CN115181789A

CN115181789A - Single cell detection method based on full-automatic digital PCR all-in-one machine

Info

Publication number: CN115181789A
Application number: CN202211116213.1A
Authority: CN
Inventors: 李奎; 全智慧; 裘宇容; 于雪; 滕祥云
Original assignee: Guangzhou Huayinkang Medical Group Co ltd; Guangzhou Huayin Medical Laboratory Center Co Ltd
Current assignee: Guangzhou Huayinkang Medical Group Co ltd; Guangzhou Huayin Medical Laboratory Center Co Ltd
Priority date: 2022-09-14
Filing date: 2022-09-14
Publication date: 2022-10-14
Anticipated expiration: 2042-09-14
Also published as: CN115181789B

Abstract

The invention relates to the field of single cell detection and analysis, and discloses a method for single cell detection and analysis. According to the method, single cell separation, cell lysis and detection analysis are carried out on the sample by utilizing the full-automatic digital PCR all-in-one machine, so that the application range of the digital PCR all-in-one machine is expanded, the digital PCR all-in-one machine can be used for detecting trace cells, and simultaneously, the target gene in each single cell in the sample can be accurately detected. The method has the advantages of visual operation, accurate and quick detection result and simultaneous qualitative and quantitative detection.

Description

Single cell detection method based on full-automatic digital PCR all-in-one machine

Technical Field

The invention relates to a single cell detection method, in particular to a method for detecting a single cell by using a digital PCR instrument, and particularly relates to a method for detecting a single cell by using a full-automatic digital PCR all-in-one machine.

Background

With the development of modern biology and the increasing demand of society, the need of analyzing the nucleic acid sequence of cell population is not satisfied, and the detection and sequencing of single cells are necessary.

To meet the above needs, qPCR technology has been developed, the basic principle of which is to collect individual cells, perform lysis and collect and purify nucleic acid material, amplify and sequence by fluorescence activated cell sorting, immunomagnetic enrichment, microsampling, flow cytometry, laser capture microdissection or various automated devices, such as Drop-sequencing technology (Drop-seq) of harvard university.

However, the above operations are still very complicated, and the separated cells need to be transferred to a corresponding micro-reaction system for lysis and extraction and purification of nucleic acid substances, and then transferred to a corresponding micro-reaction system, or amplified by adding amplification raw materials. When the above operation is required for a large number of cells, the efficiency is low even if the operation is completed fully automatically. The enzymes and starting materials used in the different steps may also interfere with each other, making it difficult to perform a one-stop procedure.

The digital PCR is an absolute nucleic acid molecule quantitative technology, and is characterized in that a micro-fluidic or micro-droplet method is adopted, a diluted nucleic acid solution is dispersed into a micro-reactor or a micro-droplet of a chip, the number of nucleic acid templates in each reactor is less than or equal to one, after PCR circulation, the reactors with the nucleic acid molecule templates give out fluorescence signals, and the reactors without the templates do not have the fluorescence signals, so that the nucleic acid concentration of the original solution can be estimated.

Disclosure of Invention

In order to solve the above problems, the present invention provides a single cell detection method, which is characterized in that:

the method comprises the following steps: preparing a single cell separation system;

the single cell separation system comprises a cell to be detected after fluorescent staining, an amplification raw material, a primer, a probe and Taq enzyme;

step two: dispersing the single cell separation system into micro-droplets containing at most one cell by using a full-automatic digital PCR all-in-one machine, and flatly paving the micro-droplets on a digital PCR detection plate;

step three: observing in a bright field and a fluorescence field, and counting the micro-droplets wrapped with the cells;

step four: thermally cracking cells, observing the micro-droplets under fluorescence, counting the micro-droplets still capable of observing the cell morphology in real time, and calculating the cell lysis rate until the cell lysis rate reaches a preset value, wherein the cell lysis rate is = (the number of micro-droplets including cells in the third step-the number of micro-droplets still having the cell morphology)/the number of micro-droplets including cells in the third step;

step five: heating to 95 ℃ for 10-20 minutes, preferably 15 minutes, to start Taq enzyme, and cooling and annealing to complete the first amplification; and (5) circularly performing amplification for a preset number of times, and counting the number of highlighted micro-droplets.

The full-automatic digital PCR all-in-one machine is a digital PCR instrument which can realize full-automatic micro-droplet generation, PCR amplification and fluorescence signal collection on one device. Exemplary models are as follows: DQ-24 of Sniper, applied Biosystems. QuantStudio. Q. Cells of Sammerfei, etc., models of different manufacturers differ in their processing capabilities, but do not affect the implementation of the technical solution of the present invention.

Preferably, in the second step, the volume of the micro-droplets is set to a value between 0.3 and 1.5nL, more preferably, to a value between 0.6 and 1.0 nL by using a control program of a full-automatic digital PCR all-in-one machine.

Preferably, when overlapping of the microdroplets is observed in step two, the microdroplets are heated to 60 ℃ to be tiled. In the experiment, it was found that the microdroplets can be efficiently spread within 5 minutes by heating, and therefore the heating time can be uniformly set to 5 minutes, or the heating can be stopped after the disappearance of the overlap is observed.

In the third step, the fourth step and the fifth step, the counting can be carried out manually or automatically.

Preferably, the temperature of the thermally lysed cells is 85 ℃. If during the lysis procedure more microdroplets are found which still contain intact cell morphology, it is evident that lysis has not been completed, the lysis time should be prolonged, and/or the temperature should be increased. Until the cracking rate reaches the preset standard. As an example, the cracking rate may be set to not less than 80%, 90%, 95%, or other values as the predetermined standard, or other values may be set according to the detection accuracy requirement. Another advantage of measuring the lysis rate is that even if the lysis effect is not good, the final measurement result can be corrected numerically and predicted error according to the value, for example, the lysis rate is 80%, and the highlighted vacuole number in the final result is divided by 0.8 when calculating the total number of the mutant cells, and if the mutation rate needs to be calculated, the calculation method in example 6 of the present invention can be used for correction. Those skilled in the art can also perform various later data correction and estimation according to the detected data. If the field has a fluorescent signal after lysis is complete, the lysis is successful and the cells of interest are present, and the lysis, amplification and detection process is initiated. Even if micro-droplets with overall high-brightness fluorescent signals are not found, the fluorescent signals are not strong enough, and the number of the high-brightness micro-droplets in the micro-droplets needs to be counted after a plurality of rounds of amplification. And finally counting the number of the high-brightness micro-droplets, namely the number of the cells with the specific genes corresponding to the primers on the detection plate.

When the test cells contain two or more types of cells, as a scheme, at least one of the cells is individually stained so that it can be distinguished from the other types of cells under fluorescence and counted individually. The single staining is to perform fluorescent staining on only one of the cells to be detected, or to make the fluorescent staining color of a certain cell to be detected different from that of other cells to be detected. In the present invention, in order to demonstrate technical effects, experiments are performed using cells individually stained in advance, and it is easily conceived by those skilled in the art that, in the case of a mixed plurality of cells, a certain cell of interest therein may be individually stained by, for example, coupling a specific antibody to a fluorescent stain. Such technical means are conventional and therefore no separate examples are given. Alternatively, the single-cell separation system may be configured to include primers and corresponding probes that are capable of detecting a specific site that is unique to one of the cells to be detected. The method can identify different types of cells and can also identify a small number of cells with specific gene mutation in the same type of cells, thereby further improving the detection precision and expanding the application range of the technology. Preferably, when no single fluorescently stained cells are found in step three (no fluorescent spots throughout the field), detection by the digital PCR detection plate is stopped and no subsequent lysis and amplification is performed. This saves detection time and machine occupancy.

When the cells to be detected may contain various types of expression or mutation, various primers and probes for detecting different sites can be arranged in the single cell separation system, and the light emitting colors of the various probes corresponding to the different sites are different. The current commercial full-automatic digital PCR all-in-one machine generally has a plurality of fluorescence detection channels, and can detect fluorescence with different wavelengths. By this preferred method, different types of expression or mutations can be distinguished.

The invention further provides the single-cell separation system, which comprises the cells to be detected after fluorescent staining, amplification raw materials, primers, probes and Taq enzyme. The fluorescent staining is a routine operation in a biological laboratory, and other raw materials are commercially available products, can be purchased respectively, and can also be purchased as a premixed solution containing a plurality of components for dilution and mixing preparation. The skilled person knows how to select primers and probes when detecting different cells.

Compared with the prior art, the technical scheme of the invention at least has the following beneficial effects:

first, through the real-time monitoring, guarantee the success rate of parcel and schizolysis, prevent to appear lou examining. And the intermediate data may be used to correct the final data and estimate the error range.

Secondly, when various cells exist, the cells of interest can be identified in time for lysis and amplification, so that the detection time is shortened, and the pressure of subsequent data processing is reduced.

Thirdly, the quantitative and qualitative detection can be simultaneously completed, and not only the mutation type can be detected, but also the number of the mutation cells of the type can be detected.

Drawings

Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:

FIG. 1 is a photograph showing the results of observing the staining of HEK293T cells by a fluorescence microscope in example 1.

FIG. 2 is a photograph showing the cells encapsulated in the micro-droplets observed in the bright field and the fluorescent field in example 1.

FIG. 3 is a photograph showing the GADPH amplification of HEK293T cells observed by the full-automatic digital PCR machine in example 2.

FIG. 4 is a photograph showing the results of observing the staining of H-1975 cells by a fluorescence microscope in example 3.

FIG. 5 is a photograph showing the micro-droplets encapsulating H-1975 cells observed in bright field and fluorescent field in example 3.

FIG. 6 is a photograph showing the mutation of T790M gene in H-1975 cells observed by the full-automatic digital PCR machine in example 4.

FIG. 7 is a photograph of stained and unstained cells observed using the full-automatic digital PCR all-in-one machine in example 5.

Detailed Description

The present application will be described in further detail with reference to the drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. It should be noted that, for convenience of description, only the portions related to the related invention are shown in the drawings.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.

Example 1

HEK293T cell single cell isolation and validation

1. Pretreatment of living cell staining

The cells used in this study were HEK293T cells stored in the laboratory, and cell suspension was prepared when the cells entered exponential growth phase, followed by staining of live cells with Calcein AM (Calcein AM). The specific operation steps are as follows:

the cell state was observed and the preparation of the cell suspension was performed as it entered the exponential growth phase. The medium was removed, and the cells were washed twice with 2 mL of 1 XPBS, and removed. Add 500. Mu.L of trypsin (a commercially available reagent) and digest for 1 minute at room temperature, gently tap the side wall of the flask to detach the digested cells from the bottom of the flask. Digestion was stopped by adding 2 mL of fresh medium DMEM (Gibco, USA) (10% FBS,1% penicillin-streptomycin mixture) and transferred to a 15 mL centrifuge tube. Centrifuging at 125 g for 5 min at room temperature, and removing supernatant; cells were washed with 1 mL of 1 × PBS to remove residual medium. The cells were resuspended in fresh 1 XPBS and cell number measurements were performed using a CountessTM 3 series automatic cell counter (Thermofisiher, USA).

2. Fluorescent staining of cells

The experiment was performed using Calcein (Calcein AM) (Beyotime, C2012) for live cell fluorescent staining. The operation method comprises the following steps: calcein AM, 2 mM, was diluted to a concentration of 2. Mu.M staining working solution with 1 XPBS buffer. According to 1 × 10 ⁶ Viable cell staining was performed at a concentration of one/mL. The staining conditions were observed with a fluorescence microscope (Ni-U model of Nikon corporation) (the results are shown in fig. 1, where the photograph marked a in fig. 1 is the bright field observation result and the photograph marked B in fig. 1 is the fluorescence field observation result, it can be seen from fig. 1 that the staining of HEK293T living cells can be achieved with a staining rate close to 100% by using the Calcein AM staining method.

3. A single cell separation system (X = 0.5) was prepared according to the components and amounts shown in Table 1, and then single cell separation was performed using a digital PCR integrated machine (model DQ-24 from Sniper) according to the procedure shown in Table 2. The observation of the micro-droplets encapsulating the cells in the bright field and the fluorescent field is shown in fig. 2, wherein the photograph marked as a in fig. 2 is the bright field observation result, and the photograph marked as B in fig. 2 is the fluorescent field observation result. As can be seen from FIG. 2, the method provided by the invention can wrap the single cell in the micro-droplet by using the full-automatic digital PCR all-in-one machine, thereby successfully realizing the separation of the single cell, and the number of the cells in each micro-droplet is 0 or 1.

TABLE 1 Single cell separation System

TABLE 2 Single cell isolation procedure

Example 2:

preparing a single cell separation system according to the components and the dosage in the table 1, then performing single cell separation by using a digital PCR all-in-one machine (model DQ-24 of Snipe corporation) (the separation program refers to the table 2), and performing GADPH amplification, wherein the amplification program is shown in the table 3, and the primer sequence is 5' -; 5 'CGACCAAATCCGTTGACTCC-3'; the probe sequence was 5 '-FAM-CGCCAGCCGAGCCACCATCGC-MGB-NFQ-3'. The amplification of the cells was observed using a full-automatic digital PCR machine (model DQ-24 from Sniper), and the non-cell samples and 1 XPBS were used as negative controls. The results are shown in FIG. 3, where the photographs labeled A/C/D in FIG. 3 represent GADPH expansion in HEK293T cell samples. In the photograph labeled A in FIG. 3, the absence of clearly visible encapsulated fluorescent spots within the microdroplets, rather than the cell sample, may be caused by insufficient cell lysis, and the expanded cells, which are still morphologically distinct, emit fluorescent spots of similar size. The photograph labeled B in fig. 3 shows the GADPH amplification in the non-cell sample, and the microdroplets are all highlighted droplets, and no fluorescent spot of cell size is seen. The 1 × PBS amplification results show that no luminescence was observed for FAM channels and no further pictures were given. The cells shown in the photograph labeled C in fig. 3 were fully lysed and amplified to form highlighted droplets, which were recognized as positive signals by the fully automated digital PCR integrator as shown in the photograph labeled D. As can be seen from FIG. 3, the method provided by the invention can realize the monitoring of the expression condition of the cell target gene by using a full-automatic digital PCR all-in-one machine.

TABLE 3 amplification procedure

Example 3

Example 1 was repeated using H-1975 cells, and the results are shown in FIGS. 4 and 5, and FIG. 4 shows the staining of the cells, wherein the photograph labeled A in FIG. 4 is the bright field observation and the photograph labeled B in FIG. 4 is the fluorescent field observation. The indicator also can cause H-1975 to emit green fluorescence, and the luminous rate is close to 100%. FIG. 5 shows that the microdroplets are also capable of packing larger H-1975 cells, and that there are 0-1 cells in each microdroplet. Wherein the photograph labeled a in fig. 5 is the bright field observation result, and the photograph labeled B in fig. 5 is the fluorescence field observation result.

Example 4

Example 2 was repeated using H-1975 cells to detect the mutation status of the T790M gene and T790M gene, and the primer sequence was 5-; 5' TCCCCTCCCGTATCTCCT-. The probe sequence is 5 '-VIC-ATGAGCTGTCGTGATAG-MGB-NFQ-3'; 5 '-FAM-ATGAGCTGCTGCATGATG-MGB-NFQ-3'. FAM fluorescence channels were selected for detection.

The results are shown in FIG. 6, and the amplification of the T790M gene mutation in H-1975 cell samples is shown in FIG. 6. In the photograph labeled a in fig. 6, the encapsulated fluorescent dots are clearly visible within the microdroplets containing the cells without the fluorescent dots of the cells. The photograph labeled B in FIG. 6 shows the amplification of the T790M gene mutation in the non-cellular sample, and the microdroplets are all highlighted droplets, and no fluorescent spot of cell size is seen. The 1 × PBS amplification results showed no luminescence observed for FAM channels and no longer gave a picture. The cells shown in the photograph labeled C in fig. 6 were fully lysed and expanded to form highlight droplets, which are identified as positive signals by the fully automated digital PCR ensemble machine as shown in the photograph labeled D. The method provided by the invention can be used for monitoring the gene mutation condition of the cell by utilizing a full-automatic digital PCR all-in-one machine.

As can be seen from examples 1 to 4, the method of the present invention is applicable to a variety of cells.

Example 5

In this example, single cell isolation was performed on live cell-stained and unstained HEK293T cells using the system and procedure of example 1, respectively. The results show that stained cells can detect fluorescent spots encapsulated within microdroplets through the fluorescent channel of the digital PCR integrator (photograph labeled a in fig. 7), whereas unstained cells do not (photograph labeled B in fig. 7).

Example 5 shows an application scenario of performing fluorescent staining on only a part of cells, and when the cells to be detected contain a plurality of cells, only a part of the cells can be subjected to fluorescent staining, so that cell types which are not concerned by a detector are ignored during statistics. An exemplary application scenario is: tumor cells are detected in human peripheral blood and may be labeled with specific fluorochromes or antibody-conjugated fluorochromes to exclude interference with normal blood cells. When detecting, if only blood cells exist and no fluorescent spot exists on the detection plate, the detection can be stopped directly, thereby saving the detection time.

Example 6

100 stained HEK293T cells and stained H-1975 cells were mixed in the ratio of 1.

The primer probe sequences were used to detect T790M gene mutations specific to H-1975 cells, as configured according to Table 1, with reference to example 4. The number of highlighted spots was observed by isolation and amplification according to tables 2 and 3. 186 microdroplets (with fluorescent spots) containing cells were detected in total by each detection plate, and the cells in 22 microdroplets maintained the cell morphology (with fluorescent spots remaining) after lysis. Finally, 87 high-brightness micro-droplets under the FAM fluorescence channel are detected. According to the traditional calculation, the T790M gene mutation rate in the cells to be detected is 87/200 multiplied by 100% =43.5%, and the deviation from the theoretical value of 50% is large.

After the results of the correction using the actually counted number of cells, the mutation rate of the T790M gene was 87/(186-22) × 100% =53%, which was closer to the theoretical value than before the correction. It can be seen that in the case of total loss of cells detected during the test (probably due to cell adhesion to the container or the sample injector, the wall of the pipeline, the smaller the number of cells is, the more obvious the cell number is), and the rate of cell lysis is not ideal, the instant monitoring of the invention can obtain the value of each stage, and still correct the detection result and improve the accuracy of the detection.

By adopting the method, when a specific cell needs to be screened from a large number of cells, the specific cell can be quickly identified and counted, and only the specific cell is sequenced. Compared with the traditional method which only can detect and count the positive rate after amplification, the method can save the detection time and reduce the later data processing work.

Claims

1. A method of single cell detection, comprising:

step four: thermally cracking the cells, observing the micro-droplets in a fluorescent field, immediately counting the micro-droplets still capable of observing the cell morphology, and calculating the cell lysis rate until the cell lysis rate reaches a preset value, wherein the cell lysis rate is = (the number of micro-droplets including the cells in the third step-the number of micro-droplets still having the cell morphology)/the number of micro-droplets including the cells in the third step;

step five: heating to 95 ℃ and keeping for 10-20 minutes to start Taq enzyme, cooling and annealing to finish the first amplification, circularly performing amplification for a preset number of times, and counting the number of highlighted micro-droplets.

2. The method of claim 1, wherein: in the second step, a control program of the full-automatic digital PCR all-in-one machine is used for setting the volume of the micro-droplets to be 0.3-1.5 nL.

3. The method of claim 1, wherein: and in the second step, when the micro droplets are observed to be overlapped, heating to 60 ℃ to flatly lay the micro droplets.

4. The method of claim 1, wherein: in the third step, the fourth step and the fifth step, an automatic counting scheme is adopted.

5. A method for single cell detection as claimed in claim 1 wherein: when the cells to be tested contain two or more types of cells, at least one of the cells is individually stained in step one so that it can be distinguished from other types of cells and individually counted in a bright field or a fluorescent field.

6. The method of claim 5, wherein: the independent staining refers to that only one of the cells to be detected is subjected to fluorescent staining, or that a certain cell to be detected is different from other cells to be detected in fluorescent staining color.

7. A method of single cell detection as claimed in claim 6 wherein: when no single fluorescently stained cells were found in step three, detection by the digital PCR detection plate was stopped.

8. A method for single cell detection as claimed in claim 1 wherein: when the cells to be detected contain two or more types of cells, the primers and the corresponding probes contained in the single-cell separation system can detect a specific site which is specific to one of the cells to be detected.

9. A method for single cell detection as claimed in claim 8 wherein: the single cell separation system comprises primers and probes for detecting different sites, and the light emitting colors of the probes corresponding to the different sites are different.

10. A single cell separation system comprises a cell to be detected after fluorescent staining, an amplification raw material, a primer, a probe and Taq enzyme.