A nucleic acid absolute quantification system and nucleic acid absolute quantification method based on nucleic acid isothermal amplification

Abstract

The present invention discloses a nucleic acid absolute quantification system and a nucleic acid absolute quantification method based on nucleic acid isothermal amplification, and belongs to the field of nucleic acid detection and quantification technology. The nucleic acid absolute quantification method provided by the present invention combines the use of nucleic acid synchronous isothermal amplification method and digital micro-droplet quantification technology, including preparing a reaction system based on nucleic acid isothermal amplification, dividing the reaction system into a plurality of micro-reaction units, performing isothermal amplification reaction on the micro-reaction units, and performing fluorescence signal detection on each of the micro-reaction units after the isothermal amplification reaction, and performing absolute quantification of the number of nucleic acid copies in the sample according to the positive and negative numbers of the detected micro-reaction units. The nucleic acid absolute quantification method provided by the present invention has the advantages of stable system, high compatibility, low equipment requirements, high sensitivity and rapid detection, and is particularly suitable for the design and development of a fully automatic digital nucleic acid quantification platform, and is suitable for nucleic acid quantitative detection in clinical testing and basic research.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection and quantification, and specifically relates to a nucleic acid absolute quantification system and a nucleic acid absolute quantification method based on nucleic acid isothermal amplification, and in particular relates to a real-time nucleic acid absolute quantification system and a real-time nucleic acid absolute quantification method based on nucleic acid isothermal amplification. Background Art

[0002] Currently, nucleic acid molecules (DNA / RNA) are quantified mainly through the relative quantification method of real-time quantitative polymerase chain reaction (RT-qPCR). This method requires the use of nucleic acid standards with fixed values ​​to draw a standard curve, thereby performing relative quantification of nucleic acid molecules. The error of this method is within the range of ±0.5log value, the quantitative accuracy is poor, and the operation is cumbersome.

[0003] In recent years, digital drop polymerase chain reaction (ddPCR, hereinafter also referred to as digital PCR) technology has developed rapidly. ddPCR technology is a nucleic acid quantitative detection technology based on PCR (polymerase chain reaction). It divides the reaction system into tens of thousands of tiny reaction units, calculates the number of positive and negative reactions after the reaction unit amplification, and uses the Poisson distribution algorithm for analysis and calculation to achieve absolute quantification of the target nucleic acid molecule. Digital PCR technology has extremely high sensitivity and accuracy and is suitable for nucleic acid detection of various complex samples. However, due to its limitations on the methodological requirements of the polymerase chain reaction itself, it generally takes a long time, and generally takes 2.5-4 hours to complete the quantification of the sample. At the same time, since PCR requires repeated temperature rise and fall operations, it has very high requirements for the stability of the tiny system. Once there is a problem with stability, problems such as detection failure and leakage and contamination of amplified DNA products are prone to occur. Summary of the invention

[0004] In view of one or more problems existing in the prior art, one aspect of the present invention provides a method for absolute quantification of nucleic acid based on isothermal amplification of nucleic acid, which comprises the following steps:

[0005] T1) preparing a reaction system based on isothermal amplification of nucleic acid;

[0006] T2) dividing the reaction system into a number of micro-reaction units and performing constant temperature amplification of nucleic acid; and

[0007] T3) After the nucleic acid isothermal amplification, the fluorescence signal of each of the micro-reaction units is detected, and the positive and negative signals are determined accordingly, and the number of nucleic acid copies in the nucleic acid sample to be tested is absolutely quantified according to the number of positive and negative micro-reaction units detected;

[0008] The reaction system based on isothermal amplification of nucleic acid in step T1) comprises the following components:

[0009] a. Nucleic acid sample to be tested;

[0010] b. a first primer, whose 3' end can hybridize with the 3' end or near the 3' end of the target sequence in the nucleic acid sample to be tested, and whose 5' end is a promoter sequence; optionally, the promoter sequence is a T7, T3, M13 or SP6 promoter sequence;

[0011] c. a second primer, which cooperates with the first primer to amplify the target sequence;

[0012] d. one or more fluorescent probes for detecting the target sequence; optionally, the one or more fluorescent probes are selected from the group consisting of molecular beacon probes, hydrolysis probes, dual hybridization probes, fluorescence resonance (probes based on fluorescence resonance energy transfer), scorpion probes and fluorescence amplification (fluorescence amplification molecular probes);

[0013] e, RNA-dependent DNA polymerase; and

[0014] f. RNA polymerase.

[0015] Another aspect of the present invention provides a real-time nucleic acid qualitative and / or absolute quantitative method based on nucleic acid isothermal amplification, which comprises the following steps:

[0016] S1) preparing a reaction system based on isothermal amplification of nucleic acid;

[0017] S2) dividing the reaction system into a plurality of micro-reaction units and performing isothermal amplification of nucleic acid; and

[0018] S3) During the process of isothermal amplification of nucleic acid in step S2), fluorescence signal detection is performed on each of the micro-reaction units at multiple time points, and real-time fluorescence signal detection can be optionally performed, and a real-time qualitative determination of the positive and negative results of the nucleic acid sample to be tested is performed based on the fluorescence signal detection results, and / or the number of nucleic acid copies in the nucleic acid sample to be tested is further performed in real time absolute quantification based on the number of positive and negative micro-reaction units detected;

[0019] The reaction system based on isothermal amplification of nucleic acid in step S1) comprises the following components:

[0020] a. Nucleic acid sample to be tested;

[0021] b. a first primer, whose 3' end can hybridize with the 3' end or near the 3' end of the target sequence in the nucleic acid sample to be tested, and whose 5' end is a promoter sequence; optionally, the promoter sequence is a T7, T3, M13 or SP6 promoter sequence;

[0022] c. a second primer, which cooperates with the first primer to amplify the target sequence;

[0023] d. one or more fluorescent probes for detecting the target sequence; optionally, the one or more fluorescent probes are selected from the group consisting of molecular beacon probes, hydrolysis probes, dual hybridization probes, fluorescence resonance (probes based on fluorescence resonance energy transfer), scorpion probes and fluorescence amplification (fluorescence amplification molecular probes);

[0024] e, RNA-dependent DNA polymerase; and

[0025] f. RNA polymerase.

[0026] In some embodiments, the operation of preparing the reaction system based on isothermal nucleic acid amplification in step T1) or step S1) includes:

[0027] (1) Mix components b, c and d to obtain a first mixed solution; optionally, the first mixed solution contains: 10-50 mM Tris, 5-40 mM KCl, 10-40 mM MgCl 2 , 1-20 mM NTP, 0.1-10 mM dNTPs, 1-10% PVP40, 10-250 pmol / mL of b, 10-350 pmol / mL of c, 10-250 pmol / mL of d;

[0028] (2) adding a to the first mixed solution, and incubating at 55-90° C. for 2-30 minutes to obtain a second mixed solution; and

[0029] (3) adding the enzyme components containing e and f to the second mixed solution to obtain the reaction system; optionally, the volume ratio of the second mixed solution to the enzyme components is (1-50):1; further optionally, the enzyme components containing e and f include: 16000-160000U / mL of RNA-dependent DNA polymerase, 8000-80000U / mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM N-acetyl-L-cysteine, 0.04-0.4mM zinc acetate, 10-100mM trehalose, 40-200mM Tris-HCl pH 8.0, 40-200mM KCl, 0.01-0.5mM EDTA, 0.1-1% (v / v) Triton X-100 and 20-50% (v / v) glycerol.

[0030] In some embodiments, the nucleic acid sample to be tested in step T1) or step S1) is an RNA (including mRNA, rRNA, lncRNA, miRNA) or a DNA sample, which can be influenza A virus 2009H1N1 (NIFDC2301-01) RNA, human LncRNA PCA3 or hepatitis B virus DNA.

[0031] In some embodiments, the RNA-dependent DNA polymerase is MMLV reverse transcriptase or AMV reverse transcriptase containing RaseH activity, or MMLV reverse transcriptase or AMV reverse transcriptase without RaseH activity, and RaseH enzyme is additionally added.

[0032] In some embodiments, the RNA polymerase is T3, T7, M13 or SP6 RNA polymerase, and the RNA polymerase corresponds to the promoter sequence used.

[0033] In some embodiments, in step T2) or step S2), the random distribution of nucleic acid molecules in the micro-reaction units conforms to Poisson distribution or the number of nucleic acid molecules contained in each micro-reaction unit is at most 1; optionally, the number of nucleic acid molecules contained in some micro-reaction units is at most 1, and the random distribution of nucleic acid molecules in the micro-reaction units conforms to Poisson distribution.

[0034] In some embodiments, in step T2) or step S2), the reaction system is divided into several micro-reaction units using microfluidics or other technologies; optionally, the reaction system is divided into several micro-reaction units using droplet segmentation technology in the form of micro-droplets or physical micro-pores; further optionally, the reaction system is divided into several micro-reaction units using oil-in-water micro-droplets or physical micro-pore chips; further optionally, the reaction system is divided into several micro-reaction units using a digital PCR system. For example, Thermo Fisher QuantStudio 3D digital PCR system, Thermo Fisher QuantStudio AbsoluteQ, QIAGEN digital PCR all-in-one machine QIAcuity, Shanghai Little Turtle Biotechnology chip digital PCR BioDigital, Shanghai Little Turtle Biotechnology digital PCR all-in-one machine SCIDigital, digital PCR system Accu One-100 (Shanghai Zhenzhun Biology), etc.

[0035] In some embodiments, the conditions for isothermal amplification of the nucleic acid in step T2) or step S2) are: isothermal incubation at 42-65°C for 5-60 minutes, which can be 20-60 minutes, 30-60 minutes, 20-50 minutes, 30-50 minutes or 20-40 minutes.

[0036] In some embodiments, the method for detecting the fluorescence signal of each of the micro-reaction units in step T3) or step S3) is flow fluorescence detection or fluorescence imaging; it can be optionally oil-in-water droplet fluorescence flow detection, oil-in-water chip fluorescence imaging or microwell chip imaging.

[0037] In some embodiments, the criterion for judging the positive and negative in step T3) or step S3) is: the presence of a fluorescent signal is judged as positive, and the absence of a fluorescent signal is judged as negative.

[0038] In some embodiments, in step T3) or step S3), the absolute quantification of the nucleic acid copy number in the nucleic acid sample to be tested based on the number of positive and negative micro-reaction units detected is to calculate the nucleic acid copy number using the Poisson distribution formula, or directly count the number of positive reaction units, thereby determining the starting copy number of the nucleic acid template molecule.

[0039] In some embodiments, the nucleic acid samples to be tested include medical samples and non-medical samples whose sources include food, blood products, dairy products, and the environment.

[0040] In some embodiments, in step S3), the fluorescence signal detection at multiple time points for each of the micro reaction units is to detect the fluorescence signal every 0.5-10 minutes during the nucleic acid isothermal amplification process, and can optionally be to detect the fluorescence signal every 0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

[0041] In another aspect, the present invention further provides a system for nucleic acid qualitative and / or absolute quantitative determination based on nucleic acid isothermal amplification, comprising:

[0042] 1) Reaction system based on isothermal amplification of nucleic acids;

[0043] 2) A device for dividing a reaction system based on isothermal nucleic acid amplification into several micro-reaction units (e.g., a digital PCR system, or an oil-in-water micro-droplet preparation device);

[0044] 3) a device for performing constant temperature amplification of nucleic acid on each micro-reaction unit (e.g., a device capable of performing constant temperature amplification, such as a plate thermostat); and

[0045] 4) a device for detecting the fluorescence signal of each of the micro-reaction units after and / or during the isothermal amplification of nucleic acid; optionally, the device for detecting the fluorescence signal of each of the micro-reaction units during the isothermal amplification of nucleic acid is a real-time fluorescence signal detection device (e.g., a flow fluorescence detection device or a fluorescence imaging device);

[0046] The reaction system based on isothermal amplification of nucleic acid comprises the following components:

[0047] A. A first primer, whose 3' end can hybridize with the 3' end or near the 3' end of the target nucleic acid to be detected, and whose 5' end is a promoter sequence;

[0048] B. a second primer, which cooperates with the first primer to amplify the target nucleic acid to be detected;

[0049] C. a target fluorescent probe, which is used to detect the target nucleic acid to be detected. Optionally, the fluorescent probe can hybridize with the negative strand of the target nucleic acid to be detected and release a fluorescent signal;

[0050] D. RNA-dependent DNA polymerase; and

[0051] E. RNA polymerase.

[0052] In some embodiments, the target nucleic acid to be detected includes RNA and DNA. Optionally, the target nucleic acid to be detected is selected from: influenza A virus 2009H1N1 (NIFDC2301-01) RNA, human LncRNA PCA3 and hepatitis B virus DNA; wherein:

[0053] When the target nucleic acid to be detected is influenza A virus 2009H1N1 (NIFDC2301-01) RNA, the nucleotide sequences of the first primer, the second primer and the target fluorescent probe are shown in SEQ ID NO: 1 to SEQ ID NO: 3, respectively;

[0054] When the target nucleic acid to be detected is human LncRNA PCA3, the nucleotide sequences of the first primer, the second primer and the target fluorescent probe are shown in SEQ ID NO: 4 to SEQ ID NO: 6, respectively;

[0055] When the target nucleic acid to be detected is hepatitis B virus DNA, the nucleotide sequences of the first primer, the second primer and the target fluorescent probe are respectively shown as SEQ ID NO:21-SEQ ID NO:23.

[0056] The use of the above system in preparing a kit for absolute quantification of nucleic acids also belongs to the content of the present invention.

[0057] The present invention provides a method for absolute quantitative determination of nucleic acid, which is a method for absolute quantitative determination of nucleic acid by digital droplet constant temperature amplification (digital drop SAT, referred to herein as ddSAT) based on the patent (CN101333565B) of the applicant for simultaneous isothermal amplification (SAT) of nucleic acid. The method combines reverse transcriptase and RNA polymerase with fluorescence detection technologies such as molecular beacon probes, hydrolysis probes, double hybridization probes, fluorescence resonance, scorpion probes and fluorescence amplification, and based on the amplification detection method of digital droplets, quantitative detection or real-time quantitative detection (referred to herein as real-time ddSAT) of nucleic acid samples is achieved after or during constant temperature amplification of nucleic acid. The principle of constant temperature amplification of nucleic acid in the method is based on the patent (CN101333565B) of the applicant, and the principle of absolute quantitative determination of nucleic acid is based on Poisson distribution theory technology.

[0058] The Poisson distribution theory technology randomly disperses the sample template in tens of thousands of micro-reaction units in the limiting dilution mode. Each reaction unit can be assigned to zero, one or more template nucleic acid molecules, and the dispersion of the template molecules conforms to the Poisson distribution. After the amplification is completed, the fluorescent signal unit is recorded as 1, and the fluorescent signal is recorded as 0, that is, the reaction unit is defined as positive and negative according to the presence or absence of the fluorescent signal. By counting the total number of reaction units and the number of positive reaction units, the starting concentration of the nucleic acid template molecule can be calculated according to the Poisson distribution formula. Ideally, when the nucleic acid concentration of the sample to be tested is diluted to a very low level, the sample solution is dispersed into a large number of reaction units, so that each reaction unit contains at most one nucleic acid molecule. Under this condition, the starting number of nucleic acid template molecules can be directly determined by counting the number of positive reaction units. However, when measuring high-concentration templates, some reaction units contain two or more nucleic acid molecules, and the random distribution of a large number of nucleic acid template molecules conforms to the Poisson distribution. Therefore, the absolute concentration of nucleic acid template molecules can be calculated using the Poisson distribution probability formula.

[0059]

[0060] In formula (1), λ is the average copy number of the template molecule in each reaction unit, and p is the probability that the reaction unit contains k copies of the template molecule. λ is equivalent to the initial copy number (c) of the template molecule in the sample diluted m times (m is the dilution factor, equivalent to the volume of an independent reaction unit), λ = cm; when k = 0, that is, there is no template molecule in the reaction unit, formula (1) can be simplified to:

[0061] p(x=0)=e -λ =e -cm (2)

[0062] At the same time, when k=0, the probability p(x=0) is equivalent to the ratio of the number of reaction units without nucleic acid template molecules to the total number of reaction units, that is:

[0063]

[0064] In formula (3), n is the total number of reaction units, and f is the number of positive reaction units. Taking the logarithm of both sides of formula (3) yields:

[0065]

[0066] Therefore, when the method of the present invention is used for quantitative analysis of nucleic acid, the starting copy number of the nucleic acid template molecule in the sample can be obtained under the condition that the total number of reaction units, the dilution factor and the number of positive reaction units are known. The main advantages of the nucleic acid absolute quantitative detection method of the present invention are as follows:

[0067] 1. Stable system: Since the amplification is carried out under constant temperature conditions, the stability of the microdroplets and the sealed chip is minimal, and it is not easy to destroy the sealing performance of the microdroplets and the chip, so the amplification reaction can proceed more smoothly.

[0068] 2. Multiple detection systems are available: Existing digital PCR quantitative detection methods require detailed optimization of reagent components in different digital PCR systems, from chemical components to primer probe concentrations, all of which need to be readjusted and adapted. However, due to its constant temperature characteristics, the ddSAT technology of the present invention is compatible with various digital quantitative systems of oil-in-water droplets and microporous chips without the need to adjust the system. Users can choose from a variety of detection systems based on their own experience or existing technical knowledge and experimental conditions.

[0069] 3. Rapid detection: Nucleic acid amplification is carried out at a constant temperature without repeated heating and cooling, which greatly shortens the amplification time and greatly improves the detection efficiency. The results of the examples show that compared with the existing real-time quantitative detection methods, the real-time ddSAT method provided by the present invention has a significantly earlier positive detection time.

[0070] 4. The equipment requirements are simple and easy to automate: Compared with ordinary digital PCR technology, the equipment system required by the present invention does not require a temperature rise and fall process, which not only reduces the design and production costs, but also makes it easy to design integrated equipment.

[0071] 5. The detection limit of the present invention is lower. The results of the examples show that the detection limit of the real-time ddSAT method provided by the present invention is better than that of the conventional real-time fluorescent PCR method, and the detection of RNA will not be affected by the DNA in the system, and the detection result is more accurate.

[0072] In summary, the method of the present invention has the advantages of stable system, high compatibility, low equipment requirements and rapid detection, and is particularly suitable for the design and development of a fully automatic digital nucleic acid quantification platform. This technology, in conjunction with the digital nucleic acid quantification platform that has been launched on the market, is suitable for the detection of high-sensitivity nucleic acid qualitative or high-precision nucleic acid quantitative in the fields of clinical testing, nucleic acid standard assignment, tumor gene detection, basic molecular biology research, etc., and is suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 The photo (A) and output result (B) of the chip fluorescence detection for absolute quantification of influenza A virus RNA in Example 1 are shown.

[0074] Figure 2 The statistical results of the absolute quantification of influenza A virus RNA by 20 laboratories in Example 1 are shown.

[0075] Figure 3 The chip fluorescence detection photo (A) and output result (B) for absolute quantification of PCA3 RNA in Example 2.

[0076] Figure 4 Schematic diagram of the production process of the microfluidic oil-in-water chip.

[0077] Figure 5 The results of oil-in-water microdroplets taken under bright field and dark field for absolute quantification of influenza A virus RNA (A1 and A2) and the fluorescence detection photo of the physical microwell chip (B).

[0078] Figure 6 Real-time fluorescence PCR amplification curves for influenza A virus RNA at different dilutions. DETAILED DESCRIPTION

[0079] The content of the present invention is described in detail below with reference to specific embodiments.

[0080] Hereinafter, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the description of the embodiments is considered to be exemplary and non-restrictive in nature.

[0081] The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0082] The methods for obtaining various biological materials described in the examples are only provided as a way of obtaining them in an experiment to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0083] All primers and probes mentioned in the present invention are synthesized using existing techniques.

[0084] Example 1: Absolute quantitative detection of pathogen nucleic acid

[0085] This embodiment takes the detection of influenza A virus RNA as an example, and uses the method of the present invention to perform absolute quantitative detection of influenza A virus mimics. The specific method includes the following steps.

[0086] 1.1. Sample Source

[0087] China Food and Drug Inspection and Quarantine Institute, Institute for In Vitro Diagnostic Testing, the first batch of inactivated cultures of influenza A 2009H1N1 for pathogen dPCR (also referred to as ddPCR in this article) capability comparison testing in 2023, number NIFDC2301-01.

[0088] 1.2. Extraction of influenza A virus RNA

[0089] Take 20 μL of the influenza A 2009 H1N1 (NIFDC2301-01) inactivated culture reconstitution solution and use the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 kit to extract RNA.

[0090] 1.3. Design of the first primer, the second primer and the target detection probe (also referred to as the target fluorescent probe in this article)

[0091] Specific primers and target detection probe sequences were designed according to the influenza A virus M gene specific sequence (GenBank: MT241814.1), as shown below:

[0092] First primer:

[0093] 5'-aatttaatacgactcactatagggagaTTTGGACAAAGCGTCTACGCTGC-3' (SEQ ID NO: 1);

[0094] Second primer: 5'-ACTGGAAAGTGTCTTTGCA-3' (SEQ ID NO: 2);

[0095] Target detection probe: 5'-CGAGCACCUCUGACUAAGGGAGCUCG-3' (SEQ ID NO: 3); the 5' end of the target detection probe is fluorescently labeled with FAM, and the 3' end is fluorescently labeled with DABCYL;

[0096] 1.4. Amplification reaction system and digital SAT chip preparation and amplification

[0097] (1) Add 30 μL of detection solution (containing 15 mM Tris, MgCl 2 15mM, dNTP2.5mM, NTP 3mM, PVP40 1%, KCl 10mM, 200pmol / mL of the first primer (SEQ ID NO: 1), 300pmol / mL of the second primer (SEQ ID NO: 2), 200pmol / mL of the target detection probe (SEQ ID NO: 3);

[0098] (2) Add 2 μL of the extracted influenza A virus RNA sample to the above-mentioned treatment tube, shake and mix, react at 60° C. for 5-10 min, and then cool to room temperature.

[0099] (3) Add 10 μL of SAT enzyme solution (preheated at 42°C, containing 60,000 U / mL M-MLV reverse transcriptase, 40,000 U / mL T7 RNA polymerase, 10 mM HEPES pH 7.5, 15 mM N-acetyl-L-cysteine, 0.15 mM zinc acetate, 20 mM trehalose, 100 mM Tris-HCl pH 8.0, 80 mM KCl, 0.25 mM EDTA, 0.5% (v / v) Triton) to the treated tube after the above reaction. X-100 and 30% (v / v) glycerol); use a pipette to quickly blow and mix, take 20 μL for digital SAT chip preparation, for example, the sample pretreatment system of the Zhenzhun Biochip Digital PCR System can be used for digital SAT chip preparation, specifically: take 20 μl of the mixed solution, add it to the scraper (single row), immediately start the sample pretreatment system, and prepare the chip.

[0100] (4) After the chip is prepared, use a flat plate thermostat to heat the reaction at 42°C for 40 minutes.

[0101] 1.5 Digital Droplet SAT (ddSAT) Detection

[0102] Place the chip that has been amplified in step 1.4 into the chip reader for endpoint fluorescence photography and reading, and select the FAM fluorescence channel. Count the number of positive and negative spots in the chip and use software to analyze (based on Poisson distribution theory) the RNA copy number in the sample. Figure 1 As shown in the middle A, the positive and negative are clearly distinguished, indicating that the amplification results are valid. Figure 1 As shown in panel B, the output result is 318.37 copies / μL. Based on the volume conversion, the RNA copy number concentration in the original virus inactivated culture is 8.80E+07 copies / mL.

[0103] 1.6. Results comparison

[0104] Influenza A 2009H1N1 (NIFDC2301-01) inactivated culture was uniformly distributed by the China Food and Drug Inspection Institute and the Institute for In Vitro Diagnostic Reagents to 20 companies (referred to as lab 1-lab 20 in this article) that have digital PCR (also referred to as ddPCR) calibration equipment and influenza A detection systems. The statistical data of all test results are as follows Figure 2As shown, the comparison results of the digital droplet SAT method valuation results of the present invention and the statistical data from lab 1-lab 20 are shown in Table 1 below. It can be seen that the valuation of influenza A virus RNA by the digital droplet SAT method (also referred to as ddSAT in this article) provided by the present invention is basically consistent with the results of the ddPCR method, with no significant difference.

[0105] Table 1: Comparison of the results of 20 digital PCR tests with the digital droplet SAT method of the present invention

[0106]

[0107] Example 2: Absolute quantitative detection of human RNA

[0108] This Example 2 takes the detection of prostate-specific target LncRNA PCA3 as an example, and uses the digital droplet SAT method to perform absolute quantitative detection of LncRNA PCA3 in human urine. The specific method includes the following steps.

[0109] 2.1. Obtaining human urine RNA samples

[0110] The urine sample was a 400 μL random preoperative urine sample from a prostate cancer patient, and RNA was extracted using the TaKaRa MiniBESTUniversal RNA Extraction Kit.

[0111] 2.2. Design of first primer, second primer and target detection probe

[0112] Specific primers and target detection probe sequences were designed according to the human LncRNA PCA3 sequence (NCBI Reference Sequence: NR_132312.1), as shown below:

[0113] First primer:

[0114] 5'-aatttaatacgactcactataggggagaGGGACCAGGCACAGGGCGAG-3' (SEQ ID NO: 4);

[0115] Second primer: 5'-CACAGGAAGCACAAAAGGA-3' (SEQ ID NO: 5);

[0116] Target detection probe: 5'-ACCCGGCCGCCAUCUUGGGU-3' (SEQ ID NO: 6); the 5' end of the target detection probe is fluorescently labeled with FAM, and the 3' end is fluorescently labeled with DABCYL;

[0117] 2.3. Amplification reaction system and digital SAT chip preparation and amplification

[0118] (1) Add 30 μL of detection solution (containing 15 mM Tris, MgCl 2 15mM, dNTP2.5mM, NTP 3mM, PVP40 1%, KCl 10mM, 200pmol / mL of the first primer (SEQ ID NO:4), 300pmol / mL of the second primer (SEQ ID NO:5), 200pmol / mL of the target detection probe (SEQ ID NO:6);

[0119] (2) Add 2 μL of extracted human urine RNA sample to the above treatment tube, shake and mix, react at 60°C for 5-10 minutes, and then cool to room temperature.

[0120] (3) Add 10 μL of SAT enzyme solution (preheated at 42° C., containing 60,000 U / mL M-MLV reverse transcriptase, 40,000 U / mL T7 RNA polymerase, 10 mM HEPES pH 7.5, 15 mM N-acetyl-L-cysteine, 0.15 mM zinc acetate, 20 mM trehalose, 100 mM Tris-HCl pH 8.0, 80 mM KCl, 0.25 mM EDTA, 0.5% (v / v) Triton X-100 and 30% (v / v) glycerol) to the treated tube after the above reaction; use a pipette to quickly pipette and mix, and take 20 μL for digital SAT chip preparation. The specific preparation method can be found in Example 1.

[0121] (4) After the chip is prepared, heat it at 42°C for 40 minutes using a plate thermostat.

[0122] 2.4 Digital Droplet SAT (ddSAT) Detection Place the chip that has been amplified in step 2.3 into the chip reader for endpoint fluorescence photography and reading, and select the FAM fluorescence channel. Count the number of positive and negative spots in the chip and use the software to analyze the RNA copy number in the sample. Figure 3 As shown in the middle A, the positive and negative are clearly distinguished, indicating that the amplification results are valid. Figure 3 As shown in panel B, the output result is 110.47 copies / μL. Based on the volume conversion, the PCA3 RNA concentration in the original human urine sample is 5.80E+5 copies / mL.

[0123] 2.5. Comparison of detection performance between ddPCR and ddSAT

[0124] To further confirm whether the ddSAT method differs from the ddPCR method in human RNA detection, specific ddPCR primers and taqman fluorescent probes were designed based on the LncRNAPCA3 sequence. The sequence information is shown in Table 2 below, where the ddPCR reaction procedure is: 50℃15min—95℃15s—(95℃15s—60℃30s—72℃1min) 40 cycles. At the same time, the human urine RNA sample extracted in step 2.1 above was used for ddSAT and ddPCR absolute quantitative detection. The results are shown in Table 3 below, which shows that the ddSAT test results are lower than ddPCR. To further confirm whether the ddPCR fixed value is high due to the presence of incompletely digested DNA fragments in the extracted nucleic acid product, the extracted human urine RNA sample product was digested with RNase and then tested using ddSAT and ddPCR. The test results are shown in Table 3. The results show that the ddPCR method cannot avoid the interference of residual genomic DNA. When the human urine RNA sample product is digested with RNase, a certain amount of target nucleic acid can still be detected using ddPCR, while the target nucleic acid cannot be detected using the ddSAT method. Therefore, the ddSAT method provided by the present invention is not interfered by genomic DNA and the quantification is more accurate.

[0125] At the same time, in order to confirm the difference between the isothermal amplification LAMP and RPA methods and ddSAT, the digital LAMP and digital RPA detection systems for the LncRNA PCA3 sequence in human urine samples were also designed in this embodiment, wherein the LAMP reaction procedure was: 65°C 40 min; the RPA reaction procedure was: 37°C 40 min, and the genomic DNA interference test was performed as described above. The results are shown in Table 3. Limited by the methodological principles, the digital LAMP and digital RPA methods, like the ddPCR method, cannot avoid the interference of residual genomic DNA.

[0126] Table 2: ddPCR, digital LAMP and digital RPA primer and probe sequence information for detecting human LncRNA PCA3

[0127]

[0128]

[0129] Table 3: Results of quantitative detection of human LncRNA PCA3 by ddPCR, digital LAMP, digital RPA and ddSAT

[0130] Methodology Type Direct quantitative results Detection of RNAse Digestion Results ddSAT 5.80E+5copies / mL 0 ddPCR 6.33E+5copies / mL 4.2E+4copies / mL Digital LAMP 6.57E+5copies / mL 6.84E+4copies / mL Digital RPA 6.21E+5copies / mL 3.79E+4copies / mL

[0131] Example 3: Absolute quantitative detection of pathogen DNA

[0132] The above-mentioned Examples 1 and 2 have proved that the ddSAT method provided by the present invention can meet the requirements of accurate quantification of pathogens or human RNA targets. In order to achieve absolute quantitative detection of pathogens or human DNA by ddSAT, this Example 3 takes the absolute quantitative detection of hepatitis B virus (HBV) DNA as an example, and uses the method of the present invention to perform absolute quantitative detection of viral DNA. The specific method includes the following steps.

[0133] 3.1. Acquisition of HBV DNA

[0134] Based on the hepatitis B virus DNA sequence in the NCBI database (GenBank: X72213.1), Bioengineering (Shanghai) Co., Ltd. was commissioned to artificially synthesize the full-length HBV DNA sequence. The DNA content provided in its product manual was 1.34E+12 copies / μL. The DNA sample was diluted tenfold to 1.34E+4 copies / μL using enzyme-free sterile water.

[0135] 3.2. Design of first primer, second primer and target detection probe

[0136] Specific primers and target detection probe sequences were designed according to the human HBV DNA sequence (GenBank: X72213.1), as shown below:

[0137] First primer:

[0138] 5'-aatttaatacgactcactatagggagaGTTTGTATTATGCCCTGAGCC-3' (SEQ ID NO: 19);

[0139] Second primer: 5'-TCAGAGCAAATACCGCAAATCCAG-3' (SEQ ID NO: 20);

[0140] Target detection probe: 5'-CGACCAACAAGGTAGGAGTTGGTCG-3' (SEQ ID NO: 21); the 5' end of the target detection probe is fluorescently labeled with FAM, and the 3' end is fluorescently labeled with DABCYL.

[0141] 3.3. Amplification reaction system and digital SAT chip preparation and amplification

[0142] (1) Add 30 μL of detection solution (containing Tris 15 mM, MgCl2 15 mM, dNTP 2.5 mM, NTP 3 mM, PVP40 1%, KCl 10 mM, 200 pmol / mL of the first primer (SEQ ID NO: 19), 300 pmol / mL of the second primer (SEQ ID NO: 20), and 200 pmol / mL of the target detection probe (SEQ ID NO: 21)) to the sample processing tube;

[0143] (2) Add 2 μL of diluted HBV DNA sample to the above treatment tube, shake and mix, react at 90°C for 5-10 min, and then cool at room temperature (20-25°C) for 5-10 min.

[0144] (3) Add 10 μL of SAT enzyme solution (preheated at 42° C., containing 60,000 U / mL M-MLV reverse transcriptase, 40,000 U / mL T7 RNA polymerase, 10 mM HEPES pH 7.5, 15 mM N-acetyl-L-cysteine, 0.15 mM zinc acetate, 20 mM trehalose, 100 mM Tris-HCl pH 8.0, 80 mM KCl, 0.25 mM EDTA, 0.5% (v / v) Triton X-100 and 30% (v / v) glycerol) to the treated tube after the above reaction; use a pipette to quickly pipette and mix, and take 20 μL to prepare the digital SAT chip. The specific preparation method can refer to Example 1.

[0145] (4) After the chip is prepared, heat it at 42°C for 40 minutes using a plate thermostat.

[0146] 3.4 Digital Droplet SAT (ddSAT) Detection

[0147] Place the chip that has been amplified in step 3.3 into the chip reader, perform endpoint fluorescence photography and reading, and select the FAM fluorescence channel. By calculating the number of positive and negative points in the chip, the software is used to analyze the number of DNA copies in the sample. The test results are shown in Table 5 below, which are basically consistent with the nucleic acid concentration of the HBV DNA sample used (1.34E+4copies / μL), indicating that the ddSAT method provided by the present invention also has a high accuracy in absolute quantitative detection of DNA samples.

[0148] 3.5. Performance comparison between ddSAT and ddPCR for DNA quantitative detection

[0149] To further confirm whether ddSAT and ddPCR are consistent in detecting DNA, primer probes related to ddPCR were designed based on the HBV DNA sequence (GenBank: X72213.1). The sequence information is shown in Table 4, where the PCR reaction program is: 95℃15s-(95℃15s-60℃30s-72℃1min) 40 cycles. Using the same DNA sample (obtained in step 3.1 above), ddSAT and ddPCR methods were used for absolute quantitative detection, and the results are shown in Table 5 below. It can be seen that when quantifying DNA samples, the results of ddSAT and ddPCR are well consistent.

[0150] Table 4: Primer and probe sequence information related to ddPCR targeting HBV DNA sequences

[0151]

[0152] Table 5: Quantitative detection results of HBV DNA by ddSAT and ddPCR

[0153] Methodology Type Quantitative results ddSAT 12766.95 copies / μL ddPCR 13240.74 copies / μL

[0154] Example 4: ddSAT detection using microdroplets or physical microwell chips

[0155] The ddSAT detection technology used in the above-mentioned Examples 1-3 is compatible with the reaction system segmentation methods of oil-in-water microdroplets and physical micropore chips. To further illustrate the compatibility of ddSAT detection, this example uses microdroplets and physical micropore chips to compare the ddSAT compatibility of the detection system and samples in Example 1. The detection process is as follows.

[0156] 4.1 Sample Source

[0157] China Food and Drug Inspection Institute, Institute for In Vitro Diagnostic Testing, 2023 first batch of inactivated cultures of influenza A 2009H1N1 for pathogen dPCR capability comparison testing, number NIFDC2301-01.

[0158] 4.2. Extraction of influenza A virus RNA

[0159] 20 μL of the influenza A 2009 H1N1 (NIFDC2301-01) inactivated culture reconstituted solution was taken and RNA was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0.

[0160] 4.3. Design of first primer, second primer and target detection probe

[0161] The first primer, second primer and target detection probe used are the same as those in Example 1.

[0162] 4.4. Amplification reaction system and digital SAT chip preparation and amplification

[0163] (1) Add 30 μL of detection solution (containing 15 mM Tris, MgCl 2 15mM, dNTP2.5mM, NTP 3mM, PVP40 1%, KCl 10mM, 200pmol / mL of the first primer (SEQ ID NO: 1), 300pmol / mL of the second primer (SEQ ID NO: 2), 200pmol / mL of the target detection probe (SEQ ID NO: 3);

[0164] (2) Add 2 μL of the extracted influenza A virus RNA sample to the above-mentioned treatment tube, shake and mix, react at 60° C. for 5-10 min, and then cool to room temperature.

[0165] (3) Add 10 μL of SAT enzyme solution (preheated at 42°C, containing 60,000 U / mL M-MLV reverse transcriptase, 40,000 U / mL T7 RNA polymerase, 10 mM HEPES pH 7.5, 15 mM N-acetyl-L-cysteine, 0.15 mM zinc acetate, 20 mM trehalose, 100 mM Tris-HCl pH 8.0, 80 mM KCl, 0.25 mM EDTA, 0.5% (v / v) Triton X-100 and 30% (v / v) glycerol) to the treated tube after the above reaction; use a pipette to quickly pipette and mix, and take 20 μL for the following digital SAT chip preparation.

[0166] (4) The mixed amplification reaction solution is added to the microfluidic oil-in-water chip preparation well and the physical micropore chip brush to prepare the two chips. The oil-in-water generation process in the microfluidic oil-in-water chip is as follows: Figure 4As shown, the microfluidic injection lanes are divided into three, the middle lane is liquid phase, and the two sides are oil phase. Under the action of pressure, the oil phase and the liquid phase form oil-in-water droplets at the intersection, and then are evenly arranged in the interlayer of the chip through multiple 1:2 lanes. Since the thickness of the chip interlayer is only the size of a droplet diameter, a single-layer flat chip full of oil-in-water droplets is formed after the droplet generation is completed; the physical micropore chip is a Zhenzhun Bio-Digital PCR silicon-based chip, with 20,000 micropores etched on the silicon-based wafer. After the amplification solution enters the micropores, the oil phase is used to fill the cavity of the chip, thereby forming an oil-in-water microreaction unit based on physical micropores.

[0167] (5) After the chip is prepared, heat it at 42°C for 40 minutes using a plate thermostat.

[0168] 4.5 Digital Droplet SAT (ddSAT) Detection

[0169] Place the chip that has been amplified in step 4.4 into the chip reader for endpoint fluorescence photography and reading, and select the FAM fluorescence channel. Calculate the number of positive and negative spots in the chip and use the software to analyze the RNA copy number in the sample. The fluorescence photography results of the two chips (microfluidic oil-in-water chip and physical microporous chip) are shown in Figure 2. Figure 5 As shown in panels A1, A2 and B, Figure 5 A1 and A2 in the middle are the results of oil-in-water microdroplets taken in bright field and dark field, respectively. A1 shows a stable microdroplet structure, and the fluorescent spots in A2 are positive oil-in-water droplets. Figure 5 The middle B is the fluorescence shooting result of the physical microporous chip, and the bright spot is the positive micro-reaction unit. By counting the number of negative and positive micro-reaction units of the two chips and substituting them into the Poisson distribution formula, the copy number of the nucleic acid target in the amplification system can be calculated. The quantitative detection results are shown in Table 6 below. It can be seen that there is no significant difference in the detection of the two chips. The results show that ddSAT technology is compatible with two technical routes of digital nucleic acid quantification: oil-in-water microdroplets and physical microporous chips.

[0170] Table 6: Digital nucleic acid quantification results of oil-in-water microdroplets and physical microwell chips

[0171] ddSAT detection platform type Quantitative results Water-in-oil chip 311.31 copies / μL Physical microwell chip 318.72 copies / μL

[0172] Example 5: Real-time digital RNA isothermal amplification quantitative (real-time ddSAT) detection

[0173] Taking the detection of influenza A RNA in Example 1 as an example, independent fluorescence signal collection is performed at several time points during the amplification process, and real-time digital RNA isothermal amplification and absolute quantitative detection is performed. The specific method includes the following steps.

[0174] 5.1. Sample Source

[0175] China Food and Drug Inspection Institute, Institute for In Vitro Diagnostic Testing, 2023 first batch of inactivated cultures of influenza A 2009H1N1 for pathogen dPCR capability comparison testing, number NIFDC2301-01.

[0176] 5.2. Extraction of influenza A virus RNA

[0177] 20 μL of the influenza A 2009 H1N1 (NIFDC2301-01) inactivated culture reconstituted solution was taken and RNA was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0.

[0178] 5.3. Design of first primer, second primer and target detection probe

[0179] The first primer, second primer and target detection probe used are the same as those in Example 1.

[0180] 5.4. Amplification reaction system and digital SAT chip preparation and amplification

[0181] (1) Add 30 μL of detection solution (containing 15 mM Tris, MgCl 2 15mM, dNTP2.5mM, NTP 3mM, PVP40 1%, KCl 10mM, 200pmol / mL of the first primer (SEQ ID NO: 1), 300pmol / mL of the second primer (SEQ ID NO: 2), 200pmol / mL of the target detection probe (SEQ ID NO: 3);

[0182] (2) Add 2 μL of the extracted influenza A virus RNA sample to the above-mentioned treatment tube, shake and mix, react at 60° C. for 5-10 min, and then cool to room temperature.

[0183] (3) Add 10 μL of SAT enzyme solution (preheated at 42° C., containing 60,000 U / mL M-MLV reverse transcriptase, 40,000 U / mL T7 RNA polymerase, 10 mM HEPES pH 7.5, 15 mM N-acetyl-L-cysteine, 0.15 mM zinc acetate, 20 mM trehalose, 100 mM Tris-HCl pH 8.0, 80 mM KCl, 0.25 mM EDTA, 0.5% (v / v) Triton X-100 and 30% (v / v) glycerol) to the treated tube after the above reaction; use a pipette to quickly pipette and mix, and take 20 μL for digital SAT chip preparation. The specific preparation method can be found in Example 1.

[0184] (4) After the chip is prepared, a fluorescence image is taken immediately, which is recorded as the 0-min image; then the chip is heated at 42°C using a flat-plate thermostat.

[0185] 5.5 Real-time digital droplet SAT (real-time ddSAT) detection

[0186] In order to realize the detection of real-time digital droplet SAT, the present embodiment transforms the existing fluorescence signal acquisition device by replacing the chip carrier with an amplification module with a constant temperature of 42°C, so that the fluorescence signal can be collected at a fixed time while amplifying at 42°C.

[0187] The modified fluorescence acquisition device was used to independently acquire fluorescence signals from the chip in step 5.4 every 5 minutes. After the acquisition was completed, amplification was continued. By comparing the image analysis data at 9 time nodes, the fluorescence change curves of all valid micro-reaction units were made; by analyzing the fluorescence change curves of each valid micro-reaction unit, it was possible to more accurately determine which wells actually generated amplified fluorescence signals, and these wells were judged as positive, and the remaining invalid micro-wells were judged as negative. The changes in fluorescence values ​​are shown in Table 7 below. The copy number was calculated based on the number of positive and negative wells, and the results are shown in Table 11 below.

[0188] At the same time, in order to compare the detection performance, this embodiment also designed dd-RT-PCR (real-time fluorescent digital PCR), dd-RT-LAMP (real-time fluorescent digital LAMP), and dd-RT-RPA (real-time fluorescent digital RPA) influenza A virus detection systems. The primer probe sequences used in each system are shown in Table 7. The same nucleic acid sample as the real-time ddSAT was used to perform real-time digital amplification detection of dd-RT-PCR, dd-RT-LAMP, and dd-RT-RPA. Among them, dd-RT-PCR is a more commonly used amplification detection method (PCR amplification program is: 95°C 15s-(95°C 15s-60°C 30s-72°C 1min) 40 cycles), and the fluorescence signal is collected every 5 cycles. dd-RT-LAMP and dd-RT-RPA are currently known more mature constant temperature amplification methods, and the fluorescence signal is collected every 5 minutes. The LAMP reaction program is: 65°C 40min; the RPA reaction program is: 37°C 40min. The real-time fluorescence detection results of the three comparison methods are shown in Tables 8, 9, and 10, respectively. The copy number was calculated based on the number of positive and negative wells, and the results are shown in Table 11.

[0189] Table 7: Specific PCR, LAMP, and RPA sequence information for detecting influenza A virus

[0190]

[0191] Table 8: Fluorescence value changes at 9 time points detected by real-time ddSAT

[0192]

[0193] Table 9: Changes in fluorescence values ​​at 9 time points detected by dd-RT-PCR

[0194]

[0195] Table 10: Fluorescence value changes at 9 time points detected by dd-RT-LAMP

[0196]

[0197] Table 11: Fluorescence value changes at 9 time points detected by dd-RT-RPA

[0198]

[0199] Table 12: Test results

[0200]

[0201] 5.6. Results comparison and analysis

[0202] Through the changes in fluorescence values ​​at 9 time points detected by real-time ddSAT in Table 8 above, it can be found that with the increase of amplification time, the fluorescence values ​​of some micropores (such as micropores 1-3) continue to rise to the plateau brightness and stabilize to 40 minutes, and these micropores are judged as positive micropores; while some micropores (such as micropores 4-5) also have fluorescence values, but the fluorescence values ​​are low and do not reach the fluorescence value intensity of normal positive amplification, and with the increase of amplification time, the fluorescence values ​​of these micropores do not continuously rise to the plateau brightness, so these micropores should also be judged as negative micropores, and such negative micropores should be excluded in the subsequent copy number calculation. However, in the endpoint detection method (i.e., only after the nucleic acid isothermal amplification is completed, the fluorescence signal of the chip micropores is detected), the negative micropores with low fluorescence values ​​above will also be judged as positive micropores (false positives), which will cause the absolute quantitative detection results of the endpoint detection method to be high. As shown in the results recorded in Table 12 above, the quantitative results of the real-time fluorescence shooting method are lower than the quantitative results of the endpoint method. The results show that the real-time digital droplet SAT (real-time ddSAT) detection method can improve the ability to distinguish positive amplification, exclude some false positive micropores, and thus improve the accuracy of quantitative detection. It can also be seen from the results of Tables 9-12 that the three real-time absolute quantitative detection methods of dd-RT-PCR, dd-RT-LAMP, and dd-RT-RPA can also obtain absolute quantitative results with higher accuracy than the endpoint method, and the above methods have basically the same detection results in the quantitative detection of pure RNA.

[0203] In addition, by comparing the changes in fluorescence values ​​at 9 time nodes based on real-time ddSAT, dd-RT-PCR, dd-RT-LAMP, and dd-RT-RPA detection as shown in Tables 8-11, it can be found that when detecting the same RNA sample, the real-time ddSAT method can obtain positive results for the microwells (e.g., microwells 1-3) within 5-10 minutes after the amplification reaction (see Table 8), while the dd-RT-PCR method requires at least 25-30 cycles after the amplification reaction to obtain positive results for the microwells (e.g., microwells 1, 3, and 6) (see Table 9), the dd-RT-LAMP method requires 10-15 minutes after the amplification reaction to obtain positive results for the microwells (e.g., microwells 1-3) (see Table 10), and the dd-RT-RPA method requires 20-25 minutes after the amplification reaction to obtain positive results for the microwells (e.g., microwells 1-3) (see Table 11). The above results show that compared with dd-RT-PCR, dd-RT-LAMP, and dd-RT-RPA methods, the real-time ddSAT method provided by the present invention has significantly higher detection sensitivity and can detect positive microwells more quickly.

[0204] Example 6: Real-time digital RNA isothermal amplification (real-time ddSAT) detection

[0205] Taking the detection of influenza A RNA in Example 1 as an example, independent fluorescence signal collection is performed at the amplification time nodes of 0, 5, 10, 15, 20, 25, 30, 35, and 40 minutes, respectively, and real-time digital RNA isothermal amplification qualitative detection is performed. The specific method includes the following steps.

[0206] 6.1. Sample Source

[0207] China Food and Drug Inspection Institute, Institute for In Vitro Diagnostic Testing, 2023 first batch of inactivated cultures of influenza A 2009H1N1 for pathogen dPCR capability comparison testing, number NIFDC2301-01.

[0208] 6.2. Extraction and dilution of influenza A virus RNA

[0209] Take 20 μL of influenza A 2009H1N1 (NIFDC2301-01) inactivated culture reconstitution solution and use TaKaRaMiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 kit to extract RNA. After the extraction is completed, use enzyme-free sterile water to dilute the extract 10 times, 100 times, and 1000 times, and use it together with the original solution as a positive sample for standby.

[0210] 6.3. Design of first primer, second primer and target detection probe

[0211] The first primer, second primer and target detection probe used are the same as those in Example 1.

[0212] 6.4. Amplification reaction system and digital SAT chip preparation and amplification

[0213] (1) Add 30 μL of detection solution (containing 15 mM Tris, MgCl 2 15mM, dNTP2.5mM, NTP 3mM, PVP40 1%, KCl 10mM, 200pmol / mL of the first primer (SEQ ID NO: 1), 300pmol / mL of the second primer (SEQ ID NO: 2), 200pmol / mL of the target detection probe (SEQ ID NO: 3);

[0214] (2) Add 2 μL of the extracted and graded diluted influenza A virus RNA samples to the above treatment tubes, shake and mix, react at 60° C. for 5-10 min, and cool to room temperature after reaction.

[0215] (3) Add 10 μL of SAT enzyme solution (preheated at 42° C., containing 60,000 U / mL M-MLV reverse transcriptase, 40,000 U / mL T7 RNA polymerase, 10 mM HEPES pH 7.5, 15 mM N-acetyl-L-cysteine, 0.15 mM zinc acetate, 20 mM trehalose, 100 mM Tris-HCl pH 8.0, 80 mM KCl, 0.25 mM EDTA, 0.5% (v / v) Triton X-100 and 30% (v / v) glycerol) to each treated tube after the above reaction; use a pipette to quickly pipette and mix, and take 20 μL for digital SAT chip preparation. The specific preparation method can be found in Example 1.

[0216] (4) After the chip is prepared, a fluorescence image is taken immediately, which is recorded as the 0-min image; then the chip is heated at 42°C using a flat-plate thermostat.

[0217] (5) Control group setting: The amplification system prepared in step (3) was directly transferred to a 96-well PCR plate and subjected to RT-PCR (primer-probe combination, see Example 5) on ABI7500 for real-time amplification and fluorescence detection. The FAM channel was selected and fluorescence was collected once per cycle. Figure 6 The amplification curves of the RT-PCR for nucleic acid templates of different concentrations are shown.

[0218] 6.5. Real-time digital RNA isothermal amplification (real-time ddSAT) detection

[0219] The chip in step 6.4 (4) was used to collect fluorescence signals independently every 5 minutes. After the collection was completed, amplification was continued. By comparing the image analysis data at 9 time points, the number of positive points at 0min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, and 40min were counted as shown in Table 13.

[0220] In order to compare the differences between real-time ddSAT and dd-RT-PCR, dd-RT-LAMP, and dd-RT-RPA methods in the real-time qualitative process, the primer probe combination described in Example 5 and the corresponding reaction program were used to perform digital amplification detection and positive point counting analysis on the three comparative methods, and the results are shown in Tables 14, 15, and 16, respectively. In the dd-RT-PCR method, the fluorescence signal was detected every 5 cycles, and in the dd-RT-LAMP and dd-RT-RPA methods, the fluorescence signal was detected every 5 minutes.

[0221] Table 13: Real-time ddSAT statistics of positive points at 9 time points

[0222]

[0223] Table 14: dd-RT-PCR statistics of positive points at 9 time points

[0224]

[0225]

[0226] Table 15: dd-RT-LAMP counts the number of positive points at 9 time points

[0227]

[0228] Table 16: dd-RT-RPA statistics of positive points at 9 time points

[0229]

[0230] 6.6. Results comparison and analysis

[0231] Depend on Figure 6It can be seen that in the conventional real-time fluorescence PCR (RT-PCR) test results, positive amplification curves were observed only in the first two concentrations of the three 10-fold dilution (10-fold dilution, 100-fold dilution and 1000-fold dilution) concentration gradient samples, and the dT values ​​were 25 and 30, respectively, which is consistent with the positive point count results of the 9 time nodes counted by the dd-RT-PCR method listed in Table 14, that is, positive signals can only be detected 25 and 30 cycles after the start of the amplification reaction. The positive point count results of the 9 time nodes counted by the real-time ddSAT method shown in Table 13 show that positive signals can be detected in the three 10-fold dilution concentration gradient samples, and the earliest positive results can be obtained 5 minutes, 5 minutes and 15 minutes after the start of the amplification reaction. Although the dd-RT-LAMP test results shown in Table 15 and the dd-RT-RPA test results shown in Table 16 can also detect three 10-fold dilution concentration gradient samples, dd-RT-LAMP detects positive signals at 15 minutes, 15 minutes and 20 minutes after the start of the amplification reaction, and dd-RT-RPA detects positive signals at 20 minutes, 25 minutes and 25 minutes after the start of the amplification reaction. The above results show that the detection limit of the real-time ddSAT method provided by the present invention is better than that of conventional RT-PCR and dd-RT-PCR methods, and relative to dd-RT-PCR, dd-RT-LAMP and dd-RT-RPA, the real-time ddSAT method provided by the present invention has a significantly earlier positive detection time, and can report positive results with higher sensitivity and faster speed, which has significant clinical application value for nucleic acid qualitative detection.

[0232] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for absolute quantification of nucleic acid based on isothermal amplification of nucleic acid, characterized in that: The method comprises the following steps: T1) preparing a reaction system based on isothermal amplification of nucleic acids; T2) dividing the reaction system into several micro-reaction units and performing constant temperature amplification of nucleic acid; and T3) After the nucleic acid isothermal amplification, the fluorescence signal of each of the micro-reaction units is detected, and the positive and negative signals are determined accordingly, and the number of nucleic acid copies in the nucleic acid sample to be tested is absolutely quantified according to the number of positive and negative micro-reaction units detected; The reaction system based on isothermal amplification of nucleic acid in step T1) comprises the following components: a. Nucleic acid sample to be tested; b. a first primer, whose 3' end can hybridize with the 3' end or near the 3' end of the target sequence in the nucleic acid sample to be tested, and whose 5' end is a promoter sequence, wherein the promoter sequence is a T7 promoter sequence; c. a second primer, which cooperates with the first primer to amplify the target sequence; d. one or more fluorescent probes for detecting the target sequence, wherein the fluorescent probe is a molecular beacon probe; e. an RNA-dependent DNA polymerase, which is M-MLV reverse transcriptase; and f. RNA polymerase, which is T7 RNA polymerase.

2. A real-time nucleic acid qualitative and / or absolute quantitative method based on nucleic acid isothermal amplification, characterized in that: The method comprises the following steps: S1) preparing a reaction system based on isothermal amplification of nucleic acid; S2) dividing the reaction system into a number of micro-reaction units and performing constant temperature amplification of nucleic acid; and S3) During the isothermal amplification of nucleic acid in step S2), the fluorescence signal of each micro-reaction unit is detected at multiple time points, and the positive and negative results of the nucleic acid sample to be tested are qualitatively determined according to the fluorescence signal detection results, and / or the number of nucleic acid copies in the nucleic acid sample to be tested is absolutely quantified in real time according to the number of positive and negative micro-reaction units detected; The reaction system based on isothermal amplification of nucleic acid in step S1) comprises the following components: a. Nucleic acid sample to be tested; b. a first primer, whose 3' end can hybridize with the 3' end or near the 3' end of the target sequence in the nucleic acid sample to be tested, and whose 5' end is a promoter sequence, wherein the promoter sequence is a T7 promoter sequence; c. a second primer, which cooperates with the first primer to amplify the target sequence; d. one or more fluorescent probes for detecting the target sequence, wherein the fluorescent probe is a molecular beacon probe; e. an RNA-dependent DNA polymerase, which is M-MLV reverse transcriptase; and f. RNA polymerase, which is T7 RNA polymerase.

3. The method according to claim 2, characterized in that In step S3), real-time fluorescence signal detection is performed, and a real-time qualitative determination of the positive or negative result of the nucleic acid sample to be tested is performed based on the fluorescence signal detection result.

4. The method according to any one of claims 1 to 3, characterized in that The operation of preparing the reaction system based on isothermal nucleic acid amplification in step T1) or step S1) includes: (1) mixing components b, c and d to obtain a first mixed solution; (2) adding a to the first mixed solution, and incubating at 55-90° C. for 2-30 minutes to obtain a second mixed solution; and (3) Adding the enzyme components e and f to the second mixed solution to obtain the reaction system.

5. The method according to any one of claims 1 to 3, characterized in that: The nucleic acid sample to be tested in step T1) or step S1) is an RNA or DNA sample.

6. The method according to claim 4, characterized in that The volume ratio of the second mixed solution to the enzyme component is (1-50):

1.

7. The method according to claim 5, characterized in that The RNA is selected from mRNA, rRNA, lncRNA, and miRNA.

8. The method according to claim 5, characterized in that The nucleic acid sample to be tested is influenza A virus 2009H1N1 RNA, human LncRNA PCA3 or hepatitis B virus DNA.

9. The method according to any one of claims 1 to 3, characterized in that: In step T2) or step S2), the random distribution of nucleic acid molecules in the micro-reaction units is made to conform to Poisson distribution or the number of nucleic acid molecules contained in each micro-reaction unit is at most one; and / or In step T2) or step S2), the reaction system is divided into a plurality of small reaction units using microfluidics technology or other technologies; and / or The conditions for the isothermal amplification of the nucleic acid in step T2) or step S2) are: isothermal incubation at 42-65°C for 5-60 minutes.

10. The method according to claim 9, characterized in that In step T2) or step S2), the number of nucleic acid molecules contained in some micro-reaction units is at most one, and the random distribution of nucleic acid molecules in the micro-reaction units conforms to Poisson distribution.

11. The method according to claim 9, characterized in that In step T2) or step S2), the reaction system is divided into a number of micro-reaction units using a droplet segmentation technique in the form of micro-droplets or physical micro-pores.

12. The method according to claim 11, characterized in that In step T2) or step S2), the reaction system is divided into a number of micro-reaction units using oil-in-water micro-droplets or physical micro-pore chips.

13. The method according to claim 11, characterized in that In step T2) or step S2), a digital PCR system is used to divide the reaction system into a plurality of micro-reaction units.

14. The method according to any one of claims 1 to 3, characterized in that The method of performing fluorescence signal detection on each of the micro-reaction units in step T3) or step S3) is flow fluorescence detection or fluorescence imaging; and / or The criterion for judging the positive and negative in step T3) or step S3) is: the presence of a fluorescent signal is judged as positive, and the absence of a fluorescent signal is judged as negative; and / or In step T3) or step S3), the absolute quantification of the nucleic acid copy number in the nucleic acid sample to be tested according to the number of positive and negative micro-reaction units detected is to calculate the nucleic acid copy number using the Poisson distribution formula, or directly count the number of positive reaction units, thereby determining the starting copy number of the nucleic acid template molecule.

15. The method according to claim 14, characterized in that The method for detecting the fluorescence signal of each of the micro-reaction units in step T3) or step S3) is oil-in-water droplet fluorescence flow detection, oil-in-water chip fluorescence photography or microwell chip photography.

16. The method according to any one of claims 1 to 3, characterized in that The nucleic acid samples to be tested include medical samples and non-medical samples whose sources include food, blood products, and the environment.

17. The method according to claim 2 or 3, characterized in that: In step S3), the fluorescence signal detection at multiple time points is performed on each of the micro-reaction units, that is, the fluorescence signal is detected once every 0.5-10 minutes during the nucleic acid isothermal amplification process.

18. The method according to claim 17, characterized in that In step S3), the fluorescence signal detection at multiple time points for each of the micro-reaction units is performed every 0.5 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min during the nucleic acid isothermal amplification process.

19. A system for qualitative and / or absolute quantitative determination of nucleic acid based on isothermal amplification of nucleic acid, comprising: 1) Reaction system based on isothermal amplification of nucleic acids; 2) A device for dividing a reaction system based on isothermal amplification of nucleic acids into a number of micro-reaction units; 3) A device for isothermal amplification of nucleic acids in each micro-reaction unit; and 4) a device for detecting the fluorescence signal of each of the micro-reaction units after and / or during the isothermal amplification of nucleic acid; The reaction system based on isothermal amplification of nucleic acid comprises the following components: A. a first primer, whose 3' end can hybridize with the 3' end or near the 3' end of the target nucleic acid to be detected, and whose 5' end is a promoter sequence, wherein the promoter sequence is a T7 promoter sequence; B. a second primer, which cooperates with the first primer to amplify the target nucleic acid to be detected; C. a target fluorescent probe, which is used to detect the target nucleic acid to be detected, wherein the target fluorescent probe can hybridize with the negative strand of the target nucleic acid to be detected and release a fluorescent signal, wherein the target fluorescent probe is a molecular beacon probe; D. an RNA-dependent DNA polymerase, which is M-MLV reverse transcriptase; and E. RNA polymerase, which is T7 RNA polymerase.

20. The system according to claim 19, wherein the device for detecting the fluorescence signal of each of the micro-reaction units during the isothermal amplification of nucleic acid is a real-time fluorescence signal detection device.

21. The system according to claim 19 or 20, wherein the target nucleic acid to be detected includes RNA and DNA.

22. The system according to claim 21, wherein the target nucleic acid to be detected is selected from: influenza A virus 2009 H1N1 RNA, human LncRNA PCA3 and hepatitis B virus DNA; wherein: When the target nucleic acid to be detected is influenza A virus 2009H1N1 RNA, the nucleotide sequences of the first primer, the second primer and the target fluorescent probe are shown in SEQ ID NO:1-SEQ ID NO:3 respectively; When the target nucleic acid to be detected is human LncRNA PCA3, the nucleotide sequences of the first primer, the second primer and the target fluorescent probe are shown in SEQ ID NO:4-SEQ ID NO:6 respectively; When the target nucleic acid to be detected is hepatitis B virus DNA, the nucleotide sequences of the first primer, the second primer and the target fluorescent probe are respectively shown as SEQ ID NO: 19 to SEQ ID NO:

21.

23. Use of the system according to any one of claims 19 to 22 in preparing a kit for absolute quantification of nucleic acids.

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