A detection primer combination for DNA degradation degree based on droplet digital PCR and application thereof

Abstract

The application discloses a detection primer combination for DNA degradation degree based on a micro-droplet digital PCR and application thereof, 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. The application utilizes digital PCR technology to detect three targets with different lengths, i.e., a large target, a medium target and a small target, and utilizes the number of the three targets to accurately evaluate the DNA degradation degree, help determine a subsequent STR typing method, and provide a new simple, economical, rapid and effective technical means for evaluation and detection of highly degraded forensic samples.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to a primer combination for detecting the degree of DNA degradation based on droplet digital PCR and its application. Background Technology

[0002] The success of DNA typing often depends on the quantity and quality of the DNA. In forensic cases, DNA samples can become highly degraded due to environmental factors or natural contaminants, rendering traditional testing methods ineffective. This is one of the major challenges facing forensic cases today. Currently, short tandem repeat (STR) typing based on capillary electrophoresis (CE) is one of the most powerful tools in forensic genetics, but DNA degradation reduces the amplification efficiency of genetic markers and can even lead to electrophoresis failure, especially for larger alleles in STR typing. These degraded samples can provide crucial information in court, making the ability to utilize them extremely important. Meanwhile, in many cases, the quantity and quality of human DNA in forensic samples are predictable. For example, in real-world cases, DNA extracted from hair shafts, bones, formalin-fixed tissues, and old case samples can all have reduced quantity and quality of the final target DNA. In forensic cases, determining the quantity and quality of DNA helps forensic workers select more appropriate testing methods. Sensitive and accurate assessments of DNA degradation can support the interpretation of forensic evidence and help explain whether low-quality STR profiles are due to problems in the STR analysis process, the DNA extraction process, or other reasons. Furthermore, accurate assessment of the quantity and quality of DNA before STR amplification can improve the first-time success rate, making this step a crucial one in the DNA analysis workflow of forensic laboratories.

[0003] Currently, real-time quantitative PCR (qPCR) and agarose gel electrophoresis are commonly used to assess the degree of DNA degradation. Agarose gel electrophoresis offers the advantage of visually determining the length and distribution of degraded fragments, but it is not precise and requires high DNA concentrations. Therefore, forensic laboratories currently use qPCR kits to assess DNA degradation. Most commercially available forensic human DNA quantification kits assess degradation by detecting the concentrations of two different amplified fragments of varying lengths, calculating the degradation index (DI), i.e., small fragment concentration / large fragment concentration. This method provides a relatively coarse assessment of degradation and does not effectively help in selecting other detection techniques. Furthermore, qPCR is more susceptible to the effects of PCR inhibitors, especially in complex samples, where PCR inhibitors co-extracted during DNA extraction can severely hinder PCR amplification. Additionally, the "absolute quantification" of qPCR relies on a standard curve, which is derived from serial dilutions of known amounts of template DNA standards. The reliability and consistency of the standard DNA significantly impact the accuracy and stability of qPCR quantification of unknown samples, especially when copy numbers are already low, making it difficult to detect minute differences in copy numbers between samples.

[0004] Furthermore, agarose gel electrophoresis, one of the aforementioned detection techniques, cannot detect highly imbalanced mixed samples. Agarose gel electrophoresis has low accuracy, requires high DNA concentrations, and is unsuitable for quantifying degraded DNA. qPCR kits for assessing DNA degradation are easily affected by PCR inhibitors. The "absolute quantification" of qPCR relies on a standard curve, resulting in poor reproducibility, especially when copy numbers are already very low, making it difficult to detect minute differences in copy numbers between samples. Therefore, there is an urgent need for a method that can accurately quantify the degree of degradation in highly degraded DNA samples and effectively assist in subsequent DNA genotyping. Summary of the Invention

[0005] The purpose of this invention is to provide a primer combination for detecting the degree of DNA degradation based on droplet digital PCR and its application, thereby solving the problems existing in the prior art. This invention utilizes digital PCR technology to detect targets of three different lengths: large, medium, and small. By utilizing the quantity of these three targets, the degree of DNA degradation can be accurately assessed, helping to determine subsequent STR typing methods. This provides a new, simple, economical, rapid, and effective technical means for the evaluation and detection of highly degraded forensic samples.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a primer set for detecting the degree of DNA degradation, the primer set including primer pairs and probes for detecting a 235bp target, primer pairs and probes for detecting a 145bp target, and primer pairs and probes for detecting a 74bp target.

[0008] The nucleotide sequences of the primer pairs and probes for detecting the 235bp target are shown in SEQ ID NO.1-3, respectively.

[0009] The nucleotide sequences of the primer pair and probe for detecting the 145bp target are shown in SEQ ID NO.4-6, respectively.

[0010] The nucleotide sequences of the primer pair and probe for detecting the 74bp target are shown in SEQ ID NO.7-9, respectively.

[0011] Optionally, the probe for detecting a 235bp target is labeled with purple CY5 fluorescence; the probe for detecting a 145bp target is labeled with blue FAM fluorescence; and the probe for detecting a 74bp target is labeled with green VIC fluorescence.

[0012] The present invention also provides the application of the above-described detection primer combination in the preparation of products for assessing the degree of DNA degradation.

[0013] Optionally, the product may include reagents or kits.

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

[0015] Optionally, the droplet digital PCR kit may also include a multiplex amplification reaction mixture.

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

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

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

[0019] (2) Using the DNA from step (1) as a template, perform droplet digital PCR using the above-mentioned droplet digital PCR kit. Calculate the degradation ratios DR1 and DR2 based on the PCR detection results to assess 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 indicates that the DNA is extremely degraded.

[0023] The reaction system for the droplet digital PCR is as follows: 10.0 µL of multiplex amplification reaction mixture, 0.9 µL of primer mixture for detecting a 235 bp target, 0.9 µL of primer mixture for detecting a 145 bp target, 0.9 µL of primer mixture for detecting a 74 bp target, 0.5 µL of probe for detecting a 235 bp target, 0.5 µL of probe for detecting a 145 bp target, 0.5 µL of probe for detecting a 74 bp target, 1.0 µL of template DNA, and deionized water to a final volume of 20 µL.

[0024] The reaction program for the droplet digital PCR is as follows: 95°C for 10 minutes; 95°C for 30 seconds, 58°C for 60 seconds, 45 cycles; then 98°C for 10 minutes; store at 16°C.

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

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

[0027] The concentrations of both the forward and reverse primers in the primer mixture for detecting the 74bp target were 20 μM / L.

[0028] The concentration of the probe used to detect the 235bp target is 100 μM / L;

[0029] The concentration of the probe used to detect the 145bp target is 100 μM / L;

[0030] The concentration of the probe used to detect the 74bp target is 100 μM / L.

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

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

[0033] Based on specific primers and probes for three different target lengths, this invention establishes a method for assessing the degree of degradation by combining DNA quantity and degradation ratio (DR). First, the quantity of the three target lengths is detected using specific primers and probes of different lengths. Then, the degradation ratio is calculated, and the degree of DNA degradation is determined based on the degradation ratio. This optimizes the STR genotyping process, increasing the DNA input amount or the number of PCR cycles to improve the STR genotyping success rate. This invention links the quantity of the three different target lengths, the degradation ratio, and the quality of the obtained STR map, providing a more intuitive way to determine the genotyping success rate of traditional STR testing kits. Therefore, this invention can be used for rapid, simple, and accurate detection and assessment of the degree of degradation of highly degraded DNA, thereby helping forensic laboratories select STR genotyping or other degraded DNA detection technologies and providing evidence interpretation. It offers a new technical means for the detection of highly degraded samples in the forensic field and has broad application prospects. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The image shows the detection results of DNA NIM-RM4035-1 at a concentration of 78.0 ng / µL. Channel 1 is the FAM fluorescence channel, with the vertical axis representing the FAM fluorescence signal intensity; points above the threshold line are positive droplets, and points below the threshold line are negative droplets. Channel 2 is the VIC fluorescence channel, with the vertical axis representing the VIC fluorescence signal intensity; points above the threshold line are positive droplets, and points below the threshold line are negative droplets. Channel 3 is the CY5 fluorescence channel, with the vertical axis representing the CY5 fluorescence signal intensity; points above the threshold line are positive droplets, and points below the threshold line are negative droplets.

[0036] Figure 2 The image shows the detection results of DNA NIM-RM4035-1 at a concentration of 3.89 ng / µL. Channel 1 is the FAM fluorescence channel, with the vertical axis representing the FAM fluorescence signal intensity; points above the threshold line are positive droplets, and points below the threshold line are negative droplets. Channel 2 is the VIC fluorescence channel, with the vertical axis representing the VIC fluorescence signal intensity; points above the threshold line are positive droplets, and points below the threshold line are negative droplets. Channel 3 is the CY5 fluorescence channel, with the vertical axis representing the CY5 fluorescence signal intensity; points above the threshold line are positive droplets, and points below the threshold line are negative droplets.

[0037] Figure 3 This is a graph showing the detection results of a highly degraded DNA simulated sample. Channel 1 is the FAM fluorescence channel, with the vertical axis representing the FAM fluorescence signal intensity; points above the threshold line are positive droplets, and points below the threshold line are negative droplets. Channel 2 is the VIC fluorescence channel, with the vertical axis representing the VIC fluorescence signal intensity; points above the threshold line are positive droplets, and points below the threshold line are negative droplets. Channel 3 is the CY5 fluorescence channel, with the vertical axis representing the CY5 fluorescence signal intensity; points above the threshold line are positive droplets, and points below the threshold line are negative droplets. 1, 2, 3, 4, and 5 represent boiling water bath times of 0, 20, 40, 60, and 80 minutes, respectively.

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

[0039] Figure 5 The image shows the STR typing results of five paraffin-embedded sections; from top to bottom, these are the STR typing results of samples numbered 158-1, 158-2, 158-7, 58-18, and 59-20; the unselected peaks are dye peaks.

[0040] Figure 6 The image shows the STR typing results of five paraffin-embedded sections. From top to bottom, these are the STR typing results of samples numbered 66-19, 77-22, 116-11, 126-19, and 136-13. The unselected peaks are dye peaks. Detailed Implementation

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

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0046] Example 1

[0047] This embodiment selected three target lengths—large, medium, and small—from the non-coding region of chromosome 3, and involved specific primers and probes for detecting these three targets. Primer information is shown in Table 1, and the primers were synthesized by Aikerui Biotechnology Co., Ltd.

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

[0049]

[0050] Based on the specific primers and probes listed in Table 1, this embodiment prepared a kit for detecting highly degraded samples based on three target lengths. The kit contains specific primer pairs and probes for amplifying large targets (235 bp), specific primer pairs and probes for amplifying medium targets (145 bp), specific primer pairs and probes for amplifying small targets (74 bp), DNA standards, and a multiplex amplification reaction mixture.

[0051] The specific primer pairs and probes for amplifying the large target (235bp) were prepared with ultrapure water at a concentration of 100μM / L. Then, the forward and reverse primers were prepared with ultrapure water to form a primer mixture with a final concentration of 20μM / L. The probe was labeled with purple CY5 fluorescent markers.

[0052] The specific primer pair and probe for amplifying the target (145bp) were prepared with ultrapure water at a concentration of 100 μM / L. Then, the forward and reverse primers were prepared with ultrapure water to a final concentration of 20 μM / L. The probe was labeled with blue FAM fluorescent label.

[0053] The specific primer pairs and probes for amplifying the small target (74bp) were prepared with ultrapure water to a concentration of 100μM / L. Then, the forward and reverse primers were prepared with ultrapure water to a final concentration of 20μM / L. The probe was labeled with green VIC fluorescent label.

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

[0055] The DNA standards are standard DNA9948 and DNA NIM-RM4035-1.

[0056] Example 2

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

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

[0059] (2) Using the DNA from step (1) as the amplification template, the sample is mixed with the multiplex amplification primers, probe mixture and multiplex amplification reaction mixture to generate droplets using the droplet generation chip and droplet generator of Yongnuo Company of China.

[0060] (3) Perform multiplex PCR amplification on the generated droplets in the following amplification system:

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

[0062] The cycling parameters for the multiplex amplification PCR reaction were: 95℃ for 10 minutes; 95℃ for 30 seconds, 58℃ for 60 seconds, 45 cycles; then 98℃ for 10 minutes; and stored at 16℃.

[0063] (4) Analysis by digital PCR analyzer

[0064] The data were analyzed using the MicroDrop-20A biochip analyzer from Yongnuo Biotechnology Co., Ltd. (China). QuantaSoft 1.7 software (Bio-Rad Laboratories) was used for data analysis.

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

[0066] Example 3

[0067] This embodiment uses the kit prepared in Example 1 to detect HMY10 DNA sample (collected and extracted by the Forensic Evidence Laboratory of Xiangya School of Basic Medical Sciences, Central South University) to verify the kit's detection performance on highly degraded DNA and its STR typing effect. The specific detection process is as follows:

[0068] (1) Constructing highly degraded DNA simulation samples: 10 ng / μL DNA sample HMY10 was degraded by treating it in a boiling water bath at 100°C for specific times (0 min, 20 min, 40 min, 60 min and 80 min).

[0069] (2) The detection kit of Example 1 was used to detect the highly degraded DNA model. The detection process is described in steps (2)-(4) of Example 2. Allele loci were divided into three categories: large, medium and small according to locus size of 74bp, 145bp and 235bp. The gene copy number and allele detection rate were compared under different boiling water bath treatment times (different degrees of degradation).

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

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

[0072]

[0073] From Table 2 and Figure 3 It can be seen that there are significant differences in gene copy number among DNA samples treated with different boiling water baths, indicating that the highly degraded DNA model has been successfully constructed. Furthermore, when using the kit of this invention to detect DNA samples with different degrees of degradation, there are also significant differences in gene copy number and allele detection rate, indicating that this invention is highly effective in detecting highly degraded DNA, and can sensitively assess the degree of degradation and determine the absolute amount of products of different lengths.

[0074] Based on this, this embodiment optimizes the detection method for simulated degraded DNA according to the absolute quantitative results of DNA samples with different degrees of degradation. By doubling the DNA input volume of samples subjected to boiling water baths for 20, 40, and 60 minutes, genotyping was performed using the SureID® PanGlobal Human DNA Identity Kit (Haier Gene, Ningbo, China). Results are as follows... Figure 4 As shown, all samples from 20 and 40 minutes were detected, and the number of observed DNA sites increased by 66.7% in the 60-minute sample. However, doubling the DNA input amount for the 80-minute sample did not significantly improve the detection rate. This indicates that, based on the detection results of the kit of this invention on highly degraded DNA samples, the STR typing detection process for degraded DNA samples can be optimized in actual testing, reducing the detection steps and lowering the detection cost.

[0075] Example 4

[0076] This embodiment used the kit from Example 1 to detect the degradation degree of paraffin-embedded samples and blood samples from actual forensic cases, and optimized the STR typing process for these samples based on the test results. The samples came from 23 sources: paraffin-embedded samples from 10 forensic cases, 1 blood sample, and paraffin-embedded samples from 6 forensic cases, along with a blood sample from the same individual. The specific testing process is as follows:

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

[0078] (2) DNA quantification was performed using a conventional NanoDrop™ one spectrometer, followed by STR typing. No STR typing was found. This indicates that the DNA in these samples was highly degraded.

[0079] (3) The degradation degree of paraffin-embedded samples in 10 forensic cases was assessed using the kit of the present invention and a commercially available qPCR kit (purchased from Thermo Fisher Scientific, USA). The detection process is described in steps (2)-(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 Detection 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 to increase the amount of DNA template to increase the copy number of medium-length targets to over 15.0 copies, or the copy number of long-length targets to over 5.0 copies, and to increase the PCR cycle by 2 times to meet the copy number requirements. The typing results of the optimized paraffin-embedded samples are shown below. Figure 5 and Figure 6 As shown, the optimization yielded more allele loci typing results, and sex chromosome allele loci were detected in all samples.

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

[0085] By analyzing the correlation between degradation degree detection results and typing results, a method was established to assess degradation degree using a combination of DNA quantity and degradation ratio (DR). Degradation ratio 1 (DR1) is the ratio of 235bp target copy number to 74bp target copy number, and degradation ratio 2 (DR2) is the ratio of 145bp target copy number to 74bp target copy number. The correlation between DR1, DR2, and site detection rate was analyzed, and the results are shown in Table 4. We concluded that if the evaluated DR1 of the sample is less than 0.8 and DR2 is greater than 0.02, the DNA is mildly to moderately degraded; if the evaluated DR1 of the sample is less than 0.02 and DR2 is greater than 0.05, the DNA is highly degraded; and if the evaluated DR2 of the sample is less than 0.05, the DNA is extremely degraded.

[0086] Table 4. Degradation degree detection results and allele locus detection rate of paraffin-embedded samples and blood samples.

[0087]

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

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer set for detecting the degree of DNA degradation, characterized in that, The detection primer combination includes primer pairs and probes for detecting a 235bp target, primer pairs and probes for detecting a 145bp target, and primer pairs and probes for detecting a 74bp target. The nucleotide sequences of the primer pairs and probes for detecting the 235bp target are shown in SEQ ID NO.1-3, respectively. The nucleotide sequences of the primer pair and probe for detecting the 145bp target are shown in SEQ ID NO.4-6, respectively. The nucleotide sequences of the primer pair and probe for detecting the 74bp target are shown in SEQ ID NO.7-9, respectively.

2. The detection primer combination according to claim 1, characterized in that, The probe for detecting a 235bp target is labeled with purple CY5 fluorescence; the probe for detecting a 145bp target is labeled with blue FAM fluorescence; and the probe for detecting a 74bp target is labeled with green VIC fluorescence.

3. The use of the detection primer combination according to claim 1 or 2 in the preparation of products for assessing the degree of DNA degradation.

4. The application according to claim 3, characterized in that, The product includes reagents.

5. A droplet-based digital PCR kit for assessing the degree of DNA degradation, characterized in that, It includes the detection primer combination as described in claim 1 or 2.

6. The droplet-type digital PCR kit according to claim 5, characterized in that, The droplet digital PCR kit also includes a multiplex amplification reaction mixture.

7. The use of the detection primer combination of claim 1 or 2 or the droplet digital PCR kit of claim 5 or 6 in assessing the degree of DNA degradation.

8. A method for assessing the degree of DNA degradation, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested; (2) Using the DNA from step (1) as a template, perform droplet digital PCR using the droplet digital PCR kit described in claim 5 or 6, and calculate the degradation ratios DR1 and DR2 based on the PCR detection results to assess the degree of DNA degradation. DR1 is the ratio of 235bp target copy number to 74bp target copy number; DR2 is the ratio of 145bp target copy number to 74bp target copy number. 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 indicates that the DNA is extremely degraded.

9. The method according to claim 8, characterized in that, The reaction system for the droplet digital PCR is as follows: 10.0 µL of multiplex amplification reaction mixture, 0.9 µL of primer mixture for detecting a 235 bp target, 0.9 µL of primer mixture for detecting a 145 bp target, 0.9 µL of primer mixture for detecting a 74 bp target, 0.5 µL of probe for detecting a 235 bp target, 0.5 µL of probe for detecting a 145 bp target, 0.5 µL of probe for detecting a 74 bp target, 1.0 µL of template DNA, and deionized water to a final volume of 20 µL. The reaction program for the droplet digital PCR is as follows: 95°C for 10 minutes; 95°C for 30 seconds, 58°C for 60 seconds, 45 cycles; then 98°C for 10 minutes; store at 16°C.

10. The method according to claim 8, characterized in that, The concentrations of both the forward and reverse primers in the primer mixture for detecting the 235bp target were 20 μM / L. The concentrations of both the forward and reverse primers in the primer mixture for detecting the 145bp target were 20 μM / L. The concentrations of both the forward and reverse primers in the primer mixture for detecting the 74bp target were 20 μM / L. The concentration of the probe used to detect the 235bp target is 100 μM / L; The concentration of the probe used to detect the 145bp target is 100 μM / L; The concentration of the probe used to detect the 74bp target is 100 μM / L.

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