CN116769884A - Application of droplet digital PCR in visual detection of HIV-positive cells - Google Patents
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Abstract
Description
技术领域Technical field
本发明涉及微滴式数字PCR应用技术领域,特别涉及一种微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用。The invention relates to the technical field of droplet digital PCR application, and in particular to the application of droplet digital PCR in the visual detection of HIV-positive cells.
背景技术Background technique
人类免疫缺陷病毒(Human Immunodeficiency Virus,HIV),即艾滋病(AIDS,获得性免疫缺陷综合征)病毒,是造成人类免疫系统缺陷的一种病毒。HIV病毒是一种RNA病毒,主要感染人体中的免疫细胞,进入人体的HIV病毒经逆转录后将自身序列整合至人基因组序列上,之后再利用人宿主细胞的转录翻译系统进行大量病毒复制,严重破坏人体的免疫系统。Human Immunodeficiency Virus (HIV), also known as AIDS (Acquired Immunodeficiency Syndrome) virus, is a virus that causes defects in the human immune system. HIV is an RNA virus that mainly infects immune cells in the human body. The HIV virus that enters the human body integrates its own sequence into the human genome sequence after reverse transcription, and then uses the transcription and translation system of the human host cell to replicate a large number of viruses. Seriously damage the body's immune system.
目前HIV的检测还是以全血中RNA检测为主,市面上有很多RNA的定量定性检测方式,例如普通PCR或者q-PCR技术,但是这些技术只是从核酸层面对病毒基因进行相对定量检测,而不能够反应艾滋病毒感染的阳性细胞数量或者比例。At present, HIV detection is still based on RNA detection in whole blood. There are many quantitative and qualitative detection methods of RNA on the market, such as ordinary PCR or q-PCR technology. However, these technologies only perform relatively quantitative detection of viral genes from the nucleic acid level. The number or proportion of positive cells that cannot reflect HIV infection.
因此,现有技术还有待于改进和发展。Therefore, the existing technology still needs to be improved and developed.
发明内容Contents of the invention
鉴于上述现有技术的不足,本发明的目的在于提供一种微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,旨在解决现有PCR技术无法实现对艾滋病毒感染的阳性细胞进行可视化检测的问题。In view of the shortcomings of the above-mentioned existing technologies, the purpose of the present invention is to provide an application of droplet digital PCR in the visual detection of HIV-positive cells, aiming to solve the problem that existing PCR technology cannot realize the visualization of HIV-positive cells. Detection issues.
本发明的技术方案如下:The technical solution of the present invention is as follows:
一种微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,其中,包括步骤:An application of droplet digital PCR in visual detection of HIV-positive cells, which includes the following steps:
预先制备艾滋病毒PCR反应液,所述艾滋病毒PCR反应液包括预处理的艾滋患者外周血单个核细胞样本、艾滋病毒基因引物、艾滋病毒基因探针、内参基因引物、内参基因探针;Preparing an HIV PCR reaction solution in advance, which includes pretreated peripheral blood mononuclear cell samples of AIDS patients, HIV gene primers, HIV gene probes, internal reference gene primers, and internal reference gene probes;
在微滴生成区,将艾滋病毒PCR反应液加入微滴生成卡的加样孔中,将微滴生成油加至微滴生成卡的加油孔中;In the droplet generation area, add the HIV PCR reaction solution to the sampling hole of the droplet generation card, and add the droplet generation oil to the oil filling hole of the droplet generation card;
将微滴生成卡中的艾滋病毒PCR反应液与微滴生成油混合均匀后,用密封垫对所述微滴生成卡进行密封并置入微滴发生仪中;After the HIV PCR reaction solution and the droplet generation oil in the droplet generation card are evenly mixed, the droplet generation card is sealed with a sealing gasket and placed in the droplet generator;
启动微滴发生仪使微滴生成油包裹艾滋患者外周血单个核细胞样本形成微滴悬浊液;Start the microdroplet generator to make the microdroplet generate oil to wrap the peripheral blood mononuclear cell sample of the AIDS patient to form a microdroplet suspension;
将所述微滴悬浊液转移至96孔PCR板中并进行ddPCR扩增;Transfer the droplet suspension to a 96-well PCR plate and perform ddPCR amplification;
待ddPCR扩增结束后,将96孔PCR板放置于微滴分析仪中进行荧光信号读取,使用分析软件对荧光信号的强度和数目进行分析,实现艾滋病毒阳性细胞可视化检测。After the ddPCR amplification is completed, place the 96-well PCR plate in a droplet analyzer to read the fluorescence signal, and use analysis software to analyze the intensity and number of the fluorescence signal to achieve visual detection of HIV-positive cells.
所述微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,其中,预先制备艾滋病毒PCR反应液的步骤包括:The application of droplet digital PCR in visual detection of HIV-positive cells, wherein the steps of pre-preparing HIV PCR reaction solution include:
采用常温PBS对获取的艾滋患者外周血液样品进行稀释,得到稀释血液样品;Use normal temperature PBS to dilute the peripheral blood samples obtained from AIDS patients to obtain diluted blood samples;
对所述稀释血液样品在Ficoll-Paque PLUS中进行梯度离心,得到单个核细胞层;Perform gradient centrifugation on the diluted blood sample in Ficoll-Paque PLUS to obtain a mononuclear cell layer;
向单个核细胞层中加入红细胞裂解液,吹打混匀后再加入PBS进行离心处理,弃上清得外周血单个核细胞(PBMC);Add red blood cell lysis solution to the mononuclear cell layer, mix by pipetting, then add PBS for centrifugation, and discard the supernatant to obtain peripheral blood mononuclear cells (PBMC);
用0.9%的生理盐水溶液对PBMC重悬稀释,调整细胞浓度得艾滋患者外周血单个核细胞样本;Resuspend and dilute PBMC in 0.9% physiological saline solution, and adjust the cell concentration to obtain peripheral blood mononuclear cell samples from AIDS patients;
将艾滋患者外周血单个核细胞样本、艾滋病毒基因引物、艾滋病毒基因探针、内参基因引物,以及内参基因探针进行混合,得到艾滋病毒PCR反应液。Mix the peripheral blood mononuclear cell samples of AIDS patients, HIV gene primers, HIV gene probes, internal reference gene primers, and internal reference gene probes to obtain an HIV PCR reaction solution.
所述微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,其中,所述艾滋患者外周血单个核细胞样本中的细胞浓度为6×106个/mL-2×107个/mL。The application of the droplet digital PCR in the visual detection of HIV-positive cells, wherein the cell concentration in the peripheral blood mononuclear cell sample of the AIDS patient is 6×10 6 /mL-2×10 7 /mL .
所述微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,其中,所述艾滋病毒基因引物包括如SEQ ID NO.1所示的正向引物和如SEQ ID NO.2所示的反向引物;所述艾滋病毒基因探针的序列如SEQ ID NO.3所示;所述内参基因引物包括如SEQ ID NO.4所示的正向引物和如SEQ ID NO.5所示的反向引物;所述内参基因探针的序列如SEQ ID NO.6所示。Application of the droplet digital PCR in visual detection of HIV-positive cells, wherein the HIV gene primers include the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2 forward primer; the sequence of the HIV gene probe is shown in SEQ ID NO.3; the internal reference gene primer includes the forward primer shown in SEQ ID NO.4 and the reverse primer shown in SEQ ID NO.5. primer; the sequence of the internal reference gene probe is shown in SEQ ID NO. 6.
所述微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,其中,将所述微滴悬浊液转移至96孔PCR板中并进行ddPCR扩增的步骤中,ddPCR扩增的条件设置如下所示:The application of the droplet digital PCR in the visual detection of HIV-positive cells, wherein in the step of transferring the droplet suspension to a 96-well PCR plate and performing ddPCR amplification, the conditions for ddPCR amplification are set As follows:
变性温度,94℃,30s,1次;Denaturation temperature, 94°C, 30s, 1 time;
退火温度,94℃,5s;Annealing temperature, 94°C, 5s;
延伸温度,60℃,35s,循环49次。Extension temperature, 60°C, 35s, 49 cycles.
有益效果:本发明提供了一种微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,先通过微滴生成油将含艾滋患者外周血单个核细胞样本的PCR反应液均匀包裹生成具有细胞的微滴,然后通过ddPCR程序中的变性与退火,由此实现对其所包裹的单个细胞中所有核酸的PCR,即实现在细胞层面上可视化的观测每个细胞内部的HIV核酸扩增情况。Beneficial effects: The present invention provides an application of droplet digital PCR in the visual detection of HIV-positive cells. First, the PCR reaction solution containing the peripheral blood mononuclear cell sample of an AIDS patient is evenly wrapped with oil generated by the droplets to generate a cell-like reaction solution. The droplets are then denatured and annealed in the ddPCR program to achieve PCR of all the nucleic acids in the single cells they are wrapped in, that is, to visually observe the HIV nucleic acid amplification inside each cell at the cellular level.
附图说明Description of drawings
图1为本发明提供的一种微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用流程图。Figure 1 is a flow chart of the application of droplet digital PCR in visual detection of HIV-positive cells provided by the present invention.
图2为油包水微滴生成示意图。Figure 2 is a schematic diagram of the generation of water-in-oil droplets.
图3中A为内参基因GAPDH的阳性液滴/阴性液滴散点图,B为HIV+细胞/HIV-细胞散点图。In Figure 3, A is a scatter plot of positive droplets/negative droplets of the internal reference gene GAPDH, and B is a scatter plot of HIV + cells/HIV - cells.
图4中A为1号样本细胞上样总数为30000个/孔时检测细胞内参GAPDH的液滴可视化呈现图,B为1号样本细胞上样总数为100000个/孔时检测细胞内参GAPDH的液滴可视化呈现图。In Figure 4, A is a visual representation of the liquid droplets used to detect the internal reference GAPDH when the total number of cells loaded in sample No. 1 is 30,000 cells/well, and B is the liquid droplet used to detect the internal reference GAPDH in the cells when the total number of cells loaded in sample No. 1 is 100,000 cells/well. Drop visualization diagram.
图5中A为1号样本细胞上样总数为30000个/孔时检测HIV+细胞(LTR-Gag引物)的液滴可视化呈现图,B为1号样本细胞上样总数为100000个/孔时检测HIV+细胞(LTR-Gag引物)的液滴可视化呈现图。In Figure 5, A is a visual presentation of droplets for detecting HIV + cells (LTR-Gag primer) when the total number of cells loaded in sample No. 1 is 30,000 cells/well, and B is when the total number of cells loaded in sample No. 1 is 100,000 cells/well. Visual representation of droplets detecting HIV + cells (LTR-Gag primer).
图6中A为5号样本细胞上样总数为30000个/孔时检测细胞内参GAPDH的液滴可视化呈现图,B为1号样本细胞上样总数为100000个/孔时检测细胞内参GAPDH的液滴可视化呈现图。In Figure 6, A is a visual representation of the droplet used to detect the internal reference GAPDH when the total number of cells loaded in sample No. 5 is 30,000 cells/well, and B is the liquid droplet used to detect the internal reference GAPDH in the cell when the total number of cells loaded in sample No. 1 is 100,000 cells/well. Drop visualization diagram.
图7中A为5号样本细胞上样总数为30000个/孔时检测HIV+细胞(LTR-Gag引物)的液滴可视化呈现图,B为5号样本细胞上样总数为100000个/孔时检测HIV+细胞(LTR-Gag引物)的液滴可视化呈现图。In Figure 7, A is a visual presentation of droplets for detecting HIV + cells (LTR-Gag primer) when the total number of cells loaded in sample No. 5 is 30,000 cells/well, and B is when the total number of cells loaded in sample No. 5 is 100,000 cells/well. Visual representation of droplets detecting HIV + cells (LTR-Gag primer).
具体实施方式Detailed ways
本发明提供一种微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,为使本发明的目的、技术方案及效果更加清楚、明确,以下对本发明进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。The present invention provides an application of droplet digital PCR in the visual detection of HIV-positive cells. In order to make the purpose, technical solution and effect of the present invention clearer and clearer, the present invention is further described in detail below. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.
普通PCR或者q-PCR技术只是从核酸层面对病毒基因或者genomic DNA进行相对定量检测,现有的ddPCR也都是直接对HIV的核酸进行扩增,这些PCR技术都没有通过液滴直接包裹细胞且在油包水的液滴环境中对其所包裹的DNA进行PCR,因此无法在细胞层面上可视化的观测每个细胞内部的HIV核酸扩增情况。Ordinary PCR or q-PCR technology only performs relatively quantitative detection of viral genes or genomic DNA from the nucleic acid level. The existing ddPCR also directly amplifies HIV nucleic acid. These PCR technologies do not directly wrap cells through droplets and PCR is performed on the wrapped DNA in a water-in-oil droplet environment, so it is not possible to visually observe the HIV nucleic acid amplification inside each cell at the cellular level.
基于此,本发明提供了一种微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,如图1所示,其包括步骤:Based on this, the present invention provides an application of droplet digital PCR in visual detection of HIV-positive cells, as shown in Figure 1, which includes the steps:
S10、预先制备艾滋病毒PCR反应液,所述艾滋病毒PCR反应液包括预处理的艾滋患者外周血单个核细胞样本、艾滋病毒基因引物、艾滋病毒基因探针、内参基因引物、内参基因探针;S10. Prepare an HIV PCR reaction solution in advance. The HIV PCR reaction solution includes pretreated peripheral blood mononuclear cell samples of AIDS patients, HIV gene primers, HIV gene probes, internal reference gene primers, and internal reference gene probes;
S20、在微滴生成区,将艾滋病毒PCR反应液加入微滴生成卡的加样孔中,将微滴生成油加至微滴生成卡的加油孔中;S20. In the droplet generation area, add the HIV PCR reaction solution to the sampling hole of the droplet generation card, and add the droplet generation oil to the oil filling hole of the droplet generation card;
S30、将微滴生成卡中的艾滋病毒PCR反应液与微滴生成油混合均匀后,用密封垫对所述微滴生成卡进行密封并置入微滴发生仪中;S30. After mixing the HIV PCR reaction solution and the droplet generation oil in the droplet generation card evenly, seal the droplet generation card with a sealing gasket and place it in the droplet generation instrument;
S40、启动微滴发生仪使微滴生成油包裹艾滋患者外周血单个核细胞样本形成微滴悬浊液;S40. Start the microdroplet generator to generate oil that wraps the AIDS patient's peripheral blood mononuclear cell sample to form a microdroplet suspension;
S50、将所述微滴悬浊液转移至96孔PCR板中并进行ddPCR扩增;S50. Transfer the droplet suspension to a 96-well PCR plate and perform ddPCR amplification;
S60、待ddPCR扩增结束后,将96孔PCR板放置于微滴分析仪中进行荧光信号读取,使用分析软件对荧光信号的强度和数目进行分析,实现艾滋病毒阳性细胞可视化检测。S60. After the ddPCR amplification is completed, place the 96-well PCR plate in a droplet analyzer to read the fluorescence signal, and use analysis software to analyze the intensity and number of the fluorescence signal to achieve visual detection of HIV-positive cells.
本发明先通过微滴生成油将含有艾滋患者外周血单个核细胞样本的PCR反应液均匀包裹生成具有细胞的微滴,然后通过ddPCR程序中的变性与退火,即在94℃环境中,使得液滴中的细胞膜裂解,开始进入PCR的工作流程,由此实现对其所包裹的单个细胞中所有核酸的PCR,即实现在细胞层面上可视化的观测每个细胞内部的HIV核酸扩增情况。The present invention first uses microdroplets to generate oil to evenly wrap the PCR reaction liquid containing peripheral blood mononuclear cell samples of AIDS patients to generate microdroplets with cells, and then through denaturation and annealing in the ddPCR program, that is, in a 94°C environment, the liquid is The cell membrane in the droplet is lysed and begins to enter the PCR workflow, thereby realizing PCR of all nucleic acids in the single cell it is wrapped in, that is, the amplification of HIV nucleic acid inside each cell can be visually observed at the cellular level.
在一些实施方式中,如图2所示,预先制备艾滋病毒PCR反应液的步骤包括:采用常温PBS对获取的艾滋患者外周血液样品进行稀释,得到稀释血液样品;对所述稀释血液样品在Ficoll-Paque PLUS中进行梯度离心,得到单个核细胞层;向单个核细胞层中加入红细胞裂解液,吹打混匀后再加入PBS进行离心处理,弃上清得PBMC;用0.9%的生理盐水溶对PBMC进行重悬稀释,调整浓度得到艾滋患者外周血单个核细胞样本;将艾滋患者外周血单个核细胞样本、艾滋病毒基因引物、艾滋病毒基因探针、内参基因引物,以及内参基因探针进行混合,得到含艾滋患者外周血单个核细胞的艾滋病毒PCR反应液。In some embodiments, as shown in Figure 2, the steps of pre-preparing the HIV PCR reaction solution include: diluting the peripheral blood samples obtained from AIDS patients with PBS at room temperature to obtain diluted blood samples; diluting the diluted blood samples in Ficoll -Carry out gradient centrifugation in Paque PLUS to obtain a mononuclear cell layer; add red blood cell lysis solution to the mononuclear cell layer, mix by pipetting, then add PBS for centrifugation, discard the supernatant to obtain PBMC; dissolve in 0.9% normal saline PBMC are resuspended and diluted, and the concentration is adjusted to obtain peripheral blood mononuclear cell samples from AIDS patients; mix the peripheral blood mononuclear cell samples from AIDS patients, HIV gene primers, HIV gene probes, internal reference gene primers, and internal reference gene probes. , to obtain the HIV PCR reaction solution containing peripheral blood mononuclear cells of AIDS patients.
在本实施例中,所述艾滋病毒基因引物包括如SEQ ID NO.1所示的正向引物和如SEQ ID NO.2所示的反向引物,其中,SEQ ID NO.1所示的正向引物记为LTR-gag-F,其序列为:GCCTCAATAAAGCTTGCCTTGA;SEQ ID NO.2所示的反向引物记为LTR-gag-R,其序列为:GGCGCCACTGCTAGAGTTTT;所述艾滋病毒基因探针的序列如SEQ ID NO.3所示,记为LTR-gag-探针,其序列为:TGTGACTCTGGTAACTAGAACCCTCAGAC;所述内参基因引物包括如SEQ ID NO.4所示的正向引物和如SEQ ID NO.5所示的反向引物,其中,SEQ ID NO.4所示的正向引物记为PDH-1,其序列为:TGAAAGTTATACAAAATTGAGGTCACTGTT,SEQ ID NO.5所示的反向引物记为PDH-2,其序列为:TCCACAGCCCTCGACTAACC;所述内参基因探针的序列如SEQ ID NO.6所示,记为PDH-探针,其序列为:CCCCCAGATACACTTAAGGGATCAACTCTTAATTGT。In this embodiment, the HIV gene primers include a forward primer shown in SEQ ID NO.1 and a reverse primer shown in SEQ ID NO.2, wherein the forward primer shown in SEQ ID NO.1 The forward primer is recorded as LTR-gag-F, and its sequence is: GCCTCAATAAAGCTTGCCTTGA; the reverse primer shown in SEQ ID NO.2 is recorded as LTR-gag-R, and its sequence is: GGCGCCACTGCTAGAGTTTT; the sequence of the HIV gene probe As shown in SEQ ID NO.3, it is recorded as LTR-gag-probe, and its sequence is: TGTGACTCTGGTAACTAGAACCCTCAGAC; the internal reference gene primer includes the forward primer as shown in SEQ ID NO.4 and the forward primer as shown in SEQ ID NO.5 The reverse primer shown in SEQ ID NO.4 is marked as PDH-1, and its sequence is: TGAAAGTTATACAAAATTGAGGTCACTGTT. The reverse primer shown in SEQ ID NO.5 is marked as PDH-2, and its sequence is: It is: TCCACAGCCCTCGACTAACC; the sequence of the internal reference gene probe is shown in SEQ ID NO. 6, recorded as PDH-probe, and its sequence is: CCCCCAGATACACTTAAGGGATCAACTCTTAATTGT.
在本实施例中,所述艾滋患者外周血单个核细胞样本中的细胞浓度为:6×106个/mL-2×107个/mL。In this embodiment, the cell concentration in the peripheral blood mononuclear cell sample of the AIDS patient is: 6×10 6 cells/mL-2×10 7 cells/mL.
下面通过具体实施例对本发明作进一步的解释说明:The present invention will be further explained below through specific examples:
一种微滴式数字PCR在艾滋病毒阳性细胞可视化检测中的应用,其包括以下步骤:An application of droplet digital PCR in visual detection of HIV-positive cells, which includes the following steps:
1、采用Ficoll-Paque PLUS密度梯度离心的方法分离艾滋病患者的外周血单个核细胞(PBMC),其包括以下步骤:1. Use Ficoll-Paque PLUS density gradient centrifugation to isolate peripheral blood mononuclear cells (PBMC) from AIDS patients, which includes the following steps:
血液稀释:加常温PBS将艾滋患者的外周血液样本进行1:1稀释(在采血管中),使用胶头滴管轻轻混匀,得到稀释血液样品,将稀释血液样品缓慢加在5mL的Ficoll-PaquePLUS上;Blood dilution: Dilute the peripheral blood sample of an AIDS patient 1:1 (in a blood collection tube) with PBS at room temperature, mix gently with a rubber dropper to obtain a diluted blood sample, and slowly add the diluted blood sample to 5 mL of Ficoll -PaquePLUS;
梯度离心:在18-20℃下以2000rpm对稀释血液样品离心20分钟,之后离心机设置为4℃预冷(program 3),尽可能将离心机的升降加速度调至最低,参数可调整为:升速为1,降速为0,可以防止离心结束后刹车震动,扰乱分层;Gradient centrifugation: Centrifuge the diluted blood sample at 2000 rpm for 20 minutes at 18-20°C, then set the centrifuge to 4°C pre-cooling (program 3), and adjust the centrifuge's lifting acceleration to the lowest possible level. The parameters can be adjusted as follows: The speed increase is 1 and the speed decrease is 0, which can prevent brake vibration and disrupt stratification after centrifugation;
吸白膜:使用无菌滴管小心将梯度离心得到的单个核细胞层转移至含6mL的预冷PBS的15mL离心管中,再加入预冷PBS补充到15mL;Aspirate the white film: Use a sterile dropper to carefully transfer the mononuclear cell layer obtained by gradient centrifugation into a 15mL centrifuge tube containing 6mL of pre-cooled PBS, and then add pre-cooled PBS to make up to 15mL;
离心:在4℃,1500rpm离心5分钟,留下层细胞;Centrifuge: Centrifuge at 1500 rpm for 5 minutes at 4°C to leave a layer of cells;
裂解红细胞:向细胞中加入3mL红细胞裂解液(EL buffer),吹打混匀室温裂红细胞5min后,加入PBS至10mL,离心1500rpm/min,5min;Lyse red blood cells: Add 3 mL of red blood cell lysis buffer (EL buffer) to the cells, mix by pipetting and lyse red blood cells at room temperature for 5 minutes, add PBS to 10 mL, and centrifuge at 1500 rpm/min for 5 minutes;
用0.9%生理盐水洗过后重悬,计数,200μL/管分装;Wash with 0.9% physiological saline, resuspend, count, and aliquot into 200 μL/tube;
最后再用0.9%的生理盐水溶液分别按6×106个/mL、2×107个/mL稀释PBMC,得到艾滋患者外周血单个核细胞样本。Finally, the PBMC were diluted with 0.9% normal saline solution at 6×10 6 cells/mL and 2×10 7 cells/mL respectively to obtain peripheral blood mononuclear cell samples from AIDS patients.
2、微滴式ddPCR实验:2. Droplet ddPCR experiment:
将12.5μL的HIV外周血液样本+1.25μL LTR-gag-F(10μM)+1.25μL LTR-gag-R(10μM)+0.625μL LTR-gag-探针(10μM)+1.25μL PDH-1(10μM)+1.25μL PDH-2(10μM)+0.625μLPDH-探针(10μM)+5μL细胞模板(细胞浓度为6×106个/mL、2×107个/mL)+1.25μL ddPCR水混合,制得艾滋病毒PCR反应液;12.5 μL of HIV peripheral blood sample + 1.25 μL LTR-gag-F (10 μM) + 1.25 μL LTR-gag-R (10 μM) + 0.625 μL LTR-gag-probe (10 μM) + 1.25 μL PDH-1 (10 μM )+1.25μL PDH-2 (10μM)+0.625μL PDH-probe (10μM)+5μL cell template (cell concentration is 6×10 6 /mL, 2×10 7 /mL)+1.25μL ddPCR water mixed, Prepare HIV PCR reaction solution;
其中,艾滋病毒基因LTR区段引物及LTR探针与内参GAPDH基因引物及GAPDH探针的序列如表1所示:Among them, the sequences of HIV gene LTR segment primers and LTR probes and internal reference GAPDH gene primers and GAPDH probes are shown in Table 1:
3、微滴的生成:3. Generation of microdroplets:
在微滴生成区,将25μL艾滋病毒PCR反应液加入微滴生成卡的加样孔,继而,70μL微滴生成油加至微滴生成卡的加油孔。微滴的反应液与油混合均匀后,用密封垫密封微滴生成卡,置入微滴发生仪,启动微滴发生仪并生成微滴;大约2分钟后,微滴制备完成,取出卡槽,将40μL的微滴悬浊液从最上排孔中移出至96孔PCR板。In the droplet generation area, add 25 μL of HIV PCR reaction solution to the sampling hole of the droplet generation card, and then add 70 μL of droplet generation oil to the oil filling hole of the droplet generation card. After the droplet reaction solution and oil are evenly mixed, seal the droplet generation card with a sealing gasket, insert the droplet generator, start the droplet generator and generate droplets; after about 2 minutes, the droplet preparation is completed, take out the card slot , remove 40 μL of microdroplet suspension from the top row of wells to the 96-well PCR plate.
4、扩增读取条件,及dd PCR条件设置:4. Amplification reading conditions, and dd PCR condition settings:
PCR程序:PCR procedure:
变性温度,94℃,30s,1次;Denaturation temperature, 94°C, 30s, 1 time;
退火温度,94℃,5s;Annealing temperature, 94°C, 5s;
延伸温度,60℃,35s,循环49次;Extension temperature, 60℃, 35s, 49 cycles;
待PCR扩增结束,将96孔板放置于微滴分析仪中选择FAM/HEX通道进行信号读取。After the PCR amplification is completed, place the 96-well plate in the droplet analyzer and select the FAM/HEX channel for signal reading.
5、分析统计:5. Analyze statistics:
使用QuantaSoft分析软件对荧光信号的强度、数目进行分析;可以根据荧光信号的有无及强度,判断阴性/阳性微滴所占比例。Use QuantaSoft analysis software to analyze the intensity and number of fluorescent signals; the proportion of negative/positive droplets can be determined based on the presence and intensity of fluorescent signals.
本实施利用了液滴发生器当中的微流控技术能够产生“油包水”微滴的方法,使用该方法将含外周血单个核细胞(PBMC)的病毒PCR反应液进行包裹形成微滴;再通过PCR程序中的94℃变性步骤,对单个液滴中所包裹的细胞进行裂解暴露核酸成分作为下一步PCR的模板;接着,通过PCR反应体系中酶与模板及引物互相结合发生反应,产生在单个细胞所包裹的微滴内进行PCR核酸扩增反应。微滴PCR体系核酸产物中的激光信号通过类似于流式细胞仪器的激发检测器逐一通过每个微滴,形成数字及图片信号。最终,通过上述反应及技术的应用,来实现对核酸目的片段阳性细胞的可视化,以及其与阴性细胞比例的区分。又因为具备液滴PCR多种荧光通道,因而可以实现对同一细胞的多靶标多位点检测。This implementation uses the microfluidic technology in the droplet generator to produce "water-in-oil" droplets. This method is used to wrap the viral PCR reaction solution containing peripheral blood mononuclear cells (PBMC) to form droplets; Then, through the 94°C denaturation step in the PCR program, the cells wrapped in a single droplet are lysed to expose the nucleic acid components as templates for the next step of PCR; then, the enzymes in the PCR reaction system combine with the templates and primers to react, producing PCR nucleic acid amplification reactions are performed within microdroplets encapsulated by individual cells. The laser signal in the nucleic acid product of the droplet PCR system passes through each droplet one by one through an excitation detector similar to a flow cytometry instrument, forming digital and picture signals. Finally, through the application of the above reactions and technologies, it is possible to visualize the cells that are positive for the nucleic acid target fragment and to distinguish the proportion of cells that are positive for the nucleic acid target fragment. And because it has multiple fluorescence channels of droplet PCR, it can realize multi-target and multi-site detection of the same cell.
采用实施例1中微滴式数字PCR方法对1-8号样本进行可视化检测中,1-8号样本均为临床确认的艾滋患者的外周血单个核细胞样本。其中,1-4号样本来自同一个病人样本,5-8号来自另一个病人样本。并且样本1号,2号,5号,6号细胞上样总数为30000个/孔。样本3号,4号,7号,8号细胞上样总数为100000个/孔。In the visual detection of samples No. 1-8 using the droplet digital PCR method in Example 1, samples No. 1-8 are peripheral blood mononuclear cell samples of clinically confirmed AIDS patients. Among them, samples No. 1-4 are from the same patient sample, and No. 5-8 are from another patient sample. And the total number of cells loaded in samples No. 1, 2, 5, and 6 is 30,000 cells/well. The total number of cells loaded in samples No. 3, 4, 7, and 8 is 100,000 cells/well.
图3中A为内参基因GAPDH的阳性液滴/阴性液滴散点图,图3中B为HIV+细胞/HIV-细胞液滴散点图。从图中可以看出ddPCR能够对PBMC实现HIV病毒可视化。A in Figure 3 is a scatter plot of positive droplets/negative droplets of the internal reference gene GAPDH, and B in Figure 3 is a scatter plot of HIV + cells/HIV - cells. It can be seen from the figure that ddPCR can visualize HIV virus in PBMC.
图4中A为1号样本细胞上样总数为30000个/孔的艾滋患者样本PB MC中试验数据,B为1号样本细胞上样总数为100000个/孔的艾滋患者样本PBMC中试验数据,所述试验数据具体为ddPCR检测内参GAPDH的细胞液滴可视化呈现图,蓝色为阴性液滴,橘红色为阳性液滴。In Figure 4, A is the experimental data in the PBMC of the AIDS patient sample with a total number of sample No. 1 cells loaded at 30,000 cells/well, and B is the experimental data in the PBMC of the AIDS patient sample loaded with a total number of sample No. 1 cells, 100,000 cells/well. The test data is specifically a visual presentation of cell droplets using ddPCR to detect the internal reference GAPDH. Blue indicates negative droplets, and orange indicates positive droplets.
图5中A为1号样本细胞上样总数为30000个/孔的艾滋患者样本PB MC中试验数据,B为1号样本细胞上样总数为100000个/孔的艾滋患者样本PBMC中试验数据,所述试验数据具体为ddPCR检测HIV(LTR-Gag引物)的细胞液滴可视化呈现图,蓝色为阴性液滴,橘红色为阳性液滴。In Figure 5, A is the experimental data in the PBMC of the AIDS patient sample with the total number of cells loaded in sample No. 1 being 30,000 cells/well, and B is the experimental data in the PBMC of the AIDS patient sample with the total number of cells loaded in sample No. 1 being 100,000 cells/well. The test data is specifically a visual presentation of cell droplets for ddPCR detection of HIV (LTR-Gag primer), with blue as negative droplets and orange as positive droplets.
图6中A为5号样本细胞上样总数为30000个/孔的艾滋患者样本PB MC中试验数据,B为5号样本细胞上样总数为100000个/孔的艾滋患者样本PBMC中试验数据,所述试验数据具体为ddPCR检测内参GAPDH的细胞液滴可视化呈现图,蓝色为阴性液滴,橘红色为阳性液滴。In Figure 6, A is the experimental data in the PBMC of the AIDS patient sample with the total number of cells loaded in sample No. 5 being 30,000 cells/well, and B is the experimental data in the PBMC of the AIDS patient sample with the total number of cells loaded in sample No. 5 being 100,000 cells/well. The test data is specifically a visual presentation of cell droplets using ddPCR to detect the internal reference GAPDH. Blue indicates negative droplets, and orange indicates positive droplets.
图7中A为5号样本细胞上样总数为30000个/孔的艾滋患者样本PB MC中试验数据,B为5号样本细胞上样总数为100000个/孔的艾滋患者样本PBMC中试验数据,所述试验数据具体为ddPCR检测HIV(LTR-Gag引物)的细胞液滴可视化呈现图,蓝色为阴性液滴,橘红色为阳性液滴。图4-图7结果均说明ddPCR能够直接可视化的检测艾滋患者的单个核细胞内部的HIV核酸扩增情况。In Figure 7, A is the test data in the PBMC of the AIDS patient sample with the total number of cells loaded in sample No. 5 being 30,000 cells/well, and B is the test data in the PBMC of the AIDS patient sample with the total number of cells loaded in sample No. 5 being 100,000 cells/well. The test data is specifically a visual presentation of cell droplets for ddPCR detection of HIV (LTR-Gag primer), with blue as negative droplets and orange as positive droplets. The results in Figures 4 to 7 all illustrate that ddPCR can directly and visually detect HIV nucleic acid amplification inside the mononuclear cells of AIDS patients.
应当理解的是,本发明的应用不限于上述的举例,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,所有这些改进和变换都应属于本发明所附权利要求的保护范围。It should be understood that the application of the present invention is not limited to the above examples. Those of ordinary skill in the art can make improvements or changes based on the above descriptions. All these improvements and changes should fall within the protection scope of the appended claims of the present invention.
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