Nucleic-acid absolute-quantification system and nucleic-acid absolute-quantification method based on nucleic-acid isothermal amplification

Abstract

Disclosed in the present invention are a nucleic-acid absolute-quantification system and nucleic-acid absolute-quantification method based on nucleic-acid isothermal amplification. In combination with a nucleic-acid synchronous isothermal amplification method and digital micro-droplet quantification technology, the nucleic-acid absolute-quantification method comprises: preparing a reaction system which is based on nucleic-acid isothermal amplification; segmenting the reaction system into several micro reaction units, and performing an isothermal amplification reaction on the micro reaction units; and after the isothermal amplification reaction, performing fluorescence signal detection on each of the micro reaction units, and on the basis of the number of negative micro reaction units after detection and the number of positive micro reaction units after detection, performing absolute quantification on the copy number of nucleic acids in a sample. The nucleic-acid absolute-quantification method provided in the present invention has the advantages of realizing a stable system and a high compatibility, involving low device requirements, and realizing high sensitivity and quick detection, is applicable in particular to the design and development of a fully automatic digital nucleic-acid quantification platform, and is applicable to the quantitative detection of nucleic acids in clinical tests and basic research.

Description

A nucleic acid absolute quantification system and nucleic acid absolute quantification method based on nucleic acid constant temperature amplification 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 constant temperature amplification, and more particularly to a real-time nucleic acid absolute quantification system and a real-time nucleic acid absolute quantification method based on nucleic acid constant temperature amplification. Background Art

[0002] Currently, nucleic acid molecules (DNA / RNA) are quantified primarily through relative quantification using real-time quantitative polymerase chain reaction (RT-qPCR). This method requires the use of fixed nucleic acid standards to plot a standard curve for relative quantification. This method has an error within the ±0.5 log value range, resulting in poor quantitative accuracy and cumbersome operation.

[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 units are amplified, and uses a 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 in various complex samples. However, due to its limitations in the methodological requirements of the polymerase chain reaction itself, it generally takes a long time, usually 2.5-4 hours to complete sample quantification. At the same time, because PCR requires repeated temperature increases and decreases, it places very high demands on the stability of the tiny system. Once stability problems occur, problems such as detection failure and leakage and contamination of the amplified DNA product are prone to occur.

[0004] Summary of the Invention

[0005] In response to 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 nucleic acid amplification, which comprises the following steps:

[0006] T1) preparing a reaction system based on isothermal nucleic acid amplification;

[0007] T2) dividing the reaction system into several small reaction units and performing isothermal nucleic acid amplification; and

[0008] T3) After the nucleic acid isothermal amplification, performing fluorescence signal detection on each of the micro-reaction units to determine the positive / negative signal, and performing absolute quantification of the nucleic acid copy number in the nucleic acid sample to be tested based on the number of positive / negative micro-reaction units detected;

[0009] The reaction system based on isothermal nucleic acid amplification in step T1) includes the following components:

[0010] a. Nucleic acid sample to be tested;

[0011] b. A first primer, whose 3' end is capable of hybridizing 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;

[0012] c. a second primer, which cooperates with the first primer to amplify the target sequence;

[0013] 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);

[0014] e, RNA-dependent DNA polymerase; and

[0015] f. RNA polymerase.

[0016] 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:

[0017] S1) preparing a reaction system based on isothermal nucleic acid amplification;

[0018] S2) dividing the reaction system into a plurality of small reaction units and performing isothermal nucleic acid amplification; and

[0019] S3) During the isothermal amplification of nucleic acid in step S2), fluorescence signal detection is performed on each of the micro-reaction units at multiple time points, optionally performing real-time fluorescence signal detection, and performing real-time qualitative determination of the positive / negative result of the nucleic acid sample to be tested based on the fluorescence signal detection result, and / or further performing real-time absolute quantification of the nucleic acid copy number in the nucleic acid sample to be tested based on the number of positive / negative micro-reaction units detected;

[0020] The reaction system based on isothermal nucleic acid amplification in step S1) includes the following components:

[0021] a. Nucleic acid sample to be tested;

[0022] b. A first primer, whose 3' end is capable of hybridizing 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;

[0023] c. a second primer, which cooperates with the first primer to amplify the target sequence;

[0024] 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);

[0025] e, RNA-dependent DNA polymerase; and

[0026] f. RNA polymerase.

[0027] In some embodiments, the operation of preparing the reaction system based on isothermal nucleic acid amplification in step T1) or step S1) includes:

[0028] (1) Mixing components b, c, and d to obtain a first mixed solution; optionally, the first mixed solution comprises: 10-50 mM Tris, 5-40 mM KCl, 10-40 mM MgCl2, 1-20 mM NTP, 0.1-10 mM dNTPs, 1-10% PVP40, 10-250 pmol / mL of b, 10-350 pmol / mL of c, and 10-250 pmol / mL of d;

[0029] (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

[0030] (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: 16000-160000 U / mL of RNA-dependent DNA polymerase, 8000-80000 U / mL of RNA polymerase, 2-10 mM HEPES pH7.5, 10-100 mM N-acetyl-L-cysteine, 0.04-0.4 mM zinc acetate, 10-100 mM trehalose, 40-200 mM Tris-HCl pH 8.0, 40-200 mM KCl, 0.01-0.5 mM EDTA, 0.1-1% (v / v) Triton X-100 and 20-50% (v / v) glycerol.

[0031] 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.

[0032] 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.

[0033] In some embodiments, the RNA polymerase is T3, T7, M13 or SP6 RNA polymerase, and the RNA polymerase corresponds to the promoter sequence used.

[0034] In some embodiments, in step T2) or step S2), the random distribution of nucleic acid molecules in the micro-reaction units conforms to the 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 the Poisson distribution.

[0035] In some embodiments, in step T2) or step S2), the reaction system is divided into several small reaction units using microfluidics or other technologies; alternatively, the reaction system is divided into several small reaction units using droplet segmentation technology in the form of microdroplets or physical micropores; further alternatively, the reaction system is divided into several small reaction units using the form of oil-in-water droplets or physical micropore chips; further alternatively, the reaction system is divided into several small 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-type digital PCR BioDigital, Shanghai Little Turtle Biotechnology digital PCR all-in-one machine SCIDigital, digital PCR system Accu One-100 (Shanghai Zhenzhun Biotechnology), etc.

[0036] In some embodiments, the conditions for the isothermal amplification of the nucleic acid in step T2) or step S2) are: constant 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.

[0037] 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 micropore chip imaging.

[0038] In some embodiments, the criterion for judging the positive or 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.

[0039] 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 microreaction units detected is performed by calculating the nucleic acid copy number using a Poisson distribution formula, or directly counting the number of positive reaction units, thereby determining the starting copy number of the nucleic acid template molecule.

[0040] 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.

[0041] 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 constant temperature amplification process, and optionally 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.

[0042] In another aspect, the present invention further provides a system for qualitative and / or absolute quantitative determination of nucleic acids based on isothermal nucleic acid amplification, comprising:

[0043] 1) Reaction system based on isothermal nucleic acid amplification;

[0044] 2) Devices for dividing a nucleic acid isothermal amplification reaction system into several micro-reaction units (e.g., digital PCR systems or water-in-oil microdroplet preparation devices);

[0045] 3) a device for performing constant-temperature amplification of nucleic acids on each micro-reaction unit (e.g., a device capable of constant-temperature amplification, such as a plate-type thermostat); and

[0046] 4) a device for detecting a fluorescent signal from each of the micro-reaction units after and / or during constant-temperature nucleic acid amplification; optionally, the device for detecting a fluorescent signal from each of the micro-reaction units during constant-temperature nucleic acid amplification is a real-time fluorescent signal detection device (e.g., a flow fluorescence detection device or a fluorescence imaging device);

[0047] The reaction system based on nucleic acid isothermal amplification comprises the following components:

[0048] A. A first primer, whose 3' end is capable of hybridizing to or near the 3' end of the target nucleic acid to be detected, and whose 5' end is a promoter sequence;

[0049] B. a second primer, which cooperates with the first primer to amplify the target nucleic acid to be detected;

[0050] 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;

[0051] D. RNA-dependent DNA polymerase; and

[0052] E.RNA polymerase.

[0053] 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:

[0054] 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 as SEQ ID NO: 1 to SEQ ID NO: 3, respectively;

[0055] 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 as SEQ ID NO: 4 to SEQ ID NO: 6, respectively;

[0056] 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 shown as SEQ ID NO: 21 to SEQ ID NO: 23, respectively.

[0057] The application of the above system in preparing a kit for absolute quantification of nucleic acids also falls within the scope of the present invention.

[0058] The present invention provides a method for absolute quantification of nucleic acids, which is a digital micro-droplet constant temperature amplification method for absolute quantification of nucleic acids (digital drop SAT, referred to herein as ddSAT) based on the simultaneous isothermal amplification (Simultaneous Amplification and Testing, SAT) method of the applicant's existing patent (CN101333565B). 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 at the same time, based on the amplification detection mode of digital micro-droplets, after or during constant temperature amplification of nucleic acids, quantitative detection or real-time quantitative detection (referred to herein as real-time ddSAT) of nucleic acid samples is achieved. The principle of constant temperature amplification of nucleic acids in this method is based on the applicant's existing patent (CN101333565B), and the principle of absolute quantification of nucleic acids is based on Poisson distribution theory technology.

[0059] This Poisson distribution theory technique randomly disperses the sample template across tens of thousands of microreaction units in a limiting dilution mode. Each reaction unit can be assigned zero, one, or multiple template nucleic acid molecules, and the distribution of the template molecules follows a Poisson distribution. After amplification, a unit with a fluorescent signal is scored as 1, while one without is scored as 0. In other words, the presence or absence of a fluorescent signal defines a reaction unit as positive or negative, respectively. By counting the total number of reaction units and the number of positive reaction units, the starting concentration of the nucleic acid template molecules can be calculated using the Poisson distribution formula. Ideally, when the nucleic acid concentration of the sample is diluted to a very low level, the sample solution is dispersed across a large number of reaction units, ensuring that each reaction unit contains at most one nucleic acid molecule. Under these conditions, the starting number of nucleic acid template molecules can be directly determined by counting the number of positive reaction units. However, when measuring high template concentrations, some reaction units contain two or more nucleic acid molecules, and the random distribution of the large number of nucleic acid template molecules follows a Poisson distribution. Therefore, the absolute concentration of the nucleic acid template molecules can be calculated using the Poisson distribution probability formula.

[0060] In formula (1), λ is the average copy number of the template molecule in each reaction unit, and p is the probability that a reaction unit contains k copies of the template molecule. λ is equivalent to diluting the starting copy number of the template molecule in the sample (c) by a factor of m (m is the dilution factor, equivalent to the volume of an independent reaction unit), λ = cm. When k = 0, that is, when there are no template molecules 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] 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:

[0064] Therefore, when using the method of the present invention for nucleic acid quantitative analysis, under the conditions of knowing the total number of reaction units, dilution factor, and number of positive reaction units, the starting copy number of the nucleic acid template molecule in the sample can be obtained. The main advantages of the nucleic acid absolute quantitative detection method of the present invention are as follows:

[0065] 1. Stable system: Since amplification is carried out under constant temperature conditions, the stability of the microdroplets and the sealed chip is minimally affected, and the sealing performance of the microdroplets and the chip is not easily destroyed, so the amplification reaction can proceed more smoothly.

[0066] 2. Multiple detection systems available: Existing digital PCR quantitative detection methods require detailed optimization of reagent components, from chemical composition to primer and probe concentrations, for different digital PCR systems. However, the ddSAT technology of the present invention, due to its constant temperature properties, is compatible with various digital quantitative systems for water-in-oil droplets and microwell chips, eliminating the need for system adjustments. Users can choose from a variety of detection systems based on their experience, existing technical knowledge, and experimental conditions.

[0067] 3. Rapid detection: Nucleic acid amplification is performed at a constant temperature, eliminating the need for repeated temperature increases and decreases. This greatly shortens the amplification time and significantly improves detection efficiency. The results of the examples show that the real-time ddSAT method provided by the present invention can detect positive results significantly earlier than existing real-time quantitative detection methods.

[0068] 4. Simple equipment requirements and easy automation: Compared with conventional digital PCR technology, the equipment system required by the present invention does not require a temperature rise and fall process, which not only reduces design and production costs but also facilitates the design of integrated equipment.

[0069] 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 fluorescence PCR method, and the detection of RNA is not affected by the DNA in the system, and the detection results are more accurate.

[0070] In summary, the method of the present invention offers advantages such as system stability, high compatibility, minimal equipment requirements, and rapid detection, making it particularly suitable for the design and development of fully automated digital nucleic acid quantification platforms. This technology, when combined with commercially available digital nucleic acid quantification platforms, is suitable for high-sensitivity nucleic acid qualitative or high-precision nucleic acid quantitative detection in fields such as clinical testing, nucleic acid standard assignment, tumor gene testing, and basic molecular biology research, making it suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG1 is a photograph (A) and output result (B) of the chip fluorescence detection for absolute quantification of influenza A virus RNA in Example 1.

[0072] FIG2 is the statistical results of absolute quantification of influenza A virus RNA by 20 laboratories in Example 1.

[0073] FIG3 is a photo (A) and output result (B) of the chip fluorescence detection for absolute quantification of PCA3 RNA in Example 2.

[0074] FIG4 is a schematic diagram of the production process of the microfluidic oil-in-water chip.

[0075] Figure 5 shows the results of absolute quantification of influenza A virus RNA using water-in-oil microdroplets (A1 and A2) captured in bright field and dark field, and a fluorescence detection photograph of a physical microwell chip (B).

[0076] FIG6 is a real-time fluorescence PCR amplification curve of influenza A virus RNA at different dilutions. DETAILED DESCRIPTION

[0077] The present invention is described in detail below with reference to specific embodiments.

[0078] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, 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 to be considered as illustrative and non-restrictive in nature.

[0079] Unless otherwise specified, the methods used in the following examples are conventional methods. 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).

[0080] The methods for obtaining the various biological materials described in the examples merely provide experimental methods for achieving the disclosed objectives and should not be construed as limiting the sources of the biological materials used in the present invention. In fact, the sources of the biological materials used are diverse, and any legally and ethically accessible biological material may be substituted for and used as indicated in the examples.

[0081] All primers and probes mentioned in the present invention were synthesized using existing techniques.

[0082] Example 1: Absolute quantitative detection of pathogen nucleic acids

[0083] 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.

[0084] 1.1 Sample Source

[0085] China Food and Drug Inspection and Quarantine Institute, Institute for In Vitro Diagnostic Testing, 2023, first batch of inactivated cultures of influenza A 2009H1N1 for pathogen dPCR (also referred to as ddPCR in this article) capability comparison testing, number NIFDC2301-01.

[0086] 1.2. Extraction of influenza A virus RNA

[0087] 20 μL of the inactivated culture solution of influenza A 2009 H1N1 (NIFDC2301-01) was taken and RNA was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0.

[0088] 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)

[0089] Specific primers and target detection probe sequences were designed based on the influenza A virus M gene specific sequence (GenBank: MT241814.1), as shown below:

[0090] First primer:

[0091] 5'-aatttaatacgactcactatagggagaTTTGGACAAAGCGTCTACGCTGC-3' (SEQ ID NO: 1);

[0092] Second primer: 5′-ACTGGAAAGTGTCTTTGCA-3′ (SEQ ID NO: 2);

[0093] 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;

[0094] 1.4. Amplification reaction system and digital SAT chip preparation and amplification

[0095] (1) Add 30 μL of detection solution (containing 15 mM Tris, 15 mM MgCl2, 2.5 mM dNTP, 3 mM NTP, 1% PVP40, 10 mM KCl, 200 pmol / mL of the first primer (SEQ ID NO: 1), 300 pmol / mL of the second primer (SEQ ID NO: 2), and 200 pmol / mL of the target detection probe (SEQ ID NO: 3)) to the sample processing tube;

[0096] (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 minutes, and then cool to room temperature.

[0097] (3) Add 10 μL of SAT enzyme solution (preheated at 42°C, containing 60,000 U / mL of M-MLV reverse transcriptase, 40,000 U / mL of 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, and 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 pipette to mix, and 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), and immediately start the sample pretreatment system to prepare the chip.

[0098] (4) After the chip is prepared, use a flat plate thermostat to heat the reaction at 42°C for 40 minutes.

[0099] 1.5 Digital Droplet SAT (ddSAT) Detection

[0100] Place the chip, which has completed amplification in step 1.4, into a chip reader and perform an endpoint fluorescence reading, selecting the FAM fluorescence channel. By counting the number of positive and negative spots on the chip, use software to analyze (based on Poisson distribution theory) the RNA copy number in the sample. The positive and negative spots of the fluorescence amplification are shown in Figure 1, Panel A. The clear distinction between positive and negative indicates that the amplification result is valid. The calculated results are shown in Figure 1, Panel B, with an output of 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.

[0101] 1.6. Results Comparison

[0102] Inactivated cultures of influenza A 2009 H1N1 (NIFDC2301-01) were distributed by the China Food and Drug Administration and the National Institute for In Vitro Diagnostic Testing to 20 companies (referred to herein as Lab 1-Lab 20) equipped with digital PCR (herein also referred to as ddPCR) calibration equipment and influenza A detection systems. The statistical data for all test results are shown in Figure 2 . A comparison of the calibration results using the digital droplet SAT method of the present invention with the statistical data from Lab 1-Lab 20 is shown in Table 1 . It can be seen that the digital droplet SAT method (herein also referred to as ddSAT) provided by the present invention achieves essentially the same calibration results for influenza A virus RNA as the ddPCR method, with no significant differences.

[0103] Table 1: Comparison of 20 digital PCR test results and the digital droplet SAT method of the present invention

[0104] Example 2: Absolute quantitative detection of human RNA

[0105] 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.

[0106] 2.1. Acquisition of human urine RNA samples

[0107] The urine sample was a 400 μL random preoperative urine sample from a prostate cancer patient, and RNA was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit.

[0108] 2.2 Design of first primer, second primer and target detection probe

[0109] Specific primers and target detection probe sequences were designed based on the human LncRNA PCA3 sequence (NCBI Reference Sequence: NR_132312.1), as shown below:

[0110] First primer:

[0111] 5'-aatttaatacgactcactataggggagaGGGACCAGGCACAGGGCGAG-3' (SEQ ID NO: 4);

[0112] Second primer: 5′-CACAGGAAGCACAAAAGGA-3′ (SEQ ID NO: 5);

[0113] 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;

[0114] 2.3 Amplification reaction system and digital SAT chip preparation and amplification

[0115] (1) Add 30 μL of detection solution (containing 15 mM Tris, 15 mM MgCl2, 2.5 mM dNTP, 3 mM NTP, 1% PVP40, 10 mM KCl, 200 pmol / mL of the first primer (SEQ ID NO: 4), 300 pmol / mL of the second primer (SEQ ID NO: 5), and 200 pmol / mL of the target detection probe (SEQ ID NO: 6)) to the sample processing tube;

[0116] (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.

[0117] (3) Add 10 μL of SAT enzyme solution (preheated at 42°C, containing 60,000 U / mL of M-MLV reverse transcriptase, 40,000 U / mL of 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.

[0118] (4) After the chip is prepared, use a flat plate thermostat to heat it at 42°C for 40 minutes.

[0119] 2.4 Digital Droplet SAT (ddSAT) Assay: Place the chip, amplified in step 2.3, into a chip reader for endpoint fluorescence imaging and reading, selecting the FAM fluorescence channel. Count the number of positive and negative spots on the chip, and use software to analyze the RNA copy number in the sample. The positive and negative spots of the fluorescence amplification are shown in Figure 3, Panel A. The clear distinction between positive and negative spots indicates valid amplification. The calculated results are shown in Figure 3, Panel B, with an output of 110.47 copies / μL. Based on volume conversion, the PCA3 RNA concentration in the original human urine sample is 5.80E+5 copies / mL.

[0120] 2.5. Comparison of ddPCR and ddSAT Detection Performance

[0121] To further confirm whether the ddSAT method differs from the ddPCR method in detecting human RNA, specific ddPCR primers and Taqman fluorescent probes were designed based on the LncRNA PCA3 sequence. The sequence information is shown in Table 2 below. The ddPCR reaction procedure was: 50°C 15 min—95°C 15 s—(95°C 15 s—60°C 30 s—72°C 1 min) 40 cycles. The human urine RNA sample extracted in step 2.1 above was also used for ddSAT and ddPCR absolute quantitative detection. The results are shown in Table 3 below, which shows that the ddSAT test results were lower than those of ddPCR. To further confirm whether the high ddPCR values ​​were 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 detected using ddSAT and ddPCR. The test results are shown in Table 3. The results showed that the ddPCR method cannot avoid the interference of residual genomic DNA. When the human urine RNA sample product was digested with RNase, a certain amount of target nucleic acid could still be detected using ddPCR, while the target nucleic acid could not be detected using the ddSAT method. Therefore, the ddSAT method provided by the present invention is not affected by genomic DNA and is more accurate in quantification.

[0122] At the same time, to confirm the differences between the isothermal amplification LAMP and RPA methods and ddSAT, this example also designed digital LAMP and digital RPA detection systems for the lncRNA PCA3 sequence in human urine samples. The LAMP reaction procedure was: 65°C for 40 minutes; the RPA reaction procedure was: 37°C for 40 minutes. The interference test of genomic DNA was also performed as described above. The results are shown in Table 3. Due to methodological limitations, the digital LAMP and digital RPA methods, like the ddPCR method, cannot avoid the interference of residual genomic DNA.

[0123] Table 2: ddPCR, digital LAMP, and digital RPA primer and probe sequence information for detecting human LncRNA PCA3

[0124] Table 3: Results of quantitative detection of human LncRNA PCA3 by ddPCR, digital LAMP, digital RPA, and ddSAT

[0125] Example 3: Absolute quantitative detection of pathogen DNA

[0126] The above Examples 1 and 2 have demonstrated that the ddSAT method provided by the present invention can meet the requirements for 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.

[0127] 3.1. Acquisition of HBV DNA

[0128] Based on the hepatitis B virus DNA sequence in the NCBI database (GenBank: X72213.1), the full-length HBV DNA sequence was artificially synthesized by Bioengineering (Shanghai) Co., Ltd. The DNA content provided in the 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.

[0129] 3.2 Design of first primer, second primer and target detection probe

[0130] Specific primers and target detection probe sequences were designed based on the human HBV DNA sequence (GenBank: X72213.1), as shown below:

[0131] First primer:

[0132] 5'-aatttaatacgactcactatagggagaGTTTGTATTATGCCCTGAGCC-3' (SEQ ID NO: 19);

[0133] Second primer: 5′-TCAGAGCAAATACCGCAAATCCAG-3′ (SEQ ID NO: 20);

[0134] 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.

[0135] 3.3 Amplification reaction system and digital SAT chip preparation and amplification

[0136] (1) Add 30 μL of detection solution (containing 15 mM Tris, 15 mM MgCl2, 2.5 mM dNTP, 3 mM NTP, 1% PVP40, 10 mM KCl, 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;

[0137] (2) Add 2 μL of diluted HBV DNA sample to the above treatment tube, shake and mix, react at 90°C for 5-10 minutes, and then cool at room temperature (20-25°C) for 5-10 minutes.

[0138] (3) Add 10 μL of SAT enzyme solution (preheated at 42°C, containing 60,000 U / mL of M-MLV reverse transcriptase, 40,000 U / mL of 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 referred to Example 1.

[0139] (4) After the chip is prepared, use a flat plate thermostat to heat it at 42°C for 40 minutes.

[0140] 3.4 Digital Droplet SAT (ddSAT) Detection

[0141] The chip, after amplification in step 3.3, was placed in a chip reader and an endpoint fluorescence reading was performed, selecting the FAM fluorescence channel. The number of positive and negative spots on the chip was counted, and the DNA copy number in the sample was analyzed using software. The test results are shown in Table 5 below, which are generally consistent with the nucleic acid concentration of the HBV DNA sample used (1.34E+4 copies / μL), demonstrating that the ddSAT method provided by the present invention also has high accuracy for absolute quantitative detection of DNA samples.

[0142] 3.5. Performance comparison of ddSAT and ddPCR methods for DNA quantitative detection

[0143] To further confirm the consistency between ddSAT and ddPCR in DNA detection, ddPCR-related primers and probes were designed based on the HBV DNA sequence (GenBank: X72213.1). The sequence information is shown in Table 4. The PCR reaction procedure was: 95°C for 15 seconds—(95°C for 15 seconds—60°C for 30 seconds—72°C for 1 minute) for 40 cycles. Using the same DNA sample (obtained in step 3.1 above), absolute quantification was performed using both ddSAT and ddPCR. The results are shown in Table 5 below, demonstrating good consistency between the ddSAT and ddPCR methods for quantifying DNA samples.

[0144] Table 4: Primer and probe sequence information related to ddPCR targeting HBV DNA sequences

[0145] Table 5: Quantitative detection results of HBV DNA by ddSAT and ddPCR

[0146] Example 4: ddSAT detection using microdroplets or physical microwell chips

[0147] The ddSAT detection technology used in the above 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 sample in Example 1. The detection process is as follows.

[0148] 4.1 Sample Source

[0149] China Food and Drug Inspection Institute, Institute for In Vitro Diagnostic Testing, 2023 First Batch of Influenza A 2009H1N1 Inactivated Cultures for Pathogen dPCR Capability Comparison Testing, No. NIFDC2301-01.

[0150] 4.2. Extraction of influenza A virus RNA

[0151] 20 μL of the inactivated culture solution of influenza A 2009 H1N1 (NIFDC2301-01) was taken and RNA was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0.

[0152] 4.3 Design of first primer, second primer and target detection probe

[0153] The first primer, second primer and target detection probe used are the same as those in Example 1.

[0154] 4.4 Amplification Reaction System and Digital SAT Chip Preparation and Amplification

[0155] (1) Add 30 μL of detection solution (containing 15 mM Tris, 15 mM MgCl2, 2.5 mM dNTP, 3 mM NTP, 1% PVP40, 10 mM KCl, 200 pmol / mL of the first primer (SEQ ID NO: 1), 300 pmol / mL of the second primer (SEQ ID NO: 2), and 200 pmol / mL of the target detection probe (SEQ ID NO: 3)) to the sample processing tube;

[0156] (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 minutes, and then cool to room temperature.

[0157] (3) Add 10 μL of SAT enzyme solution (preheated at 42°C, containing 60,000 U / mL of M-MLV reverse transcriptase, 40,000 U / mL of 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 to mix, and take 20 μL for the following digital SAT chip preparation.

[0158] (4) The mixed amplification reaction solution was added to the microfluidic oil-in-water chip preparation hole and the physical microporous chip brush respectively to prepare the two chips. The oil-in-water generation process in the microfluidic oil-in-water chip is shown in Figure 4, wherein the microfluidic injection lane is divided into 3, 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 into the chip interlayer through multiple 1-to-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 microporous chip is a Zhenzhun biological digital PCR silicon-based chip, with 20,000 micropores etched on the silicon-based thin film. 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 micro-reaction unit based on physical micropores.

[0159] (5) After the chip is prepared, use a flat plate thermostat to heat it at 42°C for 40 minutes.

[0160] 4.5 Digital Droplet SAT (ddSAT) Detection

[0161] After amplification in step 4.4, place the chip in a chip reader and perform an endpoint fluorescence reading, selecting the FAM fluorescence channel. By counting the number of positive and negative spots on the chip, the RNA copy number in the sample is analyzed using software. Fluorescence imaging results for two chips (a microfluidic oil-in-water chip and a physical microwell chip) are shown in Figures 5 (A1, A2, and B). Figures 5 (A1 and A2) show the oil-in-water microdroplet images taken under brightfield and darkfield conditions, respectively. Figure 5 (B) shows the fluorescence image of the physical microwell chip, with the bright spots representing positive microreaction units. By counting the number of negative and positive microreaction units on both 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. No significant differences were observed between the two chips, demonstrating that ddSAT technology is compatible with both oil-in-water microdroplet and physical microwell chip approaches for digital nucleic acid quantification.

[0162] Table 6: Digital nucleic acid quantification results using oil-in-water microdroplets and physical microwell chips

[0163] Example 5: Real-time digital RNA constant temperature amplification and quantitative detection (real-time ddSAT)

[0164] 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 are performed. The specific method includes the following steps.

[0165] 5.1 Sample Source

[0166] China Food and Drug Inspection Institute, Institute for In Vitro Diagnostic Testing, 2023 First Batch of Influenza A 2009H1N1 Inactivated Cultures for Pathogen dPCR Capability Comparison Testing, No. NIFDC2301-01.

[0167] 5.2. Extraction of influenza A virus RNA

[0168] 20 μL of the inactivated culture solution of influenza A 2009 H1N1 (NIFDC2301-01) was taken and RNA was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0.

[0169] 5.3 Design of first primer, second primer and target detection probe

[0170] The first primer, second primer and target detection probe used are the same as those in Example 1.

[0171] 5.4 Amplification Reaction System and Digital SAT Chip Preparation and Amplification

[0172] (1) Add 30 μL of detection solution (containing 15 mM Tris, 15 mM MgCl2, 2.5 mM dNTP, 3 mM NTP, 1% PVP40, 10 mM KCl, 200 pmol / mL of the first primer (SEQ ID NO: 1), 300 pmol / mL of the second primer (SEQ ID NO: 2), and 200 pmol / mL of the target detection probe (SEQ ID NO: 3)) to the sample processing tube;

[0173] (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 minutes, and then cool to room temperature.

[0174] (3) Add 10 μL of SAT enzyme solution (preheated at 42°C, containing 60,000 U / mL of M-MLV reverse transcriptase, 40,000 U / mL of 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.

[0175] (4) After the chip is prepared, a fluorescence image is taken immediately, which is recorded as the 0-min image; then a flat-plate thermostat is used to heat it at 42°C.

[0176] 5.5 Real-time digital droplet SAT (real-time ddSAT) detection

[0177] To achieve real-time digital droplet SAT detection, this embodiment modified the existing fluorescence signal acquisition device by replacing the chip carrier with an amplification module with a constant temperature of 42°C, which can achieve timed fluorescence signal acquisition while amplifying at 42°C.

[0178] Using the modified fluorescence acquisition device, the chip from step 5.4 was independently sampled every 5 minutes. Amplification was then continued after the acquisition was complete. By comparing the image analysis data at each of the nine time points, a fluorescence change curve for all valid microreaction units was generated. By analyzing the fluorescence change curve for each valid microreaction unit, it was possible to more accurately determine which wells had truly generated amplified fluorescence signals, and these wells were read as positive, while the remaining invalid microwells were read as negative. The fluorescence value changes are shown in Table 7 below. Copy number calculations were performed based on the number of positive and negative wells, with the results shown in Table 11 below.

[0179] At the same time, in order to compare detection performance, this embodiment also designed influenza A virus detection systems including 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). The primer and probe sequences used in each system are shown in Table 7. The same nucleic acid samples as those used for real-time ddSAT were used for real-time digital amplification detection using dd-RT-PCR, dd-RT-LAMP, and dd-RT-RPA. dd-RT-PCR is a more commonly used amplification detection method (the PCR amplification program is: 95°C for 15 s—(95°C for 15 s—60°C for 30 s—72°C for 1 min) for 40 cycles), with fluorescence signals collected every 5 cycles. dd-RT-LAMP and dd-RT-RPA are currently known and more mature isothermal amplification methods, with fluorescence signals collected every 5 minutes. The LAMP reaction program is: 65°C for 40 minutes; the RPA reaction program is: 37°C for 40 minutes. 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.

[0180] Table 7: Specific PCR, LAMP, and RPA sequence information for detecting influenza A virus

[0181] Table 8: Fluorescence value changes at 9 time points detected by real-time ddSAT

[0182] Table 9: Fluorescence value changes at 9 time points detected by dd-RT-PCR

[0183] Table 10: Fluorescence value changes at 9 time points detected by dd-RT-LAMP

[0184] Table 11: Fluorescence value changes at 9 time points detected by dd-RT-RPA

[0185] Table 12: Test results

[0186] 5.6. Results comparison and analysis

[0187] The fluorescence value changes at the nine time points detected using real-time ddSAT in Table 8 above reveal that, as amplification time increases, the fluorescence values ​​of some microwells (e.g., microwells 1-3) continuously increase to a plateau brightness, stabilizing at 40 minutes. These microwells are therefore classified as positive. However, some microwells (e.g., microwells 4-5) also exhibit fluorescence, but at low levels, failing to reach the intensity expected for positive amplification. Furthermore, the fluorescence values ​​of these microwells do not continuously rise to a plateau brightness as amplification time increases. Therefore, these microwells should also be classified as negative and excluded from subsequent copy number calculations. However, in endpoint detection (i.e., fluorescence signal detection of chip microwells only after completion of isothermal nucleic acid amplification), these negative microwells with low fluorescence values ​​can also be classified as positive (false positives). This can result in an inflated absolute quantitative result using the endpoint method. As shown in Table 12 above, the quantitative results using the real-time fluorescence imaging method are lower than those using the endpoint method. These results demonstrate that the real-time digital droplet SAT (ddSAT) detection method can improve the ability to discriminate positive amplifications, eliminate some false-positive microwells, and thus enhance the accuracy of quantitative detection. The results in Tables 9-12 also demonstrate that the three real-time absolute quantitative detection methods, dd-RT-PCR, dd-RT-LAMP, and dd-RT-RPA, can also achieve absolute quantitative results with higher accuracy than endpoint methods. Furthermore, these methods provide essentially identical results for pure RNA quantitative detection.

[0188] In addition, by comparing the changes in fluorescence values ​​at nine time points 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.

[0189] Example 6: Real-time digital RNA constant temperature amplification (real-time ddSAT) detection

[0190] 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, and real-time digital RNA isothermal amplification qualitative detection is performed. The specific method includes the following steps.

[0191] 6.1 Sample Source

[0192] China Food and Drug Inspection Institute, Institute for In Vitro Diagnostic Testing, 2023 First Batch of Influenza A 2009H1N1 Inactivated Cultures for Pathogen dPCR Capability Comparison Testing, No. NIFDC2301-01.

[0193] 6.2. Extraction and dilution of influenza A virus RNA

[0194] Extract RNA from 20 μL of the inactivated influenza A 2009 H1N1 (NIFDC2301-01) culture solution using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver. 5.0. After extraction, dilute the extract 10-fold, 100-fold, and 1000-fold with enzyme-free sterile water. This dilution, along with the original solution, is used as a positive sample.

[0195] 6.3 Design of first primer, second primer and target detection probe

[0196] The first primer, second primer and target detection probe used are the same as those in Example 1.

[0197] 6.4 Amplification Reaction System and Digital SAT Chip Preparation and Amplification

[0198] (1) Add 30 μL of detection solution (containing 15 mM Tris, 15 mM MgCl2, 2.5 mM dNTP, 3 mM NTP, 1% PVP40, 10 mM KCl, 200 pmol / mL of the first primer (SEQ ID NO: 1), 300 pmol / mL of the second primer (SEQ ID NO: 2), and 200 pmol / mL of the target detection probe (SEQ ID NO: 3)) to each sample processing tube;

[0199] (2) Add 2 μL of the extracted and gradient diluted influenza A virus RNA samples to the above treatment tubes, shake and mix, react at 60°C for 5-10 minutes, and then cool to room temperature.

[0200] (3) Add 10 μL of SAT enzyme solution (preheated at 42°C, containing 60,000 U / mL of M-MLV reverse transcriptase, 40,000 U / mL of 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.

[0201] (4) After the chip is prepared, a fluorescence image is taken immediately, which is recorded as the 0-min image; then a flat-plate thermostat is used to heat it at 42°C.

[0202] (5) Control group setup: The amplification system prepared in step (3) was directly transferred to a 96-well PCR plate and RT-PCR was performed on an ABI 7500 (for primer and probe combinations, see Example 5). Real-time amplification fluorescence detection was performed using the FAM channel, and fluorescence was collected once per cycle. Figure 6 shows the amplification curves of the RT-PCR for different nucleic acid template concentrations.

[0203] 6.5. Real-time digital RNA isothermal amplification (real-time ddSAT) detection

[0204] 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 of the 9 time nodes, the number of positive points at 0min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, and 40min were counted as shown in Table 13.

[0205] To compare the differences in real-time qualitative analysis between real-time ddSAT and dd-RT-PCR, dd-RT-LAMP, and dd-RT-RPA methods, digital amplification detection and positive point counting analysis were performed on the three comparative methods using the primer-probe combination and the corresponding reaction procedure described in Example 5. 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.

[0206] Table 13: Real-time ddSAT statistics of positive points at 9 time points

[0207] Table 14: dd-RT-PCR statistics of positive points at 9 time points

[0208] Table 15: dd-RT-LAMP statistics of positive points at 9 time points

[0209] Table 16: dd-RT-RPA statistics of positive points at 9 time points

[0210] 6.6. Results comparison and analysis

[0211] As shown in Figure 6, in the conventional real-time fluorescence PCR (RT-PCR) test results, positive amplification curves were only observed for the first two concentrations of the three 10-fold dilution gradient samples (10-fold, 100-fold, and 1000-fold dilutions), with dT values ​​of 25 and 30, respectively. This is consistent with the positive point counts at nine time points calculated by the dd-RT-PCR method listed in Table 14, indicating that positive signals were detected 25 and 30 cycles after the start of the amplification reaction. In contrast, the positive point counts at nine time points calculated by the real-time ddSAT method shown in Table 13 show that positive signals were detected in all three 10-fold dilution gradient samples, with positive results being obtained as early as 5 minutes, 5 minutes, and 15 minutes after the start of the amplification reaction, respectively. 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 as early as 15 minutes, 15 minutes, and 20 minutes after the start of the amplification reaction, and dd-RT-RPA detects positive signals as early as 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 more sensitively and quickly, which has significant clinical application value for qualitative nucleic acid detection.

[0212] The embodiments described herein are intended to be illustrative and any modifications or variations made by skilled artisans based on the embodiments should also be included within the substantive scope of the patent application.

[0213] Industrial Applicability

[0214] The present invention provides a nucleic acid absolute quantification system and method based on nucleic acid isothermal amplification, which can combine the synchronous isothermal amplification method of nucleic acid and digital microdroplet quantification technology to achieve the purpose of absolute quantification of the nucleic acid copy number in a sample. It has the advantages of system stability, high compatibility, low equipment requirements, high sensitivity and rapid detection, and is suitable for industrial application.

Claims

1. A method for absolute quantification of nucleic acid based on isothermal amplification of nucleic acid, wherein the method comprises the following steps: T1) preparing a reaction system based on isothermal amplification of nucleic acid; T2) dividing the reaction system into a number of 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; 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; e, RNA-dependent DNA polymerase; and f. RNA polymerase.

2. A real-time nucleic acid qualitative and / or absolute quantitative method based on nucleic acid isothermal amplification, wherein 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), 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 or negative result of the nucleic acid sample to be tested is performed based on the fluorescence signal detection result, 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 or 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; 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; e, RNA-dependent DNA polymerase; and f. RNA polymerase.

3. The method according to claim 1 or 2, wherein the promoter sequence is selected from T7, T3, M13 and SP6 promoter sequences; and / or The one or more fluorescent probes are selected from the group consisting of molecular beacon probes, hydrolysis probes, dual hybridization probes, fluorescence resonance, scorpion probes and fluorescence amplification.

4. The method according to claim 1 or 2, wherein in step T1) or step S1), the operation of preparing the reaction system based on isothermal nucleic acid amplification comprises: (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; optionally, the volume ratio of the second mixed solution to the enzyme components is (1-50):1; and / or 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; and / or 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; and / or The RNA polymerase is T3, T7, M13 or SP6 RNA polymerase, and the RNA polymerase corresponds to the promoter sequence used.

5. The method according to claim 4, wherein the nucleic acid sample to be tested is selected from influenza A virus 2009H1N1 (NIFDC2301-01) RNA, human LncRNA PCA3 and hepatitis B virus DNA.

6. The method according to claim 1 or 2, wherein 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 made to be at most 1; optionally, the number of nucleic acid molecules contained in some micro-reaction units is made to be at most 1, and the random distribution of nucleic acid molecules in the micro-reaction units conforms to Poisson distribution; and / or Wherein in step T2) or step S2), the reaction system is divided into several tiny reaction units using microfluidics technology or other technology; optionally, the reaction system is divided into several tiny reaction units using droplet division technology in the form of microdroplets or physical micropores; further optionally, the reaction system is divided into several tiny reaction units using oil-in-water microdroplets or physical micropore chips; further optionally, the reaction system is divided into several tiny reaction units using a digital PCR system; and / or Wherein in step T2) or step S2), the conditions for isothermal amplification of the nucleic acid are: isothermal incubation at 42-65°C for 5-60 minutes.

7. The method according to claim 1 or 2, wherein in step T3) or step S3), the method for detecting the fluorescence signal of each of the micro-reaction units is flow fluorescence detection or fluorescence imaging; it can be selected from oil-in-water droplet fluorescence flow detection, oil-in-water chip fluorescence imaging or micropore chip imaging; and / or Wherein, in step T3) or step S3), the criterion for judging whether the test result is positive or negative is: the test result with a fluorescent signal is positive, and the test result without a fluorescent signal is 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 is performed according to the number of positive and negative micro-reaction units detected, which 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.

8. The method according to claim 1 or 2, wherein 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.

9. The method according to claim 2, wherein 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 optionally, the fluorescence signal is detected 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.

10. A system for qualitative and / or absolute quantitative determination of nucleic acids based on isothermal amplification of nucleic acids, comprising: 1) Reaction system based on isothermal amplification of nucleic acid; 2) A device for dividing a reaction system based on isothermal nucleic acid amplification into a plurality of micro-reaction units; 3) A device for isothermal amplification of nucleic acids in each micro-reaction unit; and 4) After and / or during the isothermal amplification of nucleic acid, each of the micro-reaction units A device for detecting fluorescence signals; optionally, the device for detecting fluorescence signals of each of the micro-reaction units during the isothermal amplification of nucleic acids is a real-time fluorescence signal detection device; 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; 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, and the fluorescent probe can hybridize with the negative strand of the target nucleic acid to be detected and release a fluorescent signal; D. RNA-dependent DNA polymerase; and E. RNA polymerase. The system according to claim 10 , wherein the target nucleic acid to be detected includes RNA and DNA.

12. The system according to claim 11, wherein 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: 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; 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.

Images