Eight-color fluorescence composite amplification kit for on-site DNA detection of a case

Abstract

The application discloses an eight-color fluorescent composite amplification kit for case scene DNA detection, which comprises a composite primer group of 66 specific primers for simultaneously amplifying 33 STR loci and a hot-start DNA polymerase reaction mixture; the 66 specific primers comprise 33 different upstream primers labeled with fluorescent dyes and 33 downstream primers without fluorescent labeling; the eight-group double-fluorescent-dye labeling method is adopted, and the 66 specific primers are divided into seven groups; and the amplification fragments of each autosomal locus are all less than 380 bp. The eight-group double-fluorescent-dye labeling method is adopted to establish an eight-color case composite amplification detection system, 33 STR loci can be simultaneously amplified, 33 STR loci information can be provided, the DNA sample with degradation, fracture or fragment loss caused by different reasons can be quickly and accurately detected, the method has the characteristics of high sensitivity, fast amplification speed and strong resistance to inhibitors, and can efficiently identify individual information.

Description

Technical Field

[0001] This invention relates to an eight-color fluorescent multiplex amplification kit for DNA detection at crime scenes, belonging to the field of forensic genetic testing technology. Background Technology

[0002] Short tandem repeats (STRs), also known as microsatellites or simple sequence repeats (SSRs), are a class of DNA tandem repeat sequences widely found in eukaryotic genomes, with a core sequence consisting of 2-6 base repeat units. STR loci are numerous and widely distributed, accounting for approximately 3% of the entire genome (International Human Genome Sequencing Consortium, 2001), and exhibit high polymorphism. This polymorphism primarily stems from individual differences in the number of core sequence repeats, and this variation follows Mendelian inheritance laws. Therefore, STR amplification detection technology is widely used for individual identification, kinship testing, and population genetics research. Conventional STR kits have low requirements for sample quality and are not suitable for case-specific kits. Case-specific kits must accurately detect trace samples, degraded biological samples, mixed biological samples, and inhibited samples. To meet these four conditions, the loci need to be arranged as centrally as possible, and the amplified fragments as small as possible, enabling rapid and accurate detection from DNA samples that have undergone degradation, breakage, or fragment loss due to various reasons.

[0003] In case detection kits, the most common detection method is a combination of multiplex PCR amplification and capillary electrophoresis. This involves using a mixture of primer complexes labeled with one or six single-fluorescein dyes to specifically amplify the STR loci using polymerase chain reaction (PCR). The specific amplification products are then mixed with a standard of known length labeled with a single fluorescein dye and simultaneously electrophoresed. On a genetic analysis sequencer, a sample with the same fluorescent dye spectrum as the labeled primers is selected for capillary electrophoresis. Simultaneously, the system's accompanying software collects data, and analysis software is used to perform genotyping analysis on the electrophoresis results.

[0004] Currently, fluorescent detection kits for genetic testing in the domestic market typically use four groups of single fluorescent dyes, such as 6-FAM, VIC, NED, and PET, or five groups, such as 6-FAM, VIC, NED, TAZ, and SID, to label primers. Among these, the excitation wavelengths of fluorescent dyes like VIC, NED, and PET are all greater than 540 nm. Because longer wavelengths result in lower energy, the products amplified using primers labeled with single fluorescent dyes like VIC, NED, and PET emit relatively weak fluorescence during electrophoresis. This is detrimental to signal collection by the laser detector in genetic analyzers and can easily lead to the loss of STR locus information. Furthermore, current market-available 8-color case kits specifically designed for small DNA fragments typically have a limited number of loci for these fragments. For example, patent number 202210146394.6 discloses an 8-color fluorescent multiplex amplification kit and its application for detecting trace amounts of degraded DNA, which includes 16 STR loci and one sex locus; patent number 202111128931.6 discloses an 8-color fluorescent STR multiplex amplification detection kit and its application, which includes 15 STR loci and one sex locus. Both of these kits can only detect 15 or 16 autosomes and one sex locus in a single test, resulting in a limited number of loci and limited recognition capabilities. This significantly limits their use in large-scale suspect DNA screening and complex case investigations. Summary of the Invention

[0005] This invention provides an eight-color fluorescent multiplex amplification kit for DNA detection at crime scenes. The invention employs an eight-group dual-fluorescent dye labeling method to establish an eight-color case multiplex amplification detection system, which can simultaneously amplify 33 STR loci and provide information on all 33 STR loci. It features high sensitivity, fast amplification speed, and strong resistance to inhibitors, enabling efficient identification of individual information.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] An eight-color fluorescent multiplex amplification kit for DNA detection at crime scenes, characterized in that it includes a composite primer set of 66 specific primers that simultaneously amplify 33 STR sites and a reaction mixture containing hot-start DNA polymerase;

[0008] The 66 specific primers consist of 33 upstream primers labeled with different fluorescent dyes and 33 downstream primers without fluorescent labels, numbered from PM NO.01 to PM NO.66: YIn1 PM NO.01-02, D3S1358 PM NO.03-04, vWA PM NO.05-06, D2S1338 PM NO.07-08, D6S477 PM NO.09-10, D19S433 PM NO.11-12, FGA PM NO.13-14, D8S1132 PM NO.15-16, TH01 PM NO.17-18, D16S539 PM NO.19-20, D7S820 PM NO.21-22, D1S1656 PM NO.23-24, D15S659 PM NO.25-26, YIn3 PM NO.01-02, D3S1358 PM NO.03-04, vWA PM NO.05-06, D2S1338 PM NO.07-08, D6S1477 PM NO.09-10, D19S1433 PM NO.11-12, FGA PM NO.13-14, D8S1132 PM NO.15-16, TH01 PM NO.17-18, D16S539 PM NO.19-20, D7S820 PM NO.21-22, D1S1656 PM NO.23-24, D15S659 PM NO.25-26, YIn3 PM NO.01-02, D15S1659 PM NO.25-26 ... NO.27-28, YIn2 PMNO.29-30, Amel PM NO.31-32, D5S818 PM NO.33-34, D21S11 PM NO.35-36, D19S253PMNO.37-38, D10S1435 PM NO.39-40, PentaD PM NO.41-42, D18S51 PM NO.43-44, D6S1043PM NO.45-46, D3S3045 PM NO.47-48, D8S1179 PM NO.49-50, D13S317 PM NO.51-52, D12S391PM NO.53-54, D10S1248 PM NO.55-56, D2S441 PM NO.57-58、CSF1PO PM NO.59-60, TPOX PM NO.61-62, PentaE PM NO.63-64, D22S1045 PM NO.65-66;

[0009] An eight-group dual-fluorescent dye labeling method was used. The 66 specific primers were divided into seven groups. The first, second and third groups were labeled with FAM, TET and HEX respectively at the first base of the 5' end of the primers. The fourth, fifth, sixth and seventh groups were labeled with AT55, AT12, AT13 and AT14 respectively at the first base of the 5' end of the primers, and then a base in the middle sequence between the 5' and 3' ends of the primers was labeled with FAM.

[0010] Group 1: YIn1, D3S1358, VWA, D2S1338, D6S477, primer numbers: PM NO.001-PM NO.010;

[0011] Group 2: D19S433, FGA, D8S1132, primer numbers: PM NO.011-PM NO.016;

[0012] Group 3: TH01, D16S539, D7S820, D1S1656, D15S659, YIn3, primer numbers: PM NO.017-PMNO.028;

[0013] Group 4: YIn2, AMEL, D5S818, D21S11, D19S253, D10S1435, primer numbers: PM NO.029-PM NO.040;

[0014] Group 5: PentaD, D18S51, D6S1043, D3S3045, primer numbers: PM NO.041-PM NO.048;

[0015] Group 6: D8S1179, D13S317, D12S391, D10S1248, D2S441, primer numbers: PM NO.049-PM NO.058;

[0016] Group 7: CSF1PO, TPOX, Penta E, D22S1045, primer numbers: PM NO.059-PM NO.066.

[0017] This invention employs an eight-group dual-fluorescent dye labeling method to establish an eight-color multiplex amplification detection system capable of simultaneously amplifying 33 STR loci. The aforementioned multiplex amplification kit simultaneously detects 33 STR gene loci, including: Yln1, D3S1358, VWA, D2S1338, D6S477, D19S433, FGA, D8S1132, TH01, D16S539, D7S820, and D... 1S1656, D15S659, Yln3, Yln2, AMEL, D5S818, D21S11, D19S253, D10S1435, PentaD, D18S51 , D6S1043, D3S3045, D8S1179, D13S317, D12S391, D10S1248, D2S441, CSF1PO, TPOX, Penta E.D22S1045.

[0018] The 33 autosomal loci mentioned above include 13 CODIS core loci: CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1779, D13S317, D16S539, D18S51, and D21S11; 4 newly added CODIS loci: D2S1338, D1S1656, D12S391, and D19S433; 2 low-stutter, high-polymorphism loci; and 1 population-high-polymorphism locus, which are beneficial for population-specific screening. The sex locus is Amel, and there are three Y-indels with low mutation rates in the population, almost close to zero mutations, which are more conducive to rapid comparison in male family pedigree screening.

[0019] Compared with the prior art, the present invention has the following advantages: (1) The kit is an eight-color fluorescent multiplex amplification kit, which can accommodate more loci in small fragments; (2) The amplified fragments of the loci in the kit are all less than 380bp, which has a good detection effect on samples with trace amounts of degradation; (3) The primers in the kit of the present invention have the advantages of high specificity, high sensitivity and accurate typing results, which can fully meet the needs of actual case inspection, DNA database construction and parentage identification.

[0020] The allele fragments selected in this application are in the range of 75-380 bp in length, which can obtain as much genetic information as possible from challenging case evidence (including touch, degradation, and inhibitor-containing samples), making them more suitable for genetic analysis of on-site biological evidence and degradation samples; the individual identification ability is above 0.999; they have regular three, four, or five base repeats; the gene mutation rate is low, close to zero mutation; the allele discrimination rate is high; and the loci have high species specificity.

[0021] To achieve reproducible, sensitive, and efficient amplification in multiplex PCR, primer design and selection for gene loci must be highly specific, eliminating analytical errors caused by homology. In this application, the entire set of primers is designed with uniform parameters to ensure that the annealing temperatures of the primers are as consistent as possible; the primer binding regions avoid SNP sites, the amplicon avoids deletion sites, and the upstream primers for each gene locus are labeled with dual fluorescent dyes.

[0022] Primer sequences are shown in Table 2.

[0023] The above-mentioned hot-start DNA polymerase reaction mixture consists of: Taq DNA polymerase 5 U / µL, MgCl2 10.25 mM, KCl 150 mM, Tris-HCl 125 Mm, BSA 13 mg / ml, dATP 4.0 mM, dTTP 4.0 mM, dCTP 4.0 mM, and dGTP 4.0 mM. Furthermore, specifically for simulating case samples, 1 M-5 M betaine, 0.5%-0.8% NaN3, 4-7% DTT, and 3.5% glycerol are added. This allows for the amplification of templates with high GC content and complex secondary structures, resulting in stronger inhibitor tolerance and higher amplification efficiency. All percentages mentioned above are by weight. Further optimization involves the addition of 4.7 M betaine, 0.6% NaN3, 5% DTT, and 3.5% glycerol.

[0024] This kit is suitable for amplification of different samples and has a wide annealing temperature range. The amplification system can be amplified using the following program on various reaction thermal cyclers: 95℃ for 3 min, 95℃ for 5 s, 60℃ for 90 s, 28-30 cycles; final extension at 60℃ for 30 min; hold at 4℃ for PCR amplification; the amplification products are then subjected to capillary electrophoresis on the Honor 1816 genetic analyzer of Suyuan Gene and the data are collected, followed by data analysis using GeneManager (developed by Nanjing Suyuan Gene Technology Co., Ltd.) analysis software.

[0025] The DNA samples extracted from the above-mentioned samples mainly include human semen stains, blood, body fluids, hair, tissues, bloodstains, etc. The main methods for DNA genome extraction are magnetic bead extraction or Chelex 100 method; filter paper, cotton swabs, saliva cards, gauze, FTA cards and other samples can also be used for direct amplification.

[0026] The above-mentioned kits can be used for individual identification and paternity testing.

[0027] This invention utilizes a fluorescence resonance energy transfer (FRET) system to address the issue of weakened fluorescence signals in the long-wavelength range, aiming to improve fluorescence intensity and reagent kit sensitivity. FRET consists of two fluorescent groups: a fluorescent donor and a fluorescent acceptor, which can transfer energy within a certain distance. When the distance between the fluorescent donor and acceptor is less than 10 nm, the fluorescent donor group is electronically excited, and energy is non-radiatively transferred to the fluorescent acceptor group through intramolecular interactions. This energy transfer quenches the fluorescence intensity of the fluorescent donor and reduces its excited-state lifetime, while simultaneously increasing the fluorescence emission intensity of the fluorescent acceptor group. This invention also employs the FRET system and an eight-set dual-fluorescent-labeled STR primer hybrid system, which not only improves detection sensitivity in the long-wavelength range but also allows for a more concentrated arrangement of gene loci, significantly reducing the size of amplified fragments.

[0028] Working concentrations of 66 specific primers: PM NO. 01: 8 μM, PM NO. 02: 10 μM, PM NO. 03: 2 μM, PM NO. 04: 2 μM, PM NO. 05: 8 μM, PM NO. 06: 8 μM, PM NO. 07: 7 μM, PM NO. 08: 9 μM, PM NO. 09: 3 μM, PM NO. 10: 3 μM, PM NO. 11: 4.5 μM, PM NO. 12: 4.5 μM, PM NO. 13: 4 μM, PM NO. 14: 4 μM, PM NO. 15: 4 μM, PM NO. 16: 4 μM, PM NO. 17: 1.3 μM, PM NO. 18: 1.3 μM, PM NO. 19: 1.5 μM, PM NO. 20: 1.5 μM, PM NO. 21: 6.5 μM, PM NO. 22: 6.5 μM, PM NO. 23: 6.7 μM, PM NO. 24: 6.7 μM, PM NO. 25: 5 μM, PM NO. 26: 5 μM, PM NO. 27: 10 μM, PM NO. 28: 12 μM, PM NO. 29: 3 μM, PM NO. 30: 3 μM, PM NO. 31: 1.5 μM, PM NO. 32: 1.5 μM, PM NO. 33: 8 μM, PM NO. 34: 8 μM, PM NO. 35: 2.5 μM, PM NO. 36: 2.5 μM, PM NO. 37: 2 μM, PM NO. 38: 2 μM, PM NO. 39: 4 μM, PM NO. 40: 4 μM, PM NO. 41: 6 μM, PM NO. 42: 6 μM, PM NO. 43: 5 μM, PM NO. 44: 5 μM, PM NO. 45: 4.5 μM, PM NO. 46: 4.5 μM, PM NO. 47: 3 μM, PM NO. 48: 3 μM, PM NO. 49: 3.5 μM, PM NO. 50: 3.5 μM, PM NO. 51: 5.5 μM, PM NO. 52: 5.5 μM, PM NO. 53: 10 μM, PM NO. 54: 10 μM, PM NO. 55: 7.5 μM, PM NO. 56: 7.5 μM, PM NO. 57: 5 μM, PM NO. 58: 5 μM, PM NO. 59: 6.5 μM, PM NO. 60: 6.5 μM, PM NO. 61: 10 μM, PM NO. 62: 10 μM, PM NO. 63: 8 μM, PM NO. 64: 12 μM, PM NO. 65: 12.5 μM, PM NO. 66: 12.5 μM.

[0029] This invention discloses a kit for the detection of 33 short tandem repeat (STR) sequences on chromosomes using a dual-fluorescent labeling method. It employs eight sets of dual-fluorescent dyes to label and prepare primer sets for the multiplex amplification of 33 loci, simultaneously amplifying 33 STR sites. This kit contains 10 mini STRs, 12 fragments smaller than 220 bp, and 21 fragments smaller than 320 bp, enabling the extraction of more genetic information from challenging crime evidence (including touched, degraded, and inhibitor-containing samples), making it more suitable for genetic analysis of on-site biological evidence and degraded samples. The use of FRET dual-fluorescent labeling technology ensures that the longer wavelength fluorescent dyes AT550, AT12, AT13, and AT14 still exhibit efficient fluorescence signals, improving detection sensitivity and amplification efficiency. It also demonstrates good species specificity and good amplification response across different brands of PCR amplification instruments, exhibiting strong applicability.

[0030] The site information for the fluorescence detection kits in this application and related domestic and international cases is shown in the table below:

[0031]

[0032]

[0033]

[0034] Note: + indicates included loci, - indicates excluded loci.

[0035] Any techniques not mentioned in this invention are based on existing technologies.

[0036] The beneficial effects of this invention are:

[0037] 1. This kit employs eight groups of fluorescent dyes, providing information on 33 STR loci, all within 380bp. This significantly improves the amplification efficiency of small fragments, resulting in better and more complete genotyping. For highly degraded DNA samples, it can enhance the success rate of individual identification and paternity testing, making it an ideal choice for handling old or newly reported cases and more challenging samples. A single experiment with this kit can meet the requirements of the anti-trafficking DNA database for autosomal loci, obtaining a large number of loci and reducing the need for supplementary kit experiments.

[0038] 2. The gene fragments are shorter, mostly below 380bp. There are 10 Mini STRs shorter than 220bp, 12 fragments shorter than 220bp, with a cumulative individual recognition rate of 0.9999999769. There are 21 fragments shorter than 350bp, with a cumulative individual recognition rate of 0.99999999999999933643. The cumulative non-paternity screening rate is above 0.999, which is more conducive to rapid and accurate identification. It covers the 29 autosomal anti-trafficking loci specified in the autosomal anti-trafficking database, making it the kit with the most loci and the smallest fragments among current case kits.

[0039] 3. It fills the gap in the current market for 8-color case kits targeting small fragments, which have fewer loci. A single experiment can meet the requirements of autosomal loci in the anti-trafficking DNA database, and obtain more loci, reducing the work of supplementing the kit. It covers the 29 autosomal anti-trafficking loci specified by the autosomal anti-trafficking database, making it the case kit with the most loci and the smallest fragments currently available.

[0040] 4. For simulating case evidence, betaine, NaN3, DTT, and glycerol were specifically added, making the kit more resistant to inhibitors and increasing amplification efficiency. Sensitivity can reach 20 pg.

[0041] 5. The use of FRET dual fluorescent labeling technology ensures that the longer wavelength fluorescent dyes AT550, AT12, AT13, and AT14 still have high-efficiency fluorescence signals, improving detection sensitivity and amplification efficiency. Through optimization of the amplification system, the overall amplification time is controlled at around 60 minutes, resulting in fast amplification speed, good uniformity, and stronger tolerance to inhibitors.

[0042] 6. The primers in this kit have good species specificity and do not amplify the genomes of animals that humans frequently come into contact with, such as chickens, ducks, dogs, cats, pigs, cattle, horses, and sheep. The primers are designed to maintain a consistent Tm value, and they have good amplification reactions with different brands of PCR amplification instruments, making them highly applicable. Attached Figure Description

[0043] Figure 1 This is a diagram showing the arrangement of gene loci in the kit of the present invention;

[0044] Figure 2 This is a standard diagram of allele typing for the kit of the present invention;

[0045] Figure 3 The typing results are for 9948 pairs of positive standards amplified by the kit of this invention;

[0046] Figure 4This is the sensitivity amplification detection spectrum of the kit of the present invention;

[0047] Figure 5-12 This is a spectrum of amplification detection of anti-inhibitor ability;

[0048] Figure 13-21 This is a chromatogram of amplified mixed samples.

[0049] Figure 22-29 This is a species-specific amplification detection map;

[0050] Figure 30 The detection pattern of genomic DNA amplified from an old blood card sample from 2015 using the kit of the present invention in Example 1;

[0051] Figure 31 This is the detection pattern of genomic DNA amplified from an old blood card sample from 2015 using an Applied Biosystems kit, as shown in Example 1.

[0052] Figure 32 In Example 1, the reagent kit of the present invention amplifies the detection spectrum of clothing at a crime scene;

[0053] Figure 33 In Example 1, the Applied Biosystems reagent kit amplified the detection spectrum of clothing at a crime scene.

[0054] Figure 34 In Example 1, the kit of the present invention amplifies the detection spectrum of semen stains at a crime scene;

[0055] Figure 35 In Example 1, an Applied Biosystems reagent kit was used to amplify the detection pattern of semen stains at a crime scene. Detailed Implementation

[0056] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0057] Example 1

[0058] Construction of the eight-color fluorescent multiplex amplification primer system:

[0059] A kit for detecting multiplex amplification of 33 chromosomal short tandem repeat sequences using a dual-fluorescent labeling method includes: a primer set of 66 specific primers for simultaneous amplification of 33 STR loci, a reaction mixture, and a hot-start Taq polymerase; a primer set for multiplex amplification of 33 STR loci prepared using an eight-set dual-fluorescent dye labeling method, simultaneously amplifying 33 STR loci; including: Yln1, D3S1358, VWA, D2S1338, D6S477, D19S433, FGA, D... 8S1132, TH01, D16S539, D7S820, D1S1656, D15S659, Yln3, Yln2, AMEL, D5S818, D21S11, D19S253, D10S1435 , PentaD, D18S51, D6S1043, D3S3045, D8S1179, D13S317, D12S391, D10S1248, D2S441, CSF1PO, TPOX, Penta E.D22S1045.

[0060] The mixture containing the hot-start DNA polymerase reaction is shown in Table 1.

[0061] Table 1. Composition of the mixture containing hot-start DNA polymerase reaction

[0062] Taq DNA polymerase 5U / uL <![CDATA[MgCl2]]> 10.25mM KCl 150mM Tris-HCl 125Mm BSA 13mg / ml dATP 4.0mM dTTP 4.0mM dCTP 4.0mM dGTP 4.0mM

[0063] The 66 specific primers were divided into seven groups. The first, second and third groups were labeled with FAM, TET and HEX respectively at the first base of the 5' end of the primers. The fourth, fifth, sixth and seventh groups were labeled with AT550, AT12, AT13 and AT14 respectively at the first base of the 5' end of the primers, and then a base in the middle sequence between the 5' and 3' ends of the primers was labeled with FAM.

[0064] Group 1: Yln1, D3S1358, VWA, D2S1338, D6S477, primer numbers: PM NO.001-PM NO.010;

[0065] Group 2: D19S433, FGA, D8S1132, primer numbers: PM NO.011-PM NO.016;

[0066] Group 3: TH01, D16S539, D7S820, D1S1656, D15S659, Yln3, Primer numbers: PM NO.017-PM NO.028;

[0067] Group 4: Yln2, AMEL, D5S818, D21S11, D19S253, D10S1435, primer numbers: PM NO.029-PM NO.040;

[0068] Group 5: PentaD, D18S51, D6S1043, D3S3045, primer numbers: PM NO.041-PM NO.048;

[0069] Group 6: D8S1179, D13S317, D12S391, D10S1248, D2S441, primer numbers: PM NO.049-PM NO.058;

[0070] Group 7: CSF1PO, TPOX, Penta E, D22S1045, primer numbers: PM NO.059-PM NO.066;

[0071] Primers were designed using the professional primer design software Oligo7 to target the flanking regions of the core repeat regions at 33 STR gene loci, ensuring that the Tm value of each primer was close to 60℃ and the amplification products ranged from 75 to 380 bp. Each primer was further optimized through testing until high amplification efficiency was achieved, mismatches were eliminated, and no nonspecific amplification peaks were generated. Multiplex amplification tests were then performed, and no nonspecific peaks were observed between 57℃ and 63℃. Primer sequences are shown in Table 2.

[0072] Table 2. Primer information for 33 loci

[0073]

[0074]

[0075]

[0076] To ensure the uniformity of the amplified gene peaks met the requirements, a series of tests were conducted on primer concentration and amplification components, ultimately determining the primer dosage and the composition of the amplification buffer.

[0077] The kit consists of two parts: a pre-reaction amplification kit and a post-reaction electrophoresis kit. The components of each kit are shown in Table 3.

[0078] Table 3. Components of each reagent kit

[0079]

[0080] The allele typing standards provided by this invention are the products of the amplification of the corresponding allele plasmids by primers of each STR locus. The DNA positive standard 9948 was purchased from Suzhou Sacman Biotechnology Co., Ltd., and the 2.5×TFS Master Mixture was purchased from Kangwei Century Co., Ltd. Figure 1 Electrophoresis pattern for Allelic Ladder.

[0081] The kit of the present invention has been verified to have good amplification efficiency in the annealing temperature range of 57℃-63℃, and therefore has good amplification efficiency on different brands of PCR instruments (ABI9700, ABI veriti, E1000, etc.).

[0082] When amplifying the 9948 positive standard, prepare the reaction mixture according to Table 4.

[0083] Table 4. Amounts of each component added in the PCR system preparation.

[0084] 2.5×TFS Master Mixture 12.5μL E33CPrimers 5.0μL 9948 positive DNA 1μL PCR Grade Water Add to a total volume of 25 μL Total reaction volume 25μL

[0085] The amplification system yielded good results using the following program on various PCR instruments (ABI 9700, ABI Veriti, E1000, etc.): 28 cycles of 95℃ for 3 min, 95℃ for 5 s, and 60℃ for 90 s; a final extension at 60℃ for 30 min; and incubation at 4℃ for PCR amplification. 1 μL of the obtained PCR product was mixed with 0.15 μL of molecular weight internal standard and 9 μL of deionized formamide, and then subjected to capillary electrophoresis on a Suyuan Gene 1816 genetic analyzer. Electrophoresis parameters: injection time 18 s, injection voltage: 3.0 kV; other parameters were default. Electrophoresis data were collected using data collection software and analyzed using GeneManager software. Figure 2 The amplification and detection chromatogram of 9948 is completely consistent with the theoretical typing.

[0086] In the case of amplifying FTA blood cards, prepare the reaction mixture according to Table 5.

[0087] Table 5. Amounts of each component added in the PCR system preparation.

[0088] 2.5×TFS Master Mixture 12.5μL E33C primers 5.0μL FTA card or filter paper 1-2 pieces with a 1.0 or 1.2 mm aperture PCR Grade Water Add to a total volume of 25 μL Total reaction volume 25μL

[0089] Amplification was performed using a GE E1000 PCR amplification instrument according to the following amplification program: 95℃ for 3 min, 95℃ for 5 s, 60℃ for 90 s, 28 cycles; final extension at 60℃ for 30 min; and incubation at 4℃ for PCR amplification. 1 μL of the obtained PCR product was mixed with 0.15 μL of molecular weight internal standard and 9 μL of deionized formamide, and then subjected to capillary electrophoresis on a Suyuan Gene 1816 genetic analyzer. Electrophoresis parameters: injection time 18 s, injection voltage: 3.0 kV; other parameters were default. Electrophoresis data were collected using data collection software and analyzed using GeneManager software. Figure 3 This is a sensitivity amplification detection spectrum. Figure 4-11 This is a spectrum for detecting the ability to resist inhibitors. Figure 12-20 This is a spectrum of amplification detection for mixed samples.

[0090] This invention also tested a series of performance parameters of the kit, such as sensitivity, inhibitor resistance, mixed sample detection rate, and genus specificity. Sensitivity was tested. By amplifying different concentrations of 9948 positive DNA, the lowest DNA concentration capable of detecting all STR loci was identified. The amplification system was configured according to Table 4, with 9948 input amounts of 625 pg, 125 pg, 31.25 pg, and 20 pg. The amplification program was 95℃ for 3 min, 95℃ for 5 s, and 60℃ for 90 s, for 28 cycles; a final extension at 60℃ for 30 min; and incubation at 4℃ for PCR amplification. The amplified products were subjected to electrophoresis and data analysis using a Suyuan Gene 1816 genetic analyzer. Figure 3 As shown, 20 pg can obtain complete typing results, with an average peak height of around 1500 rfu, indicating that the kit's sensitivity can reach 20 pg.

[0091] The detection rate of mixed samples was mainly tested by assessing whether the genotypes of each locus in the two samples could be correctly distinguished when the total template amount was 1 ng and the mixing ratio of T1 (9947) to T2 (9948) was 1:1, 1:2, 2:1, 1:4, 4:1, 1:8, 8:1, 1:16, and 16:1. Peak loss began to appear at the 1:8 and 8:1 ratios. Specific results are shown in Table 6 below.

[0092] Table 6 Detection rate of mixed samples

[0093]

[0094]

[0095] There was no amplification response in the genomes of animals that humans frequently come into contact with, such as horses, dogs, pigs, cattle, sheep, cats, chickens, and ducks. Figure 21-28Experiments showed that the individual identification ability was above 0.999, and accurate typing and detection could still be completed even when the DNA concentration was 20 pg.

[0096] Application of the eight-color fluorescent multiplex amplification primer system in detecting highly degraded DNA samples:

[0097] 1. Collect degraded samples

[0098] ① Genomic DNA extracted from an old blood card sample from 2015 using the magnetic bead extraction method.

[0099] ② Clothing extracted from a crime scene using magnetic bead extraction method.

[0100] ③ Semen stains extracted from a crime scene using magnetic bead extraction method.

[0101] 2. Perform PCR amplification

[0102] The PCR system is as follows:

[0103] 2.5×TFS Master Mixture 12.5μL E33CPrimers 5.0μL 9948 positive DNA 1μL PCR Grade Water Add to a total volume of 25 μL Total reaction volume 25μL

[0104] The amplification program was as follows: 95℃ for 3 min, 95℃ for 5 s, 60℃ for 90 s, 28 cycles; final extension at 60℃ for 30 min; and hold at 4℃.

[0105] 3. Capillary electrophoresis detection

[0106] The obtained PCR amplification product was mixed with 1 μL of the product, 0.15 μL of molecular weight internal standard, and 9 μL of deionized formamide, and then subjected to capillary electrophoresis on a Suyuan Gene 1816 genetic analyzer. Electrophoresis parameters: injection time 18 s, injection voltage 3.0 kV; other parameters were left at machine default. Electrophoresis data were collected using data collection software.

[0107] 4. Data Analysis

[0108] Use GeneManager software for data analysis.

[0109] 5. Conclusion

[0110] The above-mentioned degraded samples were amplified using this kit. The amplification results are shown below. Figure 30 , Figure 32 , Figure 34 Using VeriFiler from Applied Biosystems. TM The Plus kit was used to amplify the same sample; the amplification results are shown below. Figure 31 , Figure 33 Figure 35The results showed that the kit of the present invention had better uniformity and detection rate in amplifying degraded samples than the AB kit, and the overall peak height was higher than that of the AB kit. Furthermore, the AB kit showed peak loss when amplifying large fragments, while the kit of the present invention did not show peak loss. It also performed better on amplified sample types and obtained more gene locus information.

[0111] Example 2

[0112] The kit consists of the following components: primer mixture, 500 μL; genomic DNA, 10 ng; PCR buffer 2.5*TFSMaster Mix, 1 mL; ddH2O, 1 mL; PCR enhancer.

[0113] The PCR buffer Master Mix contains: Taq DNA polymerase 5.5 U / µL, MgCl2 12.25 mM, KCl 150 mM, Tris-HCl 135 Mm, BSA 15 mg / mL, dATP 4.0 mM, dTTP 4.0 mM, dCTP 4.0 mM, and dGTP 4.0 mM. PCR enhancers are added to the PCR buffer Master Mix with 4.7 M betaine, 0.6% NaN3, 5% DTT, and 3.5% glycerol.

[0114] The inhibitor resistance test was conducted by adding different concentrations of inhibitors to a 10 μL system containing 1 ng of 9948, with the final concentrations of each inhibitor varying in a gradient. The products after the PCR reaction were then analyzed to examine and record the kit's tolerance to different concentrations of inhibitors. A control group without PCR enhancer was also included. The E33C kit showed tolerance (with enhancer) to heme 500 μM, hemoglobin 180 μM, humic acid 80 mg / L, EDTA 0.4 mM, indigo 40 mM, tannic acid 150 ng / μL, ferric ions 12 mM, and calcium ions 1.5 mM. The E33C kit showed tolerance (without enhancer) to heme 150 μM, hemoglobin 80 μM, humic acid 40 mg / L, EDTA 0.2 mM, indigo 10 mM, tannic acid 70 ng / μL, ferric ions 5 mM, and calcium ions 0.5 mM. Specific inhibitor resistance is shown in Table 7 below.

[0115] Table 7 Inhibitor Tolerance

[0116]

[0117] The only difference between this kit and Example 1 is that "4.7M betaine, 0.6% NaN3, 5% DTT and 3.5% glycerol are added to the PCR buffer MasterMix". Everything else is the same as in Example 1. The amplification process, related effects and specific applications in this example are consistent with those in Example 1 (the difference is less than 0.001% and can be ignored).

[0118] In summary, this invention, by employing eight groups of fluorescent dyes for dual-dye labeling, not only significantly reduces the number of amplified loci but also avoids the problem of low fluorescent dye signal intensity and easy quenching in the long wavelength range. At the same time, it improves the sensitivity and uniformity of the test kit.

Claims

1. An eight-color fluorescent multiplex amplification kit for DNA detection at crime scenes, characterized in that: This includes a complex primer set of 66 specific primers that simultaneously amplify 33 STR sites and a reaction mixture containing hot-start DNA polymerase; The 66 specific primers include 33 upstream primers labeled with different fluorescent dyes and 33 downstream primers without fluorescent labels; The 33 STR loci and their corresponding 66 specific primers are as follows: An eight-group dual-fluorescent dye labeling method was used. The 66 specific primers were divided into seven groups. The first, second and third groups were labeled with FAM, TET and HEX respectively at the first base of the 5' end of the primers. The fourth, fifth, sixth and seventh groups were labeled with AT55, AT12, AT13 and AT14 respectively at the first base of the 5' end of the primers, and then FAM was used to label one base in the middle sequence between the 5' and 3' ends of the primers. Group 1: YIn1, D3S1358, VWA, D2S1338, D6S477, primer numbers: PM NO.001-PM NO.010; Group 2: D19S433, FGA, D8S1132, primer numbers: PM NO.011-PM NO.016; Group 3: TH01, D16S539, D7S820, D1S1656, D15S659, YIn3, primer numbers: PM NO.017-PMNO.028; Group 4: YIn2, AMEL, D5S818, D21S11, D19S253, D10S1435, primer numbers: PM NO.029-PM NO.040; Group 5: PentaD, D18S51, D6S1043, D3S3045, primer numbers: PM NO.041-PM NO.048; Group 6: D8S1179, D13S317, D12S391, D10S1248, D2S441, primer numbers: PM NO.049-PM NO.058; Group 7: CSF1PO, TPOX, Penta E, D22S1045, primer numbers: PM NO.059- PM NO.066; The amplified fragments at each autosomal locus were all less than 380 bp; The hot-start DNA polymerase reaction mixture also includes 1M-5M betaine, 0.5%-0.8% NaN3, 4-7% DTT, and 2-5% glycerol.

2. The eight-color fluorescent multiplex amplification kit for DNA detection at crime scene as described in claim 1, characterized in that: the amplified fragments of each autosomal locus are in the range of 75-380 bp.

3. The eight-color fluorescent multiplex amplification kit for DNA detection at crime scene as described in claim 1 or 2, characterized in that: the hot-start DNA polymerase reaction mixture is composed of: Taq DNA polymerase 5.5 U / uL, MgCl2 12.25 mM, KCl 150 mM, Tris-HCl 135 Mm, BSA 15 mg / ml, dATP 4.0 mM, dTTP 4.0 mM, dCTP 4.0 mM and dGTP 4.0 mM.

4. The eight-color fluorescent multiplex amplification kit for DNA detection at crime scene as described in claim 1 or 2, characterized in that: the working concentrations of the 66 specific primers are: PM NO.001: 8 μM, PM NO.002: 10 μM, PM NO.003: 2 μM, PM NO.004: 2 μM, PM NO.005: 8 μM, PM NO.006: 8 μM, PM NO.007: 7 μM, PM NO.008: 9 μM, PM NO.009: 3 μM, PM NO.010: 3 μM, PM NO.011: 4.5 μM, PM NO.012: 4.5 μM, PM NO.013: 4 μM, PM NO.014: 4 μM, PM NO.015: 4 μM, PM NO.016: 4 μM, PM NO.017: 1.3 μM, PM NO.018: 1.3 μM, PM NO.019: 1.3 μM, PM NO.010: 1.3 μM, PM NO.011: 1.3 μM, PM NO.012: 4.5 μM, PM NO.013: 4 μM, PM NO.014: 4 μM, PM NO.015: 4 μM, PM NO.016: 4 μM, PM NO.017: 1.3 μM, PM NO.018: 1.3 μM, PM NO.019: 1.3 μM, PM NO.010 ... NO.019: 1.5μM, PM NO.020: 1.5μM, PM NO.021: 6.5μM, PM NO.022: 6.5μM, PM NO.023: 6.7μM, PM NO.024: 6.7μM, PM NO.025: 5μM, PM NO.026: 5μM, PM NO.027: 10μM, PM NO.028: 12μM, PM NO.029: 3μM, PM NO.030: 3μM, PM NO.031: 1.5μM, PM NO.032: 1.5μM, PM NO.033: 8μM, PM NO.034: 8μM, PM NO.035: 2.5μM, PM NO.036: 2.5μM, PM NO.037: 2μM, PM NO.038: 2μM, PM NO.039: 4μM, PMNO.040: 4μM, PM NO.041: 6μM, PM NO.042: 6μM, PM NO.043: 5μM, PM NO.044: 5μM, PM NO.045: 4.5μM, PM NO.046: 4.5μM, PM NO.047: 3μM, PM NO.048: 3μM, PM NO.049: 3.5μM, PM NO.050: 3.5μM, PM NO.051: 5.5μM, PM NO.052: 5.5μM, PM NO.053: 10μM, PM NO.054: 10μM, PMNO.055: 7.5μM, PM NO.056: 7.5μM, PM NO.057: 5μM, PM NO.058: 5μM, PM NO.059: 6.5μM, PMNO.060: 6.5μM, PM NO.061: 10μM, PM NO.062: 10μM, PM NO.063: 8μM, PM NO.064:12μM,PMNO.065:12.5μM,PM NO.066:12.5μM。.

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