WO2022151537A1

WO2022151537A1 - Digital pcr micro-droplet generating device

Info

Publication number: WO2022151537A1
Application number: PCT/CN2021/074725
Authority: WO
Inventors: 于海
Original assignee: 于海
Priority date: 2021-01-13
Filing date: 2021-02-01
Publication date: 2022-07-21

Abstract

A digital PCR micro-droplet generating device, comprising a silica gel sealing gasket, at least one micro-droplet generating part, at least one micro-droplet collecting tube, and a thin film sealing member sealingly adhered to the surface of the at least one micro-droplet generating part, wherein each micro-droplet generating part comprises a sample cavity and an oil cavity that are formed in the top, a micro-droplet generating chip, and a flow guide column at the bottom.

Description

A digital PCR droplet generating device

technical field

The invention belongs to the technical field of droplet digital PCR, in particular to a digital PCR droplet generating device.

Background technique

Polymerase chain reaction (PCR) technology has become one of the most basic and routine nucleic acid detection methods in the field of life science research due to its advantages of high sensitivity, simple operation and low cost. The first generation of traditional PCR technology uses agarose electrophoresis to analyze PCR products, but only qualitative and semi-quantitative detection can be performed. The second-generation quantitative PCR (quantitative PCR, qPCR) technology calculates the content of the target nucleic acid sequence by adding fluorescent dyes to the reaction system and detecting the number of cycles at which the fluorescent signal emitted in the reaction reaches the threshold, that is, the cycle threshold. qPCR technology is currently the most widely used nucleic acid detection technology due to its rapidity, simplicity and economy. However, qPCR technology is relatively quantitative and depends on cycle thresholds and standard curves. In the case of low target sequence content, small expression differences, and a large amount of background sequences or inhibitors in the reaction system, the sensitivity and accuracy are greatly limited. Digital PCR (Digital PCR) technology is a high-sensitivity nucleic acid detection and quantification method that has developed rapidly in recent years. In each reactor, one or more copies of the target molecule (DNA template) are included or not included in each reactor to realize "single-molecule template PCR amplification". The ratio and number of reactors are statistically analyzed, and the technique of obtaining the initial copy number or concentration of the target molecule according to the Poisson distribution principle and the number and ratio of positive droplets.

Digital PCR technology can be divided into three types: microdroplet, microcavity, and microwell according to the principle. Among them, the microdroplet digital PCR system represented by the American Bio-Rad Company is the mainstream digital PCR technology in the current market due to its excellent performance. . The operation process is as follows: first, the PCR reaction material is added to the droplet generation card to generate droplets, and then the droplets are transferred to the PCR amplification tube with a pipette. After the droplets are amplified by the PCR amplifier, the samples are loaded into the droplets. The reader detects the fluorescent signal of the droplets. After the microdroplets are generated, the technical solution needs to manually transfer the microdroplets to the PCR amplification tube, which has disadvantages such as microdroplet damage, cross-contamination, and complicated operation, which greatly limits its wide application in scientific research, clinical and other fields. application.

SUMMARY OF THE INVENTION

Aiming at the deficiencies in the prior art, the present invention discloses a digital PCR droplet generating device. After the droplet is generated, it directly enters the PCR amplification tube, eliminating the need for manual transfer of the droplet, reducing the loss of the droplet and making it difficult to generate Cross-contamination.

To achieve the above object, the present invention provides the following technical solutions:

A digital PCR droplet generating device, comprising a silicone gasket, at least one droplet generating component, at least one droplet collecting tube, and a thin film seal sealed and bonded to the surface of the at least one droplet generating component; wherein each The droplet generating component includes a sample cavity and an oil cavity arranged at the top, a droplet generating chip and a guiding column at the bottom.

Preferably, the silicone sealing gasket is sealed on the ends of the sample cavity and the oil cavity, and the droplet collection tube is arranged at the bottom of the droplet generation chip.

Preferably, the sample cavity and the oil cavity are communicated with an external pressure source through the silicone gasket, so that the droplet generation oil and the sample are pressed into the droplet generation chip.

Preferably, the volume of the sample chamber is 5-100 μL, and the volume of the oil chamber is 10-200 μL.

Preferably, the droplet generation chip includes a sample hole communicating with the sample cavity, an oil hole communicating with the oil cavity, a droplet output hole, a sample pipeline meandering out of the sample hole, and a self-oil hole Meandering out of the oil phase pipeline.

Preferably, the oil phase pipeline includes a first oil phase pipeline and a second oil phase pipeline, and the first oil phase pipeline and the second oil phase pipeline and the sample pipeline are at the droplet generation position intersect at the cutoff of the pipelines and eventually merge into the droplet output pipeline.

Preferably, the channel widths of the sample pipeline, the oil phase pipeline and the droplet output pipeline are all 0.05-0.3 mm.

Preferably, the inner side of the guide column is provided with a guide groove, which is communicated with the droplet output hole, and is used for draining the generated droplet into the droplet collecting tube.

Preferably, the droplet generating member is a thermoplastic material selected from one of polymethyl methacrylate, cyclic olefin copolymer, cyclic olefin polymer, polypropylene or polycarbonate.

The beneficial effects of the present invention are as follows: the droplet generating device provided by the present invention is composed of a silicone gasket, a droplet generating component, a droplet collecting tube, etc., and the droplet generating component is one-shot injection molding of polymer material, which has good consistency and is easy to batch. It is simple and low cost, the hole spacing is the same as that of conventional culture plates, it is easy to realize array formation, it can be matched with conventional instruments, and it is easy to achieve high throughput; the device uses positive pressure to drive the generation of droplets, and it is easy to adjust the size and generation rate of the droplets; After the droplet is generated, it directly enters the PCR amplification tube through the droplet output port through the guide column, eliminating the need for manual transfer of droplets, avoiding cross-contamination, reducing droplet breakage and fusion, increasing the number of droplets detected, and improving digital PCR. detection accuracy.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

FIG. 1 is a front view of the digital PCR droplet generating device of the present invention.

FIG. 2 is a top view of the digital PCR droplet generating device of the present invention.

FIG. 3 is a schematic structural diagram of a droplet generating component in the digital PCR droplet generating device of the present invention.

4 is a structural diagram of a droplet generation chip in the digital PCR droplet generation device of the present invention.

FIG. 5 is a rear view of the droplet generating component in the digital PCR droplet generating device of the present invention.

Fig. 6 is the effect diagram of the digital PCR droplet generation device of the present invention and the matching use of the culture plate.

In the figure, 100, silicone gasket; 200, droplet generating part; 211, sample cavity; 212, oil cavity; 220, droplet generation chip; 221, sample hole; 222, oil hole; 223, droplet output hole; 224 , sample pipeline; 225, oil phase pipeline; 2251, first oil phase pipeline; 2252, second oil phase pipeline; 226, pipeline shear; 227, droplet output pipeline; 230, diversion column; 231, diversion groove; 300, droplet collection tube; 400, membrane seal.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

As shown in Fig. 1 to Fig. 5, this case lists an embodiment of a digital PCR droplet generation device, which includes:

Silicone gasket 100, at least one droplet generating part 200, at least one droplet collecting tube 300, and a film seal 400 sealingly bonded to the surface of the at least one droplet generating part 200; wherein, each droplet generating The component 200 includes a sample chamber 211 and an oil chamber 212 arranged at the top, a droplet generation chip 220 and a guide column 230 at the bottom.

In the above-mentioned embodiment, the silicone gasket 100 is sealed on the ends of the sample chamber 211 and the oil chamber 212 ; as shown in FIG. 2 , the silicone gasket 100 may be provided with air holes for It applies pressure to ensure the airtightness of the crimping between the external pressure source and the sample chamber 211 and the oil chamber 212, so as to facilitate the application of external pressure, so that the droplet generation oil and the sample are pressed into the droplet generation chip within 220.

In the above embodiment, the droplet collecting tube 300 is arranged at the bottom of the droplet generating chip 220, and the droplet collecting tube 300 is used to collect the droplets generated from the droplet generating chip 220. Droplet collection tube 300 may be a centrifuge tube or PCR tube to facilitate droplet storage and/or to perform amplification reactions.

In the above embodiment, the membrane seal 400 may be a polymer, such as polymethyl methacrylate, cycloolefin copolymer, cycloolefin polymer, polypropylene or polycarbonate, which is sealed with the droplet generating member 200 Bonding, including but not limited to thermal bonding, solvent-assisted bonding, ultrasonic bonding or adhesive bonding, is used to prevent droplets from penetrating out of the device.

In the above-mentioned embodiment, the sample cavity 211 and the oil cavity 212, the droplet generation chip 220 and the guide column 230 can be integrally formed, and the material can be a polymer, such as polymethyl methacrylate, One of cyclic olefin copolymer, cyclic olefin polymer, polypropylene or polycarbonate, the droplet generating part 200 is obtained by injection molding, the parts obtained by one injection have good consistency, are easy to batch and have low cost; in this case Among them, the number of droplet generating components 200 may be multiple, preferably two, four, eight, twelve and/or sixteen, which may be used for high-throughput droplet generation.

The volume of the sample chamber 211 is preferably 5-100 μL, and the volume of the oil chamber 212 is preferably 10-200 μL; the pressure value of the external pressure source is preferably 10-50 kPa, which can be driven by positive pressure to generate droplets, which is easy to adjust the droplets size and generation rate.

Referring to FIG. 4 , in the above embodiment, the droplet generation chip 220 includes a sample hole 221 communicated with the sample cavity 211 , an oil hole 222 communicated with the oil cavity 212 , a droplet output hole 223 and a self-sample The sample pipeline 224 meandering out of the hole 221 and the oil phase pipeline 225 meandering out from the oil hole 222 . Both the sample pipeline 224 and the oil phase pipeline 225 are designed in a meandering shape to increase the fluid resistance, facilitate the control of an external pressure source, and achieve stable and uniform generation of droplets.

In the above embodiment, the oil phase pipeline 225 includes a first oil phase pipeline 2251 and a second oil phase pipeline 2252. On a plane, the first oil phase pipeline 2251 and the second oil phase pipeline 2251 The channel 2252 is relatively confluent in the parallel direction and intersects perpendicularly with the sample pipeline 224, forming a pipeline shear 226 and extending out of the droplet output pipeline 227, where the droplet generated oil and the sample are sheared in the pipeline The water-in-oil droplets are generated by shearing at point 226 , which flow into the droplet output hole 223 through the droplet output pipeline 227 .

The channel widths of the sample pipeline 224, the oil phase pipeline 225 and the droplet output pipeline 227 are preferably 0.05-0.3 mm.

Referring to FIG. 5 , in the above-mentioned embodiment, the inner side of the guide column 230 is provided with a guide groove 231 , which is communicated with the droplet output hole 223 and is used for draining the generated droplets to the droplet collection tube 300.

As shown in Figure 6, this case presents an application of the above-mentioned embodiment. The microdroplet generation device can realize batch production and can realize arraying, and its hole spacing is consistent with that of conventional culture plates, such as conventional 48-well plates and 96-well plates. , easy to match with conventional instruments, so as to achieve high throughput.

This case provides the use process of the digital PCR droplet generating device of the following embodiment:

1) adding the droplet generated oil into the oil cavity 212;

2) Add the sample (PCR reaction system) into the sample cavity 211;

3) Seal the silicone gasket 100 on the sample chamber 211 and the oil chamber 212, and apply positive pressure to the sample chamber 211 and the oil chamber 212 from the air hole on the silicone gasket 100 through an external pressure source, and the pressure value is set as 10-50kPa;

4) After applying positive pressure, the sample flows into the sample pipeline 224 from the sample chamber 211 through the sample hole 221, and the droplet generated oil flows from the oil chamber 212 through the oil hole 222 to the oil phase pipeline 225; The cut 226 is cut into droplets, and the droplets flow into the droplet output hole 223 through the droplet output pipeline 227;

5) The droplet output hole 223 is communicated with the guide column 230, and the droplet flows into the droplet collection tube 300 from the droplet output hole 223 through the guide groove 231 of the guide column 230;

6) Take out the droplet collection tube 300 in which the droplets are collected, amplify by a PCR amplifier, and perform fluorescence detection to obtain the sample concentration.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the application listed in the description and the embodiment, and it can be applied to various fields suitable for the present invention. For those skilled in the art, it can be easily Therefore, the invention is not limited to the specific details and illustrations shown and described herein without departing from the general concept defined by the appended claims and the scope of equivalents.

Claims

A digital PCR droplet generating device, characterized in that it comprises a silicone gasket (100), at least one droplet generating component (200), at least one droplet collecting tube (300), A film seal (400) on the surface of the droplet generation part (200); wherein each of the droplet generation parts (200) comprises a sample cavity (211) and an oil cavity (212), a droplet generation chip ( 220) and the guide column (230) at the bottom.
The digital PCR droplet generating device according to claim 1, characterized in that, the silicone gasket (100) is sealed and covered on the ends of the sample chamber (211) and the oil chamber (212), and the micro A droplet collection tube (300) is arranged at the bottom of the droplet generation chip (220).
The digital PCR droplet generation device according to claim 1, wherein the sample chamber (211) and the oil chamber (212) communicate with an external pressure source through the silicone gasket (100) to generate droplets Oil and sample are pressed into the droplet generation chip (220).
The digital PCR droplet generating device according to claim 1, wherein the volume of the sample chamber (211) is 5-100 μL, and the volume of the oil chamber (212) is 10-200 μL.
The digital PCR droplet generation device according to claim 1, characterized in that, the droplet generation chip (220) comprises a sample hole (221) communicated with the sample chamber (211), and a sample hole (221) communicated with the oil chamber (211). 212) The connected oil hole (222), the droplet output hole (223), the sample pipeline (224) meandering out from the sample hole (221), and the oil phase pipe meandering out from the oil hole (222) Road (225).
The digital PCR droplet generating device according to claim 5, wherein the oil phase pipeline (225) comprises a first oil phase pipeline (2251) and a second oil phase pipeline (2252), and the oil phase pipeline (2252) The first oil phase pipeline (2251) and the second oil phase pipeline (2252) intersect the sample pipeline (224) at the pipeline shear (226) at the droplet generation position, and finally merge into the sample pipeline (224). Droplet output line (227).
The digital PCR droplet generation device according to claim 6, characterized in that, the widths of the flow channels of the sample pipeline (224), the oil phase pipeline (225) and the droplet output pipeline (227) are all 0.05 -0.3 mm.
The digital PCR droplet generation device according to claim 5, wherein a guide groove (231) is opened on the inner side of the guide column (230), which is communicated with the droplet output hole (223), Used to drain the generated droplets into the droplet collection tube (300).
The digital PCR droplet generating device according to claim 1, wherein the droplet generating component (200) is a thermoplastic material selected from the group consisting of polymethyl methacrylate, cyclic olefin copolymer, cyclic olefin polymer, One of polypropylene or polycarbonate.