CN111139292A - Biological age inference method established based on pyrosequencing - Google Patents
Biological age inference method established based on pyrosequencing Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 31
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- 238000012163 sequencing technique Methods 0.000 claims abstract description 18
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- 238000007069 methylation reaction Methods 0.000 claims abstract description 8
- 238000012408 PCR amplification Methods 0.000 claims abstract description 7
- 210000004369 blood Anatomy 0.000 claims abstract description 6
- 239000008280 blood Substances 0.000 claims abstract description 6
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- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 238000005119 centrifugation Methods 0.000 claims description 15
- 239000000872 buffer Substances 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- 239000000706 filtrate Substances 0.000 claims description 9
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- 239000012148 binding buffer Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
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- 230000003321 amplification Effects 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
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- 238000000464 low-speed centrifugation Methods 0.000 description 2
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Abstract
The invention discloses a biological age inference method established based on pyrosequencing, and particularly relates to the field of biological age inference, wherein the method comprises the following specific operation steps: and S1, preparation of an experimental sample: a tissue sample or a blood sample; s2, extracting nucleic acid from the experimental sample by a centrifugal column method; s3, performing sulfite treatment on the extracted DNA; s4, carrying out PCR amplification and sequencing on the DNA treated by the sulfite; s5, establishing a biological age prediction model of a multiple regression equation by using the methylation sites of the 4 genes. According to the invention, 4 pairs of ELOVL2, Clorf132, TRIM59 and FHL2 amplification primer pairs, sequencing primers for pyrosequencing and analysis sequences are designed, 4 pairs of primers are used for sequencing to obtain an age inference model formula of the methylation degree of 4 genes, and the model formula is used for predicting the biological age of a corresponding sample.
Description
Technical Field
The embodiment of the invention relates to the field of biological age inference, in particular to a biological age inference method established based on pyrosequencing.
Background
The individual age inference is always the key and difficult point of forensic research, and at present, in the field of forensic science, a model is established mainly according to morphological indication changes of bones or teeth generated along with the age so as to infer the biological age range of a person of origin. Practice proves that the technology for estimating the age of the forensic anthropological individual is mature, and the error is relatively small. However, when a sample such as a bone or a tooth required for detection is lost, the age of the person cannot be predicted.
Based on the defect, the method uses the biogenic biological tissues to establish the model of age inference to infer the biological age, the accuracy rate of the method for predicting the age within the deviation 5 years reaches 83%, the method can be applied to the inference of the age range of the biogenic persons without morphological indications, and an important research method is provided for the inference of the biological age.
Disclosure of Invention
Therefore, the embodiment of the invention provides a biological age inference method established based on pyrosequencing, which is characterized in that 4 pairs of ELOVL2, C1orf132, TRIM59 and FHL2 amplification primer pairs, sequencing primers for pyrosequencing and an analysis sequence are designed, 4 pairs of primers are utilized for sequencing to obtain an age inference model formula of methylation degrees of 4 pairs of genes, and the model formula is used for predicting biological ages, so that the problem that age prediction of a person of origin cannot be carried out due to material detection loss in the prior art is solved.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions: a biological age inference method established based on pyrosequencing comprises the following specific operation steps:
s1, preparation of experimental samples: samples used in the experiments include, but are not limited to: a tissue sample or a blood sample;
s2, extracting nucleic acid from the experimental sample by a centrifugal column method by using a Qiagen cell extraction kit;
s3, performing sulfite treatment on the extracted DNA by adopting an EZ DNA methylation-Gold kit D5005 kit;
s4, carrying out PCR amplification and sequencing on the DNA treated by the sulfite, and comprising the following steps:
s4.1, screening out specific primer pairs and sequencing primers of 4 genes such as ELOVL2, C1orf132, TRIM59, FHL2 and the like;
s4.2, screening out an enzyme Pyromark PCR Kit specially used for PCR amplification of the DNA treated by the sulfite, and amplifying the DNA treated by the sulfite by PCR under the following conditions: firstly, amplifying for 15min at the temperature of 95 ℃; then amplifying at 94 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 20s for 45 cycles; finally amplifying for 10min at the temperature of 72 ℃;
s4.3, sequencing the PCR products of the 4 pairs of genes;
s5, establishing a biological prediction age model equation of a multiple regression equation by using the methylation sites of the 4 genes as follows:
age prediction value of 29.766+0.178 XFHL 2-F1-0.524 XCLorf 132-C1+0.198 XEolov 2-E4+0.276 XTrim 59-T4-0.119 XFHL 2-F9+0.286 XFHL 2-F2, R20.932, wherein R2Representing the degree of confidence in the equation formula.
Further, in step S2, the nucleic acid extraction step is as follows:
s2.1, sucking 20 mu l of proteinase K to the bottom of a 1.5ml centrifuge tube, adding 200 mu l of a sample of a genome to be extracted to the centrifuge tube, adding 200 mu l of Buffer AL to the sample, carrying out vortex oscillation for 15S, uniformly mixing (completely and uniformly mixing to obtain a uniform solution is very important for ensuring the sufficient cracking of the sample), and incubating at 56 ℃ for 10min (the DNA yield reaches the maximum value under the condition, and the yield cannot be further improved by prolonging the incubation time);
s2.2, quickly centrifuging to remove liquid drops remained in the cover of the 1.5ml centrifuge tube;
s2.3, transferring the mixture obtained in the step to a Mini centrifuge column (in a 2ml collection tube), paying attention to not wet the ring of the edge, fastening a cover (closing the cover of each centrifuge tube during centrifugation to prevent aerosol generation during centrifugation), centrifuging at 6000g (8000rpm) for 1Min, selecting low-speed centrifugation for reducing noise, using higher rotation speed without affecting the final yield and purity of DNA, and when the sample is erythrocyte sedimentation or lymphocyte, preferably using the highest rotation speed to prevent blockage, placing the QIAamp Min centrifuge column into a new clean 2ml receiving tube, and discarding the filtrate together with the used collection tube;
s2.4, carefully open the Mini spin column, add 500. mu.l Buffer AW1 (taking care not to wet the edge ring), close the lid, centrifuge at 6000g (8000rpm) for 1min, transfer the spin column to a new 2ml collection tube, discard the filtrate along with the used collection tube, this step does not require an increase in Buffer AW1 even if the initial sample is added in an amount greater than 200. mu.l;
s2.5, discarding the filtrate, carefully opening the QIAamp Mini spin column, adding 500. mu.l Buffer AW2 (taking care not to wet the edge ring), closing the lid, and centrifuging at 20000g (14000rpm) for 3 min;
s2.6, transferring the centrifugal column to a new 1.5ml or 2ml collecting tube, carefully opening the centrifugal column, adding 200. mu.l of buffer AE or double distilled water (the elution volume is not suitable for the 1.5ml collecting tube when more than 200. mu.l, because the lower edge of the centrifugal column is contacted with the eluent, thereby possibly generating aerosol in the centrifugal process; the elution volume is less than 200. mu.l, the concentration of DNA in the eluent is obviously increased, but the total DNA yield is reduced; for DNA less than 1. mu.g, 50. mu.l of eluent is recommended to be used; the washing effect of 2 times by using 100. mu.l of eluent is almost the same as the washing effect of once by using 100. mu.l of eluent), and centrifuging for 1min at the maximum rotating speed.
Further, in the step S2.2, the specific centrifugation process is as follows: adding 200 mul of ethanol with the same volume as the sample, wherein the concentration of the ethanol is 96-100%, performing vortex oscillation for 15s, mixing uniformly, and performing rapid centrifugation after oscillation to remove liquid drops remained in the cover of a 1.5ml centrifuge tube.
Further, in step S3, the step of performing sulfite treatment on the DNA is as follows:
s3.1, sucking 10 mu L of DNA samples into a PCR tube, adding 10 mu L of water to complement 20 mu L, adding 130 mu L of CTconversion reagent into each sample, flicking the tube or blowing and beating the tube by a gun to be uniformly mixed, and centrifuging to ensure that liquid is settled to the bottom of the tube;
s3.2, placing the tube into a thermal cycler, centrifuging and eluting DNA.
Further, in step S3.2, the specific centrifugal elution steps are as follows:
operating at the temperature of a.98 ℃ for 10 min;
b.64 ℃ for 2.5 h;
c.4 ℃ for 20 hours;
d. add 600. mu. L M-Binding Buffer to the Zymo-spin IC Column, which is placed in a collection tube provided;
e. adding the sample from step b to Zymo-spin IC Column containing M-Binding Buffer, covering, and reversing for mixing;
f. centrifuging at full speed of more than or equal to 10000g for 30s, and pouring out the flow-through liquid;
g. add 100 u L M-Wash Buffer to the column, full speed centrifugation for 30 s;
h. adding 200 mu L M-depletion Buffer to the column, standing at room temperature of 20-30 ℃ for 15-20min, and centrifuging at full speed for 30s after incubation is finished;
i. adding 200 mu L M-Wash Buffer to the column, centrifuging for 30s at full speed, adding 200 mu L M-Wash Buffer, and centrifuging for 30 s;
j. the column was placed in a 1.5mL centrifuge tube, 20. mu. L M-Elution buffer was added vertically to the column matrix, and the DNA was eluted by centrifugation at full speed for 30 s.
Further, in step S4, the specific primer pairs and sequencing primers of the 4 genes screened out specifically are:
Elov2-F：AGGGGAGTAGGGTATGAG
Elov2-R：AACCCACCCAAACCCCTATAAAC
Elov2-seq：GGGAGGAGATTTGTAGGTTT
CLorf132-F：GAAGAAGGGGGTTAAGTTTAAGTTTTGA
CLorf132-R：ACCCAACACCAAATCTACCAACTCAC
CLorf132-seq：AAACCAAAATTTAAATCT
FHL2-F：TTTTAGGGTTTTGAGTATAGTAGT
FHL2-R：ACACCTCCTAAACTTCTCCAATCTCC
FHL2-seq：GTTTTGGGAGTATAGTAGTTA
Trim59-F：TATGGTATAGGTGGTTTGGGGAGA
Trim59-R：CTCCTATACTCAACCCATACTC
Trim59-seq：TTGGGGGAGAGGTTG。
further, in step S4.3, the sequenced gene sequence is as follows:
elov2：
CLorf132：
FHL2：
Trim59：
and in the above gene sequence, Y represents a degenerate base, Y represents a T and a C base; r represents A and G.
Further, the method is described.
The embodiment of the invention has the following advantages:
1. the invention designs 4 pairs of ELOVL2, C1orf132, TRIM59 and FHL2 amplification primer pairs, a sequencing primer for pyrosequencing and an analysis sequence, obtains an age inference model formula of the methylation degree of 4 pairs of genes by utilizing the 4 pairs of primers for sequencing, and predicts the biological age by using the model formula; the method uses the biogenic biological tissue to establish the model for the age inference to infer the biological age, the accuracy rate of the method for predicting the age within the deviation 5 years reaches 83 percent, the method can be applied to the inference of the age range of the biogenic person without morphological indications, and an important research method is provided for the inference of the biological age;
2. according to the invention, an age prediction model of a pyrosequencing method is established by using four pairs of primers, namely ELOVL2, C1orf132, TRIM59 and FHL2, biological tissue blood and the like can be used for predicting the biological age of the body source, and an important research method can be provided for the inference of the forensic age.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the method for biological age inference based on pyrosequencing establishment of the embodiment specifically comprises the following operation steps:
s1, preparation of experimental samples: a blood sample;
s2, extracting nucleic acid from the experimental sample by a centrifugal column method by using the Qiagen cell extraction kit, wherein the extraction steps are as follows:
s2.1, sucking 20 mu l of proteinase K to the bottom of a 1.5ml centrifuge tube, adding 200 mu l of a sample of a genome to be extracted to the centrifuge tube, adding 200 mu l of Buffer AL to the sample, carrying out vortex oscillation for 15S, uniformly mixing (completely and uniformly mixing to obtain a uniform solution is very important for ensuring the sufficient cracking of the sample), and incubating at 56 ℃ for 10min (the DNA yield reaches the maximum value under the condition, and the yield cannot be further improved by prolonging the incubation time);
s2.2, adding 200 mu l of ethanol with the same volume as the sample, wherein the concentration of the ethanol is 96-100%, performing vortex oscillation for 15S, uniformly mixing, and performing rapid centrifugation after oscillation to remove liquid drops remained in the cover of a 1.5ml centrifuge tube;
s2.3, transferring the mixture obtained in the step to a Mini centrifuge column (in a 2ml collection tube), paying attention to not wet the ring at the edge, fastening a cover (closing the cover of each centrifuge tube during centrifugation to prevent aerosol generation during centrifugation), centrifuging at 6000g (8000rpm) for 1Min, selecting low-speed centrifugation for reducing noise, using higher rotation speed without affecting final yield and purity of DNA, and when the sample is erythrocyte sedimentation or lymphocyte, preferably using highest rotation speed to prevent blockage, placing the QIAamp Min centrifuge column into a new clean 2ml receiving tube, and discarding the filtrate together with the used collection tube;
s2.4, carefully open the Mini spin column, add 500. mu.l Buffer AW1 (taking care not to wet the edge ring), close the lid, centrifuge at 6000g (8000rpm) for 1min, transfer the spin column to a new 2ml collection tube, discard the filtrate along with the used collection tube, this step does not require an increase in Buffer AW1 even if the initial sample is added in an amount greater than 200. mu.l;
s2.5, discarding the filtrate, carefully opening the QIAamp Mini spin column, adding 500. mu.l Buffer AW2 (taking care not to wet the edge ring), closing the lid, and centrifuging at 20000g (14000rpm) for 3 min;
s2.6, transferring the centrifugal column to a new collection tube of 2ml, carefully opening the centrifugal column, adding 200 mul of buffer AE or double distilled water (the elution volume of less than 200 mul can obviously increase the concentration of DNA in the eluent, but can reduce the total DNA yield; for DNA less than 1 mul, 50 mul of eluent is recommended to be used; the effect of washing 2 times by using 100 mul of eluent and one time by using 100 mul of eluent is almost the same), and centrifuging at the maximum rotating speed for 1 min;
s3, performing sulfite treatment on the extracted DNA by adopting an EZ DNA methylation-Gold kit D5005 kit, wherein the treatment steps are as follows:
s3.1, sucking 10 mu L of DNA samples into a PCR tube, adding 10 mu L of water to complement 20 mu L, adding 130 mu L of CTconversion reagent into each sample, flicking the tube or blowing and beating the tube by a gun to be uniformly mixed, and centrifuging to ensure that liquid is settled to the bottom of the tube;
s3.2, placing the tube into a thermal cycler, centrifuging, eluting DNA, and performing centrifugal elution according to the following steps:
operating at the temperature of a.98 ℃ for 10 min;
b.64 ℃ for 2.5 h;
c.4 ℃ for 20 hours;
d. add 600. mu. L M-Binding Buffer to the Zymo-spin IC Column, which is placed in a collection tube provided;
e. adding the sample from step b to Zymo-spin IC Column containing M-Binding Buffer, covering, and reversing for mixing;
f. centrifuging at full speed of more than or equal to 10000g for 30s, and pouring out the flow-through liquid;
g. add 100 u L M-Wash Buffer to the column, full speed centrifugation for 30 s;
h. adding 200 mu L M-depletion Buffer to the column, standing at room temperature of 20-30 ℃ for 15-20min, and centrifuging at full speed for 30s after incubation is finished;
i. adding 200 mu L M-Wash Buffer to the column, centrifuging for 30s at full speed, adding 200 mu L M-Wash Buffer, and centrifuging for 30 s;
j. the column was placed in a 1.5mL centrifuge tube, 20. mu. L M-Elution buffer was added vertically to the column matrix, and the DNA was eluted by centrifugation at full speed for 30 s;
s4, carrying out PCR amplification and sequencing on the DNA treated by the sulfite, and comprising the following steps:
s4.1, screening out specific primer pairs and sequencing primers of 4 genes such as ELOVL2, C1orf132, TRIM59, FHL2 and the like:
s4.2, screening out an enzyme Pyromark PCR Kit specially used for PCR amplification of the DNA treated by the sulfite, and amplifying the DNA treated by the sulfite by PCR under the following conditions: firstly, amplifying for 15min at the temperature of 95 ℃; then amplifying at 94 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 20s for 45 cycles; finally amplifying for 10min at the temperature of 72 ℃;
s4.3, sequencing the PCR products of the 4 pairs of genes;
wherein Y represents a degenerate base, Y represents a T and C base; r represents A and G;
s5, establishing a biological prediction age model equation of a multiple regression equation by using the methylation sites of the 4 genes as follows:
age prediction value of 29.766+0.178 XFHL 2-F1-0.524 XCLorf 132-C1+0.198 XEolov 2-E4+0.276 XTrim 59-T4-0.119 XFHL 2-F9+0.286 XFHL 2-F2, R20.932, wherein R2Representing the credibility of an equation formula;
wherein, F1, F9, F2, C1, E4 and T4 respectively represent methylation sites of FHL2, CLorf132, Elov2 and Trim59 genes; FHL2, CLorf132, Elov2 and Trim59 represent the names of different genes respectively.
Example 2:
and (3) verification of the model:
an age inference laboratory is made by using 300 samples of different age groups, accuracy judgment is carried out by using a well-established formula, and the overall accuracy rate of people with the ages within plus or minus 5 years is found to be 82%, and the overall average deviation value (MAD) is 3.09;
according to the invention, an age prediction model of a pyrosequencing method is established by using four pairs of primers, namely ELOVL2, C1orf132, TRIM59 and FHL2, biological tissue blood and the like can be used for predicting the biological age of the body source, and an important research method can be provided for the inference of the forensic age.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (7)
1. A method of biological age inference based on pyrosequencing establishment, characterized by: the specific operation steps are as follows:
s1, preparation of experimental samples: a tissue sample or a blood sample;
s2, extracting nucleic acid from the experimental sample by a centrifugal column method by using a Qiagen cell extraction kit;
s3, using EZ DNA Mehtylation-Gold KitTMPerforming sulfite treatment on the extracted DNA by using a D5005 kit;
s4, carrying out PCR amplification and sequencing on the DNA treated by the sulfite, and comprising the following steps:
s4.1, screening specific primer pairs and sequencing primers of 4 genes such as ELOVL2, Clorf132, TRIM59, FHL2 and the like;
s4.2, screening out an enzyme Pyromark PCR Kit specially used for PCR amplification of the DNA treated by the sulfite, and amplifying the DNA treated by the sulfite by PCR under the following conditions: firstly, amplifying for 15min at the temperature of 95 ℃; then amplifying at 94 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 20s for 45 cycles; finally amplifying for 10min at the temperature of 72 ℃;
s4.3, sequencing the PCR products of the 4 pairs of genes;
s5, establishing a biological prediction age model equation of a multiple regression equation by using the methylation sites of the 4 genes as follows:
age prediction value of 29.766+0.178 XFHL 2-F1-0.524 XCLorf 132-C1+0.198 XEolov 2-E4+0.276 XTrim 59-T4-0.119 XFHL 2-F9+0.286 XFHL 2-F2, R20.932, wherein R2Representing the degree of confidence in the equation formula.
2. The method of claim 1, wherein the biological age inference is based on pyrosequencing, and wherein the biological age inference comprises: in step S2, the nucleic acid extraction step is as follows:
s2.1, sucking 20 mu l of proteinase K to the bottom of a 1.5ml centrifuge tube, adding 200 mu l of a sample of a genome to be extracted into the centrifuge tube, adding 200 mu l of Buffer AL into the sample, carrying out vortex oscillation for 15S, uniformly mixing, and incubating for 10min at 56 ℃;
s2.2, quickly centrifuging to remove liquid drops remained in the cover of the 1.5ml centrifuge tube;
s2.3, transferring the mixture obtained in the step to a Mini centrifugal column, covering the Mini centrifugal column, centrifuging at 6000g (8000rpm) for 1Min, placing the QIAamp Min centrifugal column into a new clean 2ml receiving tube, and discarding the filtrate together with the used collecting tube;
s2.4, carefully opening the Mini centrifugal column, adding 500 mu l of Buffer AW1, tightly covering the cover, centrifuging at 6000g (8000rpm) for 1min, transferring the centrifugal column into a new 2ml collection tube, and discarding the filtrate together with the used collection tube;
s2.5, discarding the filtrate, carefully opening a QIAamp Mini centrifugal column, adding 500. mu.l Buffer AW2, tightly covering the cover, and centrifuging at 20000g (14000rpm) for 3 min;
s2.6, transferring the centrifugal column to a new collecting pipe of 1.5ml or 2ml, carefully opening the centrifugal column, adding 200 mu l of buffer AE or double distilled water, and centrifuging at the maximum rotating speed for 1 min.
3. The method of claim 1, wherein the biological age inference is based on pyrosequencing, and wherein the biological age inference comprises: in the step S2.2, the specific centrifugation process is as follows: adding 200 mul of ethanol with the same volume as the sample, wherein the concentration of the ethanol is 96-100%, performing vortex oscillation for 15s, mixing uniformly, and performing rapid centrifugation after oscillation to remove liquid drops remained in the cover of a 1.5ml centrifuge tube.
4. The method of claim 1, wherein the biological age inference is based on pyrosequencing, and wherein the biological age inference comprises: in step S3, the step of treating DNA with sulfite is as follows:
s3.1, sucking 10 mu L of DNA samples into a PCR tube, adding 10 mu L of water to complement 20 mu L, adding 130 mu L of CTconversion reagent into each sample, flicking the tube or blowing and beating the tube by a gun to be uniformly mixed, and centrifuging to ensure that liquid is settled to the bottom of the tube;
s3.2, placing the tube into a thermal cycler, centrifuging and eluting DNA.
5. The method of claim 4, wherein the biological age inference is based on pyrosequencing, and wherein the biological age inference comprises: in step S3.2, the specific centrifugal elution steps are as follows:
operating at the temperature of a.98 ℃ for 10 min;
b.64 ℃ for 2.5 h;
c.4 ℃ for 20 hours;
d. add 600. mu. L M-Binding Buffer to the Zymo-spin IC Column, which is placed in a collection tube provided;
e. adding the sample from step b to Zymo-spin IC Column containing M-Binding Buffer, covering, and reversing for mixing;
f. centrifuging at full speed of more than or equal to 10000g for 30s, and pouring out the flow-through liquid;
g. add 100 u L M-Wash Buffer to the column, full speed centrifugation for 30 s;
h. adding 200 mu L M-depletion Buffer to the column, standing at room temperature of 20-30 ℃ for 15-20min, and centrifuging at full speed for 30s after incubation is finished;
i. adding 200 mu L M-Wash Buffer to the column, centrifuging for 30s at full speed, adding 200 mu L M-Wash Buffer, and centrifuging for 30 s;
j. the column was placed in a 1.5mL centrifuge tube, 20. mu. L M-Elution buffer was added vertically to the column matrix, and the DNA was eluted by centrifugation at full speed for 30 s.
6. The method of claim 1, wherein the biological age inference is based on pyrosequencing, and wherein the biological age inference comprises: in step S4, the specific primer pairs and sequencing primers of the 4 screened genes are specifically:
Elov2-F：AGGGGAGTAGGGTATGAG
Elov2-R：AACCCACCCAAACCCCTATAAAC
EloV2-seq：GGGAGGAGATTTGTAGGTTT
CLorf132-F：GAAGAAGGGGGTTAAGTTTAAGTTTTGA
CLorf132-R：ACCCAACACCAAATCTACCAACTCAC
CLorf132-seq：AAACCAAAATTTAAATCT
FHL2-F：TTTTAGGGTTTTGAGTATAGTAGT
FHL2-R：ACACCTCCTAAACTTCTCCAATCTCC
FHL2-seq：GTTTTGGGAGTATAGTAGTTA
Trim59-F：TATGGTATAGGTGGTTTGGGGAGA
Trim59-R：CTCCTATACTCAACCCATACTC
Trim59-seq：TTGGGGGAGAGGTTG。
7. the method of claim 1, wherein the biological age inference is based on pyrosequencing, and wherein the biological age inference comprises: in step S4.3, the sequenced gene sequence is as follows:
elov2：
CLorf132：
FHL2：
Trim59：
and in the above gene sequence, Y represents a degenerate base, Y represents a T and a C base; r represents A and G.
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