Genetic marker for human individual recognition and / or blood identification and application thereof

Abstract

The invention provides a genetic marker for human individual recognition and / or blood identification and application thereof, and belongs to the technical field of forensic genetics. The genetic marker provided by the invention is composed of 23 blood specific miRNA-SNPs, individual identification and body fluid identification can be synchronously realized by detecting the miRNA-SNPs, whether a blood sample exists in the body fluid can be accurately identified, and when the genetic marker is used for individual identification, the accumulative individual identification rate is 0.999999999929859. The invention further provides a method for human individual recognition or blood sample identification based on 23 blood specific miRNA-SNPs and application, and the method has forensic significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of forensic genetics, and in particular relates to genetic markers and their applications for human individual identification and / or blood differentiation. Background Art

[0002] Individual identification is a crucial task in forensic evidence, used to reveal individual identities, investigate crimes, and determine compensation and compensation. DNA evidence, specifically the genotypes of genetic markers on different individuals' chromosomes, meets forensic genetic requirements. Currently, traditional individual identification methods include second-generation genetic markers based on genomic DNA short tandem repeats (STRs) and third-generation genetic markers based on single nucleotide polymorphisms (SNPs). However, these methods are unable to determine the source of body fluids or individual characteristics.

[0003] miRNAs, a class of endogenous single-stranded noncoding RNAs (16-22 nucleotides in length), have garnered widespread attention in recent years. Due to their structural properties, miRNAs are particularly well-suited for detection using massively parallel sequencing (MPS), effectively enabling the identification of newly discovered and rare SNPs. Currently, over 2,000 miRNAs and over 2,000 miRNA SNPs have been discovered in the human body, providing a foundation for identifying the source of body fluids and individual characteristics. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the first purpose of the present invention is to provide a genetic marker for human individual identification and / or blood identification, which consists of 23 blood-specific miRNA-SNPs. By detecting these miRNA-SNPs, individual identification and body fluid identification can be achieved simultaneously.

[0005] The second object of the present invention is to provide the use of the above genetic markers in identifying blood samples or preparing a kit for identifying blood samples, or in identifying human individuals or preparing a kit for identifying human individuals.

[0006] A third object of the present invention is to provide a method for identifying a blood sample.

[0007] A fourth object of the present invention is to provide a method for identifying a human individual.

[0008] A fifth object of the present invention is to provide a kit for identifying or authenticating a blood sample of a human individual.

[0009] A sixth object of the present invention is to provide the use of the above-mentioned kit in human individual identification and / or differentiation of blood samples.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a genetic marker for human individual identification and / or blood identification, which consists of 23 blood-specific miRNA-SNPs: rs568202847, rs377250380, rs771611815, rs1267954074, rs1275333653, rs1218721253, rs1423068484, rs777206553, rs556757681, rs77 3861047, rs770235403, rs749991806, rs774322884, rs1323869733, rs558661304, rs573692968, rs1555935982, rs991454083, rs765258989, rs78547906, rs939253091, rs1454719836, and rs757261126.

[0012] The present invention provides the use of the above genetic markers in identifying blood samples or preparing a kit for identifying blood samples.

[0013] The present invention provides the use of the above genetic markers in human individual identification or in the preparation of a kit for human individual identification.

[0014] The present invention provides a method for identifying a blood sample, comprising the following steps: extracting RNA from the sample, constructing a small RNA library, and sequencing the small RNA library; comparing the sequencing results with the miRNA reference sequence of miRDeep2; and determining that the blood sample is present when ≥10 sites among the above-mentioned 23 blood-specific miRNA-SNPs are positive in the comparison results.

[0015] The present invention provides a method for human individual identification, comprising the following steps: extracting RNA from a human blood sample, constructing a small RNA library, and sequencing the small RNA library; aligning the sequencing results with the miRNA reference sequence of miRDeep2; and, based on the alignment results, performing probability calculation based on the DP of positive sites in the above-mentioned 23 blood-specific miRNA-SNPs for individual identification.

[0016] Preferably, the amount of the sample or human blood sample is ≥400 μL.

[0017] Preferably, using of Multiplex small RNA library preparation kit for construction of small RNA libraries.

[0018] Preferably, the library is sequenced on an Illumina HiSeq 2500 / 2000 platform to generate 50-base single-end read sequences to complete the sequencing.

[0019] The present invention provides a kit for identifying or authenticating a blood sample of a human individual, wherein the kit comprises reagents for detecting the above-mentioned 23 blood-specific miRNA-SNPs.

[0020] The present invention provides the use of the above-mentioned kit in human individual identification and / or differentiation of blood samples.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0022] The genetic marker composed of 23 blood-specific miRNA-SNPs described in the present invention has a lower mutation rate and a higher degree of compatibility with various sequencing platforms. It can simultaneously realize individual identification and body fluid identification during detection, and can accurately identify whether blood samples are present in body fluids. When used for individual identification, the cumulative individual identification rate is 0.999999999929859, which has forensic significance. DETAILED DESCRIPTION

[0023] The present invention provides a genetic marker for human individual identification and / or blood identification, which consists of 23 blood-specific miRNA-SNPs: rs568202847, rs377250380, rs771611815, rs1267954074, rs1275333653, rs1218721253, rs1423068484, rs777206553, rs556757681, rs77 3861047, rs770235403, rs749991806, rs774322884, rs1323869733, rs558661304, rs573692968, rs1555935982, rs991454083, rs765258989, rs78547906, rs939253091, rs1454719836 and rs757261126. The genetic markers are applicable to East Asian populations, preferably to the Han population in northern China.

[0024] The 23 blood-specific miRNA-SNPs described in the present invention meet the following characteristics: (1) The SNP sites conform to the Hardy-Weinberg equilibrium and linkage equilibrium laws in the East Asian population. (2) The SNP sites should be located on different chromosomes, or when on the same chromosome, their physical distance should be greater than 5Mb. (3) The allele frequency distribution of the diallelic SNP sites in the East Asian population should be between 0.4 and 0.6. The details of the 23 blood-specific miRNA-SNPs described in the present invention are shown in the following table:

[0025] Table 123 blood-specific miRNA-SNPs

[0026]

[0027] The present invention provides the use of the above genetic markers in identifying blood samples or preparing a kit for identifying blood samples or human individual identification or preparing a kit for human individual identification.

[0028] The present invention provides a method for identifying a blood sample, comprising the following steps: extracting RNA from the sample, constructing a small RNA library, and sequencing the small RNA library; comparing the sequencing results with the miRNA reference sequence of miRDeep2; when in the comparison results, ≥10 sites among the above 23 blood-specific miRNA-SNPs are positive, it is determined that a blood sample exists. The miRNA reference sequence used in the miRDeep2 analysis comes from the miRBase database, and the positive in the present invention refers to the presence of SNP mutations at the miRNA-SNPs site relative to the reference gene in the miRBase database. The sample described in the present invention can be a liquid or a liquid spot. When the sample is liquid, the sample volume is ≥400 μL. When the sample is a spot, the sample volume for forming the spot is ≥400 μL. The extraction of the sample RNA described in the present invention can be performed using a conventional kit in the art. The present invention uses the extracted total RNA of the sample as the input material for constructing a small RNA library to construct a small RNA library, preferably using a kit for of The multiplex small RNA library preparation kit is used to construct a small RNA library. The constructed small RNA library is sequenced, preferably on an Illumina HiSeq 2500 / 2000 platform, to generate a 50-base single-end read sequence to complete the sequencing.

[0029] The present invention provides a method for human individual identification, comprising the following steps: extracting RNA from a human blood sample, constructing a small RNA library, and sequencing the small RNA library; comparing the sequencing results with the miRNA reference sequence of miRDeep2; and performing probability calculation based on the comparison results and the DP of the positive sites in the above-mentioned 23 blood-specific miRNA-SNPs for individual identification. The miRNA reference sequence used in the miRDeep2 analysis comes from the miRBase database. The positive in the present invention refers to the presence of SNP mutations at the miRNA-SNPs site relative to the reference gene in the miRBase database. The sample in the present invention can be blood or a blood spot. When the sample is a blood liquid, the sample volume is ≥400 μL. When the sample is a blood spot, the amount of blood sample to form the spot is ≥400 μL. The extraction of RNA from the human blood sample in the present invention can be performed using a conventional kit in the art. The present invention uses the extracted total RNA of the blood sample as the input material for constructing the small RNA library to construct the small RNA library, preferably using a kit for of The multiplex small RNA library preparation kit is used to construct a small RNA library. The present invention sequences the constructed small RNA library, preferably on an Illumina HiSeq 2500 / 2000 platform, to generate a 50-base single-end read sequence to complete the sequencing. The probability calculation method of the present invention comprises: calculating the cumulative individual recognition rate based on the DP (individual recognition ability) of the positive miRNA-SNPs site; the calculation formula is: (Example: Assume that three positive miRNA-SNPs are detected, and the DP of each site is 0.8, 0.7, and 0.6, respectively. Then: CDP = 1-(1-0.8)(1-0.7)(1-0.6) = 1-(0.2×0.3×0.4) = 1-0.024 = 0.976. That is, when these three sites are combined, there is a 97.6% probability of distinguishing two random individuals.)

[0030] The present invention provides a kit for identifying or authenticating a blood sample of a human individual, the kit comprising reagents for detecting the above-mentioned 23 blood-specific miRNA-SNPs, including but not limited to RNA extraction reagents, small RNA library preparation reagents, etc.

[0031] The present invention provides the use of the above-mentioned kit in human individual identification and / or identification of blood samples. During the detection, individual identification and body fluid identification can be achieved simultaneously, and the presence of blood samples in body fluids can be accurately identified. When used for individual identification, the cumulative individual identification rate is 0.999999999929859, which has forensic significance.

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] In the following examples, unless otherwise specified, all methods are conventional.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] The research subjects were 100 people (half male and half female) from Beijing and Shandong, with peripheral blood and saliva samples. All samples signed informed consent forms based on the principles of informed consent.

[0037] 1. Sample collection

[0038] 400 μl of blood or saliva were smeared on the card to obtain a blood spot or a saliva spot.

[0039] 2. RNA Extraction

[0040] RNA extraction was performed using the Tiangen RNA extraction kit. RNA extraction was performed according to the kit instructions: The sample was shaken vigorously on a shaker for 10 minutes. A 400 μL sample (blood or saliva spot) was removed and transferred to a 2 mL centrifuge tube. 1200 μL of TRIzol RNA Extraction Reagent was added and the tube was shaken vigorously until thoroughly mixed. The sample was allowed to stand at room temperature (15-30°C) for 5 minutes. Next, 240 μL of chloroform was added to the centrifuge tube. The tube was shaken vigorously for 15 seconds and incubated for another 2-3 minutes. Centrifuge at 14,200 rpm at 4°C for 15 minutes. After centrifugation, the aqueous phase was added to 1200 μL of isopropanol and mixed to precipitate the RNA. After mixing, the mixture was incubated for 10 minutes, followed by centrifugation at 14,200 rpm at 4°C for 10 minutes. The supernatant was discarded and 1200 μL of 75% ethanol was added. After shaking, the tube was centrifuged at 14,200 rpm at 4°C for 10 minutes. The supernatant was discarded again, and 1200 μl of pre-cooled (4°C) 75% ethanol was added to wash the RNA precipitate. After shaking, the mixture was centrifuged at 14,200 rpm for 10 minutes at 4°C to obtain an RNA solution.

[0041] 3. Sequencing

[0042] A total of 3 μg of total RNA from each sample was used as input for the construction of small RNA libraries. of Multiplex small RNA library preparation kit (Tiangen) was used to construct the sequence library. According to the instructions, index codes were added to attribute the sequences to each sample. The steps are as follows: First, the NEB 3′SR adapter was directly and specifically connected to the 3′ end of the RNA sequence. Subsequently, the SR reverse transcription primer was hybridized with an excess of 3′SR adapter, and the single-stranded DNA adapter was converted into a double-stranded DNA molecule. Next, the first-strand cDNA was synthesized using reverse transcriptase. Polymerase chain reaction (PCR) amplification was performed using LongAmp Taq 2×Master Mix, SR primers for Illumina, and index (X) primers. The PCR product was purified on an 8% polyacrylamide gel (100 volts, 80 minutes) and dissolved in 8 microliters of elution buffer. The library was sequenced on the Illumina HiSeq 2500 / 2000 platform to generate a 50-base single-end read sequence to complete the sequencing.

[0043] 4. Construction of miRNA-SNP analysis method

[0044] Local miRNA SNP data was obtained and converted to FASTA format. Sequencing results were then aligned with the miRDeep2 miRNA reference sequence. In this experiment, no miRNA SNPs were detected in saliva samples from the 100 subjects. While the results varied among the blood samples from the 100 subjects, all tested positive for >10 miRNA SNPs. The identification results are shown in Table 2, using blood and saliva samples from four of these subjects as examples.

[0045] Table 2 Comparison of identification results of blood samples and saliva samples

[0046] SNP number Blood sample 1 Blood sample 2 Blood sample 3 Blood sample 4 Saliva sample 1 Saliva sample 2 Saliva sample 3 Saliva sample 4 rs568202847 + + + + - - - - rs377250380 - + + - - - - - rs771611815 + + + + - - - - rs1267954074 + + + + - - - - rs1275333653 + + + + - - - - rs1218721253 + + + + - - - - rs1423068484 - + + + - - - - rs777206553 - + + + - - - - rs556757681 + - - + - - - - rs773861047 - + + - - - - - rs770235403 + + + + - - - - rs749991806 + + + - - - - - rs774322884 - + + + - - - - rs1323869733 - - - + - - - - rs558661304 - - - - - - - - rs573692968 - - + - - - - - rs1555935982 - + - + - - - - rs991454083 + + + + - - - - rs765258989 + + + + - - - - rs78547906 - + + - - - - - rs939253091 + - + - - - - - rs1454719836 + - + - - - - - rs757261126 - - - + - - - -

[0047] The results showed that no fewer than 10 miRNA SNPs were detected in each individual in blood samples, and none were detected in saliva. This indicates that the genetic marker composed of 23 blood-specific miRNA-SNPs described in the present invention can be used to accurately identify the presence of blood samples in body fluids.

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A genetic marker for human individual identification and / or blood identification, characterized in that: The miRNA-specific SNPs were 23: rs568202847, rs377250380, rs771611815, rs1267954074, rs1275333653, rs1218721253, rs1423068484, rs777206553, rs556757681, rs773861047, and rs7702 35403, rs749991806, rs774322884, rs1323869733, rs558661304, rs573692968, rs1555935982, rs991454083, rs765258989, rs78547906, rs939253091, rs1454719836, and rs757261126.

2. Use of the genetic marker according to claim 1 in identifying a blood sample or preparing a kit for identifying a blood sample.

3. Use of the genetic marker according to claim 1 in human individual identification or in the preparation of a kit for human individual identification.

4. A method for identifying a blood sample, characterized in that: The following steps are involved: Extract RNA from the sample, construct a small RNA library, and sequence the small RNA library; compare the sequencing results with the miRNA reference sequence of miRDeep2; when ≥10 sites among the 23 blood-specific miRNA-SNPs described in claim 1 are positive in the comparison results, it is determined that the blood sample is present.

5. A method for human individual identification, characterized in that: The following steps are involved: Extract RNA from human blood samples, construct small RNA libraries, and sequence the small RNA libraries; The sequencing results were compared with the miRNA reference sequence of miRDeep2; according to the comparison results, The DP of the positive sites in the 23 blood-specific miRNA-SNPs according to claim 1 is probability calculated for individual identification.

6. The method according to claim 4 or 5, characterized in that The amount of the sample or human blood sample is ≥400 μL.

7. The method according to claim 4 or 5, characterized in that Use for of Multiplex small RNA library preparation kit for construction of small RNA libraries.

8. The method according to claim 4 or 5, characterized in that The library was sequenced on the Illumina HiSeq 2500 / 2000 platform to generate 50-base single-end reads for sequencing.

9. A kit for identifying or authenticating a blood sample of a human individual, characterized in that: The kit comprises reagents for detecting the 23 blood-specific miRNA-SNPs according to claim 1.

10. Use of the kit according to claim 9 in human individual identification and / or differentiation of blood samples.

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