CN112680530A - Highly-degraded test material detection kit based on 18 multiple insertion deletion genetic markers - Google Patents
Highly-degraded test material detection kit based on 18 multiple insertion deletion genetic markers Download PDFInfo
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Abstract
The invention discloses a highly-degradable test material detection kit based on 18 multiple insertion deletion genetic markers, which comprises a composite amplification primer mixture, an allele typing standard mixture and a DNA standard 9948 which are separately packaged. The multiple insertion deletion genetic markers contained in the kit provided by the invention are length polymorphism genetic markers, and the length of amplification products of all the genetic markers contained in the kit is not more than 125 bases. The kit provides a new simple, economic, rapid and effective technical means for the individual identification of the forensic highly-degradable test material. The kit can be used for detection by utilizing a universal capillary electrophoresis platform in a forensic laboratory, has the characteristics of simplicity, economy, rapidness and accuracy, and is easy to popularize and apply in forensic practice.
Description
Technical Field
The invention relates to a highly-degradable test material detection kit based on 18 multiple insertion deletion genetic markers. Belongs to the technical field of forensic medicine detection kits.
Background
The detection of DNA genetic markers in biological samples to realize individual identification and genetic relationship identification is an important content of forensic research. Highly degraded biological materials are often collected during forensic practice, particularly in explosion sites, large disaster events, missing personnel surveys, and old cases of well-documented cases. The high degradation test material means that the biological test material is influenced by factors such as high temperature, humidity, ultraviolet radiation, microorganisms, strong acid, strong alkali and the like, and long-chain DNA molecules in the test material are broken to form a small-molecule DNA fragment of less than 150bp and are accompanied with DNA sequence damage. When these samples are subjected to capillary electrophoresis detection of conventional Short Tandem Repeat (STR), due to the high fragmentation of DNA and the large STR amplified sequences (typically greater than 150 bases), a complete STR typing pattern is often not obtained, posing a significant challenge to evidence interpretation.
In order to solve the problem of detection of degraded test materials, researchers have proposed new genetic markers such as mitochondrial DNA (mtDNA), Single Nucleotide Polymorphisms (SNPs), insertion/deletion polymorphisms (InDels) and the like for satisfying the personal identification of highly degraded test materials. Mitochondrial DNA has been shown to be an effective tool for forensic degradation assay detection, but its discrimination ability is insufficient due to maternally inherited; SNPs and InDels are widely distributed in human genomes, have the characteristics of stable heredity, low mutation rate and small amplified fragment, and are considered as ideal genetic markers for detecting highly degraded test materials. However, SNPs and InDels are the same, and the same is the same, so that the identification capability is limited, the polymorphism information content and the STR genetic marker with multiple alleles have larger difference, at least 50-60 single loci are needed to reach the cumulative individual identification rate and the non-paternal exclusion rate of 13-15 common STRs, and meanwhile, in order to improve the identification capability, one InDels panel often contains more InDels loci, which also results in larger amplification product size and is not suitable for highly degraded test material detection.
A small number of closely linked InDels markers are present in the human genome, and such haplotypes consisting of two or more InDel that are less than 200bp apart from each other are called multiple insertion/deletion genetic markers (Multi-InDels). Compared with the common STR genetic marker, the Multi-InDels has the characteristics of small amplified fragment and low mutation rate; as the number of Multi-InDels alleles is increased, the polymorphism is better than that of two-allele SNPs and InDels, and the individual identification capability and the non-paternal exclusion rate are improved. More importantly, the detection of Multi-InDels and the detection of conventional STRs are carried out on a common platform, and can be carried out in any forensic laboratory with a capillary electrophoresis platform. Therefore, the multiple insertion deletion genetic marker has outstanding capability of analyzing highly degraded materials.
Multi-InDels has gained a long interest in the field of forensics, and some of the Multi-InDels composite detection systems have been published and even patented. However, the existing research only focuses on the conventional forensic application of Multi-InDels, and the maximum amplification fragments exceed 160 bases, which does not meet the requirement of highly degraded test material detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a highly-degradable test material detection kit based on 18 multiple insertion deletion genetic markers.
In order to achieve the purpose, the invention adopts the following technical scheme:
1. a highly degradable test material detection kit based on 18 multiple insertion deletion genetic markers, comprising: separating the packaged composite amplification primer mixture, the allelic typing standard mixture and the DNA standard; wherein the composite amplification primer mixture comprises 37 amplification primers with 18 multiple insertions and deletion of genetic markers; the allelic typing standard mixture consists of 18 allelic standards with multiple insertion deletion genetic markers, and 54 DNA fragments with fluorescein markers are included; the 18 multiple insertion/deletion genetic markers comprise insertion/deletion sites shown in table 1:
TABLE 1 multiple indel genetic markers comprising insertion/deletion sites
As shown in Table 1, each multiple indel genetic marker selected by the kit comprises 2 completely linked InDels loci, which constitute multiple indel haplotypes.
Preferably, the DNA standard is standard DNA 9948.
Preferably, the nucleotide sequence of the composite amplification primer is shown in table 2:
TABLE 2 nucleotide sequences of amplification primers
In 18 genetic markers, a single nucleotide polymorphism site exists in a primer binding region upstream of a D19MIL37 site, so that two upstream primers are designed to ensure the amplification efficiency.
Preferably, the nucleotide sequence of the allelic standard comprised in the allelic standard mixture is shown in table 3:
TABLE 3 nucleotide sequence of allele standard in the allele standard mixture
Wherein the sequence before the "-" symbol is a fluorescent dye label.
In table 3, each of the 18 multiple indels has 3 alleles, which greatly increases the recognition ability and information carrying ability of a single genetic marker, and the sizes of all the allele fragments are not more than 125 bases. The number in the first column of each sequence in Table 3 corresponds to the designation of the allele, 0 being the smallest allele, 2 representing an allele having 2 bases more than the allele designated 0, 4 representing an allele having four bases more than the allele, and so on.
Preferably, the kit further comprises: the amplification mixture was QIAGEN Multiplex PCR mix manufactured by QIAGEN GmbH, Germany.
2. The kit is applied to the identification of highly degraded detection materials.
3. The highly-degradable material detection identification method based on the kit comprises the following specific steps:
1) extracting DNA of a sample to be detected;
2) carrying out single-tube multiplex amplification on the DNA extracted in the step (1) by using the kit; the cycle parameters of the reaction of the composite amplification PCR are 95 ℃ and 5 minutes; 29 cycles of 95 ℃, 30 seconds, 60 ℃, 72 ℃, 30 seconds; then 60 minutes at 72 ℃; storing at 4 deg.C;
3) and mixing the amplified product and the allele typing standard mixture with a molecular weight internal standard and Hi-Di formamide respectively, and then carrying out capillary electrophoresis analysis to obtain 18 multiple insertion deletion genetic marker typing results of the sample.
Preferably, the volume ratio of the amplification product to the molecular weight internal standard is 1: 9, the volume ratio of the allele typing standard mixture to Hi-Di formamide is 1: 0.2.
preferably, the molecular weight internal standard is Size 500 molecular weight internal standard produced by the basic point cognitive technology (Beijing) company Limited.
The invention has the beneficial effects that: aiming at the problem that the existing forensic personal identification kit has excessively long genetic marker amplification fragments and can not detect highly degraded biological detection materials on the premise of ensuring the personal identification capability, the invention designs amplification primers by screening multiple insertion-deletion (Multi-InDels) genetic markers which are closely adjacent to each other and have multiple alleles and good polymorphism, so that the maximum amplification fragments of all genetic markers do not exceed 125 bases, establishes a simple, economic and effective composite detection system and provides a new technical means for the detection of the forensic highly degraded biological detection materials.
The invention mainly solves the technical problem of realizing forensic individual identification of highly degraded human biological detection materials by utilizing multiple insertion deletion genetic markers. The kit comprises a separately packaged composite amplification primer mixture, an allelic typing standard mixture and a DNA standard 9948. The multiple insertion deletion genetic markers contained in the kit provided by the invention are length polymorphism genetic markers, and the length of amplification products of all the genetic markers contained in the kit is not more than 125 bases. The kit provides a new simple, economic, rapid and effective technical means for the individual identification of the forensic highly-degradable test material.
The invention screens multiple insertion deletion genetic markers detected by highly degradable detection materials from a public database by an original bioinformatics method, and combines DNA mixed pool high-throughput sequencing analysis to ensure that the length of 18 multiple insertion deletion genetic marker core regions is less than 40 bases, which is much shorter than that of the multiple insertion deletion genetic marker core region in the prior art; the amplification primers are designed by using a method of ' 5 ' end artificial sequence + specific complementary sequence ', so that all multiple insertion deletion genetic markers can be subjected to composite amplification in a single tube and disposable capillary electrophoresis typing, the length of amplification products does not exceed 125 bases, the detection requirement of a highly degraded detection material is met, and the maximum amplification product length of the forensic medical detection kit in the prior art exceeds 160 bases and cannot be used for detection of the highly degraded detection material in forensic medicine; the allelic typing standard mixture consists of 18 allelic standards with multiple insertion and deletion genetic markers, and comprises 54 DNA fragments with fluorescein markers.
The 18 multiple insertion deletion genetic markers screened by the invention are different from the multiple insertion deletion genetic markers screened by the existing research, the genetic markers have shorter core sequence length and better polymorphism, and the length of all amplification product fragments does not exceed 125 basic groups by combining a characteristic primer design method, thereby meeting the detection requirement of highly degraded test materials. However, the maximum amplification fragment length of all the existing STR amplification kits, Indels composite detection kits and multiple insertion deletion genetic marker composite detection kits exceeds 150 basic groups, and the effect is not ideal when the kit is used for detecting highly degraded detection materials. The 18 multiple insertion deletion genetic markers are subjected to linkage analysis, and each genetic marker is in linkage disequilibrium and has independent efficacy. The investigation experiment of 192 Hunan Han nationality groups proves that the random matching probability of the kit is 3.23 multiplied by 10-12The cumulative non-parentage exclusion rate was 0.9989.
The kit can be used for detection by utilizing a universal capillary electrophoresis platform in a forensic laboratory, has the characteristics of simplicity, economy, rapidness and accuracy, and is easy to popularize and apply in forensic practice. Meanwhile, the 18 multiple insertion deletion genetic markers contained in the kit have the characteristics of short amplified fragments and good polymorphism, meet the detection requirements of highly degraded test materials, and have system efficiency enough for forensic individual identification. The kit provides a new technical means for detecting the highly-degraded material to be detected in the forensic medicine field, and has wide application prospect in the forensic medicine field.
Drawings
FIG. 1 is a capillary electrophoresis typing map of a numbered HN141 sample with the kit of the present invention, wherein the abscissa value is the length of the amplified fragment, the nomenclature of the genetic marker is above the allele peak, the Arabic numerals below the allele peak are the nomenclature of the allele of the corresponding genetic marker, and the ordinate value represents the fluorescence signal intensity.
FIG. 2 is a comparison graph of genomic DNA detected by the kit of the present invention and the Identifier Plus STR kit manufactured by Saimer Fei USA after being subjected to water bath at 100 ℃ for 0, 20, 40, 60 and 80 minutes, respectively; wherein, A is the typing result of the DNA degraded to different degrees detected by the kit of the invention, and B is the typing result commonly used for detecting degraded test material in forensic medicine
Typing results of Plus kit, C is the typing results of degraded DNA detected by the kit of the invention in boiling water bath for 80 minutes, D is the kit of the invention and
and (3) a comparison graph of allele detection rate of the Plus kit in the process of detecting artificially degraded DNA.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.
The highly-degradable material detection kit based on 18 multiple insertion and deletion genetic markers is characterized in that ideal multiple insertion and deletion genetic markers are screened from an NCBI database, composite amplification primers with amplification fragments not more than 125 bases are designed, alleles of samples in the genetic markers are obtained by applying a composite amplification technology, allele typing results are obtained by applying a capillary electrophoresis technology, and a highly-degradable material detection system of the multiple insertion and deletion genetic markers is finally established.
The screening criteria for the multiple insertion deletion genetic marker of the present invention are:
1) the Minimum Allele Frequency (MAF) > 0.1 of a single InDel locus contained in the multiple insertion deletion genetic marker;
2) in the non-coding region;
3) the maximum physical distance between two InDel loci within a multiple insertion deletion genetic marker is less than 50 bases;
4) the segment difference between the minimum allele and the maximum allele in the same genetic marker is 12 bases;
5) the size of the PCR product is not more than 125 bp;
6) the physical distance between multiple inserted and deleted genetic markers on the same chromosome is greater than 5 Mb.
The invention screens out 18 ideal multiple insertion deletion genetic markers according to the established standard and establishes a composite system. The information of the involved 18 multiple indels genetic markers is shown in table 1, and the nucleotide sequences of the primers of the composite amplification primer mixture are respectively listed in table 2.
The working principle of the kit is as follows: firstly, extracting genome DNA of a highly degradable test material, mixing a DNA template with 18 primer mixtures of multiple insertion deletion genetic markers and an amplification reaction mixed solution, carrying out PCR amplification in a single tube, simultaneously obtaining allele amplification products of the 18 multiple insertion deletion genetic markers of the test material, finally carrying out capillary electrophoresis, and using an allele typing standard substance mixture as a control to obtain a typing result of the highly degradable test material. In addition, the DNA standard 9948 is amplified simultaneously with the test material as a positive control. And whether the detection result is reliable can be judged according to whether the typing result of the standard DNA is accurate or not.
At present, a simple and quick method for detecting the highly-degradable material to be detected does not exist, and the kit aims to establish a simple, economical, convenient and effective novel method for detecting the highly-degradable material to be detected, and is applied to forensic practice. In the invention, the multiple insertion deletion genetic marker with good polymorphism and small core region is screened, and the design of the amplification primer with the product length not more than 125bp is a difficult point for constructing a high degradation detection kit. The present invention takes into account the following factors:
1) the length ranges of the amplified fragments between the multiple insertion deletion genetic markers of the same color fluorescent marker are not overlapped so as to distinguish the allelic peaks of different genetic markers;
2) the length of the amplified fragment is between 60 and 125bp, so that the method is suitable for detecting a highly degraded test material;
3) the annealing temperature of the primers is approximate;
4) the primers, the primers and the template have no obvious mismatching, hairpin structures and dimer structures;
5) for loci with the same fluorescent label but overlapping allelic ranges, the overlap is eliminated by adding a tailing sequence GCCTCC (TCCCC) n at the 5' end of the primer.
All 18 multiple indel genetic marker multiplex amplification primer sequences were designed as shown in table 2.
The standard mixture for allele typing facilitates accurate and rapid analysis of the genotype of a sample. The allelic typing standard provided by the present invention includes all 18 multiple insertion deletion genetic marker alleles, and has 55 fluorescein marker fragments, and the sequences and fluorescent markers of the alleles are shown in Table 3.
In table 3, the number in front of each sequence indicates the allele name of each multiple insertion deletion genetic marker, the number "0" indicates the allele with the shortest fragment, 1 indicates an allele 1 bases larger than the allele designated as 0, 3 indicates an allele 3 bases larger than the allele designated as 0, and so on.
The kit specifically comprises the following components: separating the packaged composite amplification primer mixture, the allelic typing standard mixture and the DNA standard; the composite amplification primer mixture comprises 37 amplification primers with 18 multiple insertions and deletion of genetic markers; the allelic typing standard mixture consists of 18 allelic standards with multiple insertion deletion genetic markers, and 54 DNA fragments with fluorescein markers are included; the DNA standard is standard DNA 9948.
1) The mixed solution for the composite amplification reaction contains PCR buffer solution,MgCl2The amplification reaction mixture used in the present invention is QIAGEN Multiplex PCR mix produced by QIAGEN GmbH of Germany.
2) The specific information of the composite amplification primer mixture is shown in Table 2.
3) Allelic typing standard mixture: consists of 55 fluorescein-labeled DNA fragments described in Table 3, corresponding to all known 55 alleles of 18 multiple insertions deleted genetic markers.
4) Molecular weight internal standard: LIS500, Golden eye20A, beijing base company.
The kit can be used for analyzing highly degraded biological detection materials, and the specific analysis method is as follows:
1) extracting DNA of a highly degraded test material to be detected as an amplification template;
2) and (3) carrying out single-tube composite amplification on the DNA extracted in the step (1) by using the amplification primer mixture and the amplification reaction mixed solution. The composite amplification system is prepared by the following steps: 5 microliter total reaction volume, containing 2.5 microliter QIAGEN Multiplex PCR mix, 1.15 microliter composite amplification primer mixture, 0.5 microliter DNA template and 0.85 microliter deionized water. The cycle parameters of the reaction of the multiplex amplification PCR are as follows: 5 minutes at 95 ℃; 29 cycles of 95 ℃, 30 seconds, 60 ℃, 72 ℃, 30 seconds; then, the temperature is 72 ℃ for 10 minutes; storing at 4 ℃. Note that: the highly degraded DNA has random effect during amplification, and 3-5 parallel amplifications need to be carried out in PCR reaction.
3) And mixing the amplified product and the allele typing standard mixture with a molecular weight internal standard and Hi-Di formamide respectively, performing capillary electrophoresis, and obtaining the genotype of the sample according to the electrophoresis result.
The following is further specifically described with specific examples. The main reagents and instruments used in the invention are as follows:
1) automatic laser fluorescence capillary electrophoresis 3130 genetic Analyzer, ABI
2) Eppendorf PCR Amplifier, EPPENDORF Co
3) Thermo Electron LED GmbH high speed centrifuge, ThermoFisher Corp
4) NanoDrop one ultramicro spectrophotometer, Thermo Scientific
5) Pipette Eppendorf Ltd
6) Hi-Di formamide, ABI
7) Molecular weight internal Standard (Golden eye20A, LIS-500), Beijing base Co
Example 1: preparation of the kit of the invention
The highly-degradable test material detection kit for detecting the multiple insertion deletion genetic marker comprises the following reagents packaged respectively:
1) and (3) compounding the amplification primer mixture. The amplification primers shown in Table 2 were synthesized by Oncorhynchus corporation, and the synthesized amplification primers were mixed at a ratio shown in Table 4 with ultrapure water of 100 pM/. mu.L to prepare a composite amplification primer mixture.
2) And (3) mixing the amplification reaction solution. In this example, QIAGEN Multiplex PCR mix from QIAGEN was used.
3) Allelic typing standard mixture: consists of 55 fluorescence labeled DNA fragments shown in Table 3, and four colors (blue, green, black and red) of fluorescence are FAM, HEX, TAMRA and ROX, respectively.
And packaging the reagents according to respective conventional requirements to prepare the highly-degradable material detection kit based on the multiple insertion deletion genetic markers for subsequent tests.
TABLE 4 concentration of composite amplification primers
Example 2 detection of 1 Hunan Han nationality sample Using the kit of the invention
The detection of 1 Hunan Han nationality bloodstain sample is carried out by using the highly-degradable material detection kit based on the multiple insertion deletion genetic marker. The specific detection process is carried out as follows:
1) extracting DNA of the DNA by a Chelex-100 method to be used as a composite amplification template;
2) and c, performing multiplex PCR amplification on the sample in the following amplification system by using the DNA template, the multiplex amplification primer mixture and the multiplex amplification reaction mixture in the step a:
the cycle parameters of the reaction of multiplex amplification PCR are: 5 minutes at 95 ℃; 29 cycles of 95 ℃, 30 seconds, 60 ℃, 72 ℃, 30 seconds; then, the temperature is 72 ℃ for 10 minutes; storing at 4 ℃.
3) Capillary electrophoresis
mu.L of each of the amplification product and the allelic typing standard was mixed with 9. mu.L of Hi-Di formamide and 0.2. mu.L of LIS500 (golden eye 20A) molecular weight internal standard, and the mixture was electrophoresed using a genetic analyzer 3130 available from ABI USA. Performing electrophoresis for 20min under the conditions of electrophoresis voltage of 15kV and capillary of 36cm and POP4 gel; genotyping data was analyzed using GeneMapper IDX v1.5 software (Applied Biosystems, Foster City, Calif., USA).
FIG. 1 is an example of the typing results of Han nationality samples in Hunan province, all 18 multiple insertions and deletions of genetic markers are typed, interference-free peaks are avoided, and alleles of the 18 multiple insertions and deletions of genetic markers in the samples can be clearly identified.
Example 3 detection of artificially degraded DNA Using the kit of the present invention
The artificial degradation model was created using 40 μ L of 2ng/μ L DNA sample in a water bath at 100 ℃ for specific time periods (0 min, 20min, 40 min, 60 min and 80 min). Use of the kit and
the Plus kit is used for genotyping the artificial degradation products. The results are shown in FIG. 2. FIG. 2A shows the typing results of the degraded DNAs of different degrees detected by the kit of the present invention, and FIG. 2B shows the typing results of the degraded DNA detected by forensic medicine
The typing result of the Plus kit shows that the invention has obvious effect when detecting highly degraded material (boiling water bath is more than 60 minutes) through comparison, and can obtain complete typing map; while
The Plus kit can detect only a small fraction of the allelic peaks. FIG. 2C shows the result of typing the degraded DNA in a boiling water bath for 80 minutes using the kit of the present invention, and all allelic peaks are observed, but due to the random effect, the allelic peak heights are unbalanced in some loci, suggesting that 3-5 parallel tests are required for detecting degraded samples. FIG. 2D shows the kit of the present invention and
the comparison graph of the allele detection rate of the Plus kit in the process of detecting artificially degraded DNA shows that the kit is obviously caused by the fact that the kit is used for detecting highly degraded DNA
Plus kit.
As can be seen from FIG. 2, the DNA was highly degraded after 60 minutes in a 100 ℃ water bath. The Identifier Plus kit can detect only a few alleles after being subjected to water bath at 100 ℃ for 60 minutes, and the kit can detect all alleles at 18 sites when detecting DNA in water bath at 100 ℃ for 80 minutes, so that the capability of obtaining a complete type from a highly degraded test material by using the kit is remarkably superior to that of a conventional degraded test material detection kit.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, the scope of the present invention is not limited thereto, and various modifications and variations which do not require inventive efforts and which are made by those skilled in the art are within the scope of the present invention.
Sequence listing
<110> university of south-middle school
<120> highly degraded test material detection kit based on 18 multiple insertion deletion genetic markers
<130> 2021010911
<141> 2021-01-09
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<211> 109
<212> DNA
<213> Artificial Sequence
<400> 46
acactcagat ttgctgtagt aaaactgtac aaaatacaat acatagtaaa atatcaacca 60
gagagaaaag acagattagc taaaaaatag aataacaatt acagagcat 109
<210> 47
<211> 125
<212> DNA
<213> Artificial Sequence
<400> 47
agccgctatc aggtgtcaag cgtgtaaggg tcactgccag gatatacttg agttgctttc 60
agctgtcata actctctaat aggatgggga tcctgaggtc tgcatacacc agtggttcac 120
atctc 125
<210> 48
<211> 122
<212> DNA
<213> Artificial Sequence
<400> 48
agccgctatc aggtgtcaag cgtgtaaggg tcactgccag gatatacttg agttttcagc 60
tgtcataact ctctaatagg atggggatcc tgaggtctgc atacaccagt ggttcacatc 120
tc 122
<210> 49
<211> 120
<212> DNA
<213> Artificial Sequence
<400> 49
agccgctatc aggtgtcaag cgtgtaaggg tcactgccag gatatacttg agttttcagc 60
tgtcataact ctaataggat ggggatcctg aggtctgcat acaccagtgg ttcacatctc 120
<210> 50
<211> 102
<212> DNA
<213> Artificial Sequence
<400> 50
tctaattgat tgtactcgtc tcttttcatt aaaatataag ttttataaag acagtaatgt 60
tgtatgttat ttaattacta catagctttc tgagtaaaga at 102
<210> 51
<211> 100
<212> DNA
<213> Artificial Sequence
<400> 51
tctaattgat tgtactcgtc tcttttcatt aaaatataag ttttatagac agtaatgttg 60
tatgttattt aattactaca tagctttctg agtaaagaat 100
<210> 52
<211> 99
<212> DNA
<213> Artificial Sequence
<400> 52
tctaattgat tgtactcgtc tcttttcatt aaaatataag ttttataaag acagtaatgt 60
tgtatgttat ttaactacat agctttctga gtaaagaat 99
<210> 53
<211> 108
<212> DNA
<213> Artificial Sequence
<400> 53
caaaggctgg gagaaataac atggacatat aatatcataa atgatgaagt ggtataagca 60
ttactagaat gtagactgtg ataagttaga gatgtatact gtatactg 108
<210> 54
<211> 105
<212> DNA
<213> Artificial Sequence
<400> 54
caaaggctgg gagaaataac atggacatat aatatcataa atgaagtggt ataagcatta 60
ctagaatgta gactgtgata agttagagat gtatactgta tactg 105
<210> 55
<211> 103
<212> DNA
<213> Artificial Sequence
<400> 55
caaaggctgg gagaaataac atggacatat aatatcataa atgaagtggt ataagcatta 60
ctagaatgta gactgataag ttagagatgt atactgtata ctg 103
<210> 56
<211> 94
<212> DNA
<213> Artificial Sequence
<400> 56
gggttggtgg actactctga ggaccggtga accagagagg agcgccaggg tcctgtctta 60
gcatgacgtc ctgatcatca agcgtgtcca gtgc 94
<210> 57
<211> 92
<212> DNA
<213> Artificial Sequence
<400> 57
gggttggtgg actactctga ggaccggtga accagaggag cgccagggtc ctgtcttagc 60
atgacgtcct gatcatcaag cgtgtccagt gc 92
<210> 58
<211> 84
<212> DNA
<213> Artificial Sequence
<400> 58
gggttggtgg actactctga gaaccagagg agcgccaggg tcctgtctta gcatgacgtc 60
ctgatcatca agcgtgtcca gtgc 84
<210> 59
<211> 82
<212> DNA
<213> Artificial Sequence
<400> 59
agcaactcta tcagaatgcc tgcaattgca agaggtttta cctgagcaga cagcagactt 60
tcttctccta ggtcttgagc tt 82
<210> 60
<211> 80
<212> DNA
<213> Artificial Sequence
<400> 60
agcaactcta tcagaatgcc tgcttgcaag aggttttacc tgagcagaca gcagactttc 60
<210> 61
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 61
agcaactcta tcagaatgcc tgcttgcaag aggttttacc tgagcagaca gcactttctt 60
ctcctaggtc ttgagctt 78
<210> 62
<211> 95
<212> DNA
<213> Artificial Sequence
<400> 62
gtcctgagac agttcatgtg ggccctcttt atgaaataca ctagatgtaa gaggtacaac 60
tgagagagag agagagagag ggccagagaa tatga 95
<210> 63
<211> 93
<212> DNA
<213> Artificial Sequence
<400> 63
gtcctgagac agttcatgtg ggccctcttt atgaaataca ctagatgtaa gaggtacaac 60
tgagagagag agagagaggg ccagagaata tga 93
<210> 64
<211> 91
<212> DNA
<213> Artificial Sequence
<400> 64
gtcctgagac agttcatgtg ggccctcttt atgaaataca atgtaagagg tacaactgag 60
agagagagag agagagggcc agagaatatg a 91
<210> 65
<211> 119
<212> DNA
<213> Artificial Sequence
<400> 65
cttctgtctc ttgccactca ttttccccga gtgactcata gaaatcagaa ttcctctttc 60
ccaagataag tcatagaaac taaaattcct tttccccaaa gcaaaccgta aagtctaaa 119
<210> 66
<211> 117
<212> DNA
<213> Artificial Sequence
<400> 66
cttctgtctc ttgccactca ttttcccagt gactcataga aatcagaatt cctctttccc 60
aagataagtc atagaaacta aaattccttt tccccaaagc aaaccgtaaa gtctaaa 117
<210> 67
<211> 114
<212> DNA
<213> Artificial Sequence
<400> 67
cttctgtctc ttgccactca ttttccccga gtgactcata gaaatcagaa ttcctctttc 60
ccaagtcata gaaactaaaa ttccttttcc ccaaagcaaa ccgtaaagtc taaa 114
<210> 68
<211> 88
<212> DNA
<213> Artificial Sequence
<400> 68
tttccaagtg caacagagat ttcatggaga aaaaaaaaat gtcaaaataa ataaataaat 60
aaataaatct acataaactg gtgggttt 88
<210> 69
<211> 86
<212> DNA
<213> Artificial Sequence
<400> 69
tttccaagtg caacagagat ttcatggaga aaaaaaatgt caaaataaat aaataaataa 60
ataaatctac ataaactggt gggttt 86
<210> 70
<211> 84
<212> DNA
<213> Artificial Sequence
<400> 70
tttccaagtg caacagagat ttcatggaga aaaaaaaaat gtcaaaataa ataaataaat 60
aaatctacat aaactggtgg gttt 84
<210> 71
<211> 116
<212> DNA
<213> Artificial Sequence
<400> 71
taaatcatag aatggtcaca caaaggattg ttaaactgct attaaaagtt atgctatcaa 60
agaatactta atgacacagg aaaacactca tgatacagtg ttaactggaa gataat 116
<210> 72
<211> 113
<212> DNA
<213> Artificial Sequence
<400> 72
taaatcatag aatggtcaca caaaggattg ttaaactgct attaaaagtg ctatcaaaga 60
atacttaatg acacaggaaa acactcatga tacagtgtta actggaagat aat 113
<210> 73
<211> 109
<212> DNA
<213> Artificial Sequence
<400> 73
taaatcatag aatggtcaca caaaggattg ttaaactgct attaaaagtg ctatcaaaga 60
ataatgacac aggaaaacac tcatgataca gtgttaactg gaagataat 109
<210> 74
<211> 119
<212> DNA
<213> Artificial Sequence
<400> 74
gagctatgcc aaatgaaact gtattttttt aaactctgaa agaagaaata agagttgaga 60
ggtaacaaat tataaacttc tagaataaag accaagttta ctccacgact tatgcacct 119
<210> 75
<211> 117
<212> DNA
<213> Artificial Sequence
<400> 75
gagctatgcc aaatgaaact gtattttttt aaactctgaa agaagaaatg agttgagagg 60
taacaaatta taaacttcta gaataaagac caagtttact ccacgactta tgcacct 117
<210> 76
<211> 115
<212> DNA
<213> Artificial Sequence
<400> 76
gagctatgcc aaatgaaact gtattttttt aaactctgaa agaagaaatg agttgagagg 60
taacaaatta taaacttaga ataaagacca agtttactcc acgacttatg cacct 115
<210> 77
<211> 128
<212> DNA
<213> Artificial Sequence
<400> 77
gtttaacagt ctggtcagga aacgtgattt cttttcttcc atctgggtaa tgtttttctg 60
tttaaaaagt tgttacagta aatatttttt gaaggaaggg aagaatttaa tgagagggtg 120
gagcaagt 128
<210> 78
<211> 125
<212> DNA
<213> Artificial Sequence
<400> 78
gtttaacagt ctggtcagga aacgtgattt cttttcttcc atctgggtaa tgtttttctg 60
tttaaaaagt tacagtaaat attttttgaa ggaagggaag aatttaatga gagggtggag 120
caagt 125
<210> 79
<211> 124
<212> DNA
<213> Artificial Sequence
<400> 79
gtttaacagt ctggtcagga aacgtgattt cttttcttcc atctgggtaa tgtttttctg 60
tttaaaaagt tgttacagta aatatttttt gaaggaaggg aagtaatgag agggtggagc 120
aagt 124
<210> 80
<211> 89
<212> DNA
<213> Artificial Sequence
<400> 80
tggaatagat aattgagctt gttaatatat gtattacttc atattcttac tttttgtgtg 60
tgaggagaac acttaacatt ctcttaaca 89
<210> 81
<211> 86
<212> DNA
<213> Artificial Sequence
<400> 81
tggaatagat aattgagctt gttaatatat gtattacttc atattcttac tttttgtgtg 60
tgagaacact taacattctc ttaaca 86
<210> 82
<211> 82
<212> DNA
<213> Artificial Sequence
<400> 82
tggaatagat aattgagctt gttaatatat gtattacttc atattctttt tgtgtgtgag 60
aacacttaac attctcttaa ca 82
<210> 83
<211> 126
<212> DNA
<213> Artificial Sequence
<400> 83
ttggtcacct atggaaatgt ttctaatgat gatgtgaaaa taatcactct agattcattt 60
ttttattaaa aaactttttt aaatgtttaa gcaaatacaa aagactaaag gactgtagag 120
<210> 84
<211> 123
<212> DNA
<213> Artificial Sequence
<400> 84
ttggtcacct atggaaatgt ttctaatgat gtgaaaataa tcactctaga ttcatttttt 60
tattaaaaaa cttttttaaa tgtttaagca aatacaaaag actaaaggac tgtagagtga 120
gga 123
<210> 85
<211> 121
<212> DNA
<213> Artificial Sequence
<400> 85
ttggtcacct atggaaatgt ttctaatgat gtgaaaataa tcactcgatt cattttttta 60
ttaaaaaact tttttaaatg tttaagcaaa tacaaaagac taaaggactg tagagtgagg 120
a 121
<210> 86
<211> 101
<212> DNA
<213> Artificial Sequence
<400> 86
caacaccctg aagagaagac aagctgtcgg cagccggatg cctgccttgc attcggcacc 60
ccccatgctg gaggcgggtg ggaaagaagg gttgggagtc t 101
<210> 87
<211> 98
<212> DNA
<213> Artificial Sequence
<400> 87
caacaccctg aagagaagac aagctgtcgg ggatgcctgc cttgcattcg gcagcccccc 60
catgctggag gcgggtggga aagaagggtt gggagtct 98
<210> 88
<211> 96
<212> DNA
<213> Artificial Sequence
<400> 88
caacaccctg aagagaagac aagctgtcgg ggatgcctgc cttgcattcg gcacccccca 60
tgctggaggc gggtgggaaa gaagggttgg gagtct 96
<210> 89
<211> 119
<212> DNA
<213> Artificial Sequence
<400> 89
gatgttacag gtgatggaat tagctgttgt tttctccttt attggagtgg ggattttttg 60
tttgtttgtt tgtttgtttg ttttttgaga cagagttttg ctcttgttgc ccaggaggc 119
<210> 90
<211> 115
<212> DNA
<213> Artificial Sequence
<400> 90
gatgttacag gtgatggaat tagctgttgt tttctccttt attggagtgg ggattttttg 60
tttgtttgtt tgtttgtttt ttgagacaga gttttgctct tgttgcccag gaggc 115
<210> 91
<211> 113
<212> DNA
<213> Artificial Sequence
<400> 91
gatgttacag gtgatggaat tagctgttgt tttctccttt attggagtgg ggattttttg 60
tttgtttgtt tgtttgtttt ttgagacagt tttgctcttg ttgcccagga ggc 113
Claims (7)
1. A highly degradable test material detection kit based on 18 multiple insertion deletion genetic markers is characterized by comprising: separating the packaged composite amplification primer mixture, the allelic typing standard mixture and the DNA standard; wherein the composite amplification primer mixture comprises 37 amplification primers with 18 multiple insertions and deletion of genetic markers; the allelic typing standard mixture consists of 18 allelic standards with multiple insertion deletion genetic markers, and 54 DNA fragments with fluorescein markers are included; the 18 multiple insertion/deletion genetic markers comprise insertion/deletion sites shown in table 1:
TABLE 1 multiple indel genetic markers comprising insertion/deletion sites
2. The kit of claim 1, wherein the DNA standard is standard DNA 9948.
3. The kit according to claim 1, wherein the nucleotide sequence of the composite amplification primer is shown in table 2:
TABLE 2 nucleotide sequences of amplification primers
4. The kit of claim 1, wherein the nucleotide sequence of the allelic standard in the mixture of allelic standards is shown in table 3:
TABLE 3 nucleotide sequence of allele standard in the allele standard mixture
Wherein the sequence before the "-" symbol is a fluorescent dye label.
5. Use of the kit of any one of claims 1 to 4 for the identification of highly degraded materials.
6. A highly degradable test material identification method realized based on the kit of any one of claims 1 to 4 is characterized by comprising the following specific steps:
(1) extracting DNA of a sample to be detected;
(2) performing single-tube multiplex amplification on the DNA extracted in the step (1) by using the kit of any one of claims 1-4; the cycle parameters of the reaction of the composite amplification PCR are 95 ℃ and 5 minutes; 29 cycles of 95 ℃, 30 seconds, 60 ℃, 72 ℃, 30 seconds; then 60 minutes at 72 ℃; storing at 4 deg.C;
(3) and mixing the amplified product and the allele typing standard mixture with a molecular weight internal standard and Hi-Di formamide respectively, and then carrying out capillary electrophoresis analysis to obtain 18 multiple insertion deletion genetic marker typing results of the sample.
7. The method according to claim 6, wherein the volume ratio of the amplification product to the molecular weight internal standard is 1: 9, the volume ratio of the allele typing standard mixture to Hi-Di formamide is 1: 0.2.
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