Method for detecting nucleic acid

Abstract

The invention relates to the field of biological detection, in particular to a nucleic acid detection method. The invention provides a different-temperature nucleic acid detection method. According to the detection method, in one or more cycle programs, two or more temperature points exist, each temperature point corresponds to a fluorescence signal acquisition point, and the fluorescence signal value change of the same or different temperature points in the one or more cycle programs is analyzed to determine the fluorescence signal value change of the same or different temperature points in the one or more cycle programs. Therefore, the detection of one or more targets of one or more fluorescence channels is realized. According to the method, optimal annealing temperatures are set for different primer probe groups by using a step-by-step temperature optimization strategy, and detection of one or more targets of one or more fluorescence channels is realized through fluorescence signal value changes of multi-temperature fluorescence acquisition points at the same or different temperatures.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to a method for detecting nucleic acids. Background Technology

[0002] PCR, short for Polymerase Chain Reaction, is a molecular biology technique invented in 1985 by American scientist Kary B. Mullis for the rapid in vitro amplification of specific DNA fragments. It utilizes DNA polymerase to specifically amplify target DNA fragments in vitro, undergoing three cycles of denaturation, annealing, and extension to achieve exponential amplification.

[0003] Quantitative Real-time PCR (qPCR) is a second-generation PCR technique. Its core principle is the introduction of fluorescent dyes or probes during PCR amplification. As the amount of DNA increases exponentially, the fluorescence signal in the reaction system also intensifies. Throughout the PCR reaction, the intensity of the fluorescence signal is monitored in real-time to track changes in the amount of amplified product in each cycle, thereby determining the presence and quantity of the target sequence.

[0004] Based on different fluorescent substances, qPCR can be roughly divided into two types.

[0005] One method uses fluorescent dyes, primarily SYBR Green I dye. This dye binds to the DNA double strand, producing a fluorescent signal after binding. Without binding, the fluorescent signal is almost undetectable, and it intensifies with increasing copy number. While SYBR Green I dye is relatively inexpensive, easy to use, and has good versatility due to its lack of selectivity for DNA templates, it also has some drawbacks.

[0006] 1) Non-specific binding: SYBR Green I dye binds to all double-stranded DNA (dsDNA), including target gene amplification products and non-specific products (such as primer dimers). This may lead to enhanced fluorescence signal and false positive test results.

[0007] Not suitable for multiplex detection: Because it cannot distinguish the amplification products of different target sequences, the SYBR Green I dye method is difficult to implement multiplex qPCR (i.e., detecting multiple genes in the same reaction).

[0008] Another method uses fluorescent probes, also known as the TaqMan probe assay. The principle is to design a fluorescent probe that specifically binds to the target gene. This probe contains two groups: a fluorophore and a quencher. Normally, the presence of the quencher prevents the fluorophore from fluorescing. However, during amplification, the probe binds to the DNA template, and when amplification reaches the probe site, both groups are cleaved, allowing the fluorophore to fluoresce. Similarly, the fluorescence signal intensifies with increasing copy number. The advantage is high detection specificity, making it suitable for multiplex qPCR. However, it also has certain limitations:

[0009] 1) Methodological advantages and disadvantages: Compared with the SYBR Green dye method, Taqman probes have higher specificity and can effectively distinguish non-specific amplification. Compared with melting curve technology, although melting curve technology can detect more targets through a single fluorescence channel, Taqman probes provide more accurate quantification.

[0010] 2) Limitation on the number of fluorescence channels: TaqMan probe multiplexing relies on the instrument's ability to distinguish fluorescence at different wavelengths. Most qPCR instruments are typically equipped with 4-6 fluorescence detection channels, which directly limits the number of targets that can be detected simultaneously. According to patent information, currently commercially available TaqMan multiplexing instruments support a maximum of 6 targets simultaneously. For example, CN112345678A attempts to improve multicolor detection capabilities using a "dark quencher," but the quenching efficiency is still insufficient.

[0011] 3) Instrument sensitivity requirements: In multiplex detection, the intensity of each fluorescence signal may vary significantly, requiring the instrument to have high sensitivity and a wide dynamic range to accurately detect weak signals. Low-end instruments may not be able to effectively distinguish weak signals in multiplex detection.

[0012] 4) Complicated optical system: Taqman probe multiplex detection requires a precise optical system to separate fluorescence signals of different wavelengths, which increases the complexity and cost of the instrument.

[0013] 5) Sensitivity issues: As the number of targets increases, the detection sensitivity of a single target may decrease. Competitive binding between primers and probes in multiplex reactions may lead to reduced detection efficiency for some targets. Summary of the Invention

[0014] In view of this, the present invention provides a method for nucleic acid detection. The present invention provides a temperature-controlled nucleic acid detection method. This detection method has two or more temperature points in one or more loop programs, each temperature point corresponding to a fluorescence signal acquisition point. By analyzing the changes in fluorescence signal values ​​at the same or different temperature points in one or more loop programs, the detection of one or more targets in one or more fluorescence channels can be achieved.

[0015] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0016] This invention provides a method for detecting nucleic acids, comprising: setting at least two temperature points in one or more detection cycles and acquiring fluorescence signals; obtaining fluorescence signals at the same or different temperature points based on the different melting temperatures of primers and probes for different targets in the same fluorescence channel; and obtaining detection results based on changes in fluorescence signal values.

[0017] In some embodiments of the present invention, the detection cycle procedure in the above detection method includes a PCR cycle.

[0018] In some embodiments of the present invention, the PCR cycle in the above detection method includes: a denaturation phase, an annealing phase, and an extension phase; or a denaturation phase, and a combined annealing phase and an extension phase.

[0019] In some embodiments of the present invention, in the above detection method, each fluorescence collection point corresponds to the same or different temperatures, and the difference between any two adjacent temperature points is 0~30℃.

[0020] In some embodiments of the present invention, in the above detection method, each fluorescence collection point corresponds to a different temperature, and the difference between any two adjacent temperature points is 5°C.

[0021] In some embodiments of the present invention, the detection method described above, based on changes in fluorescence signal values, includes: changes in Ct values ​​and / or changes based on increases, decreases, ratios, or divisions of the original fluorescence signal values ​​in one or more detection cycles.

[0022] In some embodiments of the present invention, in the above detection method, the primers include PCR primers; and the probes include Taqman probes.

[0023] The present invention also provides a system for implementing the above-described detection method, comprising:

[0024] Temperature control module: used to set and control at least one fluorescence signal acquisition temperature point in the detection cycle program;

[0025] Fluorescence acquisition module: used to acquire signals from at least one fluorescence channel at the stated temperature point;

[0026] Data processing module: used to analyze the obtained fluorescence signal values ​​and output the detection results of the target.

[0027] The present invention also provides detection reagents, including: the primers and probes described in the above detection method and acceptable auxiliaries.

[0028] In some embodiments of the present invention, in the above-mentioned detection reagent, the primers in the primer probe include one or more of primer sets 1 to 4;

[0029] Primer set 1: The primer set used to amplify influenza A virus has: nucleotide sequences as shown in SEQ ID NO:1 and SEQ ID NO:2; and / or

[0030] Primer set 2: The primer set used to amplify influenza B virus has: nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:4; and / or

[0031] Primer set 3: The primer set for amplifying Mycoplasma pneumoniae has: nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:6; and / or

[0032] Primer set 4: The primer set for amplifying respiratory syncytial virus has the nucleotide sequences shown in SEQ ID NO:7 and SEQ ID NO:8.

[0033] In some embodiments of the present invention, the probes in the above-mentioned detection reagents include one or more of probes 1 to 4;

[0034] The probe 1: a probe for detecting influenza A virus, has: a nucleotide sequence as shown in SEQ ID NO:9; and / or

[0035] The probe 2: a probe for detecting influenza B virus, has: a nucleotide sequence as shown in SEQ ID NO:10; and / or

[0036] The probe 3: The probe for detecting Mycoplasma pneumoniae has: a nucleotide sequence as shown in SEQ ID NO:11; and / or

[0037] The probe 4: The probe for detecting respiratory syncytial virus has a nucleotide sequence as shown in SEQ ID NO:12.

[0038] In some embodiments of the present invention, the above-mentioned detection reagent further includes DNA polymerase and reverse transcriptase.

[0039] The present invention also provides a detection kit comprising: the primers and probes described in the above detection method and / or the above detection reagents, as well as an acceptable device or carrier.

[0040] The Temperature-Step Nucleic Acid Testing (TNAT) technology in this invention is a technology based on real-time fluorescence data reading. Its core principle is to set two or more multi-temperature fluorescence acquisition points in one or more loop programs, and to detect one or more targets in one or more fluorescence channels by the change of fluorescence signal values ​​at the same or different temperatures.

[0041] Compared to traditional multiplex PCR, split-temperature nucleic acid detection technology improves performance through the following innovations:

[0042] 1) A stepwise temperature optimization strategy was adopted to set the optimal annealing temperature for different primer and probe sets.

[0043] 2) Detection of one or more targets in one or more fluorescence channels is achieved by varying the fluorescence signal values ​​at multiple fluorescence acquisition points at the same or different temperatures.

[0044] The beneficial effects of this invention include:

[0045] (1) Multiple detection and quantification capabilities: Compared with traditional multiplex PCR, it does not rely on fragment length differences or probe melting curves. It can directly detect multiple targets in a single fluorescence channel or multiple targets in multiple fluorescence channels by means of the difference in fluorescence signal value changes at different temperatures. The initial concentration of target DNA can be calculated by the threshold cycle number (Ct value) of the fluorescence signal.

[0046] (2) High specificity: The multi-temperature point design reduces primer dimer interference and improves the signal-to-noise ratio.

[0047] (3) High efficiency: Breaking through the traditional detection technology that relies on fluorescence channels, the throughput is greatly improved compared with the traditional joint detection technology.

[0048] (4) Cost advantage: No need for a detection platform or instrument with a specially designed complex optical system, reducing the complexity of instrument hardware. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0050] Figure 1 The results are shown using different methodologies; where: A represents the detection results of samples with different concentrations of FluB using the methodology of this invention; B represents the detection results of samples with different concentrations of FluA using the methodology of this invention; and C represents the detection results of samples with different concentrations using conventional Taqman probe technology.

[0051] Figure 2 The results of sample amplification are shown. Detailed Implementation

[0052] This invention discloses a method for detecting nucleic acids.

[0053] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0054] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0055] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0056] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0057] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0058] This invention provides a temperature-controlled nucleic acid detection method. In one or more cyclic programs, there are two or more temperature points, each temperature point corresponding to a fluorescence signal acquisition point. By analyzing the changes in fluorescence signal values ​​at the same or different temperature points in one or more cyclic programs, the detection of one or more targets in one or more fluorescence channels can be achieved.

[0059] A reaction solution containing primer-probe sets for multiple targets was prepared in a single reaction well. The Tm values ​​of the primer-probe sets for multiple targets differed significantly, with the Tm difference between any two primer-probe sets ranging from 0 to 30 °C.

[0060] The detection method may include a PCR cycle, comprising three stages: denaturation, annealing, and extension, or the annealing and extension steps may be combined into a single temperature step.

[0061] The detection method sets two or more temperature points in one or more loop programs. These temperature points are designed based on the melting temperatures of primer-probe sets detecting multiple targets, and the melting temperatures of different targets vary significantly. The primers are PCR primers, and the probes are TaqMan probes. Each fluorescence acquisition point corresponds to the same or different temperatures, with the difference between any two adjacent temperature points ranging from 0 to 30°C. Changes in fluorescence signal values ​​at the same or different temperature points in one or more loop programs can be converted into Ct values, or can be achieved by increasing, decreasing, folding, or dividing the original fluorescence value in one or more loop programs.

[0062] A temperature-controlled nucleic acid detection method includes the following steps:

[0063] a) The Tm values ​​of the primer and probe sets used are between 0 and 30℃.

[0064] b) One or more loop programs, setting two or more fluorescence signal temperature acquisition points;

[0065] c) By analyzing the changes in fluorescence signal values ​​at the same or different temperature points in one or more cyclic programs, a positive or negative conclusion can be drawn.

[0066] The Temperature-Step Nucleic Acid Testing (TNAT) technology in this invention is a technology based on real-time fluorescence data reading. Its core principle is to set two or more multi-temperature fluorescence acquisition points in one or more loop programs, and to detect one or more targets in one or more fluorescence channels by the change of fluorescence signal values ​​at the same or different temperatures.

[0067] Compared to traditional multiplex PCR, split-temperature nucleic acid detection technology improves performance through the following innovations:

[0068] 1) A stepwise temperature optimization strategy was adopted to set the optimal annealing temperature for different primer and probe sets.

[0069] 2) Detection of one or more targets in one or more fluorescence channels is achieved by varying the fluorescence signal values ​​at multiple fluorescence acquisition points at the same or different temperatures.

[0070] In Examples 1 to 4 of this invention, all raw materials and reagents used can be purchased from the market.

[0071] The present invention will be further illustrated below with reference to the embodiments:

[0072] Example 1

[0073] In this invention, the changes in fluorescence signal values ​​at different temperatures during the cyclic program can be converted into Ct values, or can be achieved by increasing, decreasing, multiplying, or dividing the original fluorescence values ​​in different cycles. This embodiment provides a method for calculating the fluorescence value increment in temperature-step multiplex PCR (TS-PCR) based on temperature-step nucleic acid detection technology.

[0074] Two pathogens, influenza A virus (FluA) and influenza B virus (FluB), were tested in single reaction wells. The Tm values ​​for the corresponding primer and probe sets were 58℃ and 63℃, respectively. The primer and probe set sequences are shown in Table 1. The fluorescent group selected for each probe was FAM, and the reaction procedure is shown in Table 2. The "temperature-dependent nucleic acid detection method" was implemented to detect four plasmid samples: FluA, FluB, FluA+FluB, and NC.

[0075] Ct value calculation: During detection, the nucleic acids of FluA and FluB are reverse transcribed into cDNA, and then the target gene sequence is amplified through thermal cycling. Each round of amplification releases a fluorescence signal, and the instrument records the signal intensity in real time. The Ct value is the number of cycles corresponding to when the fluorescence intensity reaches a preset threshold.

[0076] Calculation method of fluorescence increment: In this detection, the number of pathogen detection targets was 2. FluA and FluB were designated as A and B, respectively. Fluorescence was collected at three temperature points: 63℃, 58℃, and 63℃. The second temperature point of 63℃ was labeled as C. In each PCR cycle, the fluorescence increment of targets A and B in each PCR cycle was calculated. Then, the fluorescence increments of each annealing temperature point in each PCR cycle were summarized. The derivative of the fluorescence increment curve was then calculated to obtain the fluorescence increment value. The curve can be used to determine the amplification status of the target gene.

[0077] The formula for calculating the fluorescence increment of target A per PCR cycle is: The formula for calculating the fluorescence increment of target B per PCR cycle is: .

[0078] Table 1 Primer and probe sequence

[0079]

[0080] Table 2 Reaction Program Settings

[0081]

[0082] In this embodiment, the detection results of Ct values ​​for each sample are shown in Table 3. The results show that this detection method can output Ct values.

[0083] Table 3 Sample Detection Results

[0084]

[0085] Example 2

[0086] This embodiment tests four pathogens—Mycoplasma pneumoniae (MP), FluA, FluB, and respiratory syncytial virus (RSV)—in a single reaction well.

[0087] In this embodiment, the Tm values ​​of the primer and probe sets corresponding to the four pathogens MP, FluA, FluB, and RSV are 53℃, 58℃, 63℃, and 68℃, respectively. The primer and probe set sequences are shown in Table 4. The fluorescent group selected for each probe is FAM. The preparation of the reaction solution is shown in Table 5, and the reaction procedure is shown in Table 6.

[0088] The procedure of "a temperature-controlled nucleic acid detection method" was adopted to detect 11 plasmid samples: MP, FluA, FluB, RSV, MP+FluA, MP+FluB, MP+RSV, FluA+FluB, FluA+RSV, FluB+RSV, and NC.

[0089] Table 4. Respiratory Tract Quadruple Primer-Probe Set Sequences

[0090]

[0091] Table 5. Reaction Solution Preparation Table

[0092]

[0093] Table 6 Reaction Program Settings

[0094]

[0095] The test results for each sample are shown in Table 7. The analysis results show that in the multiplex detection of four pathogens (MP, FluA, FluB, and RSV) under FAM fluorescent labeling, the five single-positive samples (MP, FluA, FluB, RSV, and NC) could be well distinguished between positive and negative results at different annealing temperatures. In the six mixed-positive samples (MP+FluA, MP+FluB, MP+RSV, FluA+FluB, FluA+RSV, and FluB+RSV), the positive and negative results could also be well distinguished at different annealing temperatures.

[0096] Table 7. Detection results for each sample

[0097]

[0098] Example 3

[0099] In this embodiment, three pathogens, FluA, FluB, and RSV, were tested in a single reaction well. Each pathogen was labeled with two fluorescent groups, and a six-fold test was performed.

[0100] In this embodiment, the Tm values ​​of the primer and probe sets corresponding to the three pathogens FluA, FluB, and RSV are 58℃, 63℃, and 68℃, respectively. The primer and probe set sequences are shown in Table 8. The fluorescent groups selected for each probe are FAM and VIC. The preparation of the reaction solution is shown in Table 9, and the reaction procedure is shown in Table 10.

[0101] The procedure of "a temperature-controlled nucleic acid detection method" was adopted to detect four culture samples: FluA, FluB, RSV, and NC.

[0102] Table 8. Respiratory Tract Six-Phase Primer Probe Set Sequences

[0103]

[0104] Table 9. Reaction Solution Preparation Table

[0105]

[0106] Table 10 Reaction Program Settings

[0107]

[0108] The test results for each sample are shown in Table 11. The analysis results show that in the six-fold detection of three pathogens, FluA, FluB, and RSV, under FAM and VIC fluorescent labeling, the four single-positive samples (FluA, FluB, RSV, and NC) can be well distinguished as positive or negative under different fluorescence channels and temperatures.

[0109] Table 11 Detection results for each sample

[0110]

[0111] Example 4

[0112] To evaluate the difference in sensitivity and target detection between the detection method of this invention and the conventional qPCR detection method, FluA and FluB were selected as test pathogens, with corresponding primer and probe sets having Tm values ​​of 58℃ and 63℃, respectively. The primer and probe set sequences are shown in Table 12. The fluorescent group selected for each probe is FAM, and the reaction solution preparation is shown in Table 13. The conventional TaqMan probe technology procedure (Table 14; since the conventional TaqMan probe technology cannot select multiple annealing temperatures for fluorescence collection, the lowest temperature of 58℃ was selected for fluorescence collection) and the methodological procedure described in this invention (Table 15) were used for comparative testing. Samples were set as plasmid samples of influenza A virus (H1N1 subtype) and influenza B virus (BV type) (concentration gradient: 10). 6 , 10 5 , 10 4 , 10 3 (100, 50 copies / mL). The procedure was performed using a temperature-controlled nucleic acid detection method.

[0113] Table 12 Primer and probe sequences

[0114]

[0115] Table 13 Reaction Solution Preparation Table

[0116]

[0117] Table 14 Conventional Reaction Procedure Settings

[0118]

[0119] Table 15 Reaction program settings of the present invention

[0120]

[0121] The detection results are shown in Table 16. It can be seen that both the detection method of this invention and the conventional TaqMan probe technique can detect samples of 100 copies / mL, with no difference in detection sensitivity between the two methods. However, this invention can clearly distinguish between two targets at different temperatures within the same fluorescence channel, while the conventional TaqMan probe technique cannot effectively distinguish whether the target amplified at 58℃ is FluA or FluB.

[0122] Table 16 Sample Detection Results

[0123]

[0124] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting nucleic acids, characterized in that, include: By setting at least two temperature points and acquiring fluorescence signals in one or more detection cycles, the fluorescence signals at the same or different temperature points are obtained based on the different melting temperatures of primers and probes for different targets in the same fluorescence channel. The detection results are obtained based on the changes in fluorescence signal values.

2. The detection method as described in claim 1, characterized in that, The detection cycle procedure includes a PCR cycle.

3. The detection method as described in claim 2, characterized in that, The PCR cycle includes: a denaturation phase, an annealing phase, and an extension phase; or a denaturation phase, and a combined annealing and extension phase.

4. The detection method according to any one of claims 1 to 3, characterized in that, Each fluorescence collection point corresponds to the same or different temperatures, and the difference between any two adjacent temperature points is 0~30℃.

5. The detection method according to any one of claims 1 to 4, characterized in that, The change based on the fluorescence signal value includes: the Ct value and / or the increase, decrease, ratio, or division of the original fluorescence signal value based on one or more detection cycles.

6. The detection method according to any one of claims 1 to 5, characterized in that, The primers include PCR primers; the probes include Taqman probes.

7. A system for implementing the detection method as described in any one of claims 1 to 6, characterized in that, include: Temperature control module: used to set and control at least one signal acquisition temperature point in the detection cycle program; Fluorescence acquisition module: used to acquire signals from at least one fluorescence channel at the stated temperature point; Data processing module: used to analyze the obtained fluorescence signal values ​​and output the detection results of the target.

8. A detection reagent, characterized in that, include: The primers and probes and acceptable auxiliaries in the detection method as described in any one of claims 1 to 6.

9. A test kit, characterized in that, include: The primers and probes in the detection method as described in any one of claims 1 to 6 and / or the detection reagents as described in claim 8, as well as acceptable devices or carriers.

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