Forensic multi-body fluid age prediction method based on micro-droplet digital PCR and application of forensic multi-body fluid age prediction method

Abstract

The invention discloses a forensic multi-body fluid age prediction method based on micro-droplet digital PCR and application, and relates to the technical field of biology. The invention provides an age prediction method based on microdroplet digital PCR. The age prediction method comprises the following steps: S1, extracting DNA of a detected individual; s2, the methylation level of the cg10501210, the methylation level of the cg10528482 and the methylation level of the cg05940966 are detected through a microdroplet type digital PCR (Polymerase Chain Reaction); and S3, inputting the methylation level into the multiple linear regression model to obtain the age of the detected individual. According to the age prediction method disclosed by the invention, accurate age inference can be carried out by only needing 0.5 ng of template DNA, and the age prediction method has relatively high sensitivity and relatively short time and has a good application prospect in forensic medicine.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a forensic multi-body fluid age prediction method based on droplet digital PCR and its application. Background Art

[0002] As a key technical means of forensic individual identification, physiological age inference plays a vital role in criminal investigations. By accurately assessing an individual's age, the scope of investigation can be effectively narrowed, and the age range of the suspect can be identified, thus providing key scientific evidence for solving the case. Traditional age inference methods rely primarily on morphological analysis of bones and teeth. However, such methods have significant limitations in forensic practice: on the one hand, complete bone or tooth samples are required, and on the other hand, various body fluid stains are more commonly found at crime scenes than bone tissue.

[0003] Currently, a variety of age estimation methods based on molecular markers have been developed, including telomere length measurement, T cell receptor deletion loop abundance analysis, and advanced glycation end products (AGEs) content measurement. However, these methods still have significant shortcomings: the AGEs method has poor accuracy in estimating the age of individuals under 20 years old, and other methods also suffer from insufficient sensitivity or limited applicability, seriously restricting their practical application value at crime scenes.

[0004] Epigenetic detection technologies (such as DNA methylation analysis) provide new research directions to address the above problems. Existing studies have shown that specific epigenetic modifications show regular changes with age. Currently, age prediction research in the field of forensic medicine is mostly limited to the analysis of single body fluid samples. Although the combination of machine learning algorithms such as Multiple Linear Regression (MLR), Support Vector Regression (SVR), Artificial Neural Network (ANN), and Random Forest Regression (RF) has achieved a mean absolute deviation (MAD) prediction error of 3.24-4.7 years, it still faces challenges when dealing with the diverse and micro-degraded body fluid stains commonly found at crime scenes. Using existing prediction models for age prediction can lead to problems such as unreliable results and low prediction accuracy. It is worth noting that to date, there are few universal age prediction models applicable to multiple body fluids.

[0005] Many current technologies, such as EpiTYPER, SNapshot, and droplet digital PCR, can provide relatively accurate DNA methylation level detection. The EpiTYPER assay requires a minimum DNA template of 1 μg. However, obtaining such high-quality DNA is difficult at actual crime scenes, limiting its application in routine forensic investigations. Compared to EpiTYPER, SNapshot requires only 4 ng of template DNA for detection.

[0006] The main purpose of using droplet digital PCR (ddPCR) technology for age prediction is to achieve high-precision inference of an individual's age through a single reaction, thereby significantly reducing the amount of samples required for traditional detection methods and greatly shortening the case detection cycle. The core advantage of this technology lies in its ultra-high sensitivity and absolute quantitative ability, which can accurately detect trace biomarkers and maintain stable performance even in the face of degraded samples (such as trace blood stains and saliva stains left at the crime scene). By optimizing the system, the entire process from DNA extraction to data analysis is completed within 7-8 hours, allowing forensic workers to quickly lock in the suspect's age range within the golden 72 hours of the case, providing key scientific basis for investigation direction analysis and personnel screening.

[0007] Chinese patent CN119592702A discloses the application of CpG site combination methylation levels in inferring an individual's physiological age, wherein the CpG site combination is selected from at least two of cg25410668, cg16867657, cg22454769, cg08097417, cg21572722, cg17268658, cg23606718, cg03607117, cg24724428, cg13552692, cg04875128, cg14692377, cg00481951, cg07547549, cg06639320, and cg07553761. The aforementioned CpG site combinations can be used to infer an individual's physiological age, providing a fast, convenient, and accurate method for inferring physiological age in forensic applications. However, the amount of DNA template used for this test is relatively large, requiring 200ng.

[0008] Based on this, there is an urgent need to provide a method that can accurately infer age using a small amount of DNA sample. Summary of the Invention

[0009] The purpose of the present invention is to provide a forensic multi-body fluid age prediction method and application based on droplet digital PCR.

[0010] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0011] In one aspect, the present invention provides an age prediction method based on droplet digital PCR, comprising the following steps:

[0012] S1. Extract DNA from the individual to be tested;

[0013] S2. Detect the methylation levels of cg10501210, cg10528482, and cg05940966 using droplet digital PCR;

[0014] S3. The age of the tested individual was obtained by inputting the methylation level into the multivariate linear regression model.

[0015] Specifically, the test sample of the individual in step S1 is blood, saliva and / or semen.

[0016] Furthermore, when the test sample in step S1 is blood or saliva, the incubation condition is 70° C. for 10 minutes.

[0017] Specifically, when the test sample is blood or saliva, DNA extraction includes the following steps:

[0018] (1) Add protease solution to the blood or saliva sample, add BL buffer, mix well, and incubate at 70°C for 10 min;

[0019] (2) Add anhydrous ethanol and mix well, add to a silica glass fiber column, centrifuge, discard the filtrate, add HBC buffer, centrifuge, discard the filtrate, add DNA cleaning solution to wash, and centrifuge the empty column;

[0020] (3) Add elution buffer, centrifuge to elute the DNA, add the eluate to the silica glass fiber column, and elute again.

[0021] Furthermore, when the test sample in step S1 is semen, the incubation condition is 60° C. for 45 minutes.

[0022] Specifically, when the test sample is semen, DNA extraction includes the following steps:

[0023] (1) TL buffer, protease solution, and DTT were added to the semen sample, mixed, and incubated at 60°C for 45 min;

[0024] (2) Add BL buffer, mix well, and incubate at 70°C for 10 min;

[0025] (3) Add anhydrous ethanol and mix well, add to a silica glass fiber column, centrifuge, discard the filtrate, add HBC buffer, centrifuge, discard the filtrate, add DNA cleaning solution to wash, and centrifuge the empty column;

[0026] (4) Add elution buffer, centrifuge to elute the DNA, add the eluate to the silica glass fiber column, and elute again.

[0027] Furthermore, the amount of DTT added in step (1) is 1 / 10 of the volume of the semen sample.

[0028] According to some embodiments of the present invention, DNA is extracted using the EZNA Forensic DNA Kit.

[0029] According to some embodiments of the present invention, DNA extraction can also be performed using the QIAamp DNA Investigator kit method.

[0030] Specifically, step S2 includes the following steps:

[0031] S21, performing bisulfite conversion on the extracted DNA;

[0032] S22, PCR amplification;

[0033] S23. Analyze the PCR products using a microdroplet analyzer to obtain the methylation levels of cg10501210, cg10528482, and cg05940966.

[0034] According to some embodiments of the present invention, step S21 is to mix the DNA sample with the conversion reagent and then incubate at 95° C. for 5 minutes and at 60° C. for 20 minutes.

[0035] According to some embodiments of the present invention, step S21 is to perform bisulfite conversion on the extracted DNA using the EpiTect fast DNA bisulfite kit.

[0036] Specifically, the sample amount of the DNA is 0.5ng-100ng.

[0037] Further, the bisulfite conversion comprises the following steps:

[0038] (1) Mix the bisulfite conversion reaction system and place it in a PCR reaction instrument for conversion;

[0039] (2) Add the carrier RNA to BL buffer, mix well, add anhydrous ethanol, mix well, add to the MinElute DNA column, centrifuge, and discard the waste liquid;

[0040] (3) Add BW buffer to the MinElute DNA column, centrifuge and discard the waste liquid; add BD buffer to the MinElute DNA column, centrifuge and discard the waste liquid;

[0041] (4) Add BW buffer to the MinElute DNA column, centrifuge and discard the waste liquid. Repeat once, add anhydrous ethanol to the column, centrifuge and discard the waste liquid.

[0042] (5) Incubate the MinElute DNA column in a 60°C constant temperature bath for 5 minutes, add EB buffer to the MinElute DNA column, centrifuge, and discard the MinElute DNA column.

[0043] Specifically, the bisulfite conversion reaction system in step (1) includes: DNA, bisulfite and DNA protection buffer.

[0044] Furthermore, the volume ratio of DNA to bisulfite is 1:2-3;

[0045] Furthermore, the volume ratio of DNA to bisulfite is 1:2.125.

[0046] Specifically, the conversion reaction conditions described in step (1) are: denaturation at 95°C for 5 minutes, incubation at 60°C for 20 minutes, denaturation at 95°C for 5 minutes, and incubation at 60°C for 20 minutes.

[0047] Specifically, the PCR reaction system of step S22 includes ddPCR™ Supermix for Probes, a primer-probe combination for detecting cg10501210, cg10528482, and cg05940966, a primer-probe combination for an internal reference gene, converted DNA, and water.

[0048] According to some embodiments of the present invention, the PCR reaction system (20 μL) of step S22 includes 10 μL of ddPCR™ Supermix for Probes, 1 μL of a primer-probe combination for detecting cg10501210, cg10528482, and cg05940966, 1 μL of a primer-probe combination for an internal reference gene, 2 μL of converted DNA, and 6 μL of water.

[0049] Specifically, the cg05940966 primer concentration is 600-1000 nM, and the probe concentration is 600-800 nM;

[0050] Furthermore, the concentration of the cg05940966 primer is 1000 nM, and the concentration of the probe is 800 nM.

[0051] Specifically, the cg10501210 primer concentration is 600-1000 nM, and the probe concentration is 600-900 nM;

[0052] Furthermore, the concentration of the cg10501210 primer is 800 nM, and the concentration of the probe is 900 nM.

[0053] Specifically, the concentration of the cg10528482 primer is 600-1000 nM, and the probe concentration is 600-900 nM;

[0054] Furthermore, the concentration of the cg10528482 primer is 800 nM, and the concentration of the probe is 900 nM.

[0055] Specifically, the primer concentration of the internal reference gene is 600-1200 nM, and the probe concentration is 600-1500 nM;

[0056] Furthermore, the primer concentration of the internal reference gene is 1200 nM, and the probe concentration is 1350 nM.

[0057] Furthermore, the primer pair sequences for detecting cg10501210 are shown in SEQ ID NOs: 1-2, and the probe sequence is shown in SEQ ID NO: 3;

[0058] The primer pair sequences for detecting cg10528482 are shown in SEQ ID NOs: 4-5, and the probe sequence is shown in SEQ ID NO: 6;

[0059] The primer pair sequences for detecting cg05940966 are shown in SEQ ID NOs: 7-8, and the probe sequence is shown in SEQ ID NO: 9.

[0060] Furthermore, the internal reference gene is C-LESS-C1, the sequences of the primer pairs for detecting the internal reference gene C-LESS-C1 are shown in SEQ ID NOs: 10-11, and the probe sequence for detecting the internal reference gene C-LESS-C1 is shown in SEQ ID NO: 12.

[0061] Specifically, the reaction conditions for PCR amplification in step S22 are: pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, extension-annealing at 56°C for 60 seconds, 40 cycles, and finally storage at 4°C.

[0062] Specifically, the multiple linear regression model described in step S3 is predicted age = -71.3717*DNAm1-144.8173*DNAm2-185.0615*DNAm3+182.8617;

[0063] Where, DNAm1 is the methylation value of site cg05940966;

[0064] DNAm2 is the methylation value of site cg10501210;

[0065] DNAm3 is the methylation value of site cg10528482.

[0066] In another aspect, the present invention provides use of a biomarker or a substance for detecting the methylation level of a biomarker in age prediction, wherein the biomarker consists of cg10501210, cg10528482, and cg05940966.

[0067] Specifically, the substance is a primer-probe combination for detecting the biomarker.

[0068] Furthermore, the primer-probe composition includes the following primer pairs:

[0069] (1) A primer pair for detecting cg10501210, the sequences of the primer pair are shown in SEQ ID NOs: 1-2;

[0070] (2) a primer pair for detecting cg10528482, the sequences of the primer pair are shown in SEQ ID NOs: 4-5;

[0071] (3) A primer pair for detecting cg05940966, the sequences of which are shown in SEQ ID NOs: 7-8.

[0072] Furthermore, the primer-probe composition includes at least one of the following probes: the probe shown in SEQ ID NO: 3, the probe shown in SEQ ID NO: 6, and the probe shown in SEQ ID NO: 9.

[0073] Furthermore, the primer-probe combination also includes a primer pair and a probe for detecting the internal reference gene C-LESS-C1.

[0074] Furthermore, the sequences of the primer pair for detecting the internal reference gene C-LESS-C1 are shown in SEQ ID NOs: 10-11, and the sequence of the probe for detecting the internal reference gene C-LESS-C1 is shown in SEQ ID NO: 12.

[0075] In another aspect, the present invention provides a kit for age prediction, comprising a primer-probe combination for detecting the methylation levels of cg10501210, cg10528482, and cg05940966.

[0076] The primer-probe combination includes the following primer pairs:

[0077] (1) A primer pair for detecting cg10501210, the sequences of the primer pair are shown in SEQ ID NOs: 1-2;

[0078] (2) a primer pair for detecting cg10528482, the sequences of the primer pair are shown in SEQ ID NOs: 4-5;

[0079] (3) A primer pair for detecting cg05940966, the sequences of which are shown in SEQ ID NOs: 7-8.

[0080] Furthermore, the primer-probe composition includes at least one of the following probes: the probe shown in SEQ ID NO: 3, the probe shown in SEQ ID NO: 6, and the probe shown in SEQ ID NO: 9.

[0081] Furthermore, the primer-probe combination also includes a primer pair and a probe for detecting the internal reference gene C-LESS-C1.

[0082] Furthermore, the sequences of the primer pair for detecting the internal reference gene C-LESS-C1 are shown in SEQ ID NOs: 10-11, and the sequence of the probe for detecting the internal reference gene C-LESS-C1 is shown in SEQ ID NO: 12.

[0083] The beneficial effects of the present invention are:

[0084] (1) Only 0.5ng template DNA is required, which can be used for forensic micro-degradation samples

[0085] In the present invention, accurate age inference can be performed using 0.5 ng of template DNA, which has high sensitivity and good application prospects in forensic medicine.

[0086] (2) Short time, the whole process can be completed in 8 hours

[0087] The method of the present invention can complete DNA extraction and quantification (2.5 hours), bisulfite conversion (2.5 hours), PCR (2 hours), and droplet digital PCR detection (1 hour) within 8 hours, which can effectively save time compared to other methods.

[0088] (3) Using three CpG sites to achieve accurate age prediction in multiple forensic body fluids

[0089] The present invention can still achieve good prediction accuracy using three CPG sites, and the age prediction model can be applied to a variety of body fluids, and has high application value in forensic medicine.

[0090] (4) High prediction accuracy can be achieved using only a small amount of DNA

[0091] MLR can also demonstrate stable predictive accuracy in trace samples, making it a highly promising tool in forensic evidence analysis, especially in scenarios where traditional methods are limited. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 The correlation between DNA methylation value of site cg059540966 and age.

[0093] Figure 2 The correlation between the DNA methylation value of site cg10501210 and age.

[0094] Figure 3 The correlation between the DNA methylation value of site cg10528482 and age.

[0095] Figure 4 The age prediction model (MLR) was constructed based on 117 peripheral blood samples, with a MAD of 3.77 years for the test set and a MAD of 3.88 years for the test set.

[0096] Figure 5 An age prediction model (RF) was constructed based on 117 peripheral blood samples, with a MAD of 4.2 years for the training set and 4.29 years for the test set.

[0097] Figure 6 The age prediction model (SVR) was constructed based on 117 peripheral blood samples, with a MAD of 3.28 years for the training set and a MAD of 4.14 years for the test set.

[0098] Figure 7 These are the methylation values ​​measured for site cg059540966 when the DNA input amounts were 100 ng, 10 ng, 5 ng, 1 ng, and 0.5 ng.

[0099] Figure 8 These are the methylation values ​​measured for site cg10501210 when the DNA input amounts were 100 ng, 10 ng, 5 ng, 1 ng, and 0.5 ng.

[0100] Figure 9 These are the methylation values ​​measured for site cg10528482 when the DNA input amounts were 100 ng, 10 ng, 5 ng, 1 ng, and 0.5 ng.

[0101] Figure 10 Comparison of DNA extraction from three body fluids before and after optimization.

[0102] Figure 11 This shows the separation of positive and negative droplets before and after optimization of site cg05940966.

[0103] Figure 12 This shows the separation of positive and negative droplets before and after optimization of site cg10501210.

[0104] Figure 13 This shows the separation of positive and negative droplets before and after optimization of site cg10528482.

[0105] Figure 14 The separation of positive and negative droplets before and after optimization of the reference gene C-LESS-C1. DETAILED DESCRIPTION

[0106] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0107] In the following specific implementation manners, all comparative experiments not specifically described are single-factor variable experiments.

[0108] Example 1 Experimental method

[0109] 1. DNA extraction:

[0110] High-quality DNA templates are crucial for methylation analysis. DNA extraction from samples can be completed within 2.5 hours using the EZNA Forensic DNA Kit (Omega, USA, Cat. No. D3096).

[0111] When extracting DNA from blood and saliva, the previous method was to incubate at 60°C for 15 minutes, while the improved method was to incubate at 70°C for 10 minutes to increase the amount of DNA extracted. When extracting DNA from semen samples, the previous method was to incubate at 55°C for 60 minutes, while the improved method was to incubate at 60°C for 45 minutes and add 20 μL of DL-dithiothreitol (DTT) to the original reagent to increase the amount of DNA extracted. Figure 10 ).

[0112] The specific steps for DNA extraction from blood and saliva samples are:

[0113] Precautions before operation:

[0114] (1) Centrifugation should be performed at room temperature (15-25°C).

[0115] (2) Equilibrate the sample to room temperature (15-25°C).

[0116] (3) Set the thermomixer to 70°C for step 4.

[0117] (4) Dilute DNA Wash Buffer with anhydrous ethanol and HBC Buffer with isopropanol.

[0118] (5) Before starting the experiment, preheat the Elution Buffer to 70°C.

[0119] Steps:

[0120] (1) Pipette 200 μL of blood or saliva sample into a 1.5 mL centrifuge tube.

[0121] (2) Add 25 μL OB Protease Solution.

[0122] (3) Add 200 μL BL Buffer, vortex mix for 30 seconds, and centrifuge briefly.

[0123] (4) Place the 1.5 mL centrifuge tube in a preheated thermomixer and incubate at 70°C for 10 minutes.

[0124] (5) Briefly centrifuge the 1.5 mL centrifuge tube to collect any droplets on the inner tube cap into the tube.

[0125] (6) Add 200 μL of anhydrous ethanol, vortex mix for 15 seconds, and briefly centrifuge the 1.5 mL centrifuge tube.

[0126] (7) Place the HiBind DNA Mini column into a 2 mL collection tube.

[0127] (8) Transfer all the liquid from step 6 to a HiBind DNA Mini column, centrifuge at 12,000 × g for 1 minute, and discard the filtrate.

[0128] (9) Add 500 μL of HBC Buffer to the HiBind DNA Mini column, centrifuge at 12,000 × g for 1 minute, and discard the filtrate.

[0129] (10) Add 700 μL DNA Wash Buffer to the HiBind DNA Mini column, centrifuge at 12,000 × g for 1 minute, and discard the filtrate.

[0130] (11) Repeat step 10.

[0131] (12) Return the HiBind DNA Mini column to the centrifuge and centrifuge the empty column at 12,000 × g for 2 minutes to remove residual ethanol, which may affect downstream experiments.

[0132] (13) Place the HiBind DNA Mini column in a new 1.5 mL centrifuge tube, add 40 μL of preheated Elution Buffer, let it stand at room temperature for 5 minutes, and centrifuge at 12,000 × g for 3 minutes to elute the DNA.

[0133] (14) Add the elution solution from step 13 back to the HiBind DNA Mini column and elute again.

[0134] (15) Store the DNA in a -20°C freezer.

[0135] The only difference between the comparative experiment and the DNA extraction process in the embodiment is the incubation conditions in step (4). The incubation conditions in the comparative experiment are 60° C. for 15 minutes, and the rest are the same.

[0136] Semen DNA extraction:

[0137] Precautions before operation:

[0138] (1) Centrifugation should be performed at room temperature (15-25°C).

[0139] (2) Equilibrate the sample to room temperature (15-25°C).

[0140] (3) Set the thermostat mixer to 60°C.

[0141] (4) Dilute DNA Wash Buffer with anhydrous ethanol and HBC Buffer with isopropanol.

[0142] (5) Before starting the experiment, preheat the Elution Buffer to 70°C.

[0143] (6) Place the semen sample at room temperature for 20-30 minutes to liquefy it.

[0144] Steps:

[0145] (1) Pipette 200 μL of semen sample into a 1.5 mL centrifuge tube.

[0146] (2) Add 200 μL TL Buffer, 20 μL OB, and 20 μL DTT to the centrifuge tube and vortex to mix for 30 seconds.

[0147] (3) Place the 1.5 mL centrifuge tube in a thermomixer and incubate at 60°C with shaking for 45 minutes.

[0148] (4) Briefly centrifuge the 1.5 mL centrifuge tube to collect any droplets on the inner tube cap into the tube.

[0149] (5) Add 300 μL BL Buffer and vortex to mix for 20 seconds.

[0150] (6) Place the 1.5 mL centrifuge tube in a thermomixer and incubate at 70°C for 10 minutes. During the incubation period, take out the centrifuge tube and vortex it at the highest speed for 10 seconds several times.

[0151] (7) Briefly centrifuge the 1.5 mL centrifuge tube.

[0152] (8) Add 320 μL of anhydrous ethanol, vortex for 15 seconds, and briefly centrifuge.

[0153] (9) Place the HiBind DNA Mini column into a 2 mL collection tube.

[0154] (10) Transfer all the liquid from step 8 to a HiBind DNA Mini column, centrifuge at 12,000 × g for 1 minute, discard the filtrate, and replace with a new collection tube.

[0155] (11) Add 500 μL of HBC Buffer to the HiBind DNA Mini column, centrifuge at 12,000 × g for 30 seconds, and discard the filtrate.

[0156] (12) Add 700 μL DNA Wash Buffer to the HiBind DNA Mini column, centrifuge at 12,000 × g for 1 minute, and discard the filtrate.

[0157] (13) Repeat step 12.

[0158] (14) Return the HiBind DNA Mini column to the centrifuge and centrifuge the empty column at 12,000 × g for 2 minutes to remove any residual liquid that may affect downstream experiments.

[0159] (15) Place the HiBind DNA Mini column in a new 1.5 mL centrifuge tube, add 40 μL of preheated Elution Buffer, incubate at room temperature for 5 minutes, and centrifuge the empty column at 12,000 × g for 3 minutes.

[0160] (16) Add the eluate from step 15 back to the HiBind DNA Mini column and elute again. Store the DNA in a -20°C freezer.

[0161] The only difference between the comparative experiment and the DNA extraction process in the embodiment is that DTT is not added in step (2) and the incubation conditions in step (3) are different. The incubation conditions in the comparative experiment are incubated at 55°C for 60 minutes, and the rest are the same.

[0162] 2. Site selection:

[0163] Three methylation sites, cg10501210, cg10528482, and cg05940966, located on chr1 and chr5, respectively, were selected. These sites all showed good correlation with age and similar methylation levels.

[0164] 3. Primer and probe design:

[0165] Use Primer Primer5 to design PCR primers and probes. When designing primers, try to make the target sequence contain only cytosine (C) at the target site to improve the accuracy of detection. Avoid SNPs and other polymorphisms in the target region, as they may cause deviations in the sequencing reaction. In addition, keep the GC content below 60%, and select primers with high specificity (i.e., no primer dimers are formed). No modifications are made to the PCR primers. When designing probes, different modifications are made at the 5' end and 3' end respectively. C-LESS-C1 was selected as the reference gene, and the final primers and probes are shown in the following table:

[0166] Table 1

[0167]

[0168]

[0169] 4. Droplet Digital PCR Detection of DNA Methylation

[0170] 4.1 Bisulfite conversion

[0171] The extracted DNA (100 ng) was subjected to bisulfite conversion using the EpiTect fast DNA bisulfite kit (purchased from QIAGEN, Germany, catalog number 95824).

[0172] The specific steps for bisulfite conversion using the EpiTect fast DNA bisulfite kit are as follows: Pre-experimental preparation:

[0173] (1) All operations were performed at room temperature (15-25°C).

[0174] (2) Place the reagent at room temperature (15-25°C) for ten minutes to equilibrate the reagent to room temperature.

[0175] (3) Add 30 mL of anhydrous ethanol to Buffer BW and store at room temperature.

[0176] (4) Add 27 mL of anhydrous ethanol to Buffer BD and store at 4°C.

[0177] (5) Add 310 μL of RNase-free water to the carrier RNA and store at -20°C.

[0178] (6) Preheat the Bisulfite Solution to 56°C.

[0179] (7) Preheat Buffer EB to 75℃.

[0180] Steps:

[0181] (1) Dissolve the DNA sample and convert 100 ng of DNA into bisulfite.

[0182] (2) The system is prepared as follows:

[0183] Table 2 Bisulfite conversion reaction system

[0184]

[0185]

[0186] (3) Add the following to a 200 μL EP tube in the order listed, mix thoroughly, and centrifuge briefly. The solution will turn from green to blue, indicating that it is fully mixed and has the correct pH for the bisulfite conversion reaction.

[0187] (4) Place the EP tube in a PCR reaction instrument and set the conversion reaction conditions as follows: denaturation at 95°C for 5 minutes, incubation at 60°C for 20 minutes, denaturation at 95°C for 5 minutes, and incubation at 60°C for 20 minutes.

[0188] (5) After the reaction is complete, briefly centrifuge the EP tube and transfer the solution to a new 1.5 mL centrifuge tube. (6) Add 3.1 μL of carrier RNA to 310 μL of buffer BL and mix thoroughly.

[0189] (7) Take 310 μL of the mixed solution and add it to the above 1.5 mL centrifuge tube.

[0190] (8) Add 250 μL of anhydrous ethanol, mix manually, and centrifuge briefly.

[0191] (9) Transfer the liquid in the 1.5 mL centrifuge tube to the MinElute DNA column, centrifuge at 12,000 × g for 1 minute, and discard the waste liquid. Return the MinElute DNA column to the collection tube.

[0192] (10) Add 450 μL of Buffer BW to the MinElute DNA column, centrifuge at 12,000 × g for 1 minute, and discard the waste liquid. Return the MinElute DNA column to the collection tube.

[0193] (11) Add 500 μL of Buffer BD to the MinElute DNA column and centrifuge at 12,000 × g for 1 minute. Discard the waste solution. Return the MinElute DNA column to the collection tube. To prevent oxidation of Buffer BD, remove it from the refrigerator before use and return it to the refrigerator immediately after use.

[0194] (12) Add 450 μL of Buffer BW to the MinElute DNA column, centrifuge at 12,000 × g for 1 minute, and discard the waste liquid. Return the MinElute DNA column to the collection tube.

[0195] (13) Repeat step 12.

[0196] (14) Add 250 μL of anhydrous ethanol to the column, centrifuge at 12,000 × g for 1 minute, and discard the waste liquid. Return the MinElute DNA column to the collection tube.

[0197] (15) Centrifuge the empty tube at 12000× g for 3 minutes, discard the waste liquid and the collection tube, place the MinElute DNA column in a new 1.5 mL centrifuge tube, and incubate in a 60°C constant temperature bath for 5 minutes to evaporate the residual liquid.

[0198] (16) Add 20 μL of Buffer EB to the MinElute DNA column.

[0199] (17) Incubate at room temperature for 5 minutes and centrifuge at 12,000 × g for 3 minutes. Discard the MinElute DNA column. The transformed DNA in the centrifuge tube can now be used directly in downstream experiments without further purification and stored at -20°C.

[0200] 4.2 PCR amplification

[0201] The reaction system was prepared according to the instructions (purchased from BIO-RAD Co., Ltd., USA, catalog number 1863024). The reaction system was 20 μL, including 10 μL of ddPCR. TMSupermix for Probes (No dUTP), 1 μL of a mixture of primers and probes for CpG sites (cg05940966 primer concentration is 1000 nM, probe concentration is 800 nM; cg10501210 primer concentration is 800 nM, probe concentration is 900 nM; cg10528482 primer concentration is 800 nM, probe concentration is 900 nM), 1 μL of a mixture of primers and probes for C-LESS-C1 (primer concentration is 1200 nM; probe concentration is 1350 nM), 2 μL of converted DNA, and 6 μL of dH2O.

[0202] The configured system was placed into a droplet generation card and then into a calibrated droplet generator to generate droplets. The droplets were transferred to a 96-well plate, sealed, and PCR amplification was performed. Thermal cycling conditions were as follows: pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, extension-annealing at 56°C for 60 seconds, 40 cycles, and storage at 4°C.

[0203] 4.3 Droplet analysis

[0204] PCR products were analyzed using a microdroplet analyzer (QX200, purchased from BIO-RAD, Inc., USA). The instrument was cleaned before analysis. After analysis, the 96-well plate was placed in the instrument and the program was programmed and run according to the instructions.

[0205] The separation of positive and negative droplets before and after optimization of sites cg05940966, cg10501210, cg10528482 and reference gene C-LESS-C1 is shown in Figure 2. Figure 11-14 As shown (reference gene C-LESS-C1; optimized primer concentration is 400nM; probe concentration is 450nM; site cg05940966 optimized primer concentration is 400nM; probe concentration is 400nM; site cg10501210 optimized primer concentration is 400nM; probe concentration is 450nM; site cg10528482 optimized primer concentration is 400nM; probe concentration is 450nM).

[0206] Example 2 Construction of blood age prediction model

[0207] 1 Comparison of Multiple Linear Regression (MLR), Random Forest Regression (RF) and Support Vector Regression (SVR)

[0208] In this study, methylation values ​​were measured using the experimental method described in Example 1 from 117 peripheral blood samples collected from healthy Chinese volunteers (age range 18-59 years; all donors provided informed consent. Ethical approval for this study was obtained from the Beijing Institute of Genomics, Chinese Academy of Sciences). Methylation values ​​were then processed using R Studio software to construct MLR, RF, and SVR models. 80% of the samples were randomly selected as a training dataset, and 20% as a test dataset to assess model accuracy.

[0209] The constructed multiple linear regression model is:

[0210] Predicted age = -71.3717*DNAm1 -144.8173*DNAm2 -185.0615*DNAm3 + 182.8617;

[0211] Where, DNAm1 is the methylation value of site cg05940966;

[0212] DNAm2 is the methylation value of site cg10501210;

[0213] DNAm3 is the methylation value of site cg10528482.

[0214] The constructed random forest regression model is:

[0215] PredictedAge = (1 / 1000)∑_{t=1}^{1000}Tree_t(DNAm1,DNAm2,DNAm3);

[0216] Wherein, DNAm1 is the methylation value of site cg05940966; DNAm2 is the methylation value of site cg10501210; DNAm3 is the methylation value of site cg10528482.

[0217] The constructed support vector regression model is:

[0218] PredictedAge = 1.0000*exp(-0.3333*||x-[0.2900,0.4500,0.2700]||^2);

[0219] Wherein, x represents the methylation value of site cg05940966, site cg10501210, and site cg10528482, respectively.

[0220] The MAD value of MLR constructed using the training set was 3.77 years, the MAD value of RF was 4.2 years, and the MAD value of SVR was 3.28 years ( Figure 4-Figure 6 ).

[0221] 2. Test data set to verify prediction accuracy

[0222] Using the remaining 20% ​​peripheral blood samples as the test data set, the MAD value was 3.88 years in MLR, 4.29 years in RF, and 4.14 years in SVR. After comparison, the age prediction accuracy of MLR was higher than the other two ( Figure 4-Figure 6 ).

[0223] 3Correlation analysis between DNA methylation level and age

[0224] 117 peripheral blood samples from healthy Chinese Han volunteers aged 18-59 were collected for DNA extraction and conversion, and finally methylation analysis was performed to analyze the correlation between the sites in the present invention and age. Among these three sites, the cg10521210 site has a strong correlation with age, and its R 2 The R value was 0.686, and the R 2 The values ​​are 0.629 and 0.385 ( Figure 1-Figure 3 ).

[0225] 4 Other body fluid verification

[0226] DNA was extracted and converted from 16 semen and 16 saliva samples using the same protocol as in Example 1, followed by droplet PCR analysis. The measured methylation values ​​were incorporated into the prediction model. The calculated MAD for semen was 5.92 years, and for saliva it was 4.52 years.

[0227] 5 Sensitivity Verification

[0228] This study selected one sample from each age group and performed sensitivity tests on 100ng, 10ng, 5ng, 1ng, 0.5ng and 0.1ng blood DNA samples. The experimental results showed that ( Figure 7-Figure 9 ). When the DNA input was 0.5 ng, the ddPCR test results were not significantly different from those using a starting amount of 100 ng. Substituting the methylation values ​​obtained at each site into a multivariate linear regression model, the MAD was 2.29 years when the DNA input was 100 ng, and 3.29 years when the DNA input was 0.5 ng. Therefore, this model maintains high predictive accuracy even with trace samples.

[0229] The model constructed by the present invention can be used in blood, saliva and semen, and has good prediction accuracy.

[0230] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An age prediction method based on droplet digital PCR, characterized in that: The following steps are involved: S1. Extract DNA from the individual to be tested; S2. Detect the methylation levels of cg10501210, cg10528482, and cg05940966 using droplet digital PCR; S3. The age of the tested individual was obtained by inputting the methylation level into the multivariate linear regression model.

2. The age prediction method according to claim 1, characterized in that: In step S1 , the test sample of the individual is blood, saliva and / or semen.

3. The age prediction method according to claim 2, characterized in that: When the detection sample in step S1 is blood or saliva, the incubation condition is 70° C. for 10 minutes; when the detection sample in step S1 is semen, the incubation condition is 60° C. for 45 minutes.

4. The age prediction method according to claim 1, characterized in that: Step S2 includes the following steps: S21, performing bisulfite conversion on the extracted DNA; S22, PCR amplification; S23. Analyze the PCR products using a microdroplet analyzer to obtain the methylation levels of cg10501210, cg10528482, and cg05940966.

5. The age prediction method according to claim 4, characterized in that: The sample amount of DNA in step S21 is 0.5 ng-100 ng.

6. The age prediction method according to claim 1, characterized in that: The multiple linear regression model described in step S3 is predicted age = -71.3717*DNAm1-144.8173*DNAm2-185.0615*DNAm3+182.8617; Where, DNAm1 is the methylation value of site cg05940966; DNAm2 is the methylation value of site cg10501210; DNAm3 is the methylation value of site cg10528482.

7. Use of a biomarker or a substance for detecting the methylation level of a biomarker in age prediction, characterized in that: The biomarkers consist of cg10501210, cg10528482 and cg05940966.

8. The use according to claim 7, characterized in that The substance is a primer-probe combination for detecting the biomarker; The primer-probe combination includes the following primer pairs: (1) A primer pair for detecting cg10501210, the sequences of the primer pair are shown in SEQ ID NOs: 1-2; (2) a primer pair for detecting cg10528482, the sequences of the primer pair are shown in SEQ ID NOs: 4-5; (3) a primer pair for detecting cg05940966, the sequences of the primer pair are shown in SEQ ID NOs: 7-8; The primer-probe composition includes at least one of the following probes: the probe shown in SEQ ID NO: 3, the probe shown in SEQ ID NO: 6, and the probe shown in SEQ ID NO:

9.

9. The use according to claim 7, characterized in that The primer-probe combination also includes a primer pair and a probe for detecting the internal reference gene C-LESS-C1; The sequences of the primer pair for detecting the internal reference gene C-LESS-C1 are shown in SEQ ID NOs: 10-11, and the sequence of the probe for detecting the internal reference gene C-LESS-C1 is shown in SEQ ID NO:

12.

10. A kit for age prediction, characterized in that: The kit includes a primer-probe combination for detecting the methylation levels of cg10501210, cg10528482 and cg05940966.

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