Amplicon length measurement method

Abstract

The present invention provides a method for measuring the length of an amplicon, comprising the following steps. A qPCR mixed reagent, a pre-primer, a reverse primer, a hybridization probe, a double-stranded DNA binding dye and a nucleic acid sample are added to a reaction well to perform a qPCR reaction, and the fluorescence intensity of the hybridization probe and the double-stranded DNA binding dye as the reaction cycle number changes are measured respectively. Then, an operation method is performed to calculate the length of the amplicon. The method for measuring the length of an amplicon provided by the present invention can simultaneously and accurately analyze the length and content of the amplicon in the nucleic acid sample.

Description

Technical Field

[0001] The present invention relates to a measurement method, and in particular to a method for measuring the length of an amplicon. Background Art

[0002] In the field of molecular biology, it is necessary to sequence nucleic acid targets (such as DNA or RNA) and represent them in a series with the correct base sequence, so as to study specific samples through the gene sequence that transmits genetic information. The development of gene sequencing has accelerated the research and discovery of biology and medicine, and is widely used in the fields of biological diagnosis, biotechnology, identification science and biosystematics.

[0003] In recent years, the Next Generation Sequencing (NGS) technology has been developed. This high-efficiency sequencing technology can significantly reduce the cost and time required for sequencing. The next-generation sequencing test requires the establishment of a database (library), including the use of adapters with known sequences at both ends to connect the nucleic acid template fragments. This junction is the next-generation sequencing database. Before performing the next-generation sequencing test, the quality of the database must be confirmed. If the content of the next-generation sequencing database is too high, the cluster density is too high, which may lead to poor quality of the test experiment data. On the contrary, if the content of the next-generation sequencing database is too low, the cluster density is too low, which may lead to low yield. In addition, if the length distribution in the next-generation sequencing database is too wide, it may cause the read depth to be too low. Therefore, during the operation of the next-generation sequencing technology, quality control must be performed before the machine step to confirm the length and content of the next-generation sequencing database.

[0004] Currently, the content of the next-generation sequencing database is mostly confirmed by qPCR (quantitative polymerase-chain-reaction, also known as Real-time PCR) technology, and the length of the next-generation sequencing database is confirmed by capillary electrophoresis (CE). However, qPCR technology requires the preparation of a standard curve, which is more complicated in experimental procedures, and capillary electrophoresis has disadvantages such as low sensitivity and non-specific reactions. Furthermore, the length and content of the next-generation sequencing database must be confirmed by two different experiments respectively, which also increases the cost and time required for the experiment. Therefore, developing a measurement method that can accurately analyze the length and content of the next-generation sequencing database at the same time is a goal that technicians in this field are eager to develop. Summary of the invention

[0005] The present invention provides a method for measuring the length of an amplicon, which can simultaneously and accurately analyze the length and content of the amplicon in a nucleic acid sample. More specifically, when the content of the next-generation sequencing database is detected by qPCR technology, the nucleic acid product after the next-generation sequencing database is amplified is the amplicon. The present invention further calculates the length and content of the next-generation sequencing database by detecting the length and content of the amplicon.

[0006] The present invention provides a method for measuring the length of an amplicon, comprising the following steps. First, a qPCR master mix, a forward primer, a reverse primer, a hybridization probe, a double stranded DNA dye, and an extremely diluted (3 copies / μl to 30 copies / μl) nucleic acid sample are mixed to form a qPCR reaction mixture. Next, the qPCR reaction mixture is distributed to the reaction wells of a test slide, and each reaction well can be distributed with at most one nucleic acid sample. Thereafter, a qPCR reaction is performed on the qPCR reaction mixture, and the fluorescence intensity of the hybridization probe and the double stranded DNA dye of each amplicon in the nucleic acid sample is measured as the number of reaction cycles changes. Next, an operation method is performed to calculate the length of each amplicon.

[0007] In one embodiment of the present invention, the calculation method includes determining a threshold value from the fluorescence intensity of the hybridization probe measured for each amplicon, and correspondingly obtaining a critical reaction cycle number for each amplicon when the fluorescence intensity of the hybridization probe is the threshold value. Then, correspondingly obtaining the individual fluorescence intensity of the double-stranded DNA binding dye of each amplicon at the critical reaction cycle number. Substituting the individual fluorescence intensity into the linear regression curve of the fluorescence intensity of the double-stranded DNA binding dye and the amplicon length, the length of each amplicon is calculated.

[0008] In one embodiment of the present invention, the calculation method includes converting the fluorescence intensity of the double-stranded DNA binding dye and the fluorescence intensity of the hybridization probe measured for each amplicon into the normalized fluorescence intensity of the double-stranded DNA binding dye and the normalized fluorescence intensity of the hybridized probe, respectively. Afterwards, the normalized fluorescence intensity of the double-stranded DNA binding dye and the normalized fluorescence intensity of the hybridization probe of each amplicon are divided to obtain a fluorescence intensity ratio. Next, the fluorescence intensity ratio of each amplicon at the critical reaction cycle number is substituted into the linear regression curve of the fluorescence intensity ratio and the amplicon length to calculate the length of each amplicon.

[0009] In one embodiment of the present invention, the method for converting the normalized fluorescence intensity of the double-stranded DNA binding dye and the normalized fluorescence intensity of the hybridization probe comprises dividing the fluorescence intensity of the double-stranded DNA binding dye in each reaction cycle by the average fluorescence intensity of the double-stranded DNA binding dye in the reaction cycle interval to convert the normalized fluorescence intensity of the double-stranded DNA binding dye. The fluorescence intensity of the hybridization probe in each reaction cycle is divided by the average fluorescence intensity of the hybridization probe in the reaction cycle interval to convert the normalized fluorescence intensity of the hybridization probe.

[0010] In one embodiment of the present invention, the double-stranded DNA binding dye includes SYBR green dye, Evagreen dye, LC green dye or SYTO 9 dye.

[0011] In one embodiment of the present invention, the critical value is 1.1 to 1.5.

[0012] In one embodiment of the present invention, the qPCR mixed reagent includes a reaction buffer, dNTPs, MgCl 2 and Taq polymerase.

[0013] In one embodiment of the present invention, the concentration of the double-stranded DNA binding dye in the qPCR reaction mixture is 1 uM to 10 uM.

[0014] In one embodiment of the present invention, the concentration of the hybridization probe in the qPCR reaction mixture is 0.2 uM to 1 uM.

[0015] In one embodiment of the present invention, the concentration of the forward primer in the qPCR reaction mixture is 0.03uM to 0.5uM, and the concentration of the reverse primer in the qPCR reaction mixture is 0.03uM to 0.5uM.

[0016] In one embodiment of the present invention, the volume ratio of the nucleic acid sample to the qPCR reaction mixture is 1:60 to 1:10.

[0017] In one embodiment of the present invention, the hybridization probe includes a TaqMan probe, a Molecular Beacon probe, a Dual Hybridization Probes or an Eclipse probe.

[0018] In one embodiment of the present invention, the nucleic acid sample includes a next generation sequencing database.

[0019] Based on the above, the method for measuring the length of the amplicon proposed in the present invention is to simultaneously add the hybridization probe (related to the content of the amplicon) and the double-stranded DNA binding dye (related to the length of the amplicon) to the qPCR reaction mixture for qPCR reaction, respectively measure the fluorescence intensity of the hybridization probe and the double-stranded DNA binding dye as the reaction cycle number changes, and obtain the critical reaction cycle number of the amplicon, and then calculate the length of the amplicon. Therefore, the method for measuring the length of the amplicon proposed in the present invention can accurately analyze the length and content of the amplicon in the nucleic acid sample at the same time, and is applied to the quality control of the next-generation sequencing technology.

[0020] In order to make the above features and advantages of the present invention more clearly understood, embodiments are given below with reference to the accompanying drawings for detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A schematic diagram of a process for measuring amplicon length according to an embodiment of the present invention;

[0022] Figure 2 A curve diagram showing the fluorescence intensity of the hybridization probe and the double-stranded DNA binding dye measured as the number of reaction cycles changes in the method for measuring the length of an amplicon according to an embodiment of the present invention;

[0023] Figure 3 A schematic diagram of a process for measuring amplicon length according to another embodiment of the present invention;

[0024] Figure 4 A curve diagram showing the change in the fluorescence intensity ratio of the normalized Eva Green fluorescence intensity and the normalized TaqMan fluorescence intensity as the reaction cycle number is measured in the method for measuring the amplicon length according to another embodiment of the present invention;

[0025] Figure 5 This is an experimental example of the method for measuring the length of an amplicon according to an embodiment of the present invention, and a graph showing the fluorescence intensity of the TaqMan probe and the Eva green dye as the reaction cycle number changes;

[0026] Figure 6 A histogram of normalized Eva green dye fluorescence intensities of amplicons of different lengths in an experimental example performed in a method for measuring amplicon length according to an embodiment of the present invention;

[0027] Figure 7 A graph showing the normalized fluorescence intensity of Evagreen dye and the length of amplicon in an experimental example of a method for measuring the length of an amplicon according to an embodiment of the present invention. DETAILED DESCRIPTION

[0028] Figure 1FIG. 4 is a schematic flow chart of a method for measuring amplicon length according to an embodiment of the present invention. Figure 2 FIG. 1 is a graph showing the fluorescence intensity of the hybridization probe and the double-stranded DNA binding dye measured as the number of reaction cycles changes in the method for measuring the length of the amplicon according to an embodiment of the present invention. Figure 1 and Figure 2 A method for measuring amplicon length according to an embodiment of the present invention is described in detail.

[0029] Please refer to Figure 1 , first perform step S110, add qPCR mixed reagent, front primer, reverse primer, hybridization probe, double-stranded DNA binding dye and nucleic acid sample into the reaction well of the test slide to form a qPCR reaction mixture. The test slide can be a slide plate with multiple reaction wells, and qPCR reaction can be performed in each reaction well.

[0030] More specifically, in the formed qPCR reaction mixture, the concentration of the forward primer is, for example, 0.03uM to 0.5uM, the concentration of the reverse primer is, for example, 0.03uM to 0.5uM, the concentration of the double-stranded DNA binding dye is, for example, 1uM to 10uM, and the concentration of the hybridization probe is, for example, 0.2uM to 1uM.

[0031] In the present embodiment, the double-stranded DNA binding dye may include SYBR green dye, Eva green dye, LCgreen dye or SYTO 9 dye, preferably Eva green dye, but the present invention is not limited thereto, and other fluorescent dyes that can bind to double-stranded DNA may also be used. In more detail, the amount of double-stranded DNA binding dye added is more than the amount added in the existing qPCR reaction, for example, by one times the concentration (1×) to five times the concentration (5×). The hybridization probe may include a TaqMan probe, a molecular signaling probe, a dual hybridization probe or an Eclipse probe, but the present invention is not limited thereto, and other fluorescent dyes that can emit a fluorescent signal once in each DNA replication cycle may also be used.

[0032] In this embodiment, the volume ratio of the nucleic acid sample to the qPCR reaction mixture is, for example, 1:60 to 1:10. The nucleic acid sample may include a next-generation sequencing database, that is, the amplicon length measurement method proposed in the present invention may be applied to the quality control of next-generation sequencing technology, but is not limited thereto. The measurement method proposed in the present invention may also be applied to any other technology that requires accurate analysis of the length and content of the amplicon.

[0033] In this embodiment, the qPCR mixed reagent can use commercial products, which may include reaction buffer, dNTP, MgCl2 and Taq polymerase. Commercial products of the qPCR mixed reagent are, for example, the QuantiTect Probe PCR Kit of Qiagen, the TaqMan Gene Expression Master Mix of ABI, the iTaq Supermix with ROX of Bio-Rad, and the Express qPCR SuperMix of Invitrogen, but the present invention is not limited thereto. In more detail, when forming a qPCR reaction mixture, the concentration of the qPCR mixed reagent is, for example, one-fold concentration (1×).

[0034] Next, please continue to refer to Figure 1 , and proceed to step S120, performing a qPCR reaction on the qPCR reaction mixture formed in the reaction well of the test slide in step S110. The operating condition setting of the qPCR reaction can be compared with the operating setting of the existing qPCR reaction, for example, after reacting at 50°C for 2 minutes, reacting at 95°C for 3 minutes to perform a Taq polymerase hot start step, and then performing 40 cycles as follows: reacting at 95°C for 36 seconds and reacting at 60°C for 72 seconds, but the present invention is not limited thereto, and the operating setting of the qPCR reaction can also be adjusted according to actual needs.

[0035] Afterwards, please also refer to Figure 1 and Figure 2 ,like Figure 1 As shown, step S130 is performed to measure the fluorescence intensity of the hybridization probe and double-stranded DNA binding dye of each amplicon in the nucleic acid sample as the reaction cycle number changes. Figure 2 The curve diagram shown. Figure 2 In the figure, the upper graph shows the fluorescence intensity of the hybridization probe as the number of reaction cycles changes, while the lower graph shows the fluorescence intensity of the double-stranded DNA binding dye as the number of reaction cycles changes. The two are aligned and presented side by side to facilitate the subsequent process description. It must be noted that although Figure 2 Only three groups of amplicons are shown in the figure, but Figure 2 The curve graph shown is only used to illustrate this embodiment, and the present invention is not limited thereto. The number of experimental groups of amplicons and the number of curves shown can be adjusted according to actual experimental conditions.

[0036] Next, please also refer to Figure 1 and Figure 2 , proceed to step S140, and determine a critical value T from the measured fluorescence intensity of the hybridization probe. Figure 2 As shown, the critical value T is a value of the fluorescence intensity of the hybridization probe in the section where the fluorescence intensity of the hybridization probe gradually increases as the number of reaction cycles increases. In this embodiment, the critical value T is, for example, 1.1 to 1.5, but the present invention is not limited thereto, and an appropriate value can also be determined as the critical value T in the section where the fluorescence intensity of the hybridization probe gradually increases (i.e., the linear phase) according to the actual experimental situation.

[0037] Please also refer to Figure 1 and Figure 2 , proceed to step S150, and correspondingly obtain the critical reaction cycle numbers C1, C2 and C3 of each amplicon in the nucleic acid sample when the fluorescence intensity of the hybridization probe is the critical value T. Since the reaction principle of the hybridization probe is that a fluorescent signal is emitted each time the amplicon is replicated, that is, the fluorescence intensity of the hybridization probe is positively correlated with the content of the amplicon. However, the qPCR reaction amplification efficiency of each amplicon in the nucleic acid sample may be different (based on different GC base contents), so even at the same reaction cycle number, the content of each amplicon may be different. Therefore, by determining the critical value T in the fluorescence intensity of the hybridization probe, the content of each amplicon can be normalized to be the same as each other. That is, the content of each amplicon is the same as each other at the critical reaction cycle numbers C1, C2 and C3.

[0038] Please also refer to Figure 1 and Figure 2 , step S160 is performed to obtain the individual fluorescence intensities F1, F2 and F3 of the double-stranded DNA binding dye of each amplicon at the critical reaction cycle numbers C1, C2 and C3. Since the mechanism of action of the double-stranded DNA binding dye is to bind to the amplicon and embed into the double-stranded nucleic acid, the longer the length of the amplicon, the more double-stranded DNA binding dye can be bound and embedded, and the stronger the fluorescence intensity is, that is, the fluorescence intensity of the double-stranded DNA binding dye is positively correlated with the length of the amplicon. As described above, the content of each amplicon is the same at the critical reaction cycle numbers C1, C2 and C3. Therefore, step S160 can obtain the individual fluorescence intensities F1, F2 and F3 of the double-stranded DNA binding dye of each amplicon when the content of each amplicon is the same.

[0039] Finally, please refer to Figure 1 and Figure 2, step S170 is performed to substitute the individual fluorescence intensities F1, F2 and F3 into the linear regression curve of the fluorescence intensity of the double-stranded DNA binding dye and the length of the amplicon to calculate the length of each amplicon. The linear regression curve of the fluorescence intensity of the double-stranded DNA binding dye and the length of the amplicon and the detailed calculation steps can be performed using the prior art in the art, so they are not repeated here. As described above, step S160 can obtain the individual fluorescence intensities F1, F2 and F3 of the double-stranded DNA binding dye of each amplicon when the content of each amplicon is the same. Therefore, step S170 can accurately measure the length of each amplicon when the content of each amplicon is the same.

[0040] Figure 3 FIG. 4 is a flow chart of a method for measuring amplicon length according to another embodiment of the present invention. Figure 4 This is a graph showing the change in the fluorescence intensity ratio of the normalized Eva Green fluorescence intensity and the normalized TaqMan fluorescence intensity as the reaction cycle number is measured in a method for measuring amplicon length according to another embodiment of the present invention.

[0041] It must be pointed out that Figure 3 and Figure 4 The embodiment shown is similar to Figure 1 and Figure 2 The similarity is that in Figure 3 and Figure 4 In the embodiment shown, the following is also performed Figure 1 The present embodiment is different from the above embodiment in that after performing step S130, the present embodiment does not perform steps S140, S150, S160 and S170 in the above embodiment, but performs steps S340, S350 and S360. Since the details of steps S110, S120 and S130 have been described in detail above, they will not be repeated here. Therefore, only steps S340, S350 and S360 will be directly described below.

[0042] Please refer to Figure 3 , proceed to step S340, converting the fluorescence intensity of the double-stranded DNA binding dye and the fluorescence intensity of the hybridization probe measured for each amplicon into the normalized fluorescence intensity of the double-stranded DNA binding dye and the normalized fluorescence intensity of the hybridized probe, respectively.

[0043] In more detail, the method for converting the normalized fluorescence intensity of the double-stranded DNA binding dye may include dividing the fluorescence intensity of the double-stranded DNA binding dye during each reaction cycle by the average fluorescence intensity of the double-stranded DNA binding dye within the reaction cycle interval to convert the normalized fluorescence intensity of the double-stranded DNA binding dye. On the other hand, the method for converting the normalized fluorescence intensity of the hybridization probe includes dividing the fluorescence intensity of the hybridization probe during each reaction cycle by the average fluorescence intensity of the hybridization probe within the reaction cycle interval to convert the normalized fluorescence intensity of the hybridization probe. In the present embodiment, the reaction cycle interval is, for example, the third reaction cycle to the eighth reaction cycle in the baseline region where the fluorescence intensity has not yet risen, but the present invention is not limited thereto, and an appropriate reaction cycle interval may also be determined in the baseline region according to actual experimental conditions.

[0044] Afterwards, please also refer to Figure 3 and Figure 4 ,like Figure 3 As shown, step S350 is performed to divide the normalized fluorescence intensity of the double-stranded DNA binding dye of each amplicon and the normalized fluorescence intensity of the hybridization probe to obtain a fluorescence intensity ratio. In this embodiment, it can be shown as follows Figure 4 The curve shown in Figure 1 shows the change of fluorescence intensity ratio with the number of reaction cycles. Figure 4 Only three groups of amplicons are shown in the figure, but Figure 4 The curve graph shown is only used to illustrate this embodiment, and the present invention is not limited thereto. The number of experimental groups of amplicons and the number of curves shown can be adjusted according to actual experimental conditions. The length of the amplicon and the types of double-stranded DNA binding dyes and hybridization probes used are also not limited thereto.

[0045] Next, please also refer to Figure 3 and Figure 4 ,like Figure 3 As shown, step S360 is performed to substitute the fluorescence intensity ratio of each amplicon at the critical reaction cycle number into the linear regression curve of the fluorescence intensity ratio and the amplicon length to calculate the length of each amplicon.

[0046] In this embodiment, the critical reaction cycle number may include 25 to 35. Figure 4 In the embodiment, the critical reaction cycle number is, for example, 30, but the present invention is not limited thereto. According to the actual experimental situation, the corresponding appropriate reaction cycle number in the gradually increasing section of the fluorescence intensity ratio (i.e., the linear section (linear phase)) can be determined as the critical reaction cycle number. Figure 4As shown, when the critical reaction cycle number is 30, the fluorescence intensity ratio of the 100bp amplicon is 1.4, the fluorescence intensity ratio of the 200bp amplicon is 2.1, and the fluorescence intensity ratio of the 400bp amplicon is 3.1. Afterwards, the above fluorescence intensity ratios are substituted into the linear regression curve of the fluorescence intensity ratio and the amplicon length to calculate the length of each amplicon.

[0047] The following experimental examples are used to describe in detail the method for measuring the length of the amplicon in the above embodiment. However, the following experimental examples are not intended to limit the present invention.

[0048] Experimental example

[0049] In order to prove that the method for measuring the length of an amplicon proposed in the present invention can accurately analyze the length and content of the amplicon in a nucleic acid sample at the same time, the following experimental example is specifically made.

[0050] A qPCR reaction mixture is added to the reaction well of the test slide, and the composition and content of the qPCR reaction mixture are shown in the following Table 1. The nucleic acid sample is a standard sample with a known amplicon length, which contains amplicon lengths of 100 bp (base pair), 200 bp, 400 bp, and 800 bp. Then, a qPCR reaction is performed on the qPCR reaction mixture in the reaction well.

[0051] Table 1

[0052]

[0053]

[0054] Figure 5 This is an experimental example of the method for measuring the length of an amplicon according to an embodiment of the present invention, and a graph showing the fluorescence intensity of the TaqMan probe and the Eva green dye as the number of reaction cycles changes. Figure 5 In the figure, the upper graph shows the fluorescence intensity of the TaqMan probe as the reaction cycle number changes, and the lower graph shows the fluorescence intensity of the Eva green dye as the reaction cycle number changes, wherein each point represents one reaction cycle.

[0055] Please refer to Figure 5 , a critical value of 1.3 is determined from the measured fluorescence intensity of the TaqMan probe. The critical value 1.3 is a fluorescence value in the section where the fluorescence intensity gradually increases as the number of reaction cycles increases. Next, the critical reaction cycle number of each amplicon is obtained when the fluorescence intensity of the TaqMan probe is the critical value 1.3. After that, the individual fluorescence intensity of the Eva green dye of each amplicon is obtained at the critical reaction cycle number. Figure 5As shown, the Evagreen dye fluorescence intensity of the 100 bp amplicon is 3, the Evagreen dye fluorescence intensity of the 200 bp amplicon is 4, and the Evagreen dye fluorescence intensity of the 400 bp amplicon is 6. Afterwards, the individual fluorescence intensities of the Evagreen dye are substituted into the linear regression curve of the Evagreen dye fluorescence intensity and the amplicon length to calculate the length of each amplicon.

[0056] Figure 6 A histogram of normalized Eva green dye fluorescence intensity of amplicons of different lengths in an experimental example performed by a method for measuring amplicon length according to an embodiment of the present invention. Figure 6 , the 100bp amplicon, the 200bp amplicon, the 400bp amplicon and the 800bp amplicon are clearly divided into four separate blocks. Therefore, it can be known that the amplicon length measurement method proposed by the present invention can accurately distinguish the length of each amplicon when the content of each amplicon is the same.

[0057] Figure 7 A graph showing the normalized fluorescence intensity of Evagreen dye and the length of amplicon in an experimental example of a method for measuring the length of an amplicon according to an embodiment of the present invention. Figure 7 The average value of the fluorescence intensity of the Evagreen dye measured for the 100 bp amplicon, 200 bp amplicon, 400 bp amplicon, and 800 bp amplicon in the above experimental example is plotted against the length of each amplicon. Figure 7 As shown, it presents a nearly smooth straight line, and R 2 is 0.998, which is quite close to 1. Therefore, it can be known that the method for measuring the length of the amplicon proposed in the present invention can accurately measure the length of each amplicon when the content of each amplicon is the same.

[0058] In summary, the method for measuring the length of an amplicon proposed in the present invention can accurately measure the length of each amplicon because two types of dyes (hybridization probes and double-stranded DNA binding dyes) are used simultaneously to perform qPCR reactions. On the other hand, since the nucleic acid sample is subjected to qPCR reactions in an extreme dilution range, the content can be calculated using the statistical distribution principle, and therefore, there is no need to perform serial dilution to prepare a standard curve. Therefore, the measurement method proposed in the present invention only requires a small amount of nucleic acid sample to be executed, and the detection limit is relatively low. Therefore, the method of the present invention can simultaneously analyze the length and content of the amplicon in the nucleic acid sample in the same qPCR experiment, and can be used for quality control of next-generation sequencing technology, or any other molecular biology related application that requires simultaneous and accurate analysis of the length and content of the amplicon in the nucleic acid sample.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for measuring amplicon length, characterized in that: include: Mixing qPCR mixed reagent, forward primer, reverse primer, hybridization probe, double-stranded DNA binding dye and nucleic acid sample to form a qPCR reaction mixture; Distribute the qPCR reaction mixture into the reaction wells of the test slide, and each reaction well can be distributed with at most one of the following nucleic acid samples; Performing a qPCR reaction on the qPCR reaction mixture, and measuring the fluorescence intensity of the hybridization probe and the double-stranded DNA binding dye of each amplicon in the nucleic acid sample as the reaction cycle number changes; as well as Performing an operation to calculate the length of each of the amplicon, The operation method includes: Determine a critical value from the fluorescence intensity of the hybridization probe measured for each amplicon, and correspondingly obtain a critical reaction cycle number for each amplicon when the fluorescence intensity of the hybridization probe is the critical value; The corresponding fluorescence intensity of the double-stranded DNA binding dye of each of the amplicon at the critical reaction cycle number is obtained; and Substituting the individual fluorescence intensities into the linear regression curve of the fluorescence intensity of the double-stranded DNA binding dye and the amplicon length to calculate the length of each amplicon, or The fluorescence intensity of the double-stranded DNA binding dye and the fluorescence intensity of the hybridization probe measured for each amplicon are converted into the normalized fluorescence intensity of the double-stranded DNA binding dye and the normalized fluorescence intensity of the hybridization probe, respectively; Dividing the normalized fluorescence intensity of the double-stranded DNA binding dye of each of the amplicons and the normalized fluorescence intensity of the hybridization probe to obtain a fluorescence intensity ratio; and The fluorescence intensity ratio of each amplicon at the critical reaction cycle number is substituted into the linear regression curve of the fluorescence intensity ratio and the amplicon length to calculate the length of each amplicon.

2. The method for measuring amplicon length according to claim 1, characterized in that: The critical value is 1.1 to 1.

5.

3. The method for measuring amplicon length according to claim 1, characterized in that: The method for converting the normalized fluorescence intensity of the double-stranded DNA binding dye and the normalized fluorescence intensity of the hybridization probe comprises: The fluorescence intensity of the double-stranded DNA binding dye in each reaction cycle is divided by the average fluorescence intensity of the double-stranded DNA binding dye in the reaction cycle interval to calculate the normalized fluorescence intensity of the double-stranded DNA binding dye; and The fluorescence intensity of the hybridization probe in each reaction cycle is divided by the average fluorescence intensity of the hybridization probe within the reaction cycle interval to calculate the normalized fluorescence intensity of the hybridization probe.

4. The method for measuring amplicon length according to claim 1, characterized in that: The double-stranded DNA binding dye includes SYBR green dye, Eva green dye, LC green dye or SYTO9 dye.

5. The method for measuring amplicon length according to claim 1, characterized in that: The qPCR mixed reagent includes a reaction buffer, dNTP, MgCl2 and Taq polymerase.

6. The method for measuring amplicon length according to claim 1, characterized in that: The concentration of the double-stranded DNA binding dye in the qPCR reaction mixture is 1 uM to 10 uM.

7. The method for measuring amplicon length according to claim 1, characterized in that: The concentration of the hybridization probe in the qPCR reaction mixture is 0.2uM to 1uM.

8. The method for measuring amplicon length according to claim 1, characterized in that: The concentration of the front primer in the qPCR reaction mixture is 0.03uM to 0.5uM, and the concentration of the reverse primer in the qPCR reaction mixture is 0.03uM to 0.5uM.

9. The method for measuring amplicon length according to claim 1, characterized in that: The volume ratio of the nucleic acid sample to the qPCR reaction mixture is 1:60 to 1:

10.

10. The method for measuring amplicon length according to claim 1, characterized in that: The hybridization probe includes TaqMan probe, molecular signaling probe, dual hybridization probe or Eclipse probe.

11. The method for measuring amplicon length according to claim 1, characterized in that: The nucleic acid sample includes a next generation sequencing database.

12. The method for measuring amplicon length according to claim 1, characterized in that: The concentration of the nucleic acid sample is 3 copies / μl to 30 copies / μl.

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