Six-color fluorescence labeling kit for simultaneously amplifying 41 Y STR gene loci of human and application of six-color fluorescence labeling kit

Abstract

The invention relates to the technical field of biology, in particular to a six-color fluorescence labeling kit for simultaneously amplifying 41 Y STR gene loci of human and application of the six-color fluorescence labeling kit. According to the primer group, the kit and the detection method, 41 Y-STR gene loci can be amplified and detected at a time, the number and functions of the gene loci cover gene loci of most kits in the market, meanwhile, the primer group comprises 3 Y-indel gene loci, the identification error risk caused by lack of Y chromosomes can be effectively prevented, and the detection accuracy is improved. The specificity is high, the sensitivity is high, and the anti-inhibition capability is high.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a six-color fluorescent labeling kit for simultaneously amplifying 41 human Y STR loci and an application thereof. Background Art

[0002] Short tandem repeat (STR) or simple tandem repeat sequence is a type of length polymorphic genetic marker widely present in the human genome. STR genetic markers have large differences between individuals, high polymorphism, and small amplified fragments, and are particularly suitable for some trace or degraded samples. Therefore, STR-based amplification detection technology is widely used in forensic individual identification, paternity testing, population genetics analysis, and the construction of human DNA databases.

[0003] Y chromosome STR genetic markers refer to short tandem repeat sequences present in the non-recombinant region of the human Y chromosome. Y chromosome STR genetic markers have three characteristics: male-specific, paternal inheritance, and haploid inheritance. Combining its uniqueness with the superiority of STR locus typing detection, it can be used for forensic individual identification, paternity testing, and DNA family tree construction.

[0004] Fluorescent labeling technology uses fluorescent substances to covalently bind or physically adsorb to a certain group of the molecule to be studied, and uses its fluorescent properties to provide information about the object of study. Fluorescent labeling has the advantages of no radioactive pollution and easy operation, making fluorescent markers increasingly widely used in many research fields. The six-color fluorescent labeling refers to blue, green, yellow, red, purple (primer labeling) and orange (molecular weight internal standard labeling).

[0005] A similar implementation method is to design primer pairs, each pair of primers specifically amplifies one Y-STR locus. By mixing these primer pairs, multiple Y-STR loci can be amplified at one time without causing nonspecific amplification. After amplification, the amplification results are detected by capillary electrophoresis. In the process, different concentrations of different primer pairs are added to control the height of each locus peak, so that the lowest peak / highest peak ratio is ≥0.5 in the same fluorescent marker color; the lowest average peak height / highest average peak height is ≥0.5 in different fluorescent marker colors. However, the prior art has the following problems:

[0006] A) Most Y-STR case kits on the market test fewer loci;

[0007] B) Most Y-STR test kits on the market have low sensitivity and poor detection results for trace samples;

[0008] C) Most Y-STR case kits on the market have poor resistance to suppression;

[0009] D) Most Y-STR test kits on the market do not have internal quality control and cannot assess the sample quality and whether the amplification process is normal. Summary of the invention

[0010] In view of this, the present invention provides a six-color fluorescent labeling kit for simultaneously amplifying 41 human Y STR loci and its application, which can simultaneously amplify a large number of loci, has strong specificity, high sensitivity, and strong anti-inhibition ability, and also includes internal quality control for evaluating sample quality and amplification process.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides a primer set, including a primer pair X;

[0013] The upstream primer in the primer pair X has:

[0014] (1), the nucleotide sequence shown in SEQ ID NO: (2X-1); or

[0015] (2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (1), and having the same or similar function as (1); or

[0016] (3) a nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (1) or (2);

[0017] The downstream primer in the primer pair X has:

[0018] (4), the nucleotide sequence shown in SEQ ID NO: (2X); or

[0019] (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); or

[0020] (6) a nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (4) or (5);

[0021] X is selected from any integer between 1 and 38;

[0022] The plurality is 2 to 5.

[0023] In some specific embodiments of the present invention, the primer set includes primer combination 1, primer combination 2, primer combination 3, primer combination 4 and primer combination 5;

[0024] The primer combination 1 includes primer pairs 1 to 8;

[0025] The primer combination 2 includes primer pairs 9 to 16;

[0026] The primer combination 3 includes primer pairs 17 to 23;

[0027] The primer combination 4 includes primer pairs 24 to 30;

[0028] The primer combination 5 includes primer pairs 31 to 38;

[0029] The upstream primer and / or the downstream primer of each primer pair in each primer combination has a fluorescent label, and the fluorescent labels in primer combination 1, primer combination 2, primer combination 3, primer combination 4 and primer combination 5 are all different.

[0030] The present invention also provides the use of the primer set in any of the following items:

[0031] (I) preparing a reagent or kit for detecting a Y-STR locus;

[0032] (II), detection of Y-STR loci;

[0033] The Y-STR loci include DYS393, DYS390, DYS447, DYS391, DYS527a / b, DYS596, DYS645, DYS576, DYS635, DYS392, DYF387S1a / b, DYS643, DYS533, DYS593, DYS456, DYS549, DYS19, DYS385a / b, DYS55 7, DYS518, DYS522, DYS460, DYS389I, DYS570, DYS389II, DYS449, DYS458, DYS444, DYS437, DYS438, DYS439, YGATAH4, DYS448, DYS627, DYS481, rs771783753, rs759551978, and rs199815934.

[0034] The present invention also provides a reagent, including the above primer set and acceptable auxiliary materials or additives.

[0035] The present invention also provides a kit, comprising the above primer set or the above reagent, and acceptable auxiliary materials or additives.

[0036] In some specific embodiments of the present invention, the above-mentioned kit further comprises a PCR reaction premix;

[0037] The PCR reaction premix includes Taq DNA polymerase, Tris buffer, KCl, MgCl2, (NH4)2SO4, dNTPs and BSA;

[0038] The PCR reaction premix uses deionized water as solvent.

[0039] In some specific embodiments of the present invention, in the above-mentioned kit:

[0040] The final concentration of the upstream primer or the downstream primer in primer pair 1, primer pair 2, primer pair 5, primer pair 9, primer pair 10, primer pairs 12 to 14, primer pair 18, primer pair 25, primer pair 33 or primer pair 36 is 0.1 to 0.4 μM;

[0041] The final concentration of the upstream primer or the downstream primer in primer pair 3, primer pair 6, primer pair 16, primer pair 17, primer pairs 19 to 24, primer pairs 26 to 29, primer pair 31, primer pair 34, primer pair 35 or primer pair 37 is 0.2 to 0.5 μM;

[0042] The final concentration of the upstream primer or downstream primer in primer pair 4 is 1.3 to 1.6 μM;

[0043] The final concentration of the upstream primer or the downstream primer in primer pair 7, primer pair 8, primer pair 11 or primer pair 38 is 0.3 to 0.6 μM;

[0044] The final concentration of the upstream primer or downstream primer in primer pair 15 is 0.4 to 0.7 μM;

[0045] The final concentration of the upstream primer or the downstream primer in primer pair 30 or primer pair 32 is 0.6 to 0.9 μM.

[0046] In some specific embodiments of the present invention, the amplification procedure of the above kit is: pre-denaturation at 95°C for 1 min;

[0047] Denaturation for 10 seconds, annealing and extension at 60°C for 90 seconds, 28 cycles; final extension at 60°C for 10 minutes; storage at 4°C.

[0048] The present invention also provides a method for detecting a Y-STR locus, wherein the sample to be tested is detected based on any of the following:

[0049] (i) the above primer set;

[0050] (ii) the above reagents;

[0051] (iii) the above-mentioned kit.

[0052] In some specific embodiments of the present invention, the above method comprises:

[0053] (A), mixing the above primer set with the sample to be tested and an amplification reagent, amplifying, performing electrophoresis, and obtaining the test result; or

[0054] (B), mixing the above reagents with the sample to be tested and the amplification reagent, amplifying, performing electrophoresis, and obtaining the test results; or

[0055] (C) mixing the primer set and the PCR reaction premix in the above kit with the sample to be tested and the amplification reagent, amplifying, performing electrophoresis, and obtaining the test result;

[0056] The amplification procedure includes: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 90 s, 28 cycles; final extension at 60°C for 10 min; storage at 4°C;

[0057] In the amplification, the final concentration of the upstream primer or the downstream primer in primer pair 1, primer pair 2, primer pair 5, primer pair 9, primer pair 10, primer pairs 12-14, primer pair 18, primer pair 25, primer pair 33 or primer pair 36 is 0.1-0.4 μM, and the final concentration of the upstream primer or the downstream primer in primer pair 3, primer pair 6, primer pair 16, primer pair 17, primer pairs 19-24, primer pairs 26-29, primer pair 31, primer pair 34, primer pair 35 or primer pair 37 is 0.1-0.4 μM. The final concentration of the primers is 0.2 to 0.5 μM, the final concentration of the upstream primer or downstream primer in primer pair 4 is 1.3 to 1.6 μM, the final concentration of the upstream primer or downstream primer in primer pair 7, primer pair 8, primer pair 11 or primer pair 38 is 0.3 to 0.6 μM, the final concentration of the upstream primer or downstream primer in primer pair 15 is 0.4 to 0.7 μM, and the final concentration of the upstream primer or downstream primer in primer pair 30 or primer pair 32 is 0.6 to 0.9 μM. The kit of the present invention has the following effects:

[0058] A) The present invention can amplify and detect 41 Y-STR loci at one time. The number and function of the loci cover the gene loci of most kits on the market. At the same time, the invention contains 3 Y-indel loci, which can effectively prevent the risk of identification errors caused by the lack of Y chromosomes;

[0059] B) The primer set designed by the present invention has a Tm value of 58°C ± 5°C (NCBI Blast result), good specificity, and no primer dimer;

[0060] C) The unique PCR reaction premix of the present invention has the advantages of high sensitivity, strong anti-inhibition ability and good specificity, and is applicable to various biological samples, including but not limited to blood (spots), semen (spots), saliva (spots), exfoliated cells, various tissues, hair with hair follicles (roots), etc.;

[0061] D) The PCR amplification time of the present invention is relatively short, the overall time is about 90 minutes, which is conducive to rapid detection;

[0062] E) In the present invention, the amplification balance of the primer set exceeds the standard described in "GB / T 37226 2018 Basic Quality Requirements for Forensic Human Fluorescent Labeled STR Composite Amplification Detection Reagents" (intra-color balance ≥ 50%, inter-color balance ≥ 30%). BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0064] Figure 1 Shows sensitivity test chart;

[0065] Figure 2 Shows the test chart of anti-humic acid inhibition ability;

[0066] Figure 3 The typing of sample 1 is shown;

[0067] Figure 4 The typing of sample 2 is shown;

[0068] Figure 5 Shows allele typing control figure;

[0069] Figure 6 Shows the molecular weight internal standard Size Standard NS600. DETAILED DESCRIPTION

[0070] The present invention discloses a six-color fluorescent labeling kit for simultaneously amplifying 41 human Y STR loci and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0071] The present invention discloses a composite amplification detection kit for simultaneously amplifying 41 human Y-STR loci and six-color fluorescent labeling and its application. Specifically:

[0072] A) The present invention uses six-color fluorescent labeling composite amplification technology to amplify and detect 41 human Y-STR loci at one time;

[0073] B) The present invention can simultaneously amplify and detect 41 Y-STR loci of humans, including 38 Y chromosome loci (DYS393, DYS390, DYS447, DYS391, DYS527ab, DYS596, DYS645, DYS576, DYS635, DYS392, DYF387S1ab, DYS643, DYS533, DYS593, DYS456, DYS549, DYS19, DYS385ab, DYS557, DYS51 8. DYS522, DYS460, DYS389I, DYS570, DYS389II, DYS449, DYS458, DYS444, DYS437, DYS438, DYS439, YGATAH4, DYS448, DYS627, DYS481) and three Y-indel (insertion and deletion) loci (rs771783753, rs759551978, rs199815934), which are consistent with the loci included in the mainstream kits on the market;

[0074] C) The present invention also includes two internal quality controls (IQCS and IQCL) for evaluating sample quality and PCR process (Xinhai patent: CN102134595A), which can effectively monitor whether there is inhibition in the sample and whether the PCR process is normal;

[0075] D) The present invention provides a case test kit with high sensitivity, strong anti-inhibition ability and wide application range, which can adapt to the complexity of the case scene and meet the needs of rapidity and accuracy.

[0076] Further:

[0077] (i) The present invention provides a primer set that can simultaneously amplify 41 Y-STR loci, the genome comprising 38 Y chromosome loci (DYS393, DYS390, DYS447, DYS391, DYS527a / b, DYS596, DYS645, DYS576, DYS635, DYS392, DYF387S1a / b, DYS643, DYS533, DYS593, DYS456, DYS549, DYS19, DYS 385a / b, DYS557, DYS518, DYS522, DYS460, DYS389I, DYS570, DYS389II, DYS449, DYS458, DYS444, DYS437, DYS438, DYS439, YGATAH4, DYS448, DYS627, DYS481) and three Y-indel loci (rs771783753, rs759551978, rs199815934);

[0078] (II) The primer pair sequences used to amplify 41 Y-STR genomes are shown in Table 1:

[0079] Table 1

[0080]

[0081]

[0082] (III) Preferably, the above 41 Y-STR loci are divided into 5 groups, among which:

[0083] The first group includes: DYS393, DYS390, DYS447, DYS391, rs771783753, DYS527ab, DYS596, DYS645;

[0084] The second group includes: DYS576, DYS635, DYS392, DYF387S1a / b, rs759551978, DYS643, DYS533, DYS593;

[0085] The third group includes: DYS456, DYS549, DYS19, DYS385a / b, DYS557, DYS518, DYS522;

[0086] The fourth group includes: DYS460, DYS389I, DYS570, DYS389II, DYS449, DYS458, DYS444;

[0087] The fifth group includes: rs199815934, DYS437, DYS438, DYS439, YGATAH4, DYS448, DYS627, and DYS481.

[0088] The fluorescent labeling dyes used in the five groups were different.

[0089] (iv) The fluorescent labeling dyes used in the five groups were FAM (first group), HEX (second group), LTAM (third group), LROX (fourth group), and LPUR (fifth group).

[0090] (V) The present invention also provides a kit for the above primer set, which includes: the above primer set mixed solution, PCR reaction premix, 1×Low TE, Control DNA (positive control), molecular weight internal standard, and Allelic Ladder (allele typing control) containing 41Y-STR loci. The primer set mixed solution, PCR reaction premix, 1×Low TE, and Control DNA are PCR reagents, and the molecular weight internal standard and Allelic Ladder are electrophoresis reagents.

[0091] (VI) The concentration of each primer pair in the above primer set is shown in Table 2:

[0092] Table 2

[0093] Locus name Primer pair concentration in amplification system (μM) DYS393 0.1～0.4 DYS390 0.1～0.4 DYS447 0.2～0.5 DYS391 1.3～1.6 rs771783753 0.1～0.4 DYS527a / b 0.2～0.5 DYS596 0.3～0.6 DYS645 0.3～0.6 DYS576 0.1～0.4 DYS635 0.1～0.4 DYS392 0.3～0.6 DYF387S1a / b 0.1～0.4 rs759551978 0.1～0.4 DYS643 0.1～0.4 DYS533 0.4～0.7 DYS593 0.2～0.5 DYS456 0.2～0.5 DYS549 0.1～0.4 DYS19 0.2～0.5 DYS385a / b 0.2～0.5 DYS557 0.2～0.5 DYS518 0.2～0.5 DYS522 0.2～0.5 DYS460 0.2～0.5 DYS389Ⅰ 0.1～0.4 DYS570 0.2～0.5 DYS389Ⅱ 0.2～0.5 DYS449 0.2～0.5 DYS458 0.2～0.5 DYS444 0.6～0.9 rs199815934 0.2～0.5 DYS437 0.6～0.9 DYS438 0.1～0.4 DYS439 0.2～0.5 YGATAH4 0.2～0.5 DYS448 0.1～0.4 DYS627 0.2～0.5 DYS481 0.3～0.6

[0094] (VII) The PCR reaction premix (PCR Mix) uses deionized water as solvent and includes the following components: Taq DNA polymerase, Tris buffer, KCl, MgCl2, (NH4)2SO4, dNTPs and BSA.

[0095] (VIII) Allele typing control corresponding to the locus is as follows Figure 5 , as shown in Table 3:

[0096] Table 3

[0097] Locus Allele DYS393 7,8,9,10,11,12,13,14,15,16,17 DYS390 17,18,19,20,21,22,23,24,25,26,27,28 DYS447 16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31 DYS391 6,7,8,9,10,11,12,13 rs771783753 1,2 DYS527ab 15,16,17,18,19,20,21,22,23,24,25,26,27,28 DYS596 13,14,15,16,17,18 DYS645 6,7,8,9,10 DYS576 10,11,12,13,14,15,16,17,18,19,20,21,22,23 DYS635 15,16,17,18,19,20,21,22,23,24,25,26,27 DYS392 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 DYF387S1ab 30,31,32,33,34,35,36,37,38,39,40,41,42 rs759551978 1,2 DYS643 7,8,9,10,11,12,13,14,15,16,17 DYS533 8,9,10,11,12,13,14,15,16,17 DYS593 13,14,15,16,17,18 DYS456 10,11,12,13,14,15,16,17,18,19,20,21,22 DYS549 7,8,9,10,11,12,13,14,15,16,17,18 DYS19 10,11,12,13,14,15,16,17,18,19 DYS385ab 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26 DYS557 11,12,13,14,15,16,17,18,19,20,21,22,23 DYS518 31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47 DYS522 6,7,8,9,10,11,12,13,14,15,16 DYS460 7,8,9,10,11,12,13,14 DYS389I 10,11,12,13,14,15,16 DYS570 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26 DYS389II 24,25,26,27,28,29,30,31,32,33,34 DYS449 22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40 DYS458 12,13,14,15,16,17,18,19,20,21,22,23,24 DYS444 8,9,10,11,12,13,14,15,16,17,18 rs199815934 1,2 DYS437 10,11,12,13,14,15,16,17 DYS438 7,8,9,10,11,12,13,14,15 DYS439 7,8,9,10,11,12,13,14,15 YGATAH4 7,8,9,10,11,12,13,14,15 DYS448 15,16,17,18,18.2,19,20,21,22,23,24 DYS627 14,15,16,17,18,19,20,21,22,23,24,25,26 DYS481 19,20,21,22,23,24,25,26,27,28,29,30

[0098] (IX) The PCR program is shown in Table 4 (total duration is less than 90 minutes):

[0099] Table 4

[0100] Initial denaturation Thermal Cycle 1 Final extension Insulation 95℃ / 1min (95℃ / 10s, 60℃ / 90s)×28 cycles 60℃ / 10min 4℃ / Hold

[0101] (X) The amplification system of the present invention is as described in Table 5:

[0102] Table 5

[0103] Reagent name Samples to be tested Positive Control Negative control Primer Mix 5.0μL 5.0μL 5.0μL PCR reaction master mix 5.0μL 5.0μL 5.0μL Extracted DNA 1.0~17.5μL - - Control DNA - 5μL - 1×Low TE Make up to 25.0 μL 12.5μL 17.5μL

[0104] (XI) In the primer pair of the present invention, the 5' end of the upstream / downstream primer is labeled with a fluorescent dye, which can emit a fluorescent signal of a specific wavelength under laser excitation. The genetic analyzer collects the light signal to achieve the purpose of detecting the sample.

[0105] (XII) The molecular weight internal standard used in the present invention is Size Standard NS600 produced by Suzhou Xinhai Biotechnology Co., Ltd., which contains 28 fragments and can be used to analyze fragments with a size between 60 and 600 bp. (See Figure 6 )

[0106] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0107] The present invention will be further described below in conjunction with embodiments:

[0108] Example 1: Sensitivity Verification

[0109] The sensitivity of the kit, i.e. the detection limit, was verified by testing different concentrations of 9948 standard DNA. The specific implementation method is as follows:

[0110] 1. Sample preparation

[0111] 0.1 ng / μL of 9948 standard DNA (Suzhou Xinhai Biotechnology) was used as the original template, and 1×LowTE solution (Sangon) was used for dilution according to Table 6 to obtain a total of 5 concentrations of 9948 standard DNA templates.

[0112] Table 6

[0113] Final DNA concentration 0.1ng / μL9948 standard DNA usage (μL) 1×Low TE usage (μL) 0.1ng / μL 100 0 0.05ng / μL 100 100 0.025ng / μL 100 300 0.0125ng / μL 100 700 0.01ng / μL 100 900

[0114] 2. Preparation of reaction system

[0115] Prepare the reaction system according to Table 7 below:

[0116] Table 7

[0117] name Volume μL Primer Mix 1 PCR Mix 2 9948DNA template 5 1×Low TE 2

[0118] The sequences of the primers in the Primer Mix are shown in Table 1, and their concentrations are shown in Table 8:

[0119] Table 8

[0120] Locus name Primer pair concentration in amplification system (μM) DYS393 0.27 DYS390 0.36 DYS447 0.38 DYS391 1.47 rs771783753 0.13 DYS527a / b 0.38 DYS596 0.50 DYS645 0.55 DYS576 0.17 DYS635 0.15 DYS392 0.49 DYF387S1a / b 0.19 rs759551978 0.18 DYS643 0.13 DYS533 0.51 DYS593 0.31 DYS456 0.25 DYS549 0.14 DYS19 0.22 DYS385a / b 0.36 DYS557 0.21 DYS518 0.45 DYS522 0.36 DYS460 0.20 DYS389Ⅰ 0.23 DYS570 0.4 DYS389Ⅱ 0.25 DYS449 0.31 DYS458 0.33 DYS444 0.84 rs199815934 0.21 DYS437 0.75 DYS438 0.16 DYS439 0.29 YGATAH4 0.23 DYS448 0.10 DYS627 0.34 DYS481 0.37

[0121] 3. Amplification conditions

[0122] Amplification was performed using a 9700 PCR instrument (ABI), and the amplification conditions are shown in Table 9:

[0123] Table 9

[0124] Initial denaturation Thermal Cycle 1 Final extension Insulation 95℃ / 1min (95℃ / 10s, 60℃ / 90s)×28 cycles 60℃ / 10min 4℃ / Hold

[0125] 4. Electrophoresis detection

[0126] Formamide (VWR) and internal standard Size Standard NS600 (Suzhou Xinhai) were mixed at a ratio of 10:0.1, and 9 μL was dispensed into each well, followed by the addition of 1 μL of amplified product. Electrophoresis was performed using a 3500xL DX genetic analyzer with an electrophoresis program of (1.2 kV, 24 s). One allele typing control was required for each amplification.

[0127] 5. Experimental results and analysis

[0128] After electrophoresis, the data were analyzed using GeneMapper ID-X software. Figure 1As shown in the figure, when the kit detects a template amount of 0.05 ng or more, the allele detection rate is 100%. The sensitivity of the kit is 0.05 ng.

[0129] Example 2: Anti-inhibition ability

[0130] Test the 9948 standard DNA mixed with different concentrations of inhibitors, and confirm the anti-inhibition ability of the kit through the site detection rate of the 9948 standard DNA. The specific implementation method is as follows:

[0131] 1. Sample preparation

[0132] 1 ng / μL 9948 standard DNA (Suzhou Xinhai), 2 mg / mL humic acid (Suzhou Xinhai) and 1×Low TE (Sangong) were mixed according to the table below to obtain a total of 3 templates with different inhibitor concentrations (as shown in Table 10).

[0133] Table 10

[0134] Final inhibitor concentration 1ng / μL9948 Inhibitors 1×Low TE 0ng / μL humic acid 5 0μL 2mg / mL HA 95μL 100ng / μL humic acid 5 12.5μL 2mg / mL HA 82.5μL 150ng / μL humic acid 5 18.75 μL 2 mg / mL HA 76.25μL

[0135] 2. Preparation of reaction system

[0136] Prepare the reaction system according to Table 11:

[0137] Table 11

[0138] name Volume μL Primer Mix (same as Example 1) 1 PCR Mix 2 9948DNA template 4 1×Low TE 3

[0139] 3. Amplification conditions

[0140] Amplification was performed using a 9700 PCR instrument (ABI), and the amplification conditions are shown in Table 12:

[0141] Table 12

[0142] Initial denaturation Thermal Cycle 1 Final extension Insulation 95℃ / 1min (95℃ / 10s, 60℃ / 90s)×28 cycles 60℃ / 10min 4℃ / Hold

[0143] 4. Electrophoresis detection

[0144] Formamide (VWR) and internal standard Size Standard NS600 (Suzhou Xinhai) were mixed at a ratio of 10:0.1, and 9 μL was dispensed into each well, followed by the addition of 1 μL of amplified product. Electrophoresis was performed using a 3500xL DX genetic analyzer with an electrophoresis program of (1.2 kV, 24 s). One allele typing control was required for each amplification.

[0145] 5. Experimental results and analysis

[0146] After electrophoresis, the data were analyzed using GeneMapper ID-X software. Figure 2As shown, the kit has an allele detection rate of 100% under the inhibition of humic acid at a final concentration of 150 ng / μL. The kit of the present invention has an anti-humic acid inhibition capacity of 150 ng / μL.

[0147] Example 3: Blood card sample amplification

[0148] 1. Sample preparation

[0149] (1) Two blood sample collection cards (provided by volunteers) were used to punch blood samples with a 2 mm hole size and transferred to a 1.5 mL centrifuge tube.

[0150] (2) Add 200 μL of 5% Chelex-100 solution (Suzhou Xinhai) to a 1.5 mL centrifuge tube and heat in a metal bath at 56°C and 800 rpm for 30 min;

[0151] (3) After heating, transfer to a metal bath at 100°C for 8 min;

[0152] (4) Centrifuge the tube at 13,000 rpm for 3 min and use the supernatant for PCR amplification.

[0153] 2. Preparation of reaction system

[0154] Prepare the reaction system according to Table 13:

[0155] Table 13

[0156] name Volume μL Primer Mix (same as Example 1) 1 PCR Mix 2 9948DNA template 4 1×Low TE 3

[0157] 3. Amplification conditions

[0158] Amplification was performed using a 9700 PCR instrument (ABI), and the amplification conditions are shown in Table 14:

[0159] Table 14

[0160] Initial denaturation Thermal Cycle 1 Final extension Insulation 95℃ / 1min (95℃ / 10s, 60℃ / 90s)×28 cycles 60℃ / 10min 4℃ / Hold

[0161] 4. Electrophoresis detection

[0162] Formamide (VWR) and internal standard Size Standard NS600 (Suzhou Xinhai) were mixed at a ratio of 10:0.1, and 9 μL was dispensed into each well, followed by the addition of 1 μL of amplified product. Electrophoresis was performed using a 3500xL DX genetic analyzer with an electrophoresis program of (1.2 kV, 24 s). One allele typing control was required for each amplification.

[0163] 5. Experimental results and analysis

[0164] After electrophoresis, the data were analyzed using GeneMapper ID-X software. Figure 3 ; Sample 2 typing see Figure 4 ; The typing information of sample 1 and sample 2 is shown in the table. The allele typing of sample 1 and sample 2 is exactly the same, so it can be inferred that sample 1 and sample 2 are from the same lineal family.

[0165] Table 15: Sample 1 / 2 typing comparison table

[0166]

[0167]

[0168] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set, characterized in that: including primer pair X; The upstream primer in the primer pair X has: (1), the nucleotide sequence shown in SEQ ID NO: (2X-1); or (2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (1), and having the same or similar function as (1); or (3) a nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (1) or (2); The downstream primer in the primer pair X has: (4), the nucleotide sequence shown in SEQ ID NO: (2X); or (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); or (6) a nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (4) or (5); X is selected from any integer between 1 and 38; The plurality is 2 to 5.

2. The primer set according to claim 1, characterized in that Including primer combination 1, primer combination 2, primer combination 3, primer combination 4 and primer combination 5; The primer combination 1 includes primer pairs 1 to 8; The primer combination 2 includes primer pairs 9 to 16; The primer combination 3 includes primer pairs 17 to 23; The primer combination 4 includes primer pairs 24 to 30; The primer combination 5 includes primer pairs 31 to 38; The upstream primer and / or the downstream primer of each primer pair in each primer combination has a fluorescent label, and the fluorescent labels in primer combination 1, primer combination 2, primer combination 3, primer combination 4 and primer combination 5 are all different.

3. Use of the primer set according to claim 1 or 2 in any of the following items: (I) preparing a reagent or kit for detecting a Y-STR locus; (II), detection of Y-STR loci; The Y-STR loci include DYS393, DYS390, DYS447, DYS391, DYS527a / b, DYS596, DYS645, DYS576, DYS635, DYS392, DYF387S1a / b, DYS643, DYS533, DYS593, DYS456, DYS549, DYS19, DYS385a / b, DYS55 7, DYS518, DYS522, DYS460, DYS389I, DYS570, DYS389II, DYS449, DYS458, DYS444, DYS437, DYS438, DYS439, YGATAH4, DYS448, DYS627, DYS481, rs771783753, rs759551978, and rs199815934.

4. A reagent, characterized in that The method comprises the primer set as claimed in claim 2, and acceptable auxiliary materials or additives.

5. A kit, characterized in that: The method comprises the primer set as claimed in claim 2 or the reagent as claimed in claim 4, and acceptable auxiliary materials or additives.

6. The kit according to claim 5, characterized in that It also includes PCR reaction premix; The PCR reaction premix includes Taq DNA polymerase, Tris buffer, KCl, MgCl2, (NH4)2SO4, dNTPs and BSA; The PCR reaction premix uses deionized water as solvent.

7. The kit according to claim 5 or 6, characterized in that: The final concentration of the upstream primer or the downstream primer in primer pair 1, primer pair 2, primer pair 5, primer pair 9, primer pair 10, primer pairs 12 to 14, primer pair 18, primer pair 25, primer pair 33 or primer pair 36 is 0.1 to 0.4 μM; The final concentration of the upstream primer or the downstream primer in primer pair 3, primer pair 6, primer pair 16, primer pair 17, primer pairs 19 to 24, primer pairs 26 to 29, primer pair 31, primer pair 34, primer pair 35 or primer pair 37 is 0.2 to 0.5 μM; The final concentration of the upstream primer or downstream primer in primer pair 4 is 1.3 to 1.6 μM; The final concentration of the upstream primer or the downstream primer in primer pair 7, primer pair 8, primer pair 11 or primer pair 38 is 0.3 to 0.6 μM; The final concentration of the upstream primer or downstream primer in primer pair 15 is 0.4 to 0.7 μM; The final concentration of the upstream primer or the downstream primer in primer pair 30 or primer pair 32 is 0.6 to 0.9 μM.

8. The kit according to any one of claims 5 to 7, characterized in that The amplification program was as follows: pre-denaturation at 95°C for 1 min; Denaturation at 95°C for 10 s, annealing and extension at 60°C for 90 s, 28 cycles; final extension at 60°C for 10 min; storage at 4°C.

9. A method for detecting a Y-STR locus, characterized in that: Detect samples based on any of the following: (i) a primer set according to claim 1 or 2; (ii) a reagent as claimed in claim 4; (iii) The kit according to any one of claims 5 to 8.

10. The method according to claim 9, characterized in that include: (A), mixing the primer set as claimed in claim 1 or 2 with the sample to be tested and an amplification reagent, amplifying, performing electrophoresis, and obtaining a test result; or (B), mixing the reagent as claimed in claim 4 with the sample to be tested and the amplification reagent, amplifying, performing electrophoresis, and obtaining the test result; or (C) mixing the primer set and the PCR reaction premix in the kit according to any one of claims 5 to 8 with the sample to be tested and an amplification reagent, amplifying, performing electrophoresis, and obtaining a test result; The amplification procedure includes: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 90 s, 28 cycles; final extension at 60°C for 10 min; storage at 4°C; In the amplification, the final concentration of the upstream primer or the downstream primer in primer pair 1, primer pair 2, primer pair 5, primer pair 9, primer pair 10, primer pairs 12-14, primer pair 18, primer pair 25, primer pair 33 or primer pair 36 is 0.1-0.4 μM, and the final concentration of the upstream primer or the downstream primer in primer pair 3, primer pair 6, primer pair 16, primer pair 17, primer pairs 19-24, primer pairs 26-29, primer pair 31, primer pair 34, primer pair 35 or primer pair 37 is 0.1-0.4 μM. The final concentration of the primers is 0.2 to 0.5 μM, the final concentration of the upstream primer or the downstream primer in primer pair 4 is 1.3 to 1.6 μM, the final concentration of the upstream primer or the downstream primer in primer pair 7, primer pair 8, primer pair 11 or primer pair 38 is 0.3 to 0.6 μM, the final concentration of the upstream primer or the downstream primer in primer pair 15 is 0.4 to 0.7 μM, and the final concentration of the upstream primer or the downstream primer in primer pair 30 or primer pair 32 is 0.6 to 0.9 μM.

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