WO2021147873A1

WO2021147873A1 - Method for amplifying a target nucleic acid

Info

Publication number: WO2021147873A1
Application number: PCT/CN2021/072768
Authority: WO
Inventors: Fu Xin CHU; Xiao Nan XU; Ru Yuan SONG; Kwok Fai Joseph Chow
Original assignee: Thunderbio Innovation Ltd
Priority date: 2020-01-21
Filing date: 2021-01-19
Publication date: 2021-07-29

Abstract

Provided is a method for amplifying a target nucleic acid, comprising: forming a plurality of compartments, each of which includes a part of sample from the same sample and the necessary reagents for amplification; amplifying each compartment for the first time; combining the amplified mixtures of all compartments; amplifying the mixture for the second time.

Description

METHOD FOR AMPLIFYING A TARGET NUCLEIC ACID

FIELD OF THE INVENTION

The present invention belongs to the technical field of nucleic acid amplification, more specifically, it relates to how to achieve high-sensitivity detection in the case of low-level targets in a test sample.

BACKGROUND

Polymerase Chain Reaction (PCR) is an in vitro nucleic acid amplification technique that can rapidly amplify trace amount of nucleic acids in vitro, and the technique is widely used in molecular detection and analysis.

Fluorescence Quantitative Polymerase Chain Reaction (qPCR) is a traditional PCR technique that is widely used in molecular biology on a large scale and greatly promotes the development of molecular biology in various fields. As a new generation of PCT technique, qPCR can achieve qualitative and quantitative detections. Based on different quantitative strategies, it is divided into relative quantification and absolute quantification. Relative quantification is to perform quantitative analysis of the expression of genes to be tested by using the expression of the housekeeping gene as a reference, which is often used for analysis of gene expression levels. Absolute quantification is to perform quantitative determination of nucleic acid molecules in unknown samples using the standard curves established between the standards with a known number of nucleic acid molecules and the cycle thresholds (CT) . However, there are factors such as amplification preference or susceptibility to inhibitors in the process of qPCR amplification, and it is difficult to maintain the same amplification efficiency among different samples. Therefore, the CT has a significant deviation in the nucleic acid quantitative analysis process, which leads to an obvious error of the quantity of nucleic acids in the original samples. When used for quantifying nucleic acids, qPCR has the drawbacks of poor sensitivity, accuracy, and reproducibility; and its sensitivity and precision are greatly limited when the target sequence content is low, the expression difference is small and the reaction system contains a large number of background sequences or inhibitors, etc.

The third generation of PCR, i.e. Digital PCR (dPCR) , emerges at this time. For dPCR technology, reagents are allocated into substantially equal volume of compartments, and the templates are randomly allocated to a large number of compartments with the reagents, to achieve PCR amplification of a single template molecule. Finally, the number of nucleic acid molecules is calculated based on the proportion of positive response units and Poisson distribution, to achieve direct and absolute quantification that is independent of standard curves. Compared with qPCR, it has four advantages: high sensitivity, high accuracy, high tolerance and absolute quantification, therefore, it has a broad application prospect. With these advantages, dPCR has unparalleled application prospects in rare site mutation detection, copy number variation analysis and complex sample gene detection, etc.

At the same time, the nucleic acid isothermal amplification, which is different from PCR amplification method, has developed rapidly in recent years. Nucleic acid isothermal amplification technology is a new type of molecular biology technology that is developed in recent years. With this technology, under isothermal conditions without using thermal cycling equipment, a large number of nucleic acid molecules are replicated, and a reporter can be introduced to reflect the amplification conditions. The common methods include RCA (Rolling circle amplification, RCA) , RPA (Recombinase polymerase amplification, RPA) , MDA (Multiple strand displacement amplification, MDA) , HDA (Helicase-dependent isothermal DNA amplification, HDA) , and LAMP (Loop-mediated isothermal amplification, LAMP) , etc.

Therefore, with the emergence of dPCR technology, digital isothermal nucleic acid amplification (dINAA) developed. Unlike dPCR, DINAA does not rely on thermal cycling, and only requires a constant temperature to complete digital amplification. In principle, there is no difference between them, only the PCR method is replaced by an isothermal amplification method. Therefore, with the emergence of DINAA, digital nucleic acid detection (dNAD) has the potential to achieve on-site analysis or point-of-care testing (POCT) .

Although digital nucleic acid amplification detection has great advantages, it has many limitations such as high equipment price, low experimental throughput, and long time of experiment, etc., which restricts its applications on a large scale.

With the continuous expansion and in-depth development of molecular detection applications, the requirements for analytical techniques are increasing. At present, the most widely used PCR/qPCR technology cannot meet the actual detection needs any longer, while the digital nucleic acid amplification detection technology with better performance that is represented by dPCR and droplet digital PCR (ddPCR) , cannot be promoted quickly. The specific causes may be as follows.

First of all, in terms of traditional PCR/qPCR, competition inhibition occurs during multiplex amplification due to the primer preference, which greatly increases the difficulty of multiplex amplification, especially when the NGS library is constructed, different primers have different preferences, which may cause difference in PCR amplification efficiency and greatly affects the final sequencing results. Secondly, in the detection of rare sites, the presence of a large number of background nucleic acids may interfere with the amplification of rare sites, resulting in insufficient sensitivity and accuracy. Thirdly, when detecting complex samples, such as paraffin-embedded samples and stool samples, inhibitors in the nucleic acid extraction solution may inhibit the normal running of the amplification reaction. Fourthly, when performing analysis of very low number of copies, the amplification sensitivity is insufficient and the experiment repeatability is not good.

With the emergence of digital amplification technology, although some of the above problems can be solved well, digital instruments are still in the early stage of applications and have the following objective factors that have affected their extensive citations. First, the equipment, reagents and supplies are expensive, with high threshold for use; second, the detection need long time, the experimental throughput is low, which is not suitable for high-throughput screening; third, the operators are accustomed to traditional methods such as qPCR; fourth, digital amplification techniques can only be used for endpoint fluorescence quantification and not for real-time fluorescence quantification.

Therefore, it is necessary to improve the exixting traditional methods, to meet the needs for the existing technology development and applications, aviod the shortcomings of traditional technologies and solve some disadvantages of digital PCR detection.

SUMMARY

In order to solve these traditional problems, the technical solutions of the present invention combines digital amplification and traditional amplification methods, to improve the problem of traditional multi-amplification competition inhibition and make different amplifications occur more evenly. In addition, it can obviously enhance the ability to detect rare sites, reduce background interference and improve the sensitivity and accuracy, increase the tolerance to inhibitors and improve the ability to detect complex samples. Furthermore, it can enhance the ability to amplify very low copy nucleic acids and improve the repeatability of experiments, and solve the drawbacks of long time-consuming, low-throughput and high cost of digital PCR.

To achieve this object, in a first aspect, the present invention provides a method for amplifying a target nucleic acid, comprising: forming a plurality of compartments, each of which includes a part of sample from the same sample and the necessary reagents for amplification; amplifying each compartment for the first time; combining the amplified mixtures of all compartments; amplifying the mixture for the second time.

In some preferred embodiments, the second amplification is real-time fluorescent nucleic acid amplification. In some embodiments, the second amplification is a real-time quantitative amplification, for example, real-time fluorescent PCR or real-time fluorescent quantitative PCR.

In some embodiments, a plurality of targets is included in the same sample. In some embodiments, each compartment includes one of the plurality of targets, or each compartment includes two or more of the plurality of targets. In some embodiments, the necessary reagents for amplification include the necessary reagents for amplifying one of the targets, or the necessary reagents for amplification include the necessary reagents for amplifying all targets.

In another aspect, the present invention provides a semi-digital nucleic acid amplification method, comprising following steps:

a) forming a compartment, each of which includes a part of the same mixture, and the mixture includes a plurality of targets;

b) amplifying the targets in each compartment for the first time;

c) combining the compartments, to make the same mixture of all compartments to combine into a whole;

d) amplifying the targets in the whole to the endpoint of the amplification.

In one embodiment, it further comprises collecting the data indicative of each target during the first or second amplification of the targets in the mixture or/and after the amplification.

In one embodiment, the compartment may be a compartment containing a part of the mixture, which may be substantially uniform in size, or may have different sizes. The compartments may be distributed in a predetermined size or in a random size continuously.

In one embodiment, the “forming compartment” may be carried out by any suitable procedure, in any suitable manner, having any suitable characteristics. For example: microfluidics, sample agitation (e.g., shaking, agitation) , centrifugation, membrane vibration, and so on.

In one embodiment, the compartment contains droplets and each droplet is a compartment, and further contains all continuous phases surrounding the droplet. A "droplet" refers to a small volume of fluid, generally a liquid, which is surrounded by a different fluid, typically an immiscible fluid (eg, oil) , into an emulsion that is injected into the continuous phase. "Oil" means any fluid that is immiscible with water, including but not limited to organic fluids of any combination of carbon, hydrogen, fluorine, silicon, and oxygen, for example, one of silicone oil, mineral oil, fluorocarbon oil, vegetable oil, or a combination thereof.

In one embodiment, the continuous phase further includes a surfactant.

In one embodiment, a "target" includes the analyte (or region) of interest, and may also be referred to as the analyte or identification label.

In one embodiment, a target may be a target sequence contained in a molecule or a complex, and may form part (or all) of a molecule or a complex with it, for example, nucleotide sequences (e.g., single-stranded or double-stranded sequences) , amino acid sequences, etc.

In one embodiment, a target may be a template (or may be provided by a template) ; for example, genomic DNA, free DNA, mitochondrial DNA, RNA, miRNA, etc.

In one embodiment, a mixture may be any suitable reagent.

In one embodiment, a mixture includes all reagents used for amplification, and the amplification may be performed or inspected in the amplification mixture.

In one embodiment, a mixture may further include other regents for amplification, for example, restriction endonuclease, bisulfite.

In one embodiment, a mixture may include any combination of at least one primer or one primer pair, at least one replicase and deoxynucleotide (or nucleotide) triphosphate (dNTP and NTP) targets.

In one embodiment, a mixture may include at least one target.

In one embodiment, target molecules are randomly distributed before compartment, and the random distribution can be described as a Poisson distribution.

In one embodiment, a compartment in the compartment group includes one copy without target or with at least a target, or a combination thereof.

In one embodiment, the mixture also contains a reporter. "Reporter" refers to at least one detection reagent or group of detection reagents that report a condition, such as the presence or absence of a target or abundance, and/or whether a reaction has occurred or the extent of a reaction.

In one embodiment, the reporter may include a non-specific reporter, for example, Eva Green.

In one embodiment, the reporter may include a specific reporter, such as a probe.

In one embodiment, a probe may include an oligonucleotide and is a partner that specifically binds to the target sequence and/or the sequence of an amplicon generated by amplifying the target sequence.

In one embodiment, the type of a probe can adopt various existing structures, for example, a linear probe, a stem-loop structure probe, etc.

In one embodiment, the type of fluorescent agent of a fluorescent probe is not limited, for example, FAM, HEX, VIC, ROX, CY3, CY5, CY5.5, etc.

In one embodiment, the quencher type of a fluorescent probe is not limited, for example, BHQ1, BHQ2, BHQ3, Trama, MGB, etc.

In one embodiment, the method further includes the step of designing at least one target probe based on the melting temperature algorithm.

In one embodiment, the method further includes the step of designing any target probe based on the melting temperature algorithm.

In one embodiment, the method further includes designing at least one target primer of any target primer pair based on the melting temperature algorithm.

In one embodiment, the method further includes the step of designing each target primer of any target primer pair based on the melting temperature algorithm.

In one embodiment, the amplification of the target of the compartment may be further referred to as the first amplification.

In one embodiment, the step of amplifying includes amplifying the target to form a target amplicon. “Amplification" refers to a reaction or method that generates a target, for example, a copy of a target sequence.

In one embodiment, a copy may be a strict or non-strict copy of the target.

In one embodiment, the amplification can produce an exponential or linear increase in the number of copies.

In one embodiment, the amplification of each target may optionally occur in a compartment containing at least one copy of the target.

In one embodiment, the amplicon may be single-stranded or double-stranded, or a combination thereof. In one embodiment, the form of the amplicon may be a DNA copy of a DNA target sequence, a DNA copy of an RNA target sequence, an RNA copy of a DNA target sequence, or an RNA copy of an RNA target sequence.

In one embodiment, the first amplification or the second amplification may be performed isothermally or non-isothermally.

In one embodiment, isothermal amplification includes a method of incubating reagents at a constant temperature or at room temperature, for example, RCA (Rolling circle amplification, RCA) , RPA (Recombinase polymerase amplification, RPA) , MDA (Multiple strand displacement, MDA) , HDA (Helicase-dependent isothermal DNA amplification, HDA) , LAMP (Loop-mediated isothermal amplification, LAMP) , etc.

In one embodiment, the non-isothermal amplification includes incubation by heating reagents, including: no or at least one pre-temperature, and one or more cycles containing a denaturation temperature, an annealing temperature, and/or an extension temperature. For example, polymerase chain reaction (PCR) or ligase chain reaction, etc.

In one embodiment, the method further includes a step of inspecting different annealing temperatures for the amplification procedure in different groups containing the same reaction mixture.

In one embodiment, the method further includes a step of selecting an annealing temperature based on the step of inspecting different annealing temperatures, wherein the step of determining a level is performed based on an amplification step performed at the selected annealing temperature.

In one embodiment, for amplification of the target in the compartment, a single target molecule in a sub-compartment may be amplified to generate a target amplication.

In one embodiment, for amplification of the target in the compartment, the sole target molecule in the compartment may be amplified to generate a target amplicon, to avoid being affected by the amplification of other targets. For example, when rare target molecules are amplified, they can be protected from being affected by background nucleic acid molecules.

In one embodiment, amplifying the target in the compartment may reduce the effect of the inhibitor in the mixture on the target amplification. Due to existence of a large number of compartments, the inhibitors in the mixture are dispersed. In each compartment, the concentration of the target molecule relative to the inhibitor is greatly increased, so the effect of the inhibitor on target amplification can be reduced.

In one embodiment, after pre-amplification, rare targets, and/or single-molecule targets, and/or targets with interfering substances can be fully amplified in the compartment.

In one embodiment, combining the compartments includes a method of destroying the presence of the mixture in the continuous phase to make the mixture as a whole. After the compartments are combined, the mixture can be integrated into a whole, and the target can be analyzed using existing approaches, for example, real-time PCR, fluorescent thermostatic amplification, nucleic acid gel electrophoresis, etc. This method can reflect the target information by only collecting the whole target data rather than collecting all individual target data.

In one embodiment, the method of combining compartments includes physical intervention and chemical intervention. The preferred option is a physical method, for example, by using sound and light, electricity and heat, etc, the stability of droplets will be destroyed without the addition of any substance in a system consisting of droplets and continuous phases, to achieve the combination or fusion of all compartments. Chemical intervention means that the stability of droplets is destroyed by adding substances in a system consisting of droplets and continuous phases. These chemical substances will not destroy the secondary amplification, and the template or target for the secondary amplification, the primers, and the necessary reagents are from the combined mixture.

In one embodiment, the physical intervention includes thermal (e.g., rapid temperature change) , acoustics (e.g., ultrasound) , electricity (e.g., high-voltage electric field) , mechanics (e.g., centrifugation) , etc.

In one embodiment, the chemical intervention includes the addition of chemical reagents. The addition of the chemical reagents will not substantially destroy the necessary reagents for amplification in the mixture, such as enzymes, etc.

In one embodiment, the target after the first amplification will be further amplified in the subsequent overall amplification.

In one embodiment, the method for collecting amplification data includes a method implemented by detecting light of a reporter. The method of collecting amplification data herein can be understood as detecting the presence or quantity of target nucleic acids after the end point of the second amplification. In one embodiment, the method for collecting amplification data includes gel electrophoresis and imaging. In one embodiment, collecting the data indicative of each target may include collecting luminescence from the reporter. In one embodiment, collecting the luminescence from the reporter includes the light intensity emitted by the reporter. In one embodiment, collecting the luminescence from the reporter includes the time corresponding to the light intensity emitted by the reporter. In one embodiment, the light emitting forms include photoluminescence, chemiluminescence, electroluminescence, etc. In one embodiment, photoluminescence is any light-emitting phenomenon produced in response to the radiation of excitation light, including fluorescence, phosphorescence, etc.

In one embodiment, the target information includes the level of the target. The level refers to the quantitative or qualitative and/or relative or absolute quantitative abundance of the target. The level may be present or absent, as defined in particular by thresholds, amounts and concentrations.

In one embodiment, a method for calculating target information based on the collected target data is also included. For example, the presence or absence of the target is estimated based on the fluorescence intensity and threshold; and the relative concentration of the target is estimated based on the CT.

In one embodiment, the semi-digital nucleic acid amplification and detection method can achieve better repeatability at lower concentrations.

In one embodiment, the semi-digital nucleic acid amplification and detection method can be used for the amplification and detection of extremely low copy nucleic acid molecules.

In one embodiment, the semi-digital nucleic acid amplification and detection method makes the amplification of different targets to be more balanced. The amplification mechanism of compartments allows various targets to be amplified without interfering with each other, increases the initial concentration of various target molecules in the overall amplification and reduces competition in amplification (to adjust droplet size and/or number) .

In one embodiment, the semi-digital nucleic acid amplification and detection method allows the amplification of genomic DNA to be more balanced, for example, the construction of a second-generation test gene library.

In one embodiment, the semi-digital nucleic acid amplification and detection method can be used for multiplex amplification and detection.

In one embodiment, the semi-digital nucleic acid amplification and detection method can be used for the amplification and detection of rare genes, for example, detection of cancer mutations.

In one embodiment, the semi-digital nucleic acid amplification and detection method can enhance the tolerance to inhibitors. Firstly the compartments with high tolerance are amplified, to enhance the abundance of a target gene and increase the initial concentration of the target molecule as a whole, and reduce the effect of inhibitors on the overall amplification.

In one embodiment, the semi-digital nucleic acid amplification and detection method can be used for the detection of complex samples.

In some embodiments, the preliminary pre-amplification or the first amplification is performed in multiple droplets (for example, the first ten cycles) , and the second amplification is performed after multiple droplets are fused. In some embodiments, the first amplification and the second amplification are performed in the same amplification container, for example, a test tube, a PCR tube, and an EP tube.

In some embodiments, no additional reagents are added during or before the second amplification. In some embodiments, no additional essential reagents are added, for example, one or more of primers, enzymes, nucleotides, etc.

In one embodiment, the semi-digital nucleic acid amplification and detection method can enhance the sensitivity and stability and reduce the limit of detection. The pre-amplification of a single target molecule in the compartment has raised the target molecule concentration to a higher level, and the subsequent overall target amplification can be smoothly carried out. At the same time, it is also possible to achieve a single copy of the detection limit of traditional nucleic acid amplification.

The present invention provides a nucleic acid amplification device. The device includes a PCR amplification heating element. A plurality of holes is provided on the heating element, and the holes are used to accommodate an amplification container, for example, a PCR tube. Electrodes are disposed around the holes and the electrodes are used for fusing and breaking droplets in the amplification container.

In some embodiments, a high-voltage electric field is generated between the electrodes, and the high-voltage electric field can fuse and break the droplets in the amplification container. In some embodiments, the device includes a refrigerating element, a heat dissipation element, or a fan.

In some embodiments, the amplification container is disposed between the electrodes, and the amplification container contains droplets.

In some embodiments, the droplets are in the form of a liquid.

BENEFICIAL EFFECTS

Compared with traditional amplification or detection methods, such as commonly used PCR/qPCR/isothermal amplification, etc., the technical solutions of the present invention have the following advantages.

1. It can reduce the competitive inhibition of multiple amplification, improve the performance of multiple amplification, and allow more balanced amplification of various genes. When traditional multiplex amplification is performed, due to the preference of the primers or competition inhibition in the same system, some amplifications are more likely to occur and some amplifications are more difficult to occur in multiplex amplification, therefore, the amplification of some nucleic acids will be suppressed. Furthermore, for example, when library is constructed for NGS, a large number of multiplex amplifications are needed. The existence of competition inhibition will greatly affect the accuracy of the final sequencing. In the process of multiplex amplification, it is desired to amplify multiple target target nucleic acid regions in one time, but the number of original copies of each target nucleic acid in the sample is different, thus, the traditional amplification method will cause missing of detection.

Similarly, according to the present invention, through the first digital pre-amplification, each nucleic acid molecule is independently amplified in the compartment. Only the primers corresponding to the nucleic acid molecules are effective in the compartment which reduces the competitive inhibition, and each nucleic acid can be evenly amplified, so that the concentration of those target nucleic acids that are originally with low copies can be increased. In the second amplification, because the concentration of each nucleic acid molecule has reached a relatively high concentration, competition inhibition will be relatively reduced, which will reduce the probability of missed detection and enhance the accuracy of amplification.

2. It can reduce the interference of unrelated genes, improve the performance of rare target site amplification, enable rare sites to be amplified or detected more sensitively and accurately, and the sensitivity of rare site mutation detection can be increased by ten times or even one hundred times. When detecting rare sites by traditional amplification, for example, the detection of Single Nucleotide Polymorphism (SNP) , it is difficult to perform detection due to the presence of a large number of similar background nucleic acids, and basically, the sensitivity can only reach 0.1%, or even low, difficult to meet the demands. In the first digital amplification of the present invention, the detection of rare sites is not interfered by the background detection, so that rare sites can be more accurately amplified. In the second amplification, the rare site nucleic acid molecules have been amplified to a relatively high level, therefore the amplification and detection in the second amplification is less interfered by the background genes, and finally the sensitivity can reach 0.01%.

Thecomplete digital amplification detection method uses the fluorescence of the compartmentto read the signals, and the detection is performed baseed on the number of compartments and the presence or absence of fluorescence signals. This approach has the advantage of absolute quantification. The technical solutions of the present invention, i.e. preliminary digital amplification-combining compartments (combined droplets) -secondary amplification, have the advantages that the digital amplification does not have.

1. Lower cost. Compared with expensive digital amplification instruments and reagents, only a stabilizer is added to the traditional amplification reagent, and the reagent has low cost, and no even no reagent is required. Only additional droplet generation devices and droplet fusion devices are required. Theamplification or analysis instrument is a PCR or qPCR instrument, which is provided in most laboratories, and expensive analytical instruments are not required to buy. A cost-effective electric field PCR instrument or qPCR instrument can be used to complete the whole amplifciataionm process in one time (preliminary digital amplification-combining compartments (combining droplets) -secondary amplification) . At the same time, the droplet amplification and subsequent second amplification of digital PCR can be completed on the same device without transferring the amplified reagents. In addition, the secondary amplification does not need to add any reagents necessary for amplification. The reason is that when droplet amplification is performed, because there is no target nucleic acid in a large number of droplets and the amplification reagents are not consumed, when the liquid droplets are infused, a large amount of enzymes and necessary reagents are released, which can basically meet the needs of secondary amplification and save the costs.

2. Short time consuming and high throughput. At present, the digital PCR test takes a long time (the detection can only be performed after the end of the reaction) , especially in the final signal analysis stage, it takes a lot of time. As the experimental throughput increases, the time is greatly increased; there are only a few dozens of throughput for many platforms (generally it takes 4-6hrs to complete the test, and the cost is about 80 yuan per sample) . The method of the present invention only needs to increase the droplet generation step and the droplet fusion step. With the special droplet fusion device, the entire fusion can be completed in one minute. Other time-consuming is the same as traditional PCR or qPCR. The isothermal amplification method can even complete the detection in half an hour, with high efficiency. At the same time, the throughput is no different from the current traditional methods. It can perform 96, 384 or even 1024 at a time, and it will not greatly increase the experimental time, suitable for high-throughput screening. In addition, for a large number of detections, digital PCR can only be used to detect at the end point of the amplification, which is the main reason for time-consuming. The present invention first performs digital PCR amplification and then performs real-time fluorescent PCR, which can produce results quickly. Once the results appear, the reaction can be terminated. This is particularly effective for some preliminary screening of specific genes.

3. The analytical method is more acceptable. The analysis method of the present invention completely adopts traditional amplification methods, such as electrophoretic band analysis, amplified fluorescence curve Ct analysis, etc., without additional training, so it is easily accepted and recognized.

4. Full analysis is possible for low concentration samples. When the concentration of the target nucleic acid contained in the sample is extremely low, if multiple target nucleic acid amplifications are performed, the nucleic acid of low relative content is not easily amplified. With the method of the present invention, the results can be produced very quickly. In particular, in the second step of real-time quantitative PCR, once a peak appears, it can be initially determined whether a relatively low concentration of the target nucleic acid exists, and then subsequent analysis can be performed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow chart of the analysis of the present invention.

FIG. 2 is a cross-sectional configuration diagram of a nucleic acid amplification apparatus according to an embodiment of the present invention.

FIG. 3 is a schematic diagram illustrating the principle of traditional nucleic acid amplification and the two-step amplification method of the present invention in specific embodiments of the present invention.

FIGS. 4A-4B are comparison diagrams of the experimental results of the traditional nucleic acid amplification method and the method of the present invention for detecting the target nucleic acid content in the samples when multiplex amplification is implemented in the specific embodiment of the present invention (template A: HPV16: 100000copies/uL, HPV58: 100,000 copies/uL, HPV18: 1000copies/uL) . Among them, FIG. 4A is a traditional method, and FIG. 4B is a method of the present invention.

FIGS. 5A-5B are comparison diagrams of the experimental results of the traditional nucleic acid amplification method and the method of the present invention for detecting the target nucleic acid content in the samples when multiplex amplification is implemented in the specific embodiment of the present invention (template B: HPV16: 100000copies/uL, HPV58: 100000 copies/uL, HPV18: 100copies/uL) . Among them, FIG. 5A is a traditional method, and FIG. 5B is a method of the present invention.

FIGS. 6A-6B are comparison diagrams of the experimental results of the traditional nucleic acid amplification method and the method of the present invention for detecting the target nucleic acid content in the samples when multiplex amplification is implemented in the specific embodiment of the present invention (Template C: HPV16: 100000 copies/uL, HPV58: 100000 copies/uL, HPV18: 10copies/uL) . Among them, FIG. 6A is a traditional method, and FIG. 6B is a method of the present invention.

DETAILED DESCRIPTION

The structure or the technical terms used in the present invention will be further described below. If it is not specifically stated, it will be understood and explained according to the common meanings in the art.

Embodiments:

In order to investigate the performance of semi-digital amplification methods for detecting low-concentration genes in multiplex amplification compared to traditional amplification methods, a triple PCR detection was designed (target nucleic acids: HPV16, HPV18, HPV58) . Under the higher concentration background of two gene templates, another gene template was linearly diluted. The experimental group used digital-real-time quantitative PCR (dqPCR) , and the control group used traditional qPCR.

Table 1: Reaction solution formula

Table 2: Primer sequences

Table 3: Nucleic acid concentrations in templates of different experimental groups

Ps. genes used were plasmids.

Operating steps:

1. Mix the reagents of each tube according to the above formula. After vortexing, aliquot 18uL to each PCR reaction tube (Table 1) , and a total of 24 tubes were prepared. Twelve tubes were used in the dqPCR experimental group, and each tube was added with 5uL of stabilizer. The remaining 12 tubes were used as the qPCR control group, and each tube was added with 5uL ddH2O.

2. Prepare three templates A, B, and C according to the experimental design. Add the three kinds of template solutions (table 3) to 12 tubes in the dqPCR experiment group, 2uL /tube, and one template solution for 4 tubes respectively (the final volume of each tube: 25uL) . Similarly, add the three kinds of template solutions to 12 tubes in the qPCR control group, 2uL /tube, and one template solution for 4 tubes respectively (the final volume of each tube: 25uL) . Cover the PCR tube, vortex, and centrifuge quickly for a few seconds.

3. Add the premix to the dqPCR experimental group. 25uL of premix was added to the corresponding holes of the droplet generation chip for each reaction, and the dqPCR special droplet was formed into oil (70uL was added to the corresponding holes of the droplet generation chip) . The droplet generation chip was placed in a droplet generator, and the droplet generator was then run, to generate approximately 25,000 droplets.

4. After the machine finished the running, 40uL of all droplets and oil phases were transferred to PCR tubes (40uL per tube, 12 tubes in total, three combinations: A, B, C) .

5. Place the PCR tubes containing the droplets of the dqPCR experimental group and the PCR tubes containing the premix of the qPCR control group in the PCR instrument for the first PCR reaction. Then carry out the first amplification according to the following procedure:

The first stage: 94 ℃ for 60 seconds, 1 cycle;

The second stage: 15 seconds at 95 ℃, 30 seconds at 55 ℃, 30 seconds at 72 ℃, 10 cycles;

Third stage: 4 ℃ for 10 seconds.

6. Place the PCR reaction tubes of the dqPCR group on the droplet crusher, and run the droplet crusher for 1min (effective voltage 5000V, DropX BiotechCo., Ltd. ) . After the droplets were broken, the PCR tubes were centrifuged quickly.

7. Place two groups of PCR reaction tubes into the real-time PCR instrument, run the instrument, and perform the second amplification according to the following procedure:

95 ℃ for 15 seconds; 55 ℃ for 30 seconds (fluorescence collection) ; 72 ℃ for 30 seconds; 40 cycles.

8. After the machine finished eachcycle of running, the Ct values of each group were recorded.

Results were as follows: Table 4

Results were summarized in the following Table 3.

PS. ND: No obvious fluorescence curve

In fact, in Table 3, the ratio between three different target nucleic acids was FAM: CY5: VIC =10000: 1: 10000; the ratio in the tube B was 1000: 1: 1000; the ratio in the tube C was 100: 1: 100. The ratios of CY5 to the other two types were 1: 10000, 1: 1000 and 1: 100, respectively.

From the above results, conclusions could be made as follows:

1. In the two groups of experiments, for the target genes with high copy number, the CV of the Ct value of the background gene (HPV16 /HPV58) was less than 1%, the experiment repeatability was good, and the same effect could be achieved.

2. However, for the HPV 18 template, when the HPV 16 and HPV 58 templates were in a large number of copies (2 *10 ⁵) , the HPV 18 templates had a copy number of 10, 100 and 1000 copies /uL. According to the traditional amplification method (traditional PCR instrument) , for the level of 100copies /uL, it could not achieve accurate detection, and missed detection would occur. In the case of multiplex amplifications, the relative copy number of CY5 was less and efficient amplification could not be achieved. However, using the two-step detection method of the present invention, the samples of 100copies /uL could be accurately detected, and the detection limit for dqPCR was 10 times higher than that of qPCR. In the context of high-concentration HPV16 and HPV58 genes (greater than or equal to 1000 times) , the detection limit of qPCR system on HPV18 was 1000 copies /uL. After using the dqPCR method, the detection limit could reach 100 copies /uL, which was increased by 10 times. This indicated that, using the method of the present invention, because of two-step detection, the minimum detection limit could be increased by more than 10 times than the traditional detection method. Therefore, the semi-digital amplification method could enhance the detection limit of low-concentration genes in multiplex amplification and the sensitivity of multiplex amplification compared to the traditional methods.

3. As shown from the actual fluorescence amplification curves, when the ratio of HPV18 to HPV16 and HPV58 was 1000: 10000, the traditional real-time fluorescent PCR and the method of the present invention could achieve amplification. However, when the ratio of HPV18 to HPV16 and HPV58 was 100: 10000, the traditional real-time fluorescent PCR could not amplify the HPV18 gene, and the method of the present invention could achieve the amplification and detection.

4. By this way, it could be understood that if multiplex process was implemented, such as 4-, 5-, 6-, and 7-plex, especially in the process of high-throughput detection of mutant genes or sequencing, the trace amount of mutation sites could be effectively detected by the method of the present invention.

Based on the above experimental data, when some specific target nucleic acids were found to be low in content relative to other nucleic acids, if a digital PCR method was used, a large number of droplets should be prepared, and it was desirable that each droplet contained a single copy of the sample (target nucleic acid and other nucleic acids) . After amplification reached the end point, it needed to take a long time to detect the amplification result of each droplet. Using the method of the present invention, a small number of droplets could be prepared for simple preliminary amplification according to the amount of target nucleic acid, so that the concentration of low-level target nucleic acid could be increased, and then traditional PCR amplification could be used to quickly obtain the preliminary results.

Claims

A method for amplifying a target nucleic acid, comprising: forming a plurality of compartments, each of which includes a part of sample from the same sample and the necessary reagents for amplification; amplifying each compartment for the first time; combining the amplified mixtures of all compartments; amplifying the mixture for the second time.
A method for amplifying multi-target nucleic acid, comprising: forming a plurality of compartments, each of which includes a part of sample from the same sample and the necessary reagents for amplification; amplifying each compartment for the first time; combining the amplified mixtures of all compartments; amplifying the mixture for the second time, wherein the sample contains multiple targets.
The method according to claim 2, wherein amplifying each compartment for the first time, so that templates with low copy numbers in multiple targets in the sample are amplified with other targets equally efficiently.
The method according to any one of claims 1 to 3, wherein the compartments contain droplets and each component contains a droplet.
The method according to any one of claims 1 to 3, wherein the target nucleic acid is detected at the same time as the second amplification or after the amplification is completed, and the detection result indicates the presence or quantity of the target nucleic acid.
The method according to any one of claims 1 to 3, wherein both the first amplification and the second amplification are performed in the same container.
The method according to any one of claims 1 to 3, wherein the container is a PCR tube.
The method according to any one of claims 1 to 3, wherein the method of combining droplets is a physical method.
The method according to claim 8, wherein the physical method is one or more of thermal (e.g., rapid temperature change) , acoustics (e.g., ultrasonic) , electricity (e.g., high-voltage electric field) , and mechanics (e.g., centrifugation) .
The method according to claim 1, wherein the sample contains multiple targets.

The method according to claim 1, wherein there is no need to add additional reagents necessary for amplification when performing the second amplification.