阅读说明：本技术 基于恒温扩增与基因编辑的数字微流控芯片的核酸检测方法 (Nucleic acid detection method of digital microfluidic chip based on constant-temperature amplification and gene editing ) 是由 李博安 杨朝勇 张睿 孙珍 林康凤 郭剑光 洪欣欣 于 2019-12-11 设计创作，主要内容包括：本发明公开了本发明描述了基于恒温扩增与基因编辑的数字微流控芯片的核酸检测方法,所述方法包含以下试剂和步骤,包括：将待测样品的核酸、恒温扩增混合液以及基因编辑核酸检测液同时注入数字微流控芯片中相对应的位置；接着启动程序通过电流自动驱动液滴,使待检测样品的核酸液滴先与恒温扩增混合液的液滴混合并在芯片上反复流动混匀,在37-42℃,10-15分钟来进行核酸恒温扩增；随后将基因编辑核酸检测液的液滴通过电流自动驱动,并与上述混合液滴混匀,在37℃反应10-30分钟后用荧光探测装置,从而推断目的核酸的有无；该方法显示出恒温下高灵敏度和快速检测的巨大潜能,整个检测过程缩短在1小时之内,自动化程度高,不涉及开盖,杜绝气溶胶污染。(The invention discloses a nucleic acid detection method of a digital microfluidic chip based on constant-temperature amplification and gene editing, which comprises the following reagents and steps: simultaneously injecting nucleic acid of a sample to be detected, the constant-temperature amplification mixed solution and the gene editing nucleic acid detection solution into corresponding positions in the digital microfluidic chip; then starting a program to automatically drive liquid drops through current, so that the nucleic acid liquid drops of the sample to be detected are mixed with the liquid drops of the constant-temperature amplification mixed liquid and flow on the chip repeatedly and uniformly mixed, and the nucleic acid constant-temperature amplification is carried out at 37-42 ℃ for 10-15 minutes; then automatically driving the droplets of the gene editing nucleic acid detection solution by current, uniformly mixing the droplets with the mixed droplets, reacting at 37 ℃ for 10-30 minutes, and then using a fluorescence detection device to deduce the existence of the target nucleic acid; the method shows great potential of high sensitivity and rapid detection at constant temperature, shortens the whole detection process within 1 hour, has high automation degree, does not involve uncovering, and avoids aerosol pollution.)

1. The nucleic acid detection method of the digital microfluidic chip based on constant temperature amplification and gene editing is characterized in that: the detection method comprises the following steps:

step one, simultaneously injecting nucleic acid of a sample to be detected, a constant-temperature amplification mixed solution and a gene editing nucleic acid detection solution into corresponding positions in a digital microfluidic chip;

step two, starting a program, automatically driving liquid drops through current, mixing the liquid drops of the nucleic acid of the sample to be detected with the constant-temperature amplification mixed liquid, repeatedly flowing and uniformly mixing the liquid drops and the constant-temperature amplification mixed liquid on the chip, and carrying out constant-temperature amplification on the nucleic acid for 10-15min at 37-42 ℃;

and step three, driving the droplets of the gene editing nucleic acid detection solution by current, uniformly mixing the droplets with the liquid mixed in the step two, reacting for 10-30min at 37 ℃, and detecting the fluorescence of the mixed droplets on the digital microfluidic chip by using a fluorescence detection device.

2. The nucleic acid detection method of the digital microfluidic chip based on isothermal amplification and gene editing according to claim 1, characterized in that: the nucleic acid of the sample to be detected is DNA or RNA.

3. The nucleic acid detection method of the digital microfluidic chip based on isothermal amplification and gene editing according to any one of claims 1-2, wherein: the constant-temperature amplification mixed solution comprises: recombinase, accessory protein, single-stranded DNA binding protein, DNA polymerase, forward primer, reverse primer and reaction buffer,

if the template is RNA, the isothermal amplification mixture further comprises reverse transcriptase.

4. The nucleic acid detection method of the digital microfluidic chip based on isothermal amplification and gene editing according to claim 3, wherein: the recombinase is 80-600ng/ul recombinase T4 phage uvX protein or escherichia coli RecA, the helper protein is 20-380ng/ul helper protein T4 phage uvY protein, the single-stranded DNA binding protein is 200-800ng/ul single-stranded binding protein gp32, the DNA polymerase is 10-300ng/ul DNA polymerase, the forward primer is 0.2-0.6uM, the reverse primer is 0.2-0.6uM, and the reverse transcriptase is 10-600 ng/ul.

5. The nucleic acid detection method of the digital microfluidic chip based on isothermal amplification and gene editing according to claim 3, wherein: the reaction buffer comprises: 5-20mM MgAc, 20-200mM Tris-HCl, 1-10mM DTT, 4-12% (w/v) polyethylene glycol, 20-100mM potassium acetate, 1-20mM ATP, 0.1-4mM dNTPs, 10-100mM creatine phosphate and 10-80ug/U creatine kinase, wherein the pH value of the buffer is 7.5-8.5.

6. The nucleic acid detection method of the digital microfluidic chip based on isothermal amplification and gene editing according to any one of claims 1-2, wherein: the gene editing nucleic acid detection solution comprises: guide rna (crrna), fluorescent probe, gene editing enzyme Cas12a protein.

7. The nucleic acid detection method of the digital microfluidic chip based on isothermal amplification and gene editing according to claim 6, wherein: the guide RNA (crRNA) is 100-800nM, the fluorescent probe is 10-400nM, the gene editing enzyme Cas12a protein is 50-500ng/ul,

the gene-editing enzyme is a Cas12a protein from a bacterium of the family Lachnospiraceae: lachnospiraceae bacteria ND2006, Cas12a protein gene was cloned into E.coli by genetic engineering for large scale purification.

8. The nucleic acid detection method of the digital microfluidic chip based on isothermal amplification and gene editing according to claim 6, wherein: the length of the basic group of the fluorescent probe is 5, the sequence is TTATT or TCCCT or TTCTT, and one end of the fluorescent probe contains a fluorescent group modification: 5-FAM or FITC, and the other end contains a quenching group modification: BHQ1 or TAMRA.

9. The nucleic acid detection method of the digital microfluidic chip based on isothermal amplification and gene editing according to any one of claims 1-2, wherein: the digital microfluidic chip consists of an upper polar plate and a lower polar plate which are respectively made of glass, wherein one surface of the upper polar plate is covered with an Indium Tin Oxide (ITO) film and a hydrophobic layer formed by Teflon; the lower polar plate substrate is made of glass, a metal chromium electrode, a dielectric layer of a protective electrode and a hydrophobic layer formed by Teflon are sequentially covered on the lower polar plate substrate, the distance between the upper polar plate and the lower polar plate is about 360 micrometers, and gaps are filled with silicon oil.

10. The nucleic acid detection method of the digital microfluidic chip based on isothermal amplification and gene editing according to any one of claims 1-2, wherein: the digital microfluidic chip is connected with a computer end control program through a digital microfluidic instrument.

Technical Field

The invention relates to the field of molecular biology, in particular to a nucleic acid detection method of a digital microfluidic chip based on constant-temperature amplification and gene editing.

Background

With the change of science and technology, more and more high-automation instrument platforms are invented to liberate manpower and replace human work at present. The digital microfluidic technology is based on the generation of liquid drops caused by the surface tension of the liquid, an electric field is used for generating the polar hydrophilicity of the surface of the liquid, the liquid drops are flattened, and the polarization position is controlled through the electric charge and electric field gradient to generate a tension gradient, so that the controlled liquid drops move on the surface of a microfluidic platform. Automation of complex experimental procedures is achieved by combining a series of levels in a series of steps and repeating the operations a number of times. Digital microfluidic platforms are typically constructed of dielectric materials (e.g., glass) with the bottom layer being electrodes responsible for the accumulation of charge and electric field gradients and the top layer typically coated with a hydrophobic layer to generate low surface energy at the droplet contact points. When a voltage is applied, the electrodes are activated and the droplet is manipulated by a gradient of charge and electric field along the electrode lines.

Digital microfluidic technology, also known as chip technology, has numerous advantages in the field of life science research. Including their high potential for portability, can provide a high throughput, highly automated detection platform and significant reduction in the consumption of rare or expensive reagents or samples. Digital microfluidic technology has been applied to create immunoassay devices that greatly simplify and expand the complex experimental procedures by automatically delivering, mixing, culturing, and washing analytes on a chip.

Highly automated, rapid, sensitive nucleic acid detection can be used in a variety of contexts, such as detection of epidemic pathogens, detection of specific genes in physical examination, resolution of genotyping, and various laboratory research tasks. Has great significance for preventing the spread of infectious diseases, screening diseases at early stage and accelerating the laboratory experiment progress. The existing nucleic acid detection method still remains a real-time fluorescence quantitative PCR method, and has the disadvantages of long time consumption, very complex steps and higher cost. The real-time fluorescent quantitative PCR method requires temperature setting, thereby limiting the range of use thereof. Meanwhile, the sensitivity of real-time fluorescence quantitative PCR cannot be detected by single copy, trace nucleic acid is likely to be missed, and meanwhile, the uncovering in the operation can cause very serious aerosol pollution and cause false positive interference on the next detection.

Therefore, in order to solve the problems that the existing nucleic acid detection takes long time, the steps are complex, the cost is high, and trace nucleic acid possibly can be missed, the invention provides a nucleic acid detection method of a digital microfluidic chip based on constant-temperature amplification and gene editing.

Disclosure of Invention

The invention aims to provide a nucleic acid detection method of a digital microfluidic chip based on constant-temperature amplification and gene editing, which solves the problems that the existing nucleic acid detection is long in time consumption, complex in steps, high in cost, possible to miss detection of trace nucleic acid and the like.

The invention is realized by the following steps, and describes a method for detecting nucleic acid on a Digital Microfluidics (DMF) chip platform by combining isothermal amplification and gene editing technology, wherein the method comprises the following reagents and steps: simultaneously injecting nucleic acid of a sample to be detected, the constant-temperature amplification mixed solution and the gene editing nucleic acid detection solution into corresponding positions in the digital microfluidic chip; then starting a program to automatically drive liquid drops through current, so that the nucleic acid liquid drops of the sample to be detected are mixed with the liquid drops of the constant-temperature amplification mixed liquid and flow on the chip repeatedly and uniformly mixed, and the nucleic acid constant-temperature amplification is carried out at 37-42 ℃ for 10-15 minutes; and then automatically driving the liquid drop of the gene editing nucleic acid detection liquid by current, uniformly mixing the liquid drop with the mixed liquid drop, reacting at 37 ℃ for 10-30 minutes, and detecting the fluorescence of the mixed liquid drop on the chip by using a fluorescence detection device, such as a fluorescence microscope, a blue light lamp, an ultraviolet lamp and the like, thereby deducing the existence of the target nucleic acid.

In the operating system, the digital microfluidic platform comprises a digital microfluidic instrument, a chip and computer-side control software. The chip is composed of two parts, namely an upper polar plate and a lower polar plate. The upper polar plate is made of glass, and one surface of the upper polar plate is covered with an Indium Tin Oxide (ITO) film and a hydrophobic layer formed by Teflon; the lower electrode plate substrate is made of glass, and a metal chromium electrode, a dielectric layer of a protective electrode and a hydrophobic layer formed by Teflon are sequentially covered on the lower electrode plate substrate. The distance between the upper polar plate and the lower polar plate is about 360um, and the gap is filled with the silicone oil.

The nucleic acid to be detected is DNA or RNA.

The constant-temperature amplification mixed solution comprises: recombinase, accessory protein, single-stranded DNA binding protein, DNA polymerase, forward primer, reverse primer and reaction buffer. If the template is an RNA nucleic acid amplification reaction solution, a reverse transcriptase is also required. The reverse transcriptase converts RNA into DNA, and front and back primers, uvs X protein, uvs Y protein and gp32 protein ensure constant temperature amplification of nucleic acid, so that the probability of recognition by gene editing enzyme Cas12a protein is increased.

Further, the isothermal amplification mixture comprises: 80-600ng/ul recombinase T4 phage uvX protein or Escherichia coli RecA, 20-380ng/ul helper protein T4 phage uvY protein, 200-800ng/ul single-stranded binding protein gp32, 10-300ng/ul DNA polymerase, 0.2-0.6uM forward primer, 0.2-0.6uM reverse primer and reaction buffer. If the template is an RNA nucleic acid amplification reaction solution, 10 to 600ng/ul of reverse transcriptase is also required.

The reaction buffer comprises: MgAc, Tris-HCl, DTT, polyethylene glycol, potassium acetate, ATP, dNTPs, creatine phosphate and creatine kinase.

Further, the reaction buffer comprises: 5-20mM MgAc, 20-200mM Tris-HCl, 1-10mM DTT, 4-12% (w/v) polyethylene glycol, 20-100mM potassium acetate, 1-20mM ATP, 0.1-4mM dNTPs, 10-100mM creatine phosphate and 10-80ug/U creatine kinase, wherein the pH value of the buffer solution is 7.5-8.5.

The gene editing nucleic acid detection solution comprises: guide rna (crrna), fluorescent probe, gene editing enzyme Cas12a protein.

Further, the gene-editing nucleic acid detection solution comprises: 100-800nM guide RNA (crRNA), 10-400nM fluorescent probe, 50-500ng/ul gene editing enzyme Cas12a protein. Wherein the gene editing enzyme is Cas12a protein from a bacterium of the family Lachnospiraceae: lachnospiraceae bacterium ND 2006. Cas12a protein gene is cloned into Escherichia coli for mass purification by genetic engineering.

The length of the basic group of the fluorescent probe is 5, the sequence is TTATT or TCCCT or TTCTT, and one end of the fluorescent probe contains a fluorescent group modification: 5-FAM or FITC, and the other end contains a quenching group modification: BHQ1 or TAMRA. The device for further detecting fluorescence may be a fluorescence microscope, blue light, uv lamp or any other instrument capable of reading fluorescence on the chip.

The invention has the beneficial effects that: the method adopted by the invention shows great potential of high sensitivity and rapid detection at constant temperature, and the whole detection process is shortened within 45 minutes by combining a digital microfluidic platform, the whole detection process is highly automated, the reaction is totally in a chip, the cover opening is not involved, and the aerosol pollution is avoided. And no matter the micro-fluidic chip needs the nucleic acid, enzyme or reaction liquid of the sample to be detected, the quantity of the nucleic acid, the enzyme or the reaction liquid is extremely small, so that the cost of nucleic acid detection is greatly reduced. Solves the problems that the prior nucleic acid detection takes long time, has complex steps and higher cost, and can miss detecting trace nucleic acid, and the like.

Drawings

FIG. 1 is a schematic diagram of the corresponding position of a micro-fluidic chip plate provided by the invention;

FIG. 2 is a schematic diagram of a nucleic acid detection process of the digital microfluidic chip based on isothermal amplification and gene editing provided by the present invention;

FIG. 3 is a graph showing the results of detecting Mycoplasma pneumoniae DNA in nasopharyngeal swab samples according to the present invention;

FIG. 4 is a graph showing the results of detecting influenza A and B viral RNA in respiratory secretions provided by the present invention;

FIG. 5 is a graph showing the results of detecting hepatitis B virus DNA in serum according to the present invention;

FIG. 6 is a graph showing the results of detecting tobacco mosaic virus RNA in plants according to the present invention;

FIG. 7 is a graph showing the results of sensitivity of the present invention for detecting influenza A virus in respiratory secretions;

FIG. 8 is a graph showing the results of sensitivity control for detection of influenza A virus in respiratory secretions using real-time fluorescent quantitative PCR.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

In order that the invention may be more fully understood, there now follows a more general description of the invention, as well as the preferred embodiments thereof. The invention may, however, be embodied in many different forms and the scope of the invention is not limited to the embodiments described herein. Rather, the following specific embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.