CN116083550B - A detection method, primer set, kit and application of short tandem repeat sequence - Google Patents

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, the polymorphism information of STR allele sequences 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

A detection method, primer set, kit and application of short tandem repeat sequence

Technical Field

The present invention belongs to the technical field of gene detection, and in particular relates to a detection method, a primer set, a kit and an application of a short tandem repeat sequence.

Background Art

Polymerase Chain Reaction (PCR) is an amplification technology widely used in the field of forensic DNA analysis. Using PCR technology to amplify short tandem repeats (STR) of microsatellite loci in the human genome and combining capillary electrophoresis (CE) to detect the length of fluorescently labeled amplicon fragments to obtain an individual's DNA profile is a conventional method for personal identification and paternity testing in modern forensic medicine. The PCR reaction uses specific front and back primers designed for the target locus to bind to the DNA template. After three steps of template denaturation, primer annealing and primer extension, theoretically, the target locus amplicon product doubles at the end of each cycle, thereby achieving exponential amplification of the DNA template. However, in actual situations, due to factors such as template and primer competition, substrate consumption, product accumulation, reduced enzyme activity, and changes in the pH of the reaction system, the DNA template cannot be successfully replicated in every cycle. When the number of copies of the starting DNA template is very low, the replication process of the DNA molecule is more affected by the PCR efficiency, and the PCR amplification results may show greater variability (the random effect of PCR). The GC content of alleles, allele length differences, sample DNA degradation, primer binding efficiency, and thermal cycling conditions may all lead to differential amplification of a certain allele, causing a decrease in the balance of allele peak heights in the DNA map or even allele loss.

The particularity of the DNA sequence itself will also affect the PCR amplification process. The STR locus is composed of multiple tandem repeats of a relatively constant core sequence (motif). In the DNA map, one or more smaller peaks often appear before and after the STR locus allele peaks, separated by one or more repeated motifs, which are called stutter peaks. Stutter is a common pseudo-peak in the STR amplification process. It is generally believed that it is caused by the "slippage" of the DNA chain during the replication of the STR allele, and increases with the decrease of the DNA template amount. Therefore, the STR typing results of low-copy DNA and large-proportion mixed DNA templates are more susceptible to the influence of stutter products. When the allele peak is low, the stutter peak may be mistakenly identified as the allele peak, resulting in the interpretation of the single-source DNA map as mixed DNA, thereby misleading the investigation, reconnaissance and trial process of the case. For the mixed DNA map of multiple individuals with unbalanced proportions, the stutter peak of the major contributor allele may be mistaken for the allele peak of the minor contributor, resulting in erroneous evidence results.

Polymerase chain displacement reaction (PCDR) is another efficient DNA amplification method. PCDR uses two or more pairs of primers to amplify the template sequence, which can simultaneously play the advantages of PCR and chain displacement amplification reaction. Due to the use of heat-resistant SD DNA polymerase with strong chain 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 the primer annealing process, specific inner and outer primers simultaneously bind to both sides of the target locus and are extended under the action of SD polymerase. When the outer primer extends to the inner primer binding position, the chain displacement activity of SD polymerase can displace the inner primer extension chain, allowing the outer primer extension to continue (see Figure 1). Therefore, by setting different numbers of inner and outer primer pairs, the target locus can be replicated multiple times in a single reaction, thereby bringing about a significant improvement in amplification efficiency. The successful binding and extension of primers and templates is the key link in determining whether the target locus can be successfully amplified. In the PCDR reaction, the use of nested inner and outer primers further increases the possibility of primer-template binding, allowing low-copy DNA templates to be more effectively accumulated in the early stages of the reaction and ultimately successfully detected after efficient amplification by PCDR, thereby increasing the allele detection rate of the DNA map. At the same time, studies have found that PCDR can effectively reduce the production of stutter byproducts during STR sequence replication and reduce the interference of stutter on the DNA maps of low-copy samples and mixed samples. This stutter "inhibitory effect" of PCDR may be related to the closure of the DNA template by the inner primer extension chain during the template extension process, which enhances the stability of the extension complex composed of the extension primer, template, and DNA polymerase.

Existing studies have shown that PCDR can be used for the amplification and detection of forensic STRs and is fully compatible with conventional PCR-CE detection systems. However, the introduction of strand displacement reactions during the amplification process will significantly increase the complexity of the amplified products. Taking the PCDR reaction of two pairs of inner and outer primers as an example, four amplicons of different lengths will be generated after primer extension and strand displacement. These amplicons contain unique terminal sequences (primer sequences) and common STR sequences. Conventional CE-based STR typing methods only distinguish loci and alleles based on the length of amplicon fragments. The four amplicons generated by the combination of inner and outer primers during the PCDR process will increase the complexity of the DNA map, resulting in overlap of allele and locus signal peaks. Therefore, the CE-based analysis method can only detect part of the PCDR products, and requires the use of unilateral peripheral primers for PCDR amplification, which greatly limits the primer design of PCDR and its effect on improving amplification efficiency.

Summary of the invention

The purpose of the present invention is to provide a detection method, a primer set and a kit for short tandem repeat sequences. The detection method can realize flexible setting of PCDR primers, detect more allele polymorphisms, reduce the formation of stutter products during STR amplification, effectively improve the amplification efficiency of DNA samples, and facilitate the detection of low-copy and mixed DNA samples.

The invention provides a method for detecting short tandem repeat sequences, wherein STR primers are used to perform PCDR amplification on a DNA sample to be tested, and high-throughput sequencing is performed on the products obtained by PCDR amplification to achieve complete detection of different amplicons of PCDR and obtain sequence information of STR; the STR primers include 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 ≥2; and 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 sex identification locus.

Preferably, the forensic autosomal STR locus includes 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; and the sex identification locus is Amelogenin.

Preferably, the PCDR amplification is thermal cycle amplification;

The thermal cycle amplification includes an initial denaturation phase, a pre-amplification phase, a conventional amplification phase and a final extension phase;

The initial denaturation stage: 92°C pre-denaturation for 2 minutes; 1 cycle; the pre-amplification stage: 92°C denaturation for 0.5 minutes, 62-60°C (0.2°C decrease per cycle) annealing for 1 minute, 68°C extension for 1.5 minutes; 10 cycles;

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

The final extension stage: extension at 68° C. for 10 min; 1 cycle.

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

The present invention also provides a primer set for detecting short tandem repeat sequences in the detection method described in the above technical solution, wherein the short tandem repeat sequences include forensic autosomal STR loci and/or sex identification loci; 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 sex identification locus is Amelogenin;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the CSF1PO are shown in SEQ ID NOs. 1 to 4 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D10S1248 are shown as SEQ ID NOs. 5 to 8 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D12S391 are shown in SEQ ID NOs. 9 to 12 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D13S317 are shown as SEQ ID NOs. 13 to 16 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D16S539 are shown in SEQ ID NOs. 17 to 20 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D18S51 are shown as SEQ ID NOs. 21 to 24 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D19S433 are shown as SEQ ID NOs. 25 to 28 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D1S1656 are shown as SEQ ID NOs. 29 to 32 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the D21S11 are shown in SEQ ID NOs. 33 to 36 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D22S1045 are shown as SEQ ID NOs. 37 to 40 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D2S1338 are shown as SEQ ID NOs. 41 to 44 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D2S441 are shown as SEQ ID NOs. 45 to 48 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D3S1358 are shown as SEQ ID NOs. 49 to 52 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D5S818 are shown as SEQ ID NOs. 53 to 56 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D7S820 are shown as SEQ ID NOs. 57 to 60 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D8S1179 are shown as SEQ ID NOs. 61 to 64 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the FGA are shown as SEQ ID NOs. 65 to 68 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of Penta D are shown as SEQ ID NOs. 69 to 72 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of Penta E are shown as SEQ ID NOs. 73 to 76 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of TH01 are shown in SEQ ID NOs. 77 to 80 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the TPOX are shown in SEQ ID NOs. 81 to 84 respectively;

The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the VWA are shown as SEQ ID NOs. 85 to 88 respectively;

The front primer FW and the rear primer RV of Amelogenin are shown in SEQ ID NOs. 89 to 90 respectively.

The present invention also provides a kit for detecting short tandem repeat sequences in the detection method described in the above technical solution, and the kit comprises the primer set described in the above technical solution.

The present invention also provides the use of the detection method, primer set or kit described in the above technical solution in forensic identification.

Beneficial effects:

The invention provides a method for detecting short tandem repeat sequences, wherein STR primers are used to perform PCDR amplification on a DNA sample to be tested, and high-throughput sequencing is performed on the product obtained by PCDR amplification to obtain STR sequence information; the STR primers include two pairs of nested primers.

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

1) It has a higher amplification efficiency. The present invention sets two pairs of nested specific primers for each STR locus, and uses a DNA polymerase with thermal stability and strong chain displacement activity to perform PCDR amplification reaction. During the primer extension process, the outer primer extension chain can replace the inner primer extension chain, and each reaction cycle amplifies the DNA template chain twice (see Figure 1). In the subsequent reaction, the longer amplicon formed by the extension of the outer primer can be used as a template for the short amplicon of the inner primer, further amplifying the amplification efficiency. After n rounds of PCDR cycle amplification, a single DNA molecule can theoretically produce ( ⁿ² +5n+4)× ^2n-2 amplicons, which is much higher than the ²ⁿ amplicons amplified by PCR. Therefore, compared with the traditional PCR method, the present invention has a higher amplification efficiency and is conducive to the detection and analysis of low-copy DNA samples.

2) It has lower stutter byproducts. The STR locus sequence is formed by multiple tandem repetitions of a relatively fixed motif. Low-complexity tandem repeat sequences are prone to relative sliding of the template chain and the primer extension chain during polymerase replication, forming stutter products. Stutter products usually have one less repeating motif than STR allele products, and increase as the amount of DNA template decreases. Under certain conditions, stutter products may be misjudged as alleles, thereby interfering with the correct typing of DNA samples. The present invention introduces a chain displacement process during STR allele amplification, and the inner primer is extended before the outer primer, and its extended chain temporarily blocks the STR repeat sequence, thereby enhancing the stability of the extension complex composed of the DNA template, the primer extension chain, and the DNA polymerase, and reducing the possibility of relative sliding of the template chain and the primer extension chain, thereby reducing the formation of stutter products. The reduction of stutter is conducive to the correct typing of alleles and improves the accuracy of low-copy DNA and mixed DNA analysis.

3) Complete PCDR product detection. After PCDR amplification, two pairs of nested primers can produce four amplification products, each product has a different terminal sequence (primer sequence) and the same core sequence (STR sequence). Ordinary CE-based detection methods cannot effectively distinguish these amplicons of different lengths, resulting in overlapping allele peaks within the locus and between different loci. The present invention uses high-throughput sequencing technology (MPS technology) for the first time to detect PCDR products, and all amplicon types can be fully detected by high-throughput sequencing. Based on this, the present invention does not need to use fluorescent groups to pre-label primers, nor does it need to distinguish loci and alleles by amplicon length, thereby allowing flexible primer settings and simultaneous amplification of more loci. In addition, since the sequence information of STR can be directly obtained, the detection method of the present invention can simultaneously detect sequence variations of STR alleles, thereby improving the ability to identify loci.

4) Single-tube targeted composite amplification, compatible with PCR thermal cycle reaction. The present invention can realize single-tube composite amplification of multiple loci and has wide instrument compatibility.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG1 is a technical principle of PCDR amplification of STR loci;

FIG2 shows the product concentration of gradient diluted DNA samples after amplification by the composite PCDR and composite PCR systems;

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

DETAILED DESCRIPTION

The invention provides a method for detecting short tandem repeat sequences, wherein STR primers are used to perform PCDR amplification on a DNA test sample, and high-throughput sequencing is performed on the product obtained by PCDR amplification to obtain STR sequence information; the STR primers include two pairs of nested primers.

The present invention uses STR primers to perform PCDR amplification on the DNA sample to be tested to obtain a PCDR amplification product. The number of short tandem repeat sequences described in the present invention is preferably ≥2, and more preferably 10 to 30. Each short tandem repeat sequence of the present invention includes a set of STR primer pairs, and each set of STR primer pairs includes two pairs of nested primers, preferably including a front primer FW, a rear primer RV, a front peripheral primer OF and a rear peripheral primer OR. The front peripheral primer OF described in 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 present invention has no special restrictions on the type and quantity of the short tandem repeat sequence, and any short tandem repeat sequence can be measured by the detection method of the present invention. In the specific implementation of the present invention, the short tandem repeat sequence preferably includes a forensic autosomal STR locus and/or a sex identification locus, and more preferably includes a forensic autosomal STR locus and a sex identification locus; the forensic autosomal STR locus includes 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, and more preferably 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 identification locus is preferably Amelogenin. Although the present invention takes the above-mentioned short tandem repeat sequence as an example to illustrate the detection method of the present invention, the above-mentioned types and numbers of short tandem repeat sequences cannot be identified as the entire protection scope of the present invention.

The PCDR amplification of the present invention is preferably thermal cycle amplification; the thermal cycle amplification preferably includes an initial denaturation stage, a pre-amplification stage, a conventional amplification stage and a final extension stage; the amplification program of the initial denaturation stage: 92°C pre-denaturation for 2 minutes; 1 cycle; the amplification program of the pre-amplification stage: 92°C denaturation for 0.5 minutes, 62-60°C (0.2°C decrease per cycle) annealing for 1 minute, 68°C extension for 1.5 minutes; 10 cycles; the amplification program of the conventional amplification stage: 92°C denaturation for 0.5 minutes, 60°C annealing for 1 minute, 68°C extension for 1.5 minutes; 18 cycles; the amplification program of the final extension stage: 68°C extension for 10 minutes; 1 cycle. The thermal cycle amplification of the present invention preferably also includes a maintenance stage, and the maintenance stage preferably maintains the state of the amplified product at 4°C.

The reagents used for the PCDR amplification of the present invention preferably include: SD polymerase, SD polymerase reaction buffer, MgCl ₂ , dNTP mix and water. The DNA polymerase of the present invention is preferably a heat-resistant SDDNA polymerase with strong chain displacement activity. In the implementation of the present invention, the concentration of the SD polymerase of the present invention is preferably 10-100U/μL, more preferably 50U/μL, and the SD polymerase is preferably purchased from Bioron, Germany. The SD polymerase reaction buffer of the present invention is preferably 10×SD polymerase reaction buffer, and the SD polymerase reaction buffer is preferably purchased from Bioron, Germany. The MgCl ₂ of the present invention is preferably a MgCl ₂ solution, and the concentration of the MgCl ₂ solution is preferably 10-200mM, more preferably 100mM; the MgCl ₂ is preferably purchased from Thermo Fisher Scientific, the United States; the concentration of the dNTP mix of the present invention is preferably 1-100mM, more preferably 10mM, and the dNTP mix is preferably purchased from Promega, the United States. The system for PCDR amplification of the present invention preferably includes the reagents for PCDR amplification, STR primers and genomic DNA. The system for PCDR amplification of the present invention is preferably a composite PCDR amplification system, i.e., a single-tube composite amplification of multiple STR loci; the system for PCDR amplification is preferably as shown in Table 1:

Table 1 PCDR amplification system

Element PCDR amplification system SD polymerase reaction buffer (10×) 2.0μL Genomic DNA 1.0μL (0.05～10ng) MgCl ₂ 0.75μL dNTP mix 1.0μL Primer mix 2.5μL SD polymerase 0.05μL _H2O Make up the volume to 25 μL

The primer mixture in Table 1 of the present invention is preferably a mixed primer of one or more sets of STR primers, and each set of STR primers preferably includes 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 of the present invention are preferably 0.05 to 1 μM, more preferably 0.1 to 0.8 μM. The amount of genomic DNA in Table 1 of the present invention is preferably 0.05 to 10 ng, more preferably 0.5 ng.

After obtaining the PCDR amplification product, the present invention performs high-throughput sequencing on the product obtained by PCDR amplification to obtain the sequence information of STR. Before performing the high-throughput sequencing, the present invention preferably also includes purifying the PCDR amplification product. The present invention does not specifically limit the purification method, and a conventional purification method in the art can be used. In the specific implementation process of the present invention, MinElute PCR Purification Kit is used, which is purchased from QIAGEN, Germany.

The high-throughput sequencing of the present invention preferably includes library construction, sequencing reaction and data analysis. The present invention has no special limitation on the process of library construction, and the conventional library construction process in the art can be used. In the specific implementation of the present invention, the NEBNext Ultra DNA Library Prep Kit for Illumina kit is used, which is purchased from New England Biolabs, USA. The present invention preferably mixes the single libraries constructed for a single sample into a mixed library before performing the sequencing reaction.

The sequencing reaction of the present invention is preferably double-end 250bp sequencing. The present invention has no particular limitation on the sequencer used for the sequencing reaction, and a conventional sequencer in the art can be used. In the specific implementation of the present invention, an Illumina Novaseq 6000 sequencer (Illumina, USA) is used.

After completing the sequencing reaction, the present invention preferably performs data analysis on the data obtained from the sequencing reaction to obtain the allele typing information of STR. The data analysis of the present invention preferably includes quality control, splicing, extracting amplicons and allele typing. The present invention does not specifically limit the specific process and software of the data analysis, and conventional processes and software in the art can be used. In the specific implementation process of the present invention, fastp v0.23.1 is preferably used for quality control to remove reads with a Phred quality value less than 15 or unrecognized bases greater than 5. The present invention preferably uses FLASH v1.2.11 to splice the sequence after quality control. The present invention preferably uses Seqkit v2.0.0 to extract amplicons from the spliced sequence; the amplicons preferably include amplicons obtained by amplifying four primer sequence combinations of FW+RV, OF+RV, FW+RV and OF+OR. The present invention preferably uses the FDSTools2.0 software package to perform allele typing on the extracted amplicon. The present invention preferably discriminates the data after the allele typing as follows: N-1 stutter is defined as a sequence with one less STR motif than the allele sequence; N-2 stutter is defined as a sequence with two less STR motifs than the allele. The stutter ratio (Stutter Ratio, SR) is the ratio of the number of stutter sequence reads to the number of corresponding allele reads. Heterozygote balance (HeterozygoteBalance, Hb) is defined as the ratio of the number of low coverage allele reads to the high coverage allele reads of the heterozygous locus. The difference in means between groups was statistically tested using a two-tailed Mann-Whitney U test, and if the p value was less than 0.05, the difference in the means of the two groups of data was considered to be statistically significant.

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

In the present invention, the sequences and concentrations of the front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR 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, and the sequences and concentrations of the front primer FW and rear primer RV of Amelogenin are as shown in Table 2.

Table 2 Specific sequences of the front primer FW, the rear primer RV, the front peripheral primer OF and the rear peripheral primer OR, and their concentrations in the primer mixture

The present invention also provides a kit for detecting short tandem repeat sequences in the detection method described in the above technical solution, and the kit includes the primer set described in the above technical solution. The kit of the present invention preferably also includes a reagent for PCDR amplification and/or a reagent for high-throughput sequencing, and more preferably includes a reagent for PCDR amplification and a reagent for high-throughput sequencing. The reagent for PCDR amplification of the present invention is preferably the same as that in the above technical solution, and will not be repeated here. The present invention does not specifically limit the reagent for high-throughput sequencing, and conventional reagents in the art can be used. In the specific implementation of the present invention, the reagent for high-throughput sequencing used is preferably the same as that in the above technical solution, and will not be repeated here.

The present invention also provides the use of the detection method, primer set or kit described in the above technical solution in forensic identification. The present invention is a technical solution for combining PCDR technology and high-throughput sequencing technology for the first time to identify short tandem repeat sequences, especially forensic STR identification. In the present invention, each STR genome uses two pairs of nested internal and external primers for PCDR amplification, which can effectively improve the amplification efficiency of DNA samples compared with conventional PCR amplification methods. At the same time, based on the high-throughput characteristics of MPS high-throughput sequencing and the resolution at the base level, the present invention can achieve flexible settings of PCDR primers without increasing the complexity of the map, and can detect more allele polymorphisms. In addition, the present invention effectively reduces the formation of stutter products during STR amplification, which is conducive to the detection of low-copy and mixed DNA samples. Based on this, the detection method, primer set and kit described in the present invention can be applied to forensic identification, providing a technical basis for forensic identification.

In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

Unless otherwise specified, the test methods used in the embodiments of the present invention are all conventional methods in the art, and the reagents used can be obtained through conventional purchasing channels.

Example 1

STR locus primer design:

In this embodiment, 22 autosomal STRs and 1 sex identification locus 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 sex identification locus is Amelogenin. Each STR locus uses a pair of front primers (FW) and rear primers (RV), as well as a pair of front peripheral primers (OF) and rear peripheral primers (OR) located outside the front and rear primers. Two pairs of primers are used to specifically amplify the STR loci, respectively, and two peripheral primers are used to initiate the displacement reaction of the inner primer extension chain. The PCDR amplification principle is shown in Figure 1. No peripheral primers are set for the sex identification locus Amelogenin. The corresponding primer sequences and concentrations of the 23 loci involved in the present invention are shown in Table 2 below, which will not be repeated.

Preparation of gradient dilution DNA template:

2800M standard DNA was purchased from Promega (USA). The standard was quantified using the Investigator Quantiplex Kit PCR Assay (QIAGEN, Germany) on a 7500 Real-time PCR System (Applied Biosystems, USA). Based on the quantitative results, the standard was diluted with ultrapure water to 500 pg/μL, 250 pg/μL, 125 pg/μL, 62.5 pg/μL, 31.3 pg/μL, and 15.6 pg/μL, respectively. Store at -20°C for future use.

Multiplex amplification system configuration:

According to the primers and concentrations in Table 2, a composite PCDR reaction primer set is constructed, and a corresponding composite PCR control reaction is set. Among them, the composite PCDR system is an experimental system for implementing the present invention, and the composite PCR system is a control system. The difference between the two is that the composite PCDR system contains FW, RV, OF and OR primers of the 22 STR loci in Table 2, and FW and RV primers of the Amelogenin locus; the composite PCR system only includes FW and RV primers in Table 1. Since the peripheral primers OF and OR for initiating chain displacement reactions are not contained, each locus in the composite PCR system is amplified by PCR, and other conditions are completely consistent with the composite 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: 1. SD polymerase 50U/μL (Bioron, Germany); 2. 10×SD polymerase reaction buffer (Bioron, Germany); 3. MgCl ₂ solution 100mM (Thermo Fisher Scientific, USA); dNTP mix 10mM (Promega, USA). The specific amplification system composition is shown in Table 3:

Table 3 Composite PCDR system and composite PCR system

The primers of the composite PCDR system are FW, RV, OF, OR primers and corresponding concentrations, and the primers in the composite PCR system are FW and RV primers.

Thermal Cycling Amplification:

The multiplex PCDR reaction and multiplex PCR reaction were performed in a thermal cycler. The thermal cycler amplification program is shown in Table 4.

Table 4 Thermal cycle amplification program for multiplex PCDR and multiplex PCR

The amplified product was stored at 4°C in the dark.

Sequencing library construction and sequencing:

The amplified product was purified by MinElute PCR Purification Kit (QIAGEN, Germany) and eluted in a volume of 25 μL. The purity of the purified product was determined using a NanoDrop 1000 ultra-micro spectrophotometer (Thermo Fisher Scientific, USA), and the product concentration was determined using a Qubit dsDNA HS Assay Kit (Invitrogen, USA) and a Qubit 4 fluorometer. Subsequently, 15 μL of the purified amplified product was taken for sequencing library construction.

The library was constructed using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, USA). After the amplified products were end-repaired and A-tailed, they were connected to the full-length adapter of NEBNext Multiplex Oligosfor Illumina (New England Biolabs, USA) to obtain a complete sequencing template. The library was purified and fragments were screened using 1.3× Agencourt AMPure XP magnetic beads (Beckman Coulter, USA). The library quality was evaluated using the Agilent DNA 1000 Kit (Agilent Technologies, USA) on the 2100 Bioanalyzer (Agilent Technologies, USA). The library was then accurately quantified using KAPA Library Quantification Kits (KAPA Biosystems, USA) on the 7500 Real-time PCR System (Applied Biosystems, USA).

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

Sequencing data analysis:

The sequencing data FASTQ files were quality controlled using fastp v0.23.1 to remove reads with a Phred quality value of less than 15 or unidentified bases greater than 5. Then, the double-end sequencing reads were spliced using FLASH v1.2.11. According to the four primer sequence combinations of FW+RV, OF+RV, FW+RV and OF+OR, Seqkit v2.0.0 was used to extract the four amplicons generated by PCDR amplification from the FASTQ files after double-end read splicing. The FDSTools2.0 software package was used to perform allele typing on the FASTQ files of sequencing reads.

Allele discrimination:

N-1 stutter is defined as a sequence with one less STR motif than the allele sequence, and N-2 stutter is defined as a sequence with two less STR motifs than the allele. Stutter Ratio (SR) is the ratio of the number of stutter sequence reads to the number of corresponding allele reads. Heterozygote Balance (Hb) is defined as the ratio of the number of low coverage allele reads to the high coverage allele reads of the heterozygous locus. The difference in means between groups was statistically tested using the two-tailed Mann-Whitney U test. If the p value was less than 0.05, the difference in the means of the two groups of data was considered statistically significant.

Result data:

The product concentrations of the gradient diluted DNA samples after amplification by the composite PCDR and composite PCR systems are shown in FIG2 : wherein the error bars in FIG2 indicate the standard deviation of the data.

It can be concluded from Figure 2 that the amount of products after amplification of different amounts of DNA templates by the composite PCDR system of the present invention is higher than that of the control group amplified by the composite PCR system, and the difference is statistically significant (p = 8.9E-06). Taking 500pg DNA template as an example, the product concentration after composite PCDR amplification is 67.1±3.5ng/μL, which is much higher than 8.4±0.6ng/μL after composite PCR amplification. This shows that the composite PCDR of the present invention has a higher amplification efficiency.

Example 2

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

DNA extraction and quantification

The venous blood samples in this embodiment were provided by 46 healthy volunteers. After venous blood collection, EDTA-Na ₂ was added for anticoagulation. Genomic DNA was extracted from venous blood samples using QIAmp DNA Mini Kit (QIAGEN, Germany). DNA extraction can also be performed using other widely validated methods, such as the Chelex100 method, the phenol chloroform method, etc. 9948 DNA standards were purchased from AGCU (China). DNA quantification was performed using the Qubit dsDNA HS Assay Kit (Invitrogen, USA) on the Qubit 4 fluorescence quantifier. DNA quantification operations were performed according to the instructions provided by the kit and instrument manufacturers. According to the quantitative results, the DNA samples were diluted to 0.5 ng/μL and stored at -20°C for later use.

The configuration of the composite amplification system, thermal cycle amplification reaction, sequencing library construction, sequencing and data analysis in this example are all carried out according to the method in Example 1, and will not be described in detail.

Result data:

1. Average coverage results of each locus of composite PCDR and composite PCR amplification products

The present invention first combines and analyzes the different types of amplification products produced by the combination of internal and external primers. For the composite PCDR system in the present invention, the average coverage of 22 STR loci is 26398±15997, which is higher than the average coverage of 19188±13018 of the composite PCR system in the control group. Subsequently, the present invention uses a primer sequence matching method to extract four different amplification products in the composite PCDR. As shown in Figure 3, the method proposed by the present invention successfully detected all four types of PCDR amplification products, S, M1, M2 and L, produced by primer combinations FW+RV, OF+RV, FW+OR and OF+OR. Among them, the average coverage of amplification product S is the highest, followed by M1 and M2, and L is the least. The average read number ratio of the four product types S:M1:M2:L is approximately 81:12:12:1.

In Figure 3, Gross represents the different types of amplified products of combined PCDR; S, M1, M2, and L are the amplified products of primer combinations FW+RV, OF+RV, FW+OR, and OF+OR generated by PCDR, respectively. Error bars indicate the standard deviation of the data.

2. Heterozygous balance of each locus of composite PCDR and composite PCR amplification products

After 46 single-source DNA samples were amplified by the composite PCDR system of the present invention, the average heterozygosity of the loci was 0.80±0.17, and the average heterozygosity of the loci in the composite PCR system of the control group was 0.81±0.15, and there was no statistically significant difference between the two (p=0.65).

3. Stutter ratio (SR) of each locus of composite PCDR and composite PCR amplification products

Combined analysis of different PCDR amplification product types showed that the composite PCDR system of the present invention significantly reduced the stutter product in the STR allele replication process. For 22 STR loci, the N-1SR of 46 samples after amplification by the composite PCDR system is shown in Table 4. The average N-1SR of the loci is 6.9±3.9%, while the average N-1SR of the composite PCR control system is 8.3±4.5% (p=1.4E-17). For the rarer N-2stutter, its average SR in the composite PCDR group of the present invention is 0.59±0.41%, which is significantly lower than its average value of 0.75±0.53% in the composite PCR control group (p=1.8E-11). The inhibitory effect of the present invention on stutter products in STR amplification has universal applicability to loci. Taking N-1 stutter as an example, all 22 loci in the composite PCDR amplification system of the present invention showed a reduction in SR, and the mean value of stutter in 73.3% of the loci was statistically significantly reduced relative to the PCR control group.

Table 4 Composite PCDR and composite PCR system STR locus N-1SR

Locus Multiplex PCR (mean ± standard deviation) Composite PCDR (mean ± standard deviation) Significance 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: Significant marks: ***, p<0.001; **, p<0.01; p<0.05; NS, p≥0.05.

4. Composite PCDR and composite PCR amplification products have the same alleles

The present invention uses MPS technology (massively parallel sequencing) to detect the amplified products, which can provide more STR sequence variations and improve the polymorphism of STR loci compared to the CE-based detection method. After amplification and MPS detection by the composite PCDR system of the present invention, we observed 29 isoalleles in 20 samples. As shown in Table 5, these alleles have the same fragment length, but there are variations in the repeat segments or flanking sequences. The sequence-based isoallele naming rules refer to the FDSTools software package manual.

Table 5 STR alleles in single-source DNA samples

Comparative Example 1

The EX25 fluorescent kit (AGCU, China) was used for comparison with the present invention. The kit uses PCR reaction to amplify 22 autosomal STR loci, and the amplified products are detected and typed by CE. The method of using the kit in the comparative example is:

DNA extraction and quantification: DNA of the 46 samples in Example 2 was extracted according to the method in Example 2 and the DNA was quantified and diluted;

Amplification system configuration: The amplification system was configured according to the kit instructions. The genomic DNA template used was consistent with that in Example 2;

Thermal cycle amplification reaction: Perform thermal cycle amplification according to the kit instructions;

Amplification product detection: 1.0 μL of amplification product was mixed with 8.9 μL of Hi-Di formamide (Applied Biosystems, USA) and 0.1 μL of AGCU Marker SIZ-500 molecular weight internal standard (AGCU, China), and fluorescence detection was performed using a 3500 GeneticAnalyzer (Applied Biosystems, USA) genetic analyzer;

Discrimination of allele peaks and stutter peaks: STR allele typing was performed using GeneMapper ID-X 1.5. A stutter peak was defined as a peak that was one STR motif less before the allele peak on the DNA map. The stutter ratio (SR) was the height of the stutter peak divided by the height of the corresponding allele peak.

Result data:

1. STR allele typing consistency

The complete typing of 46 single-source DNA samples was successfully obtained using the EX25 kit amplification combined with CE detection, but it can only distinguish alleles based on STR length and cannot distinguish the same alleles with only sequence differences.

Compared with Example 2, the typing accuracy of the method of the present invention using composite PCDR amplification combined with MPS detection for STR allele length polymorphism is 100%.

2. Stutter ratio (SR)

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

Table 6 Average SR of the same STR loci in EX25 as in the present invention

Locus SR (mean ± standard deviation) Locus SR (mean ± 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% vW 7.9±2.9%

It can be concluded that compared with conventional forensic STR kits based on PCR-CE, the present invention has the same typing accuracy and can distinguish STR allele sequence variations. At the same time, the present invention can effectively reduce the generation of stutter byproducts during STR amplification.

It can be concluded from the above examples that the PCDR amplification technology of the present invention can significantly improve the amplification efficiency of DNA samples, and combined with the MPS detection technology, it can effectively detect all amplification products of PCDR for the first time. Compared with the PCR-based method, the present invention can significantly reduce the stutter byproducts in the replication process of STR alleles and improve the reliability of STR locus detection; at the same time, the PCDR amplification method proposed by the present invention hardly affects the amplification balance of heterozygous locus alleles; in addition, the present invention can also effectively detect the internal variation of STR alleles and improve the degree of polymorphism of STR loci.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (3)

1. A primer set for detecting short tandem repeat sequences, characterized in that the short tandem repeat sequences include forensic autosomal STR loci and sex identification loci; the forensic autosomal STR loci include CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, PentaD, PentaE, TH01, TPOX and VWA; the sex identification locus is Amelogenin; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the CSF1PO are shown in SEQ ID NOs. 1 to 4 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D10S1248 are shown as SEQ ID NOs. 5 to 8 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D12S391 are shown in SEQ ID NOs. 9 to 12 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D13S317 are shown as SEQ ID NOs. 13 to 16 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D16S539 are shown in SEQ ID NOs. 17 to 20 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D18S51 are shown as SEQ ID NOs. 21 to 24 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D19S433 are shown as SEQ ID NOs. 25 to 28 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D1S1656 are shown as SEQ ID NOs. 29 to 32 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the D21S11 are shown in SEQ ID NOs. 33 to 36 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D22S1045 are shown in SEQ ID NOs. 37 to 40 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D2S1338 are shown as SEQ ID NOs. 41 to 44 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D2S441 are shown as SEQ ID NOs. 45 to 48 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D3S1358 are shown as SEQ ID NOs. 49 to 52 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D5S818 are shown as SEQ ID NOs. 53 to 56 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D7S820 are shown as SEQ ID NOs. 57 to 60 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of D8S1179 are shown as SEQ ID NOs. 61 to 64 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the FGA are shown as SEQ ID NOs. 65 to 68 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the PentaD are shown as SEQ ID NOs. 69 to 72 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of PentaE are shown as SEQ ID NOs. 73 to 76 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of TH01 are shown in SEQ ID NOs. 77 to 80 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the TPOX are shown in SEQ ID NOs. 81 to 84 respectively; The front primer FW, rear primer RV, front peripheral primer OF and rear peripheral primer OR of the VWA are shown as SEQ ID NOs. 85 to 88 respectively; The front primer FW and the rear primer RV of Amelogenin are shown in SEQ ID NOs. 89 to 90 respectively. 2. A kit for detecting short tandem repeat sequences, characterized in that the kit comprises the primer set according to claim 1. 3. Use of the primer set according to claim 1 or the kit according to claim 2 for non-disease diagnosis purposes in forensic identification.