CN116083550A

CN116083550A - Detection method, primer group, kit and application of short tandem repeat sequence

Info

Publication number: CN116083550A
Application number: CN202210857606.1A
Authority: CN
Inventors: 张; 王玉芳; 黄雨果; 朱强; 陈晓刚; 宋凤; 罗海玻
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-07-20
Filing date: 2022-07-20
Publication date: 2023-05-09

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a detection method, a primer group, a kit and application of a short tandem repeat sequence. Compared with the traditional STR detection method based on polymerase chain reaction amplification and capillary electrophoresis detection, the invention combines the polymerase chain reaction amplification technology with the high-throughput sequencing technology for STR detection, can realize flexible setting of PCDR primers and complete detection of different PCDR amplicons, and effectively improves the amplification efficiency of DNA samples; meanwhile, STR allele sequence polymorphism information can be obtained, the formation of stutter products in the STR amplification process is reduced, and the analysis of low-copy and mixed DNA samples is facilitated.

Description

Detection method, primer group, kit and application of short tandem repeat sequence

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a detection method, a primer group, a kit and application of a short tandem repeat sequence.

Background

Polymerase chain reaction (Polymerase Chain Reaction, PCR) is an amplification technique widely used in the forensic DNA analysis field. The PCR technology is used to amplify the short tandem repeat (Short Tandem Repeat, STR) of human genome microsatellite loci and the capillary electrophoresis (Capillary Electrophoresis, CE) is combined to detect the length of the fluorescent-labeled amplicon fragments, so that the DNA map of the individual is a conventional method for personal identification and parent identification in modern forensic science. The PCR reaction combines the specific front and back primers designed for the target locus with the DNA template, and the amplification product of the target locus is doubled after each cycle is theoretically finished through three steps of template denaturation, primer annealing and primer extension, so that the exponential amplification of the DNA template is realized. However, in practical cases, the DNA template is not successfully replicated every cycle due to the effects of competition between the template and the primer, substrate consumption, accumulation of products, reduced enzyme activity, and the like. When the copy number of the initial DNA template is low, the replication process of the DNA molecule is greatly affected by the efficiency of PCR, and the PCR amplification result may have larger variability (random effect of PCR). GC content of an allele, difference in allele length, degradation degree of sample DNA, primer binding efficiency, thermal cycling conditions may all lead to differential amplification of a certain allele, leading to reduced peak height balance of the allele in a DNA map and even loss of the allele.

The specificity of the DNA sequence itself can also affect the PCR amplification process. STR loci are made up of multiple tandem repeats of a relatively constant core sequence (motif). In DNA maps, the allele peak of the STR locus is often preceded and followed by one or more smaller peaks, known as stutter peaks, at positions spaced one or more repeat motifs. stutter is a common spurious peak during STR amplification, which is believed to be caused by DNA strand "slipping" during STR allele replication and increases as the amount of DNA template decreases. Thus, STR typing results for low copy DNA and large proportions of mixed DNA templates are more susceptible to stutter products. When allele peaks are low, stutter peaks may be erroneously identified as allele peaks, resulting in interpretation of single source DNA maps as mixed DNA, misleading the investigation, reconnaissance and interrogation process of cases. Whereas for a plurality of bulk mixed DNA maps with unbalanced proportions, the stutter peak of the major contributor allele may be mistaken for the allele peak of the minor contributor, leading to false evidence results.

Polymerase chain displacement reaction (Polymerase Chain Displacement Reaction, PCDR) is another efficient method of DNA amplification. PCDR uses two or more pairs of primers to amplify the template sequence, allowing the benefits of both PCR and strand displacement amplification reactions to be exploited. Due to the use of thermostable SD DNA polymerases with strong strand displacement activity, PCDR is compatible with the same thermal cycling conditions as PCR, and all amplification reactions are completed in the same tube system. During primer annealing, specific inner and outer primers bind simultaneously to both sides of the target locus and are extended by SD polymerase. When the outer primer extends to the inner primer binding site, the strand displacement activity of SD polymerase can displace the inner primer extension strand, allowing the outer primer extension to proceed (see FIG. 1). Therefore, the target gene locus can be replicated for multiple times in a single reaction by arranging different numbers of inner and outer primer pairs, so that remarkable amplification efficiency improvement is brought. Successful binding and extension of the primer to the template is a key element in determining whether the target locus can be successfully amplified. In PCDR reaction, the use of nested inner and outer primers further increases the likelihood of primer binding to the template, allowing for a more efficient accumulation of low copy DNA template at the early stages of the reaction and ultimately successful detection after efficient amplification of PCDR, thereby increasing the allele detection rate of the DNA map. Meanwhile, research shows that PCDR can effectively reduce the generation of stutter byproducts in the STR sequence replication process, and reduce the interference of stutter on DNA maps of low-copy samples and mixed samples. This stutter "inhibitory effect" of PCDR may be related to the blocking of the DNA template by the inner primer extension strand during template extension, resulting in an enhanced stability of the extension complex consisting of the extension primer, template, and DNA polymerase.

Studies have shown that PCDR can be used for amplification and detection of forensic STRs and achieve complete compatibility with conventional PCR-CE detection systems. However, introducing a strand displacement reaction during amplification can significantly increase the complexity of the amplified product. Taking the PCDR reaction of two pairs of inner and outer primers as an example, four amplicons of different lengths are generated after primer extension and strand displacement. These amplicons contain unique terminal sequences (primer sequences) and a consensus STR sequence. Conventional CE-based STR typing methods simply differentiate loci and alleles based on amplicon fragment length, and four amplicons generated by internal and external primer combinations during PCDR will exacerbate DNA profile complexity, resulting in overlapping allele and locus signal peaks. Thus, CE-based assays can only detect partial products of PCDR and require PCDR amplification using single-sided peripheral primers, which greatly limits the primer design of PCDR and its enhancement of amplification efficiency.

Disclosure of Invention

The invention aims to provide a detection method, a primer group and a kit for a short tandem repeat sequence, wherein the detection method can realize flexible setting of PCDR primers, can detect more allelic polymorphism, reduces the formation of stutter products in the STR amplification process, effectively improves the amplification efficiency of DNA samples, and is beneficial to detection of low-copy and mixed DNA samples.

The invention provides a detection method of a short tandem repeat sequence, which comprises the steps of carrying out PCDR amplification on a DNA sample to be detected by using STR primers, carrying out high-throughput sequencing on a product obtained by PCDR amplification, realizing complete detection of different amplicons of the PCDR, and obtaining sequence information of the STR; the STR primers comprise two pairs of nested primers.

Preferably, the STR primers include a front primer FW, a rear primer RV, a front peripheral primer OF and a rear peripheral primer OR;

the front peripheral primer OF is located upstream OF the 5 'end OF the front primer FW, and the rear peripheral primer OR is located upstream OF the 5' end OF the rear primer RV.

Preferably, the number of the short tandem repeat sequences is equal to or more than 2; each short tandem repeat sequence includes a set of STR primers.

Preferably, the short tandem repeat sequence comprises a forensic autosomal STR locus and/or a sexing locus.

Preferably, the forensic autosomal STR locus comprises any one or more of CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, penta D, penta E, TH01, TPOX and VWA; the sexing locus is Amelogenin.

Preferably, the PCDR amplification is a thermocycling amplification;

the thermal cycle amplification comprises an initial denaturation stage, a pre-amplification stage, a conventional amplification stage and a final extension stage;

the initial denaturation phase: pre-denaturation at 92 ℃ for 2min;1 cycle; the pre-amplification stage: denaturation at 92℃for 0.5min, annealing at 62℃to 60℃for 0.2℃per cycle for 1min, and extension at 68℃for 1.5min;10 cycles;

the conventional amplification stage: denaturation at 92℃for 0.5min, annealing at 60℃for 1min and extension at 68℃for 1.5min;18 cycles;

the final extension stage: extending at 68 ℃ for 10min;1 cycle.

Preferably, the reagents for the PCDR amplification include: SD polymerase, SD polymerase reaction buffer, mgCl ₂ dNTP mix and water.

The invention also provides a primer group for detecting the short tandem repeat sequence of the detection method in the technical scheme, wherein the short tandem repeat sequence comprises a forensic autosomal STR locus and/or a sex identification locus; the forensic autosomal STR loci include any one or more of CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, penta D, penta E, TH01, TPOX and VWA; the sexing locus is Amelogenin;

The front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the CSF1PO are respectively shown in SEQ ID NO. 1-4;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D10S1248 are respectively shown in SEQ ID NO. 5-8;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D12S391 are respectively shown in SEQ ID NO. 9-12;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D13S317 are respectively shown in SEQ ID NO. 13-16;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D16S539 are respectively shown in SEQ ID NO. 17-20;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D18S51 are respectively shown in SEQ ID NO. 21-24;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D19S433 are respectively shown in SEQ ID NO. 25-28;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D1S1656 are respectively shown in SEQ ID NO. 29-32;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D21S11 are respectively shown in SEQ ID NO. 33-36;

The front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D22S1045 are respectively shown in SEQ ID NO. 37-40;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D2S1338 are respectively shown in SEQ ID NO. 41-44;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D2S441 are respectively shown in SEQ ID NO. 45-48;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D3S1358 are respectively shown in SEQ ID NO. 49-52;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D5S818 are respectively shown in SEQ ID NO. 53-56;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D7S820 are respectively shown in SEQ ID NO. 57-60;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D8S1179 are respectively shown in SEQ ID NO. 61-64;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the FGA are respectively shown in SEQ ID NO. 65-68;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the Penta D are respectively shown in SEQ ID NO. 69-72;

The front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the Penta E are respectively shown in SEQ ID NO. 73-76;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the TH01 are respectively shown in SEQ ID NO. 77-80;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the TPOX are respectively shown in SEQ ID NO. 81-84;

the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the VWA are respectively shown in SEQ ID NO. 85-88;

the front primer FW and the rear primer RV of the Amelogenin are respectively shown in SEQ ID NO. 89-90.

The invention also provides a kit for detecting the short tandem repeat sequence of the detection method in the technical scheme, which comprises the primer set in the technical scheme.

The invention also provides an application of the detection method, the primer group or the kit in forensic identification.

The beneficial effects are that:

the invention provides a detection method of a short tandem repeat sequence, which comprises the steps of carrying out PCDR amplification on a DNA sample to be detected by using STR primers, and carrying out high-throughput sequencing on a product obtained by PCDR amplification to obtain sequence information of STR; the STR primers comprise two pairs of nested primers.

Compared with the traditional STR detection method based on polymerase chain reaction (Polymerase Chain Reaction, PCR) amplification and capillary electrophoresis (Capillary Electrophoresis, CE) detection, the invention combines the PCDR amplification technology with the high-throughput sequencing technology for STR detection, and has the following advantages:

1) Has higher amplification efficiency. The present invention provides two nested pairs of specific primers for each STR locus and uses DNA polymerase with thermal stability and strong strand displacement activity to perform PCDR amplification reactions. During primer extension, the outer primer extension strand can displace the inner primer extension strand, and the DNA template strand is amplified twice per reaction cycle (see FIG. 1). In subsequent reactions, longer amplicons formed by peripheral primer extension can serve as templates for inner primer short amplicons, further amplifying the amplification efficiency. A single DNA molecule can theoretically be produced after n rounds of PCDR cycle amplification (n ² +5n+4)×2 ^n-2 Individual amplicons, far higher than 2 amplified by PCR ⁿ And each amplicon. Therefore, compared with the traditional PCR method, the invention has higher amplification efficiency and is beneficial to the detection and analysis of low-copy DNA samples.

2) With lower stutter byproducts. The STR locus sequence is formed from multiple tandem repeats of a relatively fixed motif. The low complexity of the tandem repeat sequence is prone to relative slippage of the template strand and the primer extension strand during polymerase replication, forming the stutter product. stutter products are typically one repeat motif less than STR allele products and increase as the amount of DNA template decreases. Under certain conditions, the stutter product may be misjudged as an allele, thereby interfering with the correct typing of the DNA sample. The invention introduces a strand displacement process in the STR allele amplification process, the inner primer extends before the outer primer, and the extension chain temporarily seals the STR repeated sequence, thereby enhancing the stability of an extension complex formed by a DNA template, a primer extension chain and a DNA polymerase, reducing the possibility of relative sliding of the template chain and the primer extension chain, and further reducing the formation of stutter products. The reduction of stutter facilitates correct typing of alleles and improves the accuracy of low copy DNA and mixed DNA analysis.

3) Complete PCDR product detection. Through PCDR amplification, two nested pairs of primers can produce four amplification products, each with a different terminal sequence (primer sequence) and the same core sequence (STR sequence). Common CE-based detection methods do not allow for efficient discrimination between these amplicons of varying lengths, resulting in overlapping allelic peaks within and between loci. The invention adopts high-throughput sequencing technology (MPS technology) to detect PCDR products for the first time, and can completely detect all amplicon types through high-throughput sequencing. Based on this, the present invention does not require the use of fluorophores to pre-label the primers nor the need to distinguish between loci and alleles by amplicon length, thus allowing for flexible primer placement and simultaneous amplification of more loci. In addition, as the sequence information of STR can be directly obtained, the detection method can detect the sequence variation of STR alleles at the same time, and the identification capability of loci is improved.

4) Single tube targeting multiplex amplification is compatible with PCR thermal cycle reaction. The invention can realize single-tube multiplex amplification of a plurality of loci and has wide instrument compatibility.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a schematic diagram of the PCDR amplification STR locus;

FIG. 2 shows the product concentration of a gradient diluted DNA sample amplified by a multiplex PCDR and multiplex PCR system;

FIG. 3 shows the average coverage of each locus for composite PCDR and composite PCR amplification products.

Detailed Description

The invention provides a detection method of a short tandem repeat sequence, which comprises the steps of carrying out PCDR amplification on a sample to be detected by using STR primers, and carrying out high-throughput sequencing on a product obtained by PCDR amplification to obtain sequence information of STR; the STR primers comprise two pairs of nested primers.

The invention uses STR primer to carry out PCDR amplification to the DNA sample to be detected, and obtains PCDR amplified product. The number of the short tandem repeat sequences in the invention is preferably not less than 2, more preferably 10 to 30. Each short tandem repeat OF the present invention comprises a set OF STR primer pairs, each set comprising two nested pairs OF primers, preferably comprising a front primer FW, a rear primer RV, a front peripheral primer OF and a rear peripheral primer OR. The front peripheral primer OF the present invention is preferably located upstream OF the 5 'end OF the front primer FW, and the rear peripheral primer OR is located upstream OF the 5' end OF the rear primer RV.

The invention is not particularly limited in the kind and number of the short tandem repeat sequences, and any short tandem repeat sequence can be determined by the detection method. In the practice of the invention, the short tandem repeat sequences preferably comprise a forensic autosomal STR locus and/or a sexing locus, more preferably comprise a forensic autosomal STR locus and a sexing locus; the forensic autosomal STR loci include any one or more of CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, pentad, pentae, TH01, TPOX, and VWA, more preferably include CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S 117820, D8S 9, FGA, pentad 818, TPOX, and VWA; the sexing locus is preferably Amelogenin. Although the detection method of the present invention will be described by taking the above-described short tandem repeat sequences as an example, it is not intended that only the above-described types and numbers of short tandem repeat sequences be regarded as the full scope of the present invention.

The PCDR amplification of the present invention is preferably a thermocycling amplification; the thermocycling amplification preferably comprises an initial denaturation phase, a pre-amplification phase, a conventional amplification phase and a final extension phase; amplification procedure of the initial denaturation phase: pre-denaturation at 92 ℃ for 2min;1 cycle; amplification procedure of the pre-amplification stage: denaturation at 92℃for 0.5min, annealing at 62℃to 60℃for 0.2℃per cycle for 1min, and extension at 68℃for 1.5min;10 cycles; amplification procedure of the conventional amplification stage: denaturation at 92℃for 0.5min, annealing at 60℃for 1min and extension at 68℃for 1.5min;18 cycles; amplification procedure for the final extension phase: extending at 68 ℃ for 10min;1 cycle. The thermocycling amplification according to the invention preferably further comprises a maintenance phase, which maintains the state of the amplification product, preferably at 4 ℃.

The reagent for PCDR amplification of the present invention preferably comprises: SD polymerase, SD polymerase reaction buffer, mgCl ₂ dNTP mix and water. The DNA polymerase of the present invention is preferably SDDNA polymerase which is thermostable and has strong strand displacement activity. In the practice of the present invention, the concentration of the SD polymerase of the present invention is preferably 10 to 100U/. Mu.L, more preferably 50U/. Mu.L, and the SD polymerase is preferably available from Bioron, germany. The SD polymerase reaction buffer according to the invention is preferably a 10 XSD polymerase reaction buffer, preferably from Bioron, germany. MgCl according to the invention ₂ Preferably MgCl ₂ Solution of MgCl ₂ The concentration of the solution is preferably 10 to 200mM, more preferably 100mM; the MgCl ₂ Preferably from Thermo Fisher Scientific, U.S.; the concentration of dNTP mix according to the present invention is preferably 1 to 100mM, more preferably 10mM, and is preferably available from Promega, USA. The system for PCDR amplification of the present invention preferably includes the reagents for PCDR amplification, STR primers, and genomic DNA. The PCDR amplification system of the present invention is preferably a multiplex PCDR amplification system, i.e., single tube multiplex amplification of multiple STR loci; the PCDR amplification system is preferably as shown in Table 1:

TABLE 1 PCDR amplification System

Composition of the components	PCDR amplification system
		SD polySynthase reaction buffer (10×)	2.0μL
Genomic DNA	1.0μL(0.05～10ng)
		MgCl ₂	0.75μL
dNTP mix	1.0μL
		Primer mixture	2.5μL
SD polymerase	0.05μL
		H ₂ O	Make up the volume to 25 mu L

The primer mixture in Table 1 OF the present invention is preferably a mixed primer OF 1 OR more sets OF STR primers, each set OF STR primers preferably comprising a front primer FW, a rear primer RV, a front peripheral primer OF and a rear peripheral primer OR. The reaction concentrations OF the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR in the present invention are preferably 0.05 to 1. Mu.M, more preferably 0.1 to 0.8. Mu.M. The amount of the genomic DNA in Table 1 according to the present invention is preferably 0.05 to 10ng, more preferably 0.5ng.

After the PCDR amplification product is obtained, the invention carries out high-throughput sequencing on the PCDR amplification product to obtain the sequence information of STR. The present invention preferably further includes purifying the PCDR amplification product before the high throughput sequencing, and the purification method is not particularly limited, and any purification method conventional in the art may be used. MinElute PCR Purification Kit, available from QIAGEN, germany, is used in the practice of the invention.

The high throughput sequencing of the present invention preferably includes library construction, sequencing reactions, and data analysis. The process of constructing the library is not particularly limited, and conventional library construction processes in the art can be adopted, and in the specific implementation process of the invention, a NEBNext Ultra DNA Library Prep Kit for Illumina kit is adopted and is purchased from New England Biolabs, U.S.A. In the invention, a single library constructed on a single sample is preferably mixed into a mixed library and then subjected to a sequencing reaction.

The sequencing reaction according to the invention is preferably double-ended 250bp sequencing. The sequencer used in the sequencing reaction is not particularly limited, and a conventional sequencer in the art may be used, and in the specific implementation process of the present invention, an Illumina Novaseq 6000 sequencer (Illumina, usa) is used.

After the sequencing reaction is completed, the invention preferably performs data analysis on the data obtained by the sequencing reaction to obtain the allele parting information of the STR. The data analysis according to the invention preferably comprises quality control, splicing, amplicon extraction and allele typing. The specific process and software of the data analysis are not particularly limited, and conventional processes and software in the art can be adopted. In the practice of the invention, the quality control is preferably performed using fastp v0.23.1, removing reads with Phred quality values less than 15 or unidentified bases greater than 5. The invention preferably uses FLASH v1.2.11 to splice the sequences after quality control. The invention preferably employs Seqkit v2.0.0 to extract amplicons from the spliced sequences; the amplicon preferably comprises four primer sequences OF FW+RV, OF+RV, FW+RV and OF+OR, and is obtained by amplification. The invention preferably uses the FDSTools2.0 software package to genotype the extracted amplicon. The present invention preferably makes the following discrimination of the data after the allele typing: n-1 stutter is defined as a sequence 1 STR motif fewer than the allele sequence; n-2 stutter is defined as a sequence of 2 STR motifs less than the allele. The Stutter Ratio (SR) is the Ratio of the number of Stutter sequence reads to the number of corresponding allele reads. Heterozygote balance (Heterozygote Balance, hb) is defined as the ratio of the number of low coverage alleles to the number of high coverage alleles at the heterozygous locus. The mean differences between the groups were statistically tested using a two-tailed Mann-Whitney U test, and if the p-value was less than 0.05, the mean differences between the two groups were considered statistically significant.

The invention also provides a primer group for detecting the short tandem repeat sequence of the detection method in the technical scheme, wherein the short tandem repeat sequence comprises a forensic autosomal STR locus and/or a sex identification locus; the forensic autosomal STR loci include any one or more of CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, penta D, penta E, TH01, TPOX and VWA; the sexing locus is Amelogenin; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the CSF1PO are respectively shown in SEQ ID NO. 1-4; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D10S1248 are respectively shown in SEQ ID NO. 5-8; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D12S391 are respectively shown in SEQ ID NO. 9-12; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D13S317 are respectively shown in SEQ ID NO. 13-16; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D16S539 are respectively shown in SEQ ID NO. 17-20; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D18S51 are respectively shown in SEQ ID NO. 21-24; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D19S433 are respectively shown in SEQ ID NO. 25-28; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D1S1656 are respectively shown in SEQ ID NO. 29-32; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D21S11 are respectively shown in SEQ ID NO. 33-36; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D22S1045 are respectively shown in SEQ ID NO. 37-40; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D2S1338 are respectively shown in SEQ ID NO. 41-44; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D2S441 are respectively shown in SEQ ID NO. 45-48; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D3S1358 are respectively shown in SEQ ID NO. 49-52; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D5S818 are respectively shown in SEQ ID NO. 53-56; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D7S820 are respectively shown in SEQ ID NO. 57-60; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the D8S1179 are respectively shown in SEQ ID NO. 61-64; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the FGA are respectively shown in SEQ ID NO. 65-68; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the Penta D are respectively shown in SEQ ID NO. 69-72; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the Penta E are respectively shown in SEQ ID NO. 73-76; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the TH01 are respectively shown in SEQ ID NO. 77-80; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the TPOX are respectively shown in SEQ ID NO. 81-84; the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR OF the VWA are respectively shown in SEQ ID NO. 85-88; the front primer FW and the rear primer RV of the Amelogenin are respectively shown in SEQ ID NO. 89-90. The forensic autosomal STR loci of the present invention preferably include CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, penta D, penta E, TH01, TPOX and VWA. The sex determination locus Amelogenin of the present invention is not provided with peripheral primers.

In the present invention, the sequences and concentrations OF the front primer FW, the rear primer RV, the front and rear peripheral primers OF, OR, and the sequences and concentrations OF the front primer FW, the rear primer RV OF Amelogenin OF CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, penta D, penta E, TH01, TPOX, and VWA are shown in table 2.

TABLE 2 specific sequences OF the front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR, their concentrations in the primer mixture

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The invention also provides a kit for detecting the short tandem repeat sequence of the detection method in the technical scheme, which comprises the primer set in the technical scheme. The kit of the present invention preferably further comprises reagents for PCDR amplification and/or reagents for high throughput sequencing, more preferably comprises reagents for PCDR amplification and reagents for high throughput sequencing. The reagent for PCDR amplification in the present invention is preferably the same as that in the above technical scheme, and will not be described in detail. The reagent for high-throughput sequencing is not particularly limited, and can be a reagent conventional in the art, and in the specific implementation process of the invention, the reagent for high-throughput sequencing is preferably the same as that in the technical scheme, and is not described in detail herein.

The invention also provides an application of the detection method, the primer group or the kit in forensic identification. The invention is a technical scheme for combining PCDR technology and high-flux sequencing technology for the first time to identify short tandem repeat sequences, especially forensic STR. In the invention, each STR genome uses two pairs of nested inner and outer primers to carry out PCDR amplification, and compared with the conventional PCR amplification method, the amplification efficiency of the DNA sample can be effectively improved. Meanwhile, based on the high-throughput characteristic of MPS high-throughput sequencing and the resolution of the base level, the invention can realize flexible setting of PCDR primer without increasing the complexity of the map, and can detect more allelic polymorphism. In addition, the invention effectively reduces the formation of stutter products in the STR amplification process, and is beneficial to the detection of low copy and mixed DNA samples. Based on the detection method, the primer group and the kit disclosed by the invention can be applied to forensic identification, and a technical basis is provided for forensic identification.

The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.

Unless otherwise specified, the assay methods employed in the examples of the present invention are conventional in the art, and the reagents employed are available from conventional sources.

Example 1

Primer design of STR gene locus:

in this example 22 autosomal STRs and 1 individual identification loci were selected to construct a composite PCDR amplification system. The forensic STR loci involved include: CSF1PO, D10S1248, D12S391, D13S317, D16S539, 18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, penta D, penta E, TH01, TPOX, VWA. The sexing locus is Amelogenin. A pair OF front primer (FW) and rear primer (RV) is used for each STR locus, and a pair OF front peripheral primer (OF) and rear peripheral primer (OR) located outside the front and rear primers. The two pairs of primers are used for carrying out specific amplification on STR loci respectively, wherein the two peripheral primers are used for starting displacement reaction of an inner primer extension chain. The PCDR amplification principle is shown in FIG. 1. The sex determination locus Amelogenin is not provided with peripheral primers. The primer sequences and concentrations of the 23 loci of the present invention are shown in Table 2 below, and will not be described in detail.

Preparing a gradient dilution DNA template:

2800M standard DNA was purchased from Promega corporation (USA). The standard was quantified on a 7500 Real-time PCR System (Applied Biosystems, USA) using Investigator Quantiplex Kit PCR Assay (QIAGEN, germany). According to the quantitative results, the standard was diluted with ultrapure water to 500 pg/. Mu.L, 250 pg/. Mu.L, 125 pg/. Mu.L, 62.5 pg/. Mu.L, 31.3 pg/. Mu.L, 15.6 pg/. Mu.L, respectively. Preserving at-20 ℃ for standby.

Configuration of a multiplex amplification system:

a composite PCDR reaction primer set was constructed based on the primers and concentrations shown in Table 2, and a corresponding composite PCR control reaction was set. Wherein, the compound PCDR system is an experimental system for implementing the invention, and the compound PCR system is a control system, which are different from each other in that: the composite PCDR system contained FW, RV, OF and OR primers for the 22 STR loci in Table 2, and FW, RV primers for the Amelogenin locus; the multiplex PCR system included only FW and RV primers in table 1. Since there are no peripheral primers OF and OR to initiate the strand displacement reaction, each locus in the multiplex PCR system is amplified by PCR and other conditions are fully consistent with the multiplex PCDR system to illustrate the principles and advantages OF the present invention.

The reagents used in the composite PCDR and composite PCR reaction system include: SD polymerase 50U/. Mu.L (Bioron, germany); 2.10×SD polymerase reaction buffer (Bioron, germany); mgCl ₂ Solution 100mM (Thermo Fisher Scientific, USA); dNTP mix 10mM (Promega, USA). Specific amplification system configurations are shown in Table 3:

TABLE 3 composite PCDR System and composite PCR System

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Wherein the primer of the composite PCDR system is FW, RV, OF, OR primer and corresponding concentration, and the primer of the composite PCR system is FW primer and RV primer.

Thermal cycle amplification:

the multiplex PCDR reaction and multiplex PCR reaction were performed in a thermal cycler. The thermal cycler amplification procedure is shown in table 4.

TABLE 4 multiplex PCDR and multiplex PCR thermal cycle amplification procedure

The amplified product was stored at 4℃in the absence of light.

Sequencing library construction and sequencing:

the amplified product was purified by MinElute PCR Purification Kit (QIAGEN, germany) and eluted in a volume of 25. Mu.L. The purified product was assayed for purity using a NanoDrop 1000 ultramicro spectrophotometer (Thermo Fisher Scientific, U.S.) and for product concentration using Qubit dsDNA HS Assay Kit (Invitrogen, U.S.) and Qubit 4 fluorometer. Subsequently, 15. Mu.L of the purified amplified product was used for sequencing library construction.

The amplified product was subjected to end repair and A tail addition using NEBNext Ultra DNA Library Prep Kit for Illumina kit (New England Biolabs, USA) and then ligated to the full-length adapter of NEBNext Multiplex Oligos for Illumina (New England Biolabs, USA) to obtain the complete sequencing template. The library was purified and fragment screened using 1.3 x Agencourt AMPure XP magnetic beads (Beckman Coulter, usa). Library quality was assessed on a 2100 Bioanalyzer (Agilent Technologies, usa) using the Agilent DNA 1000 Kit (Agilent Technologies, usa). The library was then precisely quantified using KAPA Library Quantification Kits (KAPA Biosystems, usa) on a 7500 Real-time PCR System (Applied Biosystems, usa).

After quantification, single libraries of each sample were diluted to 10mM and mixed to the same volume to construct the final sequencing library. The mixed library was transferred to cBot Cluster Generation System (Illumina, usa) for sequencing cluster generation, and finally double-ended 250bp sequencing was performed on Illumina Novaseq 6000 sequencer (Illumina, usa).

Sequencing data analysis:

sequencing data FASTQ file quality control using fastp v0.23.1, removing reads with a value of less than 15 for the Phred quality or greater than 5 for the unidentified base. The double ended sequencing reads were then spliced using FLASH v 1.2.11. Four amplicons generated by PCDR amplification were extracted from FASTQ files spliced by double-ended read using Seqkit v2.0.0 based on FW+RV, OF+RV, FW+RV, and OF+OR. The FASTQ file of sequencing read was allele-typed using the fdstools2.0 software package.

Allele discrimination:

n-1 stutter is defined as a sequence 1 STR motif less than the allele sequence, and N-2 stutter is defined as a sequence 2 STR motifs less than the allele sequence. The Stutter Ratio (SR) is the Ratio of the number of Stutter sequence reads to the number of corresponding allele reads. Heterozygote balance (Heterozygote Balance, hb) is defined as the ratio of the number of low coverage alleles to the number of high coverage alleles at the heterozygous locus. The mean differences between the groups were statistically tested using a two-tailed Mann-Whitney U test, and if the p-value was less than 0.05, the mean differences between the two groups were considered statistically significant.

Results data:

the product concentration of the gradient diluted DNA sample amplified by the composite PCDR and composite PCR system is shown in FIG. 2: wherein the error bars in fig. 2 indicate the data standard deviation.

From fig. 2, it can be derived that: the amounts of the products of the different amounts of DNA templates amplified by the composite PCDR system of the invention were higher than the control amplified by the composite PCR system, and the differences were statistically significant (p=8.9E-06). Taking 500pg of DNA template as an example, the concentration of the product after the amplification of the composite PCDR is 67.1+/-3.5 ng/. Mu.L, which is far higher than 8.4+/-0.6 ng/. Mu.L after the amplification of the composite PCR. The composite PCDR of the invention has higher amplification efficiency.

Example 2

Amplification of 46 single source DNA samples by the detection method of the present invention

DNA extraction and quantification

Venous blood samples in this example were provided by 46 healthy volunteers. EDTA-Na is added after vein blood collection ₂ And performing anticoagulation treatment. From venous blood using QIAmp DNA Mini Kit (QIAGEN, germany)Genomic DNA was extracted from the sample. Other widely proven methods, such as the Chelex100 method, the phenol chloroform method, etc., can also be used for DNA extraction. 9948 DNA standards were purchased from AGCU (China). DNA quantification was performed using Qubit dsDNA HS Assay Kit (Invitrogen, usa) on a Qubit 4 fluorescence quantifier. DNA quantification was performed according to the instructions provided by the kit and instrument manufacturer. According to the quantitative result, the DNA samples are diluted to 0.5 ng/. Mu.L respectively and stored at-20 ℃ for standby.

The configuration of the multiplex amplification system, the thermal cycle amplification reaction, the construction of the sequencing library, the sequencing and the data analysis in this embodiment are all performed according to the method in embodiment 1, and no detailed description is given.

Results data:

1. average coverage results for each locus of composite PCDR and composite PCR amplified products

The invention firstly carries out combination analysis on different amplification product types generated by combining the inner primer and the outer primer. For the composite PCDR system of the present invention, the average coverage of 22 STR loci is 26398+ -15997, which is higher than the average coverage of the control composite PCR system of 19188+ -13018. Subsequently, the present invention utilizes a primer sequence matching method to extract four different amplification products in the composite PCDR. As shown in FIG. 3, the method OF the present invention successfully detected all four PCDR amplification product types S, M, M2 and L generated by primer combinations FW+RV, OF+RV, FW+OR and OF+OR. Wherein the average coverage of the amplified product S is highest, M1 and M2 times, and L is least. The average read number ratio S: M1: M2: L of the four product types was about 81:12:12:1.

In FIG. 3, gross represents pooled PCDR different amplification product types; s, M1, M2 and L are the amplified products OF the primer combinations FW+RV, OF+RV, FW+OR and OF+OR, respectively, produced by PCDR. Error bars indicate data standard deviation.

2. Hybrid balance of each locus of composite PCDR and composite PCR amplified product

After 46 single source DNA samples were amplified by the composite PCDR system of the invention, the average heterozygosity of the loci was 0.80±0.17, the average heterozygosity of the loci of the composite PCR system of the control group was 0.81±0.15, and the difference was not statistically significant (p=0.65).

3. Composite PCDR and ratio of stutter for each locus of composite PCR amplified product (SR)

Combining analysis of different types of amplified products of PCDR shows that the composite PCDR system of the present invention significantly reduces the stutter product during STR allele replication. For 22 STR loci, the N-1SR of 46 samples amplified by the multiplex PCDR system is shown in Table 4, with an average N-1SR of 6.9.+ -. 3.9% for the loci, and 8.3.+ -. 4.5% for the multiplex PCR control system (p=1.4E-17). For the more rare N-2stutter, its average SR in the composite PCDR group of the invention was 0.59±0.41%, significantly lower than its average value in the composite PCR control group of 0.75±0.53% (p=1.8e-11). The invention has universality of loci on the inhibition effect of stutter products in STR amplification. Taking N-1 stutter as an example, all 22 loci in the multiplex PCDR amplification system of the present invention showed a decrease in SR, with a 73.3% average decrease in locus stutter statistically significant relative to the decrease in the PCR control.

TABLE 4 composite PCDR and composite PCR System STR loci N-1SR

Gene locus	Composite PCR (mean.+ -. Standard deviation)	Composite PCDR (mean value.+ -. Standard deviation)	Significance of the invention
				CSF1PO	8.3±2.0％	7.5±1.7％	NS
D10S1248	10.3±2.9％	9.6±2.0％	*
				D12S391	12.8±4.9％	10.7±4.4％	***
D13S317	5.3±2.2％	4.3±1.9％	**
				D16S539	5.9±4.4％	4.8±3.6％	NS
D18S51	10.5±2.5％	8.3±2.1％	***
				D19S433	11.4±3.3％	9.0±2.2％	***
D1S1656	9.9±4.5％	8.4±3.9％	*
				D21S11	12.4±1.9％	10.8±1.6％	***
D22S1045	12.4±4.3％	11.5±4.2％	NS
				D2S1338	11.1±5.2％	8.6±4.7％	***
D2S441	5.8±3.2％	5.0±2.9％	NS
				D3S1358	10.6±1.4％	8.7±1.1％	***
D5S818	7.9±2.8％	6.8±2.6％	**
				D7S820	5.6±3.5％	4.7±3.1％	NS
D8S1179	9.3±3.0％	6.6±3.4％	***
				FGA	9.8±2.5％	7.9±2.0％	***
Penta D	2.5±1.0％	2.2±0.9％	NS
				Penta E	4.6±2.2％	4.3±2.1％	NS
TH01	3.3±1.5％	2.9±2.2％	*
				TPOX	4.3±1.7％	3.1±1.1％	***
VWA	7.8±5.8％	6.2±4.8％	*

Note that: significance marking: * P <0.001; * P <0.01; p <0.05; NS, p is more than or equal to 0.05.

4. Equivalent gene of composite PCDR and composite PCR amplified product

The invention uses MPS technology (large-scale parallel sequencing) to detect amplified products, and can provide more STR sequence variation and improve polymorphism of STR loci compared with a CE-based detection method. After amplification and MPS detection by the composite PCDR system of the invention, we observed 29 alleles in 20 samples. As shown in Table 5, these alleles have the same fragment length, but have variations in the repeat segment or flanking sequences. Sequence-based allele naming convention references FDSTools package insert.

TABLE 5 Single source DNA sample STR alleles

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Comparative example 1

The EX25 fluorescence kit (AGCU, china) was used for comparison with the present invention. The kit adopts a PCR reaction to amplify 22 autosomal STR loci, and amplified products are detected and typed by CE. The use method of the kit in the comparative example is as follows:

DNA extraction and quantification: the DNA of 46 samples in example 2 was extracted and quantified and diluted as in example 2;

Amplification system configuration: the amplification system was configured according to the kit instructions. The genomic DNA template used was identical to example 2;

thermocycling amplification reaction: performing thermocycling amplification according to the instruction of the kit;

detection of amplification products: 1.0. Mu.L of the amplified product was mixed with 8.9. Mu.L of Hi-Di carboxamide (Applied Biosystems, USA) and 0.1. Mu.L of LAGCU Marker SIZ-500 molecular weight internal standard (AGCU, china) and subjected to fluorescence detection by a 3500 Genetic Analyzer (Applied Biosystems, USA) gene analyzer;

discrimination of allele peaks and stutter peaks: STR allele typing was performed using GeneMapper ID-X1.5. stutter peak is defined as the peak 1 STR motif less before the peak of the DNA map allele. The Stutter Ratio (SR) is the stutter peak height divided by the corresponding allele peak height.

Results data:

1. STR allele typing identity

Complete typing of 46 single source DNA samples was successfully obtained using EX25 kit amplification in combination with CE detection, but it only allowed for discrimination of alleles based on STR length, and not sequence-differential-only alleles.

Compared with example 2, the typing accuracy of STR allele length polymorphism using the method of combined PCDR amplification and MPS detection proposed by the present invention is 100%.

2. Stutter Ratio (SR)

The average ratio of EX25 pattern N-1stutter was 7.7±3.1%, which is significantly higher than the average SR in example 2 (p=2.6e-06). The average SR at each locus is shown in Table 6.

Average SR at the same STR locus as in the present invention in Table 6 EX25

Gene locus	SR (mean value + -standard deviation)	Gene locus	SR (mean value + -standard deviation)
				CSF1PO	7.6±1.6％	D2S441		6±1.8％
D10S1248	10.1±2％	D3S1358	10.3±1.6％
				D12S391	11.8±2.2％	D5S818	8.6±1.9％
D13S317	5.1±2.1％	D7S820	5.6±1.7％
				D16S539	6.6±1.9％	D8S1179	8±2.1％
D18S51	9±2.4％	FGA	8.2±1.5％
				D19S433	7.8±1.8％	Penta D	2.2±0.9％
D1S1656	9.3±1.6％	Penta E	5.1±2％
				D21S11	11±1.7％	TH01	2.6±0.9％
D22S1045	8.8±3.5％	TPOX	4.1±1.2％
				D2S1338	10.2±1.7％	vWA	7.9±2.9％

From this, it can be derived that: compared with the conventional forensic STR kit based on PCR-CE, the kit has the same typing accuracy and can distinguish STR allele sequence variation. Meanwhile, the invention can effectively reduce the generation of stutter byproducts in the STR amplification process.

From the above embodiments it can be derived that: the PCDR amplification technology can remarkably improve the amplification efficiency of DNA samples, and can effectively detect all amplified products of PCDR for the first time by combining with the MPS detection technology. Compared with a PCR-based method, the method can remarkably reduce the stutter byproduct in the STR allele replication process and improve the reliability of STR locus detection; meanwhile, the PCDR amplification method provided by the invention hardly influences the amplification balance of heterozygous locus alleles; in addition, the invention can also effectively detect the internal variation of STR alleles and improve the polymorphism degree of STR loci.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. A detection method of short tandem repeat sequence is characterized in that STR primer is used for PCDR amplification of DNA sample to be detected, and high flux sequencing is carried out on the product obtained by PCDR amplification to obtain sequence information of STR; the STR primers comprise two pairs of nested primers.

2. The detection method OF claim 1, wherein the STR primers comprise a front primer FW, a rear primer RV, a front peripheral primer OF and a rear peripheral primer OR;

3. The method according to claim 1 or 2, wherein the number of short tandem repeats is not less than 2; each short tandem repeat sequence includes a set of STR primers.

4. The method of claim 3, wherein the short tandem repeat sequence comprises a forensic autosomal STR locus and/or a sexing locus.

5. The method of detection of claim 4, wherein the forensic autosomal STR locus comprises any one or more of CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, penta D, penta E, TH01, TPOX and VWA; the sexing locus is Amelogenin.

6. The method of detection of claim 1, wherein the PCDR amplification is thermocycling amplification;

the final extension stage: extending at 68 ℃ for 10min;1 cycle.

7. The method according to any one of claims 1 to 2 and 4 to 6, wherein the reagent for the PCDR amplification comprises: SD polymerase, SD polymerase reaction buffer, mgCl ₂ dNTPmix and water.

8. A primer set for use in the detection of a short tandem repeat according to any one of claims 1 to 7, wherein said short tandem repeat comprises a forensic autosomal STR locus and/or a sexing locus; the forensic autosomal STR loci include any one or more of CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, penta D, penta E, TH01, TPOX and VWA; the sexing locus is Amelogenin;

9. A kit for short tandem repeat detection according to any one of claims 1 to 7, wherein said kit comprises the primer set according to claim 8.

10. Use of the detection method according to any one of claims 1 to 7, the primer set according to claim 8 or the kit according to claim 9 in forensic identification.