A detection kit, method and application of a 10-color fluorescent autosomal STR composite amplification system

Abstract

The present invention discloses a detection kit, method and application thereof using a 10-color fluorescent autosomal STR composite amplification system. The detection kit uses nine fluorescent dyes, FAM, TET, HEX, ER650, ER652, ER663, ER664, ER674 and ER685, to label composite amplification primers of 48 STR loci, and uses fluorescent dye ER690 as an internal standard marker. The present invention breaks the limitations of the existing detection system's fluorescent channels and number and the number of loci, incorporates more mini STRs and STR sites with high polymorphism, and can obtain typing information of 48 STR loci through one amplification, thereby solving the problem of less detection site information in a single detection of trace samples, degraded samples, and difficult samples in complex environments, thereby effectively improving the detection efficiency of the system.

Description

Technical Field

[0001] The invention relates to a detection kit, a method and an application of a 10-color fluorescent autosomal STR composite amplification system, and belongs to the technical field of molecular genetics. Background Art

[0002] Short tandem repeats (STRs) are a type of DNA tandem repeats widely found in eukaryotic genomes, with a core sequence of 2-6 base repeat units. STR loci are large in number, widely distributed, highly polymorphic, and follow Mendelian inheritance in the genetic process. Therefore, STR amplification detection technology is widely used in individual identification, kinship identification, and population genetics research. Currently, multiplex PCR composite amplification and multicolor fluorescent labeling detection technology are mostly used for DNA genotyping of biological samples.

[0003] The application of fluorescent dye labeling in DNA detection is an important progress in the field of molecular biology. Fluorophores are a special class of molecules that can re-emit light at a longer wavelength after absorbing light of a specific wavelength. The fluorescent dye is labeled at the 5' end of the oligonucleotide primer, and one chain of the amplified PCR product carries the fluorescent dye of the labeled primer. These amplified products with specific fluorescent substances can be clearly identified on the subsequent electrophoresis separation and detection equipment, and the existence, structure and function of DNA can be detected and analyzed by measuring the changes in the fluorescent signal. In STR analysis, multi-color fluorescent-labeled primers are usually used to simultaneously amplify multiple STR loci. These fluorescent markers have different excitation and emission spectra, and multiple STR loci can be detected simultaneously in one experiment. For example, commonly used fluorescent dyes include FAM, HEX, TET and VIC, which correspond to different color channels.

[0004] At present, most of the fluorescent detection kits for STR in the domestic market use five or six groups of fluorescence for compound amplification, with large amplified fragments and more detected gene loci. However, in actual applications, it is found that when amplifying normal samples such as blood cards, hair, nails, etc., STR locus information is relatively comprehensive and will not be affected by the large number of loci and large amplified fragments. However, when amplifying degraded samples, trace samples, and difficult samples in complex environments, it is found that the amplified fragments after 450bp face the possibility of low amplification efficiency or fragment loss, which is a waste of precious samples. At the same time, due to the limitation of the fluorescence channel in the detection system, the locus information that can be provided by related products is limited and cannot fully meet actual needs. Summary of the invention

[0005] The present invention provides a detection kit, method and application of a 10-color fluorescent autosomal STR composite amplification system, which breaks the limitations of the existing detection system on the number of fluorescent channels and loci, incorporates more mini STRs and STR sites with high polymorphism, and can obtain typing information of 48 STR loci through one amplification, thereby solving the problem of less information on detected sites in a single detection of trace samples, degraded samples and difficult samples in complex environments, thereby effectively improving the detection efficiency of the system.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A detection kit using a 10-color fluorescent autosomal STR composite amplification system, using nine fluorescent dyes, FAM, TET, HEX, ER650, ER652, ER663, ER664, ER674 and ER685, to label composite amplification primers of 48 STR loci, and using fluorescent dye ER690 as an internal standard marker;

[0008] The 48 STR loci include 41 autosomal STR loci, 1 Y chromosome STR locus, 2 X chromosome STR loci, 1 gender locus Amel and 3 Y chromosome Indel; the 41 autosomal STR loci are: D3S1358, vWA, D12S391, CSF1PO, Penta E. D16S539, D7S820, D13S317, D2S1338, PentaD, D22S1045, D19S433, D18S51, D6S1043, D8S1179, D5S818, D21S11, F GA, TH01, D1S1656, TPOX, SE33, D4S2366, D6S477, D14S608, D19S253, D3S1744, D21S2055, D7S1517, D18S535, D10S1 435, D7S3048, D10S2325, D22GATA198B05, D15S659, D8S1132, D13S325, D2S441, D10S1248, D3S3045 and D5S2500; 1 Y chromosome STR locus: DYS391; 2 X chromosome STR loci: DXS8378 and DXS6810; 3 Y chromosome Indels: rs199815934, rs759551978 and rs771783753.

[0009] The 48 STR loci were divided into nine groups, and at least one of the amplification primers for each locus was labeled with a fluorescent dye at its 5' end:

[0010] The fluorescent dye FAM was used to label the amplification primers of the loci D3S1358, vWA, D12S391, CSF1PO, and Penta E, respectively;

[0011] The fluorescent dye TET was used to label the amplification primers of loci rs199815934, D22S1045, D19S433, D18S51, D6S1043, and DYS391, respectively;

[0012] The fluorescent dye HEX was used to label the amplification primers of the loci rs771783753, D16S539, D7S820, 25824841, D13S317, D2S1338, and PentaD, respectively;

[0013] The fluorescent dye ER650 was used to label the amplification primers of loci D8S1179, D5S818, D21S11, and FGA, respectively;

[0014] The fluorescent dye ER652 was used to label the amplification primers of the loci rs759551978, TH01, D1S1656, TPOX, and SE33, respectively;

[0015] The fluorescent dye ER663 was used to label the amplification primers of loci D4S2366, D6S477, D14S608, D19S253, D3S1744, and D21S2055, respectively;

[0016] The fluorescent dye ER664 was used to label the amplification primers of loci D7S1517, D18S535, D10S1435, D7S3048, and D10S2325, respectively;

[0017] The fluorescent dye ER674 was used to label the amplification primers of the gene loci AMEL, D22-GATA198B05, D15S659, D8S1132, and D13S325, respectively;

[0018] The fluorescent dye ER685 was used to label the amplification primers of loci D2S441, DXS8378, D10S1248, DXS6810, D3S3045, and D5S2500, respectively.

[0019] The fluorescent dye ER690 was used for internal standard labeling, and the labeling types of each upstream primer are described in Table 1.

[0020] The present application adopts 9 groups of fluorescent dyes to label and prepare 48 STR loci composite amplification primer sets, and simultaneously amplifies 48 STR sites.

[0021] The 10 fluorescence emission spectra of the present application can be distinguished and do not penetrate each other, the wavelength is in the range of 480-710nm, and the emission intensity is high and the balance is good. The specific chemical structure formula of each dye is as follows:

[0022]

[0023]

[0024] An in the formula - It is the fluorescent group contained in the chemical formula.

[0025] The present invention uses the honor1816 genetic analyzer as the detection platform. Based on the successful establishment of an eight-color fluorescence detection system in the early stage and the application of the eight-color fluorescence composite amplification kits Y62 and AY60, the fluorescent energy transfer dyes are further screened and the fluorescent group labeling method is optimized to establish a ten-color fluorescence analysis system.

[0026] The above-mentioned 10-color fluorescent composite amplification STR short tandem repeat sequence typing method comprises the following steps:

[0027] ① Construction of the fluorescence system: 9 fluorescent dyes were used to label the composite amplification primers of 48 STR loci, and ER690 was used as an internal standard label.

[0028] ② Construction of fluorescent composite amplification system: Amplification was performed using composite amplification primer pairs of 48 STR loci labeled with 9 fluorescent dyes to obtain amplification products.

[0029] ③STR locus typing: STR locus typing was performed on the PCR amplification products of 48 fluorescently labeled STR loci.

[0030] The sequences of the amplification primers for the above 48 STR loci are shown in Table 1 as AM NO.01-AM NO.101, including 49 upstream primers labeled with different fluorescent dyes and 52 downstream primers labeled with non-fluorescent dyes.

[0031] The working concentrations of the amplification primers in the composite amplification kit during amplification are shown in Table 1. Among them, the sequences, numbers and working concentrations of the amplification primers of 41 pairs of autosomal STR loci are as follows: D3S1358, AM NO.1-2, 0.036±0.002uM; vWA, AM NO.3-4, 0.06±0.002uM; D12S391, AM NO.5-7, 0.14±0.002uM; CSF1PO, AM NO.8-9, 0.06±0.002uM; Penta E, AM NO.10-11, 0.2±0.002uM; D22S1045, AM NO.14-15, 0.05±0.002uM; D19S433, AM NO. NO.16-17, 0.06±0.002uM; D18S51, AMNO.18-19, 0.04±0.002uM; D6S1043, AM NO.20-21, 0.08±0.002uM; D16S539, AMNO.26-27, 0.07±0.002uM; D7S820, AM NO.28-29, 0.1±0.002uM; D13S317, AMNO.30-31, 0.12±0.002uM; D2S1338, AMNO.32-33, 0.2±0.002uM; Penta D, AMNO.34-35, 0.15±0.002uM; D8S1179, AM NO.36-37, 0.06±0.002uM; D5S818, AMNO.38-39, 0.04±0.002uM; D21S11, AM NO.40-41, 0.05±0.002uM; FGA, AM NO.42-43, 0.06±0.002uM; TH01, AM NO.46-47, 0.06±0.002uM; D1S1656, AM NO.48-49, 0.1±0.002uM; TPOX, AM NO.50-51, 0.12±0.002uM; SE33, AM NO.52-53, 0.11±0.002uM; D4S2366, AM NO.54-56, 0.19±0.002uM; D6S477, AM NO.57-58, 0.12±0.002uM; D14S608, AM NO.59-61, 0.17±0.002uM; D19S253, AM NO.62-63, 0.11±0.002uM; D3S1744, AM NO.64-66, 0.12±0.002uM; D21S2055, AM NO.67-68, 0.17±0.002uM; D7S1517, AMNO.69-70, 0.17±0.002uM; D18S535, AM NO.71-72, 0.15±0.002uM; D10S1435, AM NO.73-74, 0.22±0.002uM; D7S3048, AM NO.75-76, 0.42±0.002uM; D10S2325, AM NO.77-78, 0.35±0.002uM; D22GATA198B05, AM NO.82-83, 0.6±0.002uM; D15S659, AM NO.84-85, 0.12±0.002uM; D8S1132, AM NO.86-87, 0.1±0.002uM; D13S325, AM NO.88-89, 0.07±0.002uM; D2S441, AM NO.90-91, 0.4±0.002uM; D10S1248, AM NO.94-95, 0.5±0.002uM; D3S3045, AMNO.98-99, 0.33±0.002uM; D5S2500, AM NO.100-101, 0.3±0.002uM;.

[0032] The sequences of the amplification primers for the Y chromosome STR locus were as follows: DYS391, AM NO.34-35, 0.5±0.002uM;

[0033] The sequences of the primers for amplification of the two X-chromosome STR loci were as follows: DXS8378, AM NO.92-93, 0.5±0.002uM; DXS6810, AM NO.96-97, 0.4±0.002uM;

[0034] The sequences of the amplification primers for the sex locus Amel are as follows: Amel, AM NO.79-81, 0.06±0.002uM;

[0035] The sequences of the three pairs of Y chromosome Indel amplification primers are as follows: rs199815934, AM NO.12-13, 0.3±0.002uM; rs771783753, AM NO.24-25, 0.3±0.002uM; rs759551978, AM NO.44-45, 0.4±0.002uM.

[0036] The composite amplification kit comprises a composite primer set of 101 specific primers for simultaneously amplifying 48 STR loci, a reaction mixture and a hot-start Taq polymerase.

[0037] Further preferably, the above-mentioned composite amplification kit comprises 25 μl of an amplification reaction system: 10 μl of 2.5×PCR Mix, 5 μl of T48 primer mixture, 1 μl of Taq DNA polymerase, 2 μl of template DNA, and 7 μl of deionized water; wherein the T48 primer mixture is an amplification primer mixture for 48 loci; and the components of 2.5×PCR Mix are: 90 mM Tris-HCl at pH 8.4, 10 mM MgCl2, 125 mM KCl, 10 mg / ml BSA, and 6.0 mM dNTPs.

[0038] The above-mentioned composite amplification kit also includes an allele ladder containing 48 STR loci, a DNA standard, a fluorescent molecular weight internal standard O-500 (ER690 marker) and a T10-Matrix.

[0039] The above-mentioned composite amplification reagent is suitable for amplification of different samples and has a large annealing temperature range. The amplification reaction procedure is as follows: 95°C for 3 minutes, 95°C for 5 seconds, 62°C for 90 seconds, 28-30 cycles; 60°C for final extension for 20 minutes; 4°C for maintenance. The amplified product is detected for fluorescence signal using a genetic analyzer; the fluorescence signal data is collected, and the DNA typing results of 48 loci are analyzed using genetic analysis software.

[0040] The above STR loci include 20 core loci of CODIS and 20 autosomal supplementary loci recommended by the forensic identification standards. Among them, Penta E, Penta D, and D6S1043 are sites with high polymorphism in the Chinese population, and SE33 is highly polymorphic in the European population. Among the PCR products of 48 STR locus primers, 16 loci have amplicon fragments less than 220bp, and except for D10S2325, the amplicon fragments of the remaining loci are all less than 450bp, which can be used for kinship identification, library construction, and amplification of trace samples and degraded samples at the scene of the case, as well as difficult samples in complex environments.

[0041] The above-mentioned composite amplification and detection method of the detection kit using the 10-color fluorescent autosomal STR composite amplification system comprises the following steps:

[0042] ① Sample processing: extract the genomic DNA of the sample as the amplification template or directly use the sample without extraction as the amplification template;

[0043] ② Using the amplification primers shown in AM NO.01-101 in Table 1, perform PCR amplification on the sample genomic DNA obtained in step ①;

[0044] ③ Use the honor1816 genetic analyzer to detect the fluorescence signal of the amplified product obtained in step ②;

[0045] ④ Collect fluorescence signal data and use gene analysis software to analyze the DNA typing results of 48 loci.

[0046] In the above step ①, the amplification template is an extracted sample or a direct amplification sample. The extracted sample includes human semen stains, blood, body fluids, hair, tissues or bloodstains, etc. The application samples also include difficult samples such as old samples and degraded samples. The main extraction methods for DNA genomes are magnetic bead extraction or Chelex100 method; direct amplification samples include human blood or oral cells collected by one or more carriers in filter paper, blood cards, cotton swabs or FTA cards.

[0047] The amount of DNA template in the sample is preferably 0.5 ng to 4 ng.

[0048] The above-mentioned composite amplification kit can be applied to individual identification of the Chinese population, paternity testing, forensic identification, construction of autosomal DNA database, population genetics research, etc.

[0049] Table 1 Primer sequences and final concentrations of each locus

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] The above-mentioned composite amplification kit also includes 48 loci allele ladders, DNA standards, fluorescent molecular weight internal standard O-500 (ER690 marker) and T10-Matrix.

[0056] The above-mentioned composite amplification kit can be applied to individual identification of the Chinese population, paternity testing, construction of autosomal DNA database, population genetics research, etc.

[0057] The technologies not mentioned in the present invention are all referred to the prior art.

[0058] The beneficial effects of the present invention are:

[0059] 1. For the first time, a detection kit, method and application of a 10-color fluorescent autosomal STR composite amplification system are proposed. There are currently no relevant literature or patents. The emission spectra of the ten fluorescent dyes can be distinguished and do not penetrate each other. The ten fluorescent emission intensities are high and well balanced. The 10 fluorescent dyes are optimized and screened, with wavelengths in the range of 480-710.

[0060] 2. The kit of the present invention, because it uses ten groups of fluorescent dyes for labeling, can accommodate more gene loci within the same fragment length range, and has 48 STR loci information, which not only covers the new CODIS loci recommended by the FBI of the United States, but also covers the 20 autosomal supplementary loci recommended by the judicial identification specification. It also adds 1 Y chromosome STR locus DYS391, 2 X chromosome STR loci DXS8378, DXS6810, 1 gender locus Amel and 3 Y chromosome Indelrs199815934, rs759551978, rs771783753 for auxiliary judgment, which can solve the problem of less detection site information in a single test of trace samples and degraded samples and difficult samples under complex environments, thereby effectively improving the detection efficiency of the system. Among them, Penta E, Penta D, and D6S1043 are loci with high polymorphism in the Chinese population.

[0061] 3. Among the 48 STR gene loci of the kit of the present invention, 16 STR gene loci have amplicon fragments less than 220bp, and except for D10S2325, the amplicon fragments of the remaining loci are all less than 450bp. It is applicable to a variety of test materials, and the amplification template is a direct amplification sample or an extracted sample. The extracted sample includes human semen stains, blood, body fluids, hair, tissues or bloodstains. The application samples also include old samples, degraded samples and other difficult samples. It can efficiently provide site information and has strong applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the 10-color Matrix result diagram for the successful establishment of this application;

[0063] Figure 2 This is a schematic diagram of the arrangement of the 48 STR loci in this application;

[0064] Figure 3 This is the allele typing standard diagram of the kit in the embodiment of this application;

[0065] Figure 4 It is the typing map of 48 loci of the amplified positive standard 9948 in the example of this application;

[0066] Figure 5 It is the typing map of 48 loci of the amplified positive standard 9947A in the example of this application;

[0067] Figure 6 It is the typing map of 48 loci of the positive DNA standard 9948 amplified by the kit in the embodiment of the present invention with a final concentration of 31.25 pg;

[0068] Figure 7 The typing map of 48 loci of the positive DNA standard 9947A amplified by the kit in the embodiment of the present invention with a final concentration of 31.25 pg;

[0069] Figure 8 , Fig. 9 , Fig.10 They are respectively the typing maps of 48 loci of male blood cards, male saliva cards, and male hair (with hair follicles) samples amplified by the kit in the embodiment of the present invention;

[0070] Fig.11 , Fig.12 , Fig.13 They are respectively the typing maps of 48 loci of female blood cards, female saliva cards, and female hair (with hair follicles) samples amplified by the kit in the embodiment of the present invention;

[0071] Fig.14 The typing patterns of other species samples, including pig, chicken, dog, cattle, sheep, and fish, were amplified by the kit in the embodiment of the present invention. DETAILED DESCRIPTION

[0072] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0073] Example 1

[0074] 1.1. Construction of 10-color fluorescence analysis system:

[0075] Nine fluorescent dyes were used to fluorescently label the amplification primers of 48 STR gene loci, and ER690 was used to label the internal standard. The T10-Matrix spectrum constructed by the ten-color fluorescent standard was electrophoresed by the honor1816 genetic analyzer. The results are shown in Figure 1 The nine fluorescent dyes are FAM, TET, HEX, ER650, ER652, ER663, ER664, ER674, ER685, and ER690.

[0076] The 48 STR loci include 41 autosomal STR loci: D3S1358, vWA, D12S391, CSF1PO, Penta E, D16S539, D7S820, D13S317, D2S1338, PentaD, D22S1045, D19S433, D18S51, D6S1043, D8S1179, D5S818, D21S11, FGA, TH01, D1S1656, TPOX, SE33, D4S2366, D6S477, D14S608, D19S253, D3S1744, D21S2055, D7S151 7. D18S535, D10S1435, D7S3048, D10S2325, D22GATA198B05, D15S659, D8S1132, D13S325, D2S441, D10S1248, D3S3045, D5S2500; 1 Y chromosome STR locus: DYS391; 2 X chromosome STR loci: DXS8378, DXS6810; 1 sex locus Amel and 3 Y chromosome Indels: rs199815934, rs759551978, rs771783753.

[0077] The 101 specific primers for the 48 STR loci include 49 upstream primers labeled with different fluorescent dyes and 52 downstream primers labeled with non-fluorescent dyes. The primer sequences of the composite primer set of 101 specific primers are the nucleotide sequences of AM NO.01 to AM NO.101 in Table 1, as shown in Table 1 for details.

[0078] The fluorescent labels of the composite amplification primers of 48 STR loci are as follows: Group 1: D3S1358, vWA, D12S391, CSF1PO, Penta E; Group 2: rs199815934, D22S1045, D19S433, D18S51, D6S1043, DYS391; Group 3: rs771783753, D16S539, D7S820, 25824841, D13S317, D2S1338, PentaD; Group 4: D8S1179, D5S818, D21S11, FGA; Group 5: rs759551978, TH01, D1S1656, TPOX, SE33; Group 6: D4S2366, D6S477, D14S608, D19S253, D3S1744, D21S2055; Group 7: D7S1517, D18S535, D10S1435, D7S3048, D10S2325; Group 8: AMEL, D22-GATA198B05, D15S659, D8S1132, D13S325; Group 9: D2S441, DXS8378, D10S1248, DXS6810, D3S3045, D5S2500. At least one of the primers in each pair is labeled with a fluorescent dye at its 5' end. Among them, the first group of fluorescent dyes is FAM, the second group of fluorescent dyes is TET, the third group of fluorescent dyes is HEX, the fourth group of fluorescent dyes is ER650, the fifth group of fluorescent dyes is ER652, the sixth group of fluorescent dyes is ER663, the seventh group of fluorescent dyes is ER664, the eighth group of fluorescent dyes is ER674, and the ninth group of fluorescent dyes is ER685. The internal standard uses orange fluorescent ER690. The working concentrations of each upstream primer labeling type and the amplification of the 48 STR loci composite amplification primer set are shown in Table 1. Among them, the 48 STR gene loci and the corresponding sites of the fluorescent dyes are shown in Table 1. Figure 2 shown.

[0079] 1.2. Construction of the composite amplification system:

[0080] A composite amplification primer set containing 48 STR gene loci was used for PCR amplification to obtain the amplified product. In the case of direct amplification samples such as amplified extraction samples or FTA blood cards, the reaction mixture was prepared according to Table 2.

[0081] Table 2 Quantity of each component added in the PCR system

[0082]

[0083] The components of the reaction mixture 2.5×PCR Mix are: Tris-HCl 90mM, pH 8.4, MgCl2 10mM, KCl 125mM, BSA 10mg / ml, and dNTPs 6.0mM.

[0084] Amplification was performed using a PCR amplification instrument according to the amplification program: 95°C for 3 min, 95°C for 5 s, 62°C for 90 s, 28 cycles; 60°C for final extension for 20 min; 4°C for maintenance. It has been verified that the kit of the present invention has good amplification efficiency within the annealing temperature range of 57°C-63°C, and has good amplification efficiency on PCR instruments of different brands (ABI9700, ABI veriti, E1000, etc.).

[0085] 1.3. Perform STR locus typing on PCR products:

[0086] The obtained PCR amplification product and allele standard typing material were mixed with 1ul+0.15ul molecular weight internal standard+9ul deionized formamide, and then capillary electrophoresis was performed on the Suyuan honor1816 genetic analyzer. Electrophoresis parameters: injection time 10s, injection voltage: 3.0Kv; other parameters were machine default. The electrophoresis data were collected using the collection software, and the data were analyzed using the Suyuan GeneMapper software.

[0087] The allele typing standard for analysis provided by the present invention is a product group of allele plasmids corresponding to each STRs locus primer amplification. DNA positive standard 9948 was purchased from Suzhou Xinhai Biotechnology Co., Ltd., and hot start Taq DNA polymerase was purchased from Bao Biotechnology Co., Ltd. Figure 3 It is the Allelic Ladder electrophoresis pattern.

[0088] Example 2

[0089] Sample PCR amplification test and performance test:

[0090] 2.1. Positive standard consistency test

[0091] Using 1 ng of positive standard DNA 9947A (female) and 1 ng of positive standard DNA 9948 (male) as templates, refer to the experimental process of Example 1, mix the reaction reagents according to the amplification system, shake and centrifuge, and then perform PCR amplification and STR locus typing.

[0092] Figure 4 This is the 9948 amplification detection map. Figure 5 This is the 9947A amplification detection spectrum, which is completely consistent with the theoretical typing. Figure 4 , Figure 5It can be seen from the results shown that clear and accurate typing diagrams can be obtained by amplifying different types of samples using the system of the present invention, and both the molecular weight internal standard and the allele typing standard can accurately calibrate the typing.

[0093] 2.2, Sensitivity detection:

[0094] The sensitivity of this kit was tested, and 9948 and 9947A were tested three times in parallel with different template masses (1, 0.5, 0.25, 0.125, 0.0625, 0.03125 ng) to verify the minimum detection limit of the system established in this paper. Figure 6 , Figure 7 As shown, the minimum detection sensitivity of this system is 0.03125ng, and all types can be fully detected at 31.25pg.

[0095] 2.3、Test material adaptability test:

[0096] Using male and female blood cards, saliva cards, hair (with hair follicles) and other samples as templates, refer to the experimental process of Example 1, mix the reaction reagents according to the amplification system, shake and centrifuge, and then perform PCR amplification and STR locus typing.

[0097] Figures 8 to 13 Directly amplify the spectrum of FTA cards, saliva cards, hair (with hair follicles) and other samples. Figures 8 to 13 It can be seen from the results shown that clear and accurate typing diagrams can be obtained by amplifying different types of samples using the system of the present invention, and both the molecular weight internal standard and the allele typing standard can accurately calibrate the typing.

[0098] 2.4. Species-specific detection:

[0099] This kit was used to perform species-specific detection of different species. DNA templates of chicken, pig, dog, cattle, sheep, fish and E. coli were diluted to 1 ng / μL and tested three times in parallel. Fig.14 As shown, this system did not amplify any bands in other species, indicating good species specificity.

[0100] In summary, the present invention uses ten fluorescent dye emission spectra that can be distinguished and do not penetrate each other, and the ten fluorescent emission intensities are high and well balanced, and the limitations of the existing detection system's fluorescent channels and number and the number of loci can be broken, and more mini STR and STR loci with high polymorphism can be included in the same fragment length range, not only covering the new CODIS loci recommended by the FBI of the United States, but also covering the 20 autosomal supplementary loci recommended by the judicial identification specification, and adding 1 Y chromosome STR locus DYS391, 2 X chromosome STR loci DXS8378, DXS6810, 1 gender locus Amel and 3 Y chromosome Indel rs199815934, rs759551978, rs771783753 for auxiliary judgment. The typing information of 48 STR loci can be obtained through one amplification, and the amplicon fragments of 16 STR loci are less than 220bp, and except for D10S2325, the amplicon fragments of the remaining loci are all less than 450bp. This can solve the problem of insufficient detection site information in a single test of trace samples, degraded samples, and difficult samples in complex environments. It can efficiently provide site information, thereby effectively improving the detection efficiency of the system.

Claims

1. A detection kit using a 10-color fluorescent autosomal STR composite amplification system, characterized in that it comprises a composite primer set of amplification primers for composite amplification of 48 STR loci; the 48 STR loci are divided into nine groups, the amplification primers of the 48 STR loci are respectively labeled with nine fluorescent dyes, namely, FAM, TET, HEX, ER650, ER652, ER663, ER664, ER674 and ER685, and the fluorescent dye ER690 is used as an internal standard marker; The 48 STR loci include 41 autosomal STR loci, 1 Y chromosome STR locus, 2 X chromosome STR loci, 1 gender locus Amel and 3 Y chromosome Indel; the 41 autosomal STR loci are: D3S1358, vWA, D12S391, CSF1PO, Penta E. D16S539, D7S820, D13S317, D2S1338, PentaD, D22S1045, D19S433, D18S51, D6S1043, D8S1179, D5S818, D21S11, F GA, TH01, D1S1656, TPOX, SE33, D4S2366, D6S477, D14S608, D19S253, D3S1744, D21S2055, D7S1517, D18S535, D10S1 435, D7S3048, D10S2325, D22GATA198B05, D15S659, D8S1132, D13S325, D2S441, D10S1248, D3S3045 and D5S2500; 1 Y chromosome STR locus: DYS391; 2 X chromosome STR loci: DXS8378 and DXS6810; 3 Y chromosome Indels: rs199815934, rs759551978 and rs771783753; The sequences and fluorescent markers of the 48 STR loci and their amplification primers are shown in the following table: 。 2. The detection kit according to claim 1, characterized in that the working concentrations of the amplification primers for 41 autosomal STR loci are as follows: D3S1358, 0.036±0.002μM; vWA, 0.06±0.002μM; D12S391, 0.14±0.002μM; CSF1PO, 0.06±0.002μM; Penta E, 0.2±0.002μM; D22S1045, 0.05±0.002μM; D19S433, 0.06±0.002μM; D18S51, 0.04±0.002μM; D6S104 3, 0.08±0.002μM; D16S539, 0.07±0.002μM; D7S820, 0.1±0.002μM; D13S317, 0.12±0.002μM; D2S1338, 0.2±0.002μM; PentaD, 0.15±0.002μM; D8S1179, 0.06±0.002μM; D5S818, 0.04±0.002μM; D21S11, 0.05±0.002μM; FGA, 0.06±0. 002μM; TH01, 0.06±0.002μM; D1S1656, 0.1±0.002μM; TPOX, 0.12±0.002μM; SE33, 0.11±0.002μM; D4S2366, 0.19±0.002μM; D6S D7 S1517, 0.17±0.002μM; D18S535, 0.15±0.002μM; D10S1435, 0.22±0.002μM; D7S3048, 0.42±0.002μM; D10S2325, 0.35±0.002μM; D22GATA198B05, 0.6±0.002μM; D15S659, 0.12±0.002μM; D8S1132, 0.1±0.002 μM; D13S325, 0.07±0.002μM; D2S441, 0.4±0.002μM; D10S1248, 0.5±0.002μM; D3S3045, 0.33±0.002μM; D5S2500, 0.3±0.002μM; The working concentrations of primers for amplification of 1 Y-chromosome STR locus were as follows: DYS391, 0.5 ± 0.002 μM; The working concentrations of the primers for amplification of the two X-chromosome STR loci were as follows: DXS8378, 0.5 ± 0.002 μM; DXS6810, 0.4 ± 0.002 μM; The working concentrations of the amplification primers for 1 sex locus, Amel, were as follows: Amel, 0.06 ± 0.002 μM; The working concentrations of the primers for amplification of the three Y chromosome Indels were as follows: rs199815934, 0.3 ± 0.002 μM; rs771783753, 0.3 ± 0.002 μM; rs759551978, 0.4±0.002μM.

3. The detection kit according to claim 1 or 2, characterized in that it comprises 25 μL of amplification reaction system: 10 μL of 2.5×PCR Mix, 5 μL of T48 primer mixture, 1 μL of Taq DNA polymerase, 2 μL of template DNA, and 7 μL of deionized water; wherein, The T48 primer mixture is a mixture of amplification primers for 48 loci; the components of 2.5×PCR Mix are: Tris-HCl 90 mM, pH 8.4, MgCl2 10 mM, KCl 125 mM, BSA 10 mg / ml, and dNTPs 6.0 mM.

4. The detection kit according to claim 1 or 2, characterized in that it comprises an allele ladder containing 48 STR loci, a DNA standard, a fluorescent molecular weight internal standard O-500 and a T10-Matrix.

5. The detection kit according to claim 1 or 2, characterized in that the amplification reaction procedure is as follows: 95°C for 3 min, 95°C for 5 s, 62°C for 90 s, 28-30 cycles; final extension at 60°C for 20 min; hold at 4°C.

6. The detection kit according to claim 1 or 2, characterized in that among the PCR products of the amplification primers of 48 STR loci, 16 amplicon fragments are less than 220 bp, and except for D10S2325, the amplicon fragments of the remaining loci are all less than 450 bp.

7. A method for composite amplification and detection of 48 STR loci using the detection kit according to any one of claims 1 to 6, characterized in that it comprises the following steps: ① Sample processing: extract the genomic DNA of the sample as the amplification template or directly use the sample without extraction as the amplification template; ② using the amplification primers shown in SEQ ID NO: 01-101 in claim 1 to perform PCR amplification on the sample genomic DNA obtained in step ①; ③ Use the honor1816 genetic analyzer to detect the fluorescence signal of the amplified product obtained in step ②; ④ Collect fluorescence signal data and use gene analysis software to analyze the DNA typing results of 48 loci.

8. The method according to claim 7 is characterized in that in step ①, the amplification template is an extracted sample or a direct amplification sample, the extracted sample includes human semen stains, blood, body fluids, hair, tissue or blood stains; the direct amplification sample includes human blood or oral cells collected by one or more carriers in filter paper, blood card, cotton swab or FTA card.

9. An application of the detection kit according to any one of claims 1 to 6, characterized in that it is applied to individual identification of the Chinese population, paternity testing, forensic identification, construction of autosomal DNA database and / or population genetics research.

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