DNA degradation degree detection primer combination based on microdroplet type digital PCR and application of DNA degradation degree detection primer combination

Abstract

The invention discloses a DNA (Deoxyribose Nucleic Acid) degradation degree detection primer combination based on microdroplet type digital PCR (Polymerase Chain Reaction) and application of the DNA degradation degree detection primer combination, and belongs to the technical field of molecular biology. The detection primer combination comprises a primer pair and a probe for detecting a 235bp target, a primer pair and a probe for detecting a 145bp target, and a primer pair and a probe for detecting a 75bp target. According to the method, large, medium and small targets with different lengths are detected by utilizing a digital PCR technology, the DNA degradation degree can be accurately evaluated by utilizing the quantity of the three targets, a subsequent STR typing method is helped to be determined, and a novel simple, economical, rapid and effective technical means is provided for evaluation and detection of forensic highly-degraded detection materials.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a detection primer combination for DNA degradation degree based on droplet digital PCR and application thereof. Background Art

[0002] The success of DNA typing often depends on the quantity and quality of DNA. In forensic cases, DNA samples may be highly degraded due to environmental factors or natural contaminants, making traditional detection methods ineffective, which is one of the main challenges facing forensic cases today. Short tandem repeat (STR) typing based on capillary electrophoresis (CE) detection technology is currently one of the most powerful tools in forensic genetics, but DNA degradation can reduce the amplification efficiency of genetic markers and even cause electrophoresis failure, especially for large alleles in STR typing. These degraded samples may provide critical information in court, so the ability to utilize these samples becomes very important. At the same time, in many cases, the quantity and quality of human DNA in forensic samples are predictable. For example, in actual cases, DNA extracted from hair shafts, bones, formalin-fixed tissues, and old case samples will reduce the quantity and quality of the final target DNA. In forensic cases, the determination of DNA quantity and quality can help forensic workers choose more appropriate detection methods. Sensitive and accurate DNA degradation assessment can provide support for the interpretation of forensic evidence and help explain whether the low quality of STR profiles is due to problems in the STR analysis process, problems in the DNA extraction process, or other reasons. Furthermore, accurate assessment of DNA quantity and quality prior to STR amplification improves first-time success rates, and thus this step has become a critical step in the DNA analysis workflow in forensic laboratories.

[0003] Currently, real-time quantitative PCR (qPCR) and agarose gel electrophoresis are commonly used to assess the degree of DNA degradation. The advantage of agarose gel electrophoresis is that the length and distribution of degraded fragments can be determined intuitively, but it is not accurate and requires a high DNA concentration. Therefore, forensic laboratories currently use qPCR kits to assess DNA degradation. Most commercially available forensic human DNA quantification kits assess the degree of degradation by detecting the concentration of large and small amplified fragments of different lengths, and calculate the degradation index (DI), that is, the concentration of small fragments / the concentration of large fragments. This method is a rough assessment of the degree of degradation and cannot help choose other detection techniques. In addition, qPCR is more susceptible to PCR inhibitors, especially in complex samples. PCR inhibitors co-extracted during DNA extraction may seriously hinder PCR amplification. In addition, the "absolute quantification" of qPCR relies on the standard curve, which is obtained by serial dilution of a known amount of template DNA standard. The reliability and consistency of standard DNA greatly affect the accuracy and stability of qPCR quantification of unknown samples, especially when the copy number is already very low, it is difficult to detect small differences in copy number between samples.

[0004] In addition, agarose electrophoresis in the above detection technology cannot detect extremely unbalanced mixed samples. Agarose gel electrophoresis is not very accurate, requires a higher DNA concentration, and is not suitable for the quantification of degraded DNA. The qPCR kit for assessing the degree of DNA degradation is easily affected by PCR inhibitors. The "absolute quantification" of qPCR relies on the standard curve, and the results have poor repeatability, especially when the copy number is already very low, it is difficult to detect small differences in the copy number between samples. Therefore, there is an urgent need to provide a detection method that can accurately quantify the degree of degradation of highly degraded DNA samples and effectively assist in subsequent DNA typing. Summary of the invention

[0005] The purpose of the present invention is to provide a detection primer combination of DNA degradation degree based on droplet digital PCR and its application to solve the problems existing in the above-mentioned prior art. The present invention uses digital PCR technology to detect targets of three different lengths, large, medium and small. By using the number of the three targets, the degree of DNA degradation can be accurately assessed, which helps determine the subsequent STR typing method, and provides a new simple, economical, fast and effective technical means for the assessment and detection of highly degraded forensic samples.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a primer combination for detecting the degree of DNA degradation, the primer combination comprising a primer pair and a probe for detecting a 235 bp target, a primer pair and a probe for detecting a 145 bp target, and a primer pair and a probe for detecting a 75 bp target;

[0008] The nucleotide sequences of the primer pair and probe for detecting the 235 bp target are shown in SEQ ID NOs. 1-3 respectively;

[0009] The nucleotide sequences of the primer pair and probe for detecting the 145 bp target are shown in SEQ ID NOs. 4-6 respectively;

[0010] The nucleotide sequences of the primer pair and probe for detecting the 75 bp target are shown in SEQ ID NOs. 7-9, respectively.

[0011] Optionally, the probe for detecting the 235 bp target is labeled with a purple CY5 fluorescent marker; the probe for detecting the 145 bp target is labeled with a blue FAM fluorescent marker; and the probe for detecting the 75 bp target is labeled with a green VIC fluorescent marker.

[0012] The present invention also provides the use of the above detection primer combination in preparing a product for evaluating the degree of DNA degradation.

[0013] Optionally, the product comprises a reagent or a kit.

[0014] The present invention also provides a droplet digital PCR kit for evaluating the degree of DNA degradation, comprising the above-mentioned detection primer combination.

[0015] Optionally, the droplet digital PCR kit further comprises a composite amplification reaction mixture.

[0016] The present invention also provides the use of the above detection primer combination or the above droplet digital PCR kit in evaluating the degree of DNA degradation.

[0017] The present invention also provides a method for evaluating the degree of DNA degradation, comprising the following steps:

[0018] (1) Extracting DNA from the sample to be tested;

[0019] (2) using the DNA from step (1) as a template, performing droplet digital PCR using the above-mentioned droplet digital PCR kit, and calculating degradation ratios DR1 and DR2 based on the PCR test results to evaluate the degree of DNA degradation;

[0020] If DR1 is less than 0.8 and DR1 is greater than 0.02, it indicates mild to moderate DNA degradation;

[0021] If DR1 is less than 0.02 and DR2 is greater than 0.05, it indicates that the DNA is highly degraded;

[0022] If DR2<0.05, it means that the DNA is extremely degraded.

[0023] The reaction system of the droplet digital PCR is: 10.0 μL of composite amplification reaction mixture, 0.9 μL of primer mixture for detecting 235 bp target, 0.9 μL of primer mixture for detecting 145 bp target, 0.9 μL of primer mixture for detecting 75 bp target, 0.5 μL of probe for detecting 235 bp target, 0.5 μL of probe for detecting 145 bp target, 0.5 μL of probe for detecting 75 bp target, 1.0 μL of template DNA, and deionized water to 20 μL;

[0024] The reaction procedure of the droplet digital PCR is: 95°C, 10 minutes; 95°C, 30 seconds, 58°C, 60 seconds, 45 cycles; then 98°C, 10 minutes; and stored at 16°C.

[0025] Optionally, the concentrations of the forward primer and the reverse primer in the primer mixture for detecting the 235 bp target are both 20 μM / L;

[0026] The concentrations of the forward primer and the reverse primer in the primer mixture for detecting the 145 bp target are both 20 μM / L;

[0027] The concentrations of the forward primer and the reverse primer in the primer mixture for detecting the 75 bp target are both 20 μM / L;

[0028] The concentration of the probe for detecting the 235 bp target is 100 μM / L;

[0029] The concentration of the probe for detecting the 145 bp target is 100 μM / L;

[0030] The concentration of the probe for detecting the 75 bp target is 100 μM / L.

[0031] The present invention discloses the following technical effects:

[0032] The present invention designs targets of three different lengths, large, medium and small, according to the non-coding region of chromosome 3, and designs specific primers and probes corresponding to the three targets, which have good specificity, good reproducibility, stable and reliable results, and can detect highly degraded DNA as low as 2 copies.

[0033] According to the specific primers and probes of three different length targets, the present invention establishes a method for evaluating the degree of degradation by combining the amount of DNA and the degradation ratio (DR): first, the number of three length targets is detected by using the specific primers and probes of three different length targets, and then the degradation ratio is calculated, and the degree of DNA degradation is determined according to the degradation ratio, so as to optimize the STR typing detection process, increase the DNA input amount or the number of PCR cycles, so as to improve the typing success rate of STR. The present invention links the number of three different length targets, the degradation ratio and the quality of the obtained STR spectrum, and can more intuitively determine the typing success rate of the traditional STR detection kit. Therefore, the present invention can be used to quickly, simply and accurately detect and evaluate the degree of degradation of highly degraded DNA, thereby helping forensic laboratories to select STR typing or other degraded DNA detection technologies, and provide evidence interpretation, providing a new technical means for the detection of highly degraded samples in the field of forensic medicine, and has broad application prospects in the field of forensic medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The figure is the detection result of DNA NIM-RM4035-1 at 78.0 ng / μL; wherein, channel 1 is the FAM fluorescence channel, the ordinate value represents the FAM fluorescence signal intensity, the points above the threshold line are positive droplets, and the points below the threshold line are negative droplets; channel 2 is the VIC fluorescence channel, the ordinate value represents the VIC fluorescence signal intensity, the points above the threshold line are positive droplets, and the points below the threshold line are negative droplets; channel 3 is the CY5 fluorescence channel, the ordinate value represents the CY5 fluorescence signal intensity, the points above the threshold line are positive droplets, and the points below the threshold line are negative droplets;

[0036] Figure 2 The figure is the detection result of DNA NIM-RM4035-1 at 3.89 ng / μL; wherein, channel 1 is the FAM fluorescence channel, the ordinate value represents the FAM fluorescence signal intensity, the points above the threshold line are positive droplets, and the points below the threshold line are negative droplets; channel 2 is the VIC fluorescence channel, the ordinate value represents the VIC fluorescence signal intensity, the points above the threshold line are positive droplets, and the points below the threshold line are negative droplets; channel 3 is the CY5 fluorescence channel, the ordinate value represents the CY5 fluorescence signal intensity, the points above the threshold line are positive droplets, and the points below the threshold line are negative droplets;

[0037] Figure 3 It is the detection result diagram of the highly degraded DNA simulation sample; wherein, channel 1 is the FAM fluorescence channel, the ordinate value represents the FAM fluorescence signal intensity, the points above the threshold line are positive droplets, and the points below the threshold line are negative droplets; channel 2 is the VIC fluorescence channel, the ordinate value represents the VIC fluorescence signal intensity, the points above the threshold line are positive droplets, and the points below the threshold line are negative droplets; channel 3 is the CY5 fluorescence channel, the ordinate value represents the CY5 fluorescence signal intensity, the points above the threshold line are positive droplets, and the points below the threshold line are negative droplets; 1, 2, 3, 4 and 5 represent the boiling water bath time of 0, 20, 40, 60 and 80 minutes, respectively;

[0038] Figure 4 The STR typing results of the highly degraded DNA simulation sample before and after optimization;

[0039] Figure 5 The STR typing results of 5 paraffin-embedded sections are shown in the figure; from top to bottom, they are the STR typing results of samples numbered 158-1, 158-2, 158-7, 58-18, and 59-20; the unselected ones are dye peaks;

[0040] Figure 6 The figure shows the STR typing results of 5 paraffin-embedded sections; from top to bottom, they are the STR typing results of samples numbered 66-19, 77-22, 116-11, 126-19, and 136-13; the unselected ones are dye peaks. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] Example 1

[0047] This example selected three types of targets of large, medium and small lengths in the non-coding region of chromosome 3, and involved specific primers and probes for detecting these three targets. The information of the primers is shown in Table 1, and they were synthesized by Acori Biotech.

[0048] Table 1 Nucleotide sequences of amplification primers and probes

[0049]

[0050] According to the specific primers and probes recorded in Table 1, this embodiment prepared a kit for detecting highly degraded samples based on three length targets, which included specific primer pairs and probes for amplifying large targets (235bp), specific primer pairs and probes for amplifying medium targets (145bp), specific primer pairs and probes for amplifying small targets (75bp), DNA standards, and a composite amplification reaction mixture.

[0051] Among them, the specific primer pair and probe for amplifying the large target (235bp) were configured with ultrapure water to 100μM / L, and then the forward and reverse primers were configured with ultrapure water to a primer mixture with a final concentration of 20μM / L respectively; the probe was labeled with purple CY5 fluorescence.

[0052] The specific primer pair and probe of the target (145 bp) in the amplification were prepared with ultrapure water to 100 μM / L, and then the forward and reverse primers were prepared with ultrapure water to a primer mixture with a final concentration of 20 μM / L; the probe was labeled with blue FAM fluorescence.

[0053] The specific primer pair and probe for amplifying the small target (75 bp) were prepared with ultrapure water to 100 μM / L, and then the forward and reverse primers were prepared with ultrapure water to a primer mixture with a final concentration of 20 μM / L; the probe was labeled with green VIC fluorescence.

[0054] The composite amplification reaction mixture is Yongnuo's PCR reaction mixture ddPCR TM Supermix.

[0055] The DNA standards were DNA9948 and DNANIM-RM4035-1.

[0056] Example 2

[0057] This example uses the kit prepared in Example 1 to test a DNA standard (DNANIM-RM4035-1) to verify the detection performance of the kit. The specific detection process is as follows:

[0058] (1) Dilute 78.0 ng / μL DNA 20 times to 3.9 ng / μL with ultrapure water.

[0059] (2) Using the DNA in step (1) as an amplification template, the composite amplification primer, the probe mixture and the composite amplification reaction mixture are used to generate droplets using a droplet generation chip and a droplet generation instrument of Yongnuo Company of China.

[0060] (3) The generated droplets are subjected to composite PCR amplification in the following amplification system:

[0061] ddPCR TM 10.0 μL of premix, 0.9 μL of specific primer mixture for amplifying large targets, 0.9 μL of specific primer mixture for amplifying medium targets, 0.9 μL of specific primer mixture for amplifying small targets, 0.5 μL of specific probe for amplifying large targets, 0.5 μL of specific probe for amplifying medium targets, 0.5 μL of specific probe for amplifying small targets, 1.0 μL of template DNA, and deionized water to 20 μL.

[0062] The cycle parameters of the multiplex amplification PCR reaction were 95° C., 10 minutes; 95° C., 30 seconds, 58° C., 60 seconds, 45 cycles; then 98° C., 10 minutes; and stored at 16° C.

[0063] (4) Analysis by digital PCR analyzer

[0064] The data were analyzed using MicroDrop-20A biochip analyzer from Yongnuo, China, and QuantaSoft 1.7 software (Bio-Rad Laboratories).

[0065] Test results such as Figure 1 and Figure 2 shown. Figure 1 The detection result is 78.0ng / μLDNA. It can be seen that the positive droplets of large, medium and small sites are clearly separated from the negative droplets. The positive result of the sample can be clearly identified, and the ratio of large, medium and small sites is 1:1:1. Figure 2 The detection result is 3.9ng / μL DNA. It can be seen that the positive droplets of large, medium and small sites are clearly separated from the negative droplets. The positive result of the sample can also be clearly identified, and the ratio of large, medium and small sites is 1:1:1.

[0066] Example 3

[0067] In this example, the kit prepared in Example 1 was used to test a DNA sample HMY10 (collected and extracted by the Forensic Evidence Laboratory of Xiangya School of Basic Medical Sciences, Central South University) to verify the detection performance of the kit for highly degraded DNA and the STR typing effect. The specific detection process is as follows:

[0068] (1) Construction of highly degraded DNA simulation samples: 10 ng / μL DNA sample HMY10 was treated in a 100°C boiling water bath for specific time periods (0 min, 20 min, 40 min, 60 min, and 80 min) for degradation.

[0069] (2) The highly degraded DNA model was tested using the detection kit of Example 1, and the detection process was as described in steps (2) to (4) of Example 2. The allele loci were divided into three categories: large, medium, and small according to the locus size of 75 bp, 145 bp, and 235 bp, and the gene copy number and allele detection rate under different boiling water bath treatment times (different degradation degrees) were compared.

[0070] The test results are shown in Table 2 and Figure 3 .

[0071] Table 2 Detection results of DNA samples with different degradation degrees

[0072]

[0073] From Table 2 and Figure 3 It can be seen that there are obvious differences in the gene copy numbers of DNA samples after different boiling water bath treatments, indicating that the highly degraded DNA model was successfully constructed. At the same time, when the kit of the present invention is used to detect DNA samples with different degradation degrees, there are also obvious differences in the gene copy number and allele detection rate, indicating that the present invention is effective in detecting highly degraded DNA, and can sensitively evaluate the degree of degradation and determine the absolute amount of products of different lengths.

[0074] On this basis, this example optimized the detection method of simulated degraded DNA according to the absolute quantitative results of DNA samples with different degradation degrees. The PanGlobal Human DNA Identity Kit (Helsch Gene, Ningbo, China) was used for typing test. Figure 4 As shown, it can be seen that all samples of 20 and 40 minutes were detected, and the number of sites observed in 60-minute DNA increased by 66.7%, but doubling the DNA input of 80-minute samples did not significantly improve the detection rate. This shows that based on the detection results of the kit of the present invention on highly degraded DNA samples, the STR typing detection process of degraded DNA samples can be optimized in actual detection, reducing the detection process and reducing the detection cost.

[0075] Example 4

[0076] This example uses the kit of Example 1 to detect the degradation degree of paraffin-embedded samples and blood samples in actual forensic cases, and optimizes the STR typing detection process of these samples based on the test results. The samples come from paraffin-embedded samples and 1 blood sample from 10 forensic cases, paraffin-embedded samples from 6 forensic cases and blood samples from the same individual, a total of 23. The specific detection process is as follows:

[0077] (1) Extract DNA from paraffin-embedded samples and blood samples in 2022, 2023 and 2024 respectively.

[0078] (2) Using the conventional spectrophotometer NanoDrop TM One performed DNA quantification and then STR typing, and found no STR typing, indicating that the DNA in these samples was highly degraded.

[0079] (3) The degradation degree of paraffin-embedded samples in 10 forensic cases was evaluated using the kit of the present invention and a commercially available qPCR kit (purchased from Thermo Fisher Scientific, USA). The detection process was as described in steps (2) to (4) of Example 2. The detection results are shown in Table 3. It can be seen that the detection limit of the kit of the present invention is lower than that of the commercially available qPCR kit.

[0080] Table 3 Degradation test results of paraffin-embedded samples

[0081]

[0082] Note: Und. indicates undetermined; Year indicates the year of paraffin embedding.

[0083] (4) Based on the results in Table 3, the STR typing detection system for paraffin-embedded samples was optimized. The optimization method was: by increasing the amount of DNA template, the copy number of the medium-length target was increased to more than 15.0 copies, or the copy number of the long-length target was increased to more than 5.0 copies, and the PCR cycle was increased by 2 times to meet the copy number conditions. The typing results of the optimized paraffin-embedded samples are shown in Figure 2. Figure 5 and Figure 6 As shown, it can be seen that after optimization, typing results of more allele loci were obtained, and sex chromosome allele loci were detected in all samples.

[0084] (5) The kit of the present invention was used to evaluate the degradation degree of paraffin-embedded samples and blood samples in all forensic cases, and to perform STR typing. The detection process was as described in steps (2) to (4) of Example 2. The results are shown in Table 4.

[0085] By analyzing the relationship between the degradation degree test results and the typing results, a method for evaluating the degradation degree using a combination of DNA quantity and degradation ratio (DR) was established, where degradation ratio 1 (degradationratio1, DR1) is the ratio of the 235bp target copy number to the 75bp target copy number, and degradation ratio 2 (degradationratio2, DR2) is the ratio of the 145bp target copy number to the 75bp target copy number. The relationship between DR1, DR2 and site detection rate was analyzed, and the results are shown in Table 4. We believe that if the sample to be tested is evaluated to have DR1 less than 0.8 and DR1 greater than 0.02, it indicates that the DNA is mildly or moderately degraded, if the sample to be tested is evaluated to have DR1 less than 0.02 and DR2 greater than 0.05, it indicates that the DNA is highly degraded, and if the sample to be tested is evaluated to have DR2<0.05, it indicates that the DNA is extremely degraded.

[0086] Table 4 Degradation test results and allele detection rates of paraffin-embedded samples and blood samples

[0087]

[0088] Note: BL is blood sample; PAS is paraffin-embedded sample.

[0089] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer combination for detecting the degree of DNA degradation, characterized in that: The detection primer combination includes a primer pair and a probe for detecting a 235 bp target, a primer pair and a probe for detecting a 145 bp target, and a primer pair and a probe for detecting a 75 bp target; The nucleotide sequences of the primer pair and probe for detecting the 235 bp target are shown in SEQ ID NOs. 1-3 respectively; The nucleotide sequences of the primer pair and probe for detecting the 145 bp target are shown in SEQ ID NOs. 4-6 respectively; The nucleotide sequences of the primer pair and probe for detecting the 75 bp target are shown in SEQ ID NOs. 7-9, respectively.

2. The detection primer combination according to claim 1, characterized in that: The probe for detecting the 235 bp target is labeled with a purple CY5 fluorescent marker; the probe for detecting the 145 bp target is labeled with a blue FAM fluorescent marker; and the probe for detecting the 75 bp target is labeled with a green VIC fluorescent marker.

3. Use of the detection primer combination according to claim 1 or 2 in the preparation of a product for evaluating the degree of DNA degradation.

4. The use according to claim 3, characterized in that: The product comprises a reagent or a kit.

5. A droplet digital PCR kit for evaluating the degree of DNA degradation, characterized in that: Comprising the detection primer combination according to claim 1 or 2.

6. The droplet digital PCR kit according to claim 5, characterized in that: The droplet digital PCR kit also contains a composite amplification reaction mixture.

7. Use of the detection primer combination described in claim 1 or 2 or the droplet digital PCR kit described in claim 5 or 6 in evaluating the degree of DNA degradation.

8. A method for evaluating the degree of DNA degradation, characterized in that: The steps include: (1) Extracting DNA from the sample to be tested; (2) using the DNA of step (1) as a template, performing droplet digital PCR using the droplet digital PCR kit of claim 5 or 6, and calculating degradation ratios DR1 and DR2 based on the PCR test results to evaluate the degree of DNA degradation; If DR1 is less than 0.8 and DR1 is greater than 0.02, it indicates mild to moderate DNA degradation; If DR1 is less than 0.02 and DR2 is greater than 0.05, it indicates that the DNA is highly degraded; If DR2<0.05, it means that the DNA is extremely degraded.

9. The method according to claim 8, characterized in that The reaction system of the droplet digital PCR is: 10.0 μL of composite amplification reaction mixture, 0.9 μL of primer mixture for detecting 235 bp target, 0.9 μL of primer mixture for detecting 145 bp target, 0.9 μL of primer mixture for detecting 75 bp target, 0.5 μL of probe for detecting 235 bp target, 0.5 μL of probe for detecting 145 bp target, 0.5 μL of probe for detecting 75 bp target, 1.0 μL of template DNA, and deionized water to 20 μL; The reaction procedure of the droplet digital PCR is: 95°C, 10 minutes; 95°C, 30 seconds, 58°C, 60 seconds, 45 cycles; then 98°C, 10 minutes; and stored at 16°C.

10. The method according to claim 8, characterized in that The concentrations of the forward primer and the reverse primer in the primer mixture for detecting the 235 bp target are both 20 μM / L; The concentrations of the forward primer and the reverse primer in the primer mixture for detecting the 145 bp target are both 20 μM / L; The concentrations of the forward primer and the reverse primer in the primer mixture for detecting the 75 bp target are both 20 μM / L; The concentration of the probe for detecting the 235 bp target is 100 μM / L; The concentration of the probe for detecting the 145 bp target is 100 μM / L; The concentration of the probe for detecting the 75 bp target is 100 μM / L.

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