guide rails are arranged on one side of the test tube rack along the height direction of the rack. Wen Yocao insert the test tube and carry out closed air heating, be favorable to reaching required temperature material saving fast, compare in Wen Yocao add water and heat can avoid water spill or evaporation in the incubation groove damages the machine easily or influences experimental effect. The incubation groove is made of metal material and can comprise aluminum or stainless steel.
Preferably, the magnetic separation device comprises a magnetic field fixing plate and a strong magnet arranged on the magnetic field fixing plate, the second transmission mechanism comprises 2 second linear guide rails, 2 second sliding blocks movably intersected with the second linear guide rails and second linear motors, the second sliding blocks are arranged at two ends of the magnetic field fixing plate, the second linear motors are arranged on the magnetic field fixing plate and electrically connected with the controller, and the second linear guide rails are arranged on two sides of the test tube rack along the width direction of the rack. The magnetic field fixing plate and the strong magnet are provided with a magnet fixing plate therebetween, namely, two ends of the magnet fixing plate are respectively connected with the magnetic field fixing plate and the strong magnet, so that the influence of the magnet on the second linear motor is avoided, and the positions of the strong magnets distributed on the magnet fixing plate correspond to the positions of the test tube holes of the test tube rack.
Preferably, the strong magnet is L-shaped. Facilitating sufficient contact of the test tube with the strong magnet.
The method for constructing the library by the small multichannel library constructing instrument is as follows: two kits, a DNA purification kit and a library construction kit, were used, and both kits were used in the form of a kit strip.
The first step: DNA purification, namely placing a DNA sample to be purified into a sample hole of a purification kit, opening a small multichannel library construction instrument, starting self-checking the instrument, after self-checking is qualified, placing a reagent strip and a gun head which are filled with the DNA sample into a test tube rack, starting purification after the information of the kit is read by a bar code reader, uniformly mixing and incubating magnetic beads and the DNA sample for 5 minutes, magnetically separating supernatant for the first time, washing the DNA sample by adding 80% ethanol for the first time, uniformly mixing the 80% ethanol and the magnetic beads for 1-2 minutes, magnetically separating supernatant for the second time, washing the DNA sample by adding 80% ethanol for the second time, uniformly mixing the 80% ethanol and the magnetic beads for 1-2 minutes, magnetically separating supernatant for the third time, drying the magnetic beads for 5 minutes, adding an elution buffer, uniformly incubating the elution buffer and the magnetic beads for 5 minutes, magnetically separating, keeping the magnetic separation for 2-3 minutes, and transferring the supernatant into a new sample tube, wherein the mixing and incubation of the magnetic beads and the DNA sample for 5 minutes, the mixing of 80% ethanol and the magnetic beads, and the mixing of the elution buffer and the magnetic beads are realized by continuously blowing and sucking the DNA sample by the sample adding device, the DNA sample can be mixed by moving the test tube rack by the X-direction transmission mechanism or the Y-direction transmission mechanism, the magnetic separation supernatant is realized by moving the magnetic separation device to the bottom of the sample hole to adsorb the magnetic beads and then removing the waste liquid by the sample adding device, moving the waste liquid by the Y-direction transmission mechanism to the waste liquid hole of the reagent strip, throwing the gun head into the waste liquid box, using the new gun head to move the test tube rack and the third transmission mechanism by the X-direction transmission mechanism to drive the sample adding needle to move up and down, the magnetic beads and 80% ethanol are added, the elution buffer solution is moved by the Y-direction transmission mechanism, the test tube rack and the third transmission mechanism drive the sample adding needle to move up and down, and the magnetic beads are dried at room temperature or heated by the heating device.
And a second step of: the obtained purified DNA is processed into DNA small fragments, and the DNA is cut off by enzyme or ultrasonic waves outside the instrument;
and a third step of: constructing a library, heating the heating device to 30 ℃, incubating for 30 minutes by using an End repairing reagent at 30 ℃, incubating for 30 minutes by using an A-tailing reagent at 30 ℃ and incubating for 15 minutes by using a Ligation reagent at 30 ℃, purifying the obtained mixed solution after each incubation, wherein the purification method is the same as the purification method of the first step, and finally obtaining purified DNA for direct sequencing; or sequentially carrying out the incubation for 30 minutes by an End polishing reagent at 30 ℃ and the incubation for 15 minutes by a Ligation reagent at 30 ℃, purifying the obtained mixed solution after each incubation, wherein the purification method is the same as the purification method in the first step, and finally obtaining the purified DNA for direct sequencing.
The beneficial effects of the invention are as follows: the problems of time consumption and low flux of manually preparing samples are thoroughly solved, and the whole flow of the system is automatically completed by a one-stop workstation, so that errors are avoided, and the system is more suitable for laboratory standardization.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is an isometric schematic of the present invention;
FIG. 2 is an exploded view of the present invention;
FIG. 3 is a schematic diagram of a test tube rack;
FIG. 4 is a flow chart of the construction of a gene library after purification of a DNA sample.
Wherein the figures are marked as: 1. a frame; 2. a first support plate; 21. a long through hole; 3. a second support plate; 31. a second linear bearing; 3. a controller; 4. a test tube rack; 41. tabletting a synchronous belt; 42. a first linear bearing; 5. a sample adding device; 51. a sample adding pump; 52. a loading needle assembly; 53. a lifting mechanism fixing plate; 6. a waste liquid box; 7. a heating device; 71. wen Yocao; 72. wen Yocao fixing plates; 8. a magnetic separation device; 81. a magnetic field fixing plate; 82. a magnet fixing plate; 83. strong magnet; 9. a first transmission mechanism; 91. a first linear guide rail; 92. a first slider; 93. a first linear motor; 10. a second transmission mechanism; 101. a second linear guide rail; 102. a second slider; 103. a second linear motor; 11. a third transmission mechanism; 111. a third linear motor; 12. a bar code reader; 13. a fourth transmission mechanism; 131. a second driving motor; 132. a second motor gear; 133. a second timing belt; 134. a second driven gear; 135. a third linear guide rail; 14. an X-direction transmission mechanism; 141. a fourth linear motor; 142. a second guide rail; 15. y-direction transmission mechanism; 151. a first driving motor; 152. a first motor gear; 153. a first synchronization belt; 154. a first driven gear; 155. a first guide rail.
Detailed Description
As shown in figures 1-3, a small multichannel library construction instrument comprises a frame 1, wherein a first supporting plate 2 and a controller 3 are arranged on the frame 1, a test tube rack 4 for storing reagent strips and gun heads and a sample adding device 5 are arranged on the first supporting plate 2, a waste liquid box 6 is arranged on one side of the test tube rack 4, a heating device 7 and a magnetic separation device 8 are respectively and movably arranged at the bottom of the test tube rack 4, the heating device 7 and the magnetic separation device 8 are respectively connected with a first transmission mechanism 9 and a second transmission mechanism 10, the sample adding device 5 is arranged above the test tube rack 4, the sample adding device 5 is connected with a third transmission mechanism 11, and the first transmission mechanism 9, the second transmission mechanism 10 and the third transmission mechanism 11 are electrically connected with the controller 3.
Specifically, a bar code reader 12 for scanning information of the reagent kit is movably arranged on the test tube rack 4, the bar code reader 12 is connected with a fourth transmission mechanism 13, and the fourth transmission mechanism 13 is electrically connected with the controller 3. The bar code reader 12 is used to scan the kit information to facilitate determining if the kit used is correct. The fourth transmission mechanism 13 includes a second driving motor 131, a second motor gear 132, a second synchronous belt 133, a second driven gear 134, and a third linear guide rail 135 parallel to the second synchronous belt 133 and disposed along the length direction of the frame 1, where the barcode reader 12 is connected to the second synchronous belt 133 and moves on the third linear guide rail 135.
Specifically, the sample adding device 5 includes a sample adding pump 51, a sample adding needle assembly 52 and a lifting mechanism fixing plate 53, the lifting mechanism fixing plate 53 is disposed on the first support plate 2, the sample adding pump 51 is connected with the sample adding needle assembly 52 through a hose, the third transmission mechanism 11 is a third linear motor 111 connected with the sample adding needle assembly 52 and driving the sample adding needle assembly 52 to move up and down on the lifting mechanism fixing plate 53, and the bottom of the test tube rack 4 is provided with a Y-direction transmission mechanism 15 driving the test tube rack 4 to move back and forth along the width direction of the rack 1 and an X-direction transmission mechanism 14 driving the test tube rack 4 to move left and right along the length direction of the rack 1. The sample adding pump 51 drives the sample adding needle assembly 52 to suck and discharge liquid to finish the function of sample adding or waste liquid sucking. The loading needle assembly 52 is configured to move up and down, and the rack 4 can move up and down in conjunction with the loading device 5 in a direction X or Y. The number of the sample application needles and the sample application pumps 51 may be 8, and 8 samples may be made at a time. The sample pumps 51 are placed on both sides of the frame 1 in groups of four. The test tube rack 4 and the first support plate 2 are provided with a second support plate 3, the first support plate 2 and the second support plate 3 are respectively provided with a Y-direction transmission mechanism 15 and an X-direction transmission mechanism 14, the Y-direction transmission mechanism 15 comprises a first synchronous belt 153, a first motor gear 152 and a first driven gear 154 which are arranged at two ends of the first synchronous belt 153, a first driving motor 151 and two first guide rails 155 which are parallel to each other, the first guide rails 155 are parallel to the first synchronous belt 153, the bottom surface of the test tube rack 4 is provided with a first linear bearing 42 which is used for traversing the first guide rails 155 and a synchronous belt pressing sheet 41 which is connected with the first synchronous belt 153, the second support plate 3 is provided with a first motor gear 152, a first driven gear 154 and the first guide rails 155, the bottom of the second support plate 3 is provided with a second linear bearing 31, the X-direction transmission mechanism 14 comprises a fourth linear motor 141 and two second guide rails 142 which are parallel to each other and are arranged on the first support plate 2, the second guide rails 142 traverse the second linear bearing 31, and the fourth linear motor 141 is connected with the second support plate 3, and the test tube rack 4 is driven to move by driving the second support plate 3. The second support plate 3 is provided with a long through hole 21, and the first driving motor 151 is connected with the first motor gear 152 through the long through hole 21. The first driving motor 151 is disposed under the first support plate 2 to save space while saving materials for fixing the first driven gear 154.
Specifically, the heating device 7 includes a plurality of Wen Yocao for inserting the test tube, wen Yocao fixed plate 72 for fixing the incubation groove 71 and heating resistor or heating pad for heating the incubation groove 71, the first transmission mechanism 9 includes a plurality of first linear guide 91, a plurality of first sliders 92 movably intersecting with the first linear guide 91 and a first linear motor 93 for driving the heating device 7 to move up and down, the first sliders 92 and the first linear motor 93 are all disposed on the fixed plate Wen Yocao, the first linear motor 93 is connected with the controller 3, and the first linear guide 91 is disposed on one side of the test tube rack 4 along the height direction of the rack 1. Wen Yocao 71 inserts the test tube and carries out closed air heating, is favorable to reaching required temperature saving material fast, compares in Wen Yocao that water is added and is heated can avoid the water in the incubation groove 71 to splash or evaporate out and damage the machine or influence experimental effect easily. Wen Yocao 71 is made of metal and can comprise aluminum or stainless steel.
Specifically, the magnetic separation device 8 includes a magnetic field fixing plate 81, a magnet fixing plate 82 and an L-shaped strong magnet 83, two ends of the magnet fixing plate 82 are respectively connected with the magnetic field fixing plate 81 and the strong magnet 83, the second transmission mechanism 10 includes 2 second linear guide rails 101, 2 second sliding blocks 102 movably intersected with the second linear guide rails 101, and second linear motors 103 driving the magnetic separation device 8 to move back and forth along the width direction of the rack 1, the second sliding blocks 102 are arranged at two ends of the magnetic field fixing plate 81, the second linear motors 103 are arranged on the magnetic field fixing plate 81 and electrically connected with the controller 3, and the second linear guide rails 101 are arranged at two sides of the test tube rack 4 along the width direction of the rack 1. The positions of the strong magnets 83 distributed on the magnet fixing plate 82 correspond to the positions of the test tube holes of the test tube rack 4.
As shown in fig. 4, the method for constructing the library by the small multi-channel library constructing apparatus is as follows: two kits, a DNA purification kit and a library construction kit, were used, and both kits were used in the form of a kit strip.
The first step: DNA purification, namely placing a DNA sample to be purified into a sample hole of a purification kit, opening a small multichannel library construction instrument, starting self-checking the instrument, after self-checking is qualified, placing a reagent strip and a gun head which are filled with the DNA sample into a test tube rack 4, reading kit information by a bar code reader 12, starting purification, uniformly mixing and incubating magnetic beads and the DNA sample for 5 minutes, magnetically separating supernatant for the first time, washing the DNA sample by adding 80% ethanol for the first time, uniformly mixing the 80% ethanol and the magnetic beads for 1-2 minutes, magnetically separating supernatant for the second time, washing the DNA sample by adding 80% ethanol for the second time, uniformly mixing the 80% ethanol and the magnetic beads for 1-2 minutes, magnetically separating supernatant for the third time, drying the magnetic beads for 5 minutes, adding an elution buffer solution, uniformly mixing and incubating the elution buffer solution and the magnetic beads for 5 minutes, magnetically separating, and keeping the magnetic separation for 2-3 minutes, and transferring supernatant into a new sample tube, wherein, the magnetic beads and the DNA sample are evenly mixed and incubated for 5 minutes, 80 percent ethanol and the magnetic beads are evenly mixed, and an elution buffer solution and the magnetic beads are evenly mixed by continuously blowing and sucking the DNA sample through a sample adding device 5, the magnetic separation supernatant is obtained by moving a magnetic separating device to the bottom of a sample hole to adsorb the magnetic beads and then remove waste liquid through the sample adding device 5, the waste liquid is moved into a waste liquid hole of a reagent strip by moving a test tube rack 4 through a Y-direction transmission mechanism 15, the gun head is thrown into a waste liquid box 6, the new gun head is used by moving the test tube rack 4 and a third transmission mechanism 11 through an X-direction transmission mechanism 14 to drive a sample adding needle component to move up and down, the magnetic beads, the 80 percent ethanol and the elution buffer solution are all obtained by moving the test tube rack 4 and the third transmission mechanism 11 through the Y-direction transmission mechanism 15 to drive the sample adding needle component to move up and down, the magnetic bead drying is realized by room temperature drying or heating by the heating device 7. That is, when the bar code reader 12 reads the information of the reagent kit and starts to purify, the X-direction transmission mechanism 14 drives the test tube rack 4 to move, so that the sample adding device 5 is positioned in the hole with the magnetic beads in the reagent strip, the magnetic beads are sucked, then the sample adding pump 51 drives the sample adding needle to continuously blow and suck the sample hole for 5 minutes to fully mix the DNA sample with the magnetic beads, the magnetic separation device 8 moves to the bottom of the sample hole for 1-2 minutes, the sample adding device 5 is used for sucking the supernatant, the X-direction transmission mechanism 14 drives the test tube rack 4 to move, the supernatant is discharged into the waste hole of the reagent strip, the gun head is discharged into the waste liquid box 6, the Y-direction transmission mechanism 15 drives the test tube rack 4 to move, the sample adding device 5 reuses the new gun head, the X-direction transmission mechanism 14 drives the test tube rack 4 to move, the sample adding device 5 sucks a certain amount of 80% ethanol, adding the sample into the sample hole, removing the magnetic separation device 8 by the second driving device, continuously blowing and sucking the sample hole for 1-2 minutes by using the sample adding device 5, moving the magnetic separation device 8 to the bottom of the sample hole, waiting for 1-2 minutes, sucking the supernatant by using the sample adding device 5, driving the test tube rack 4 to move by the X-direction transmission mechanism 14, discharging the supernatant into the waste hole of the reagent strip, discharging the gun head into the waste liquid box 6, driving the test tube rack 4 to move by the Y-direction transmission mechanism 15, reusing the new gun head by the sample adding device 5, repeatedly cleaning the sample hole again by taking 80% ethanol, removing the supernatant, moving the heating device 7 to the sample hole, inserting the test tube of the sample hole into Wen Yocao, heating the 5 minutes to dry the magnetic beads, driving the test tube rack 4 to move by the Y-direction transmission mechanism 15, reusing the new gun head by the sample adding device 5, the X-direction transmission mechanism 14 drives the test tube rack 4 to move, a certain amount of eluent is taken by using the sample adding device 5 and added into the sample hole, the sample hole is continuously blown and sucked by using the sample adding device 5 for 5 minutes, the eluent is fully and uniformly mixed with the magnetic beads, DNA is dissolved into the eluent from the magnetic beads, the magnetic separation device 8 is moved to the bottom of the sample adding hole, waiting for 1-2 minutes, and the purified DNA sample is sucked by using the sample adding device 5 and transferred into a new test tube.
And a second step of: the obtained purified DNA is processed into DNA small fragments, and the DNA is cut off by enzyme or ultrasonic waves outside the instrument;
and a third step of: constructing a library, heating a heating device 7 to 30 ℃, incubating for 30 minutes by using an End repairing reagent at 30 ℃, incubating for 30 minutes by using an A-tailing reagent at 30 ℃ and incubating for 15 minutes by using a Ligation reagent at 30 ℃, purifying the obtained mixed solution after each incubation, wherein the purification method is the same as the purification method in the first step, and finally obtaining purified DNA direct sequencing such as a sequencer suitable for Illumina; or sequentially carrying out the incubation for 30 minutes by an End polishing reagent at 30 ℃ and the incubation for 15 minutes by a Ligation reagent at 30 ℃, purifying the obtained mixed solution after each incubation, wherein the purification method is the same as the purification method in the first step, and finally obtaining the purified DNA direct sequencing such as a sequencer suitable for Ion Torrent.
The advantages of this embodiment are:
1. the problems of time consumption and low flux of manually preparing samples are thoroughly solved, and the whole flow of the system is automatically completed by a one-stop workstation, so that errors are avoided, and the system is more suitable for laboratory standardization;
2. the DNA sample after split charging is directly used for second generation sequencing after being processed by the small multichannel library construction instrument, and no additional processing is needed. 8 reagent strip positions can be used for simultaneously preparing 8 samples, so that the sample preparation efficiency is greatly improved.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. The utility model provides a small-size multichannel library constructs appearance, its characterized in that includes the frame, be equipped with first backup pad and controller in the frame, be equipped with test-tube rack and the application of sample device that are used for depositing reagent strip and rifle head in the first backup pad, test-tube rack one side is equipped with the waste liquid box, the test-tube rack bottom is movable heating device and magnetic separation device that are equipped with respectively, heating device with magnetic separation device is connected with first drive mechanism and second drive mechanism respectively, the top of test-tube rack is equipped with the application of sample device, the application of sample device is connected with third drive mechanism, first drive mechanism second drive mechanism and third drive mechanism all are connected electrically the controller;
the sample adding device comprises a sample adding pump, a sample adding needle assembly and a lifting mechanism fixing plate, wherein the lifting mechanism fixing plate is arranged on the first supporting plate, the sample adding pump is connected with the sample adding needle assembly through a hose, the third transmission mechanism is a third linear motor which is connected with the sample adding needle assembly and drives the sample adding needle assembly to move up and down on the lifting mechanism fixing plate, and the bottom of the test tube rack is provided with a Y-direction transmission mechanism for driving the test tube rack to move back and forth along the width direction of the rack and an X-direction transmission mechanism for driving the test tube rack to move left and right along the length direction of the rack;
the magnetic separation device comprises a magnet fixing plate, a magnetic field fixing plate and a strong magnet, wherein the magnetic field fixing plate and the strong magnet are respectively arranged at two ends of the magnet fixing plate, the second transmission mechanism comprises 2 second linear guide rails, 2 second sliding blocks movably intersected with the second linear guide rails and a second linear motor, the second sliding blocks are arranged at two ends of the magnetic field fixing plate, the second linear motor is arranged on the magnetic field fixing plate and is electrically connected with the controller, and the second linear guide rails are arranged at two sides of the test tube rack along the width direction of the rack;
a bar code reader for scanning the information of the kit is movably arranged on the test tube rack, the bar code reader is connected with a fourth transmission mechanism, and the fourth transmission mechanism is electrically connected with the controller;
a second support plate is arranged between the test tube rack and the first support plate, a Y-direction transmission mechanism and an X-direction transmission mechanism are respectively arranged on the first support plate and the second support plate, the Y-direction transmission mechanism and the X-direction transmission mechanism are respectively connected with the test tube rack and the second support plate, and the Y-direction transmission mechanism and the X-direction transmission mechanism are electrically connected with the controller;
the heating device comprises a plurality of incubation grooves for inserting test tubes, a Wen Yocao fixing plate for fixing the incubation grooves and a heating resistor or a heating pad for heating the incubation grooves, wherein the first transmission mechanism comprises a plurality of first linear guide rails, a plurality of first sliding blocks movably intersected with the first linear guide rails and a first linear motor for driving the heating device to move up and down, the first sliding blocks and the first linear motor are all arranged on the Wen Yocao fixing plate, the first linear motor is connected with the controller, and the first linear guide rails are arranged on one side of the test tube rack along the height direction of the rack.
2. The small multichannel library construction instrument according to claim 1, wherein the Y-direction transmission mechanism comprises a first synchronous belt, a first motor gear and a first driven gear which are arranged at two ends of the first synchronous belt, a first driving motor and two first guide rails which are parallel to each other, the first guide rails are parallel to the first synchronous belt, a first linear bearing which is used for crossing the first guide rails and a synchronous belt pressing sheet which is connected with the first synchronous belt are arranged on the bottom surface of the test tube rack, a second linear bearing is arranged on the bottom of the second support plate, the X-direction transmission mechanism comprises a fourth linear motor and two second guide rails which are parallel to each other and are arranged on the first support plate, the second guide rails are crossing the second linear bearing, the fourth linear motor is connected with the second support plate, a strip through hole is formed in the first support plate, and the first driving motor passes through the strip through hole and is connected with the first gear.
3. A method of constructing a library based on the small multi-channel library construction instrument of claim 1, the method comprising: two kits, namely a DNA purification kit and a library construction kit, are used, and the two kits are used in the form of reagent strips,
the first step: the DNA is purified by the following steps:
placing a DNA sample to be purified into a sample hole of a purification kit, opening a small multichannel library construction instrument, starting self-checking the instrument, after the self-checking is qualified, placing a reagent strip and a gun head which are filled with the DNA sample into a test tube rack, starting purification after the information of the kit is read by a bar code reader, incubating the magnetic beads and the DNA sample for 5 minutes after the information is confirmed, magnetically separating a supernatant for the first time, washing the DNA sample by adding 80% ethanol for the first time, evenly mixing the 80% ethanol and the magnetic beads for 1-2 minutes, magnetically separating the supernatant for the second time, washing the DNA sample by adding 80% ethanol for the second time, evenly mixing the 80% ethanol and the magnetic beads for 1-2 minutes, magnetically separating the supernatant for the third time, drying the magnetic beads for 5 minutes, adding an elution buffer, evenly mixing the elution buffer and the magnetic beads for 5 minutes, magnetically separating the fourth time, keeping the magnetic separation for 2-3 minutes and transferring the supernatant into a new sample tube, wherein the magnetic separation supernatant is obtained by continuously blowing and sucking the DNA sample by the sample adding device after the magnetic beads and the DNA sample are mixed and incubated for 5 minutes, 80% ethanol and the magnetic beads are mixed and the elution buffer and the magnetic beads are mixed, the magnetic separation supernatant is obtained by moving the magnetic separation device to the bottom of the sample hole to adsorb the magnetic beads and then removing the waste liquid by the sample adding device, the waste liquid is moved into a waste liquid hole of a reagent strip by moving the test tube rack through the Y-direction transmission mechanism, the gun head is lost into the waste liquid box, the new gun head is used for driving the sample adding needle to move up and down through the X-direction transmission mechanism, the magnetic beads, the 80% ethanol and the elution buffer are added by driving the sample adding needle to move up and down through the Y-direction transmission mechanism, the magnetic bead drying is realized by room temperature drying or heating drying by the heating device;
and a second step of: the obtained purified DNA is processed into DNA small fragments, and the DNA is cut off by enzyme or ultrasonic waves outside the instrument;
and a third step of: constructing a library, heating the heating device to 30 ℃, incubating for 30 minutes by using an End repairing reagent at 30 ℃, incubating for 30 minutes by using an A-tailing reagent at 30 ℃ and incubating for 15 minutes by using a Ligation reagent at 30 ℃, purifying the obtained mixed solution after each incubation, wherein the purification method is the same as the purification method of the first step, and finally obtaining purified DNA for direct sequencing; or sequentially carrying out the incubation for 30 minutes by an End polishing reagent at 30 ℃ and the incubation for 15 minutes by a Ligation reagent at 30 ℃, purifying the obtained mixed solution after each incubation, wherein the purification method is the same as the purification method in the first step, and finally obtaining the purified DNA for direct sequencing.
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