Isothermal pre-amplification STR detection kit and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an isothermal pre-amplification STR detection kit and application thereof. According to the STR amplification detection kit provided by the invention, the isothermal pre-amplification reagent is firstly used for amplifying a forensic trace sample, and then the miniSTR amplification reagent is used for amplifying an NDA product, so that the problem that the trace sample is low in detection rate and even cannot be detected is solved. 12 gene loci selected in the mini STR reagent can be amplified by more than two times, and trace amount and degradation samples can be detected more easily by the mini STR. Compared with a traditional STR detection method, the sensitivity is improved by more than 10 times. Therefore, the possibility of detecting old and trace samples in public security accumulated and old cases can be greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an STR detection kit for isothermal pre-amplification and its application. Background Art

[0002] Nucleic acid amplification technology has a wide range of applications in molecular biology, medicine, forensic medicine, epidemiology, paleontology, etc., and is a very important technology. Among them, the most widely used is the polymerase chain reaction (PCR) technology, which was a revolutionary breakthrough in the 1980s, greatly improving the in vitro amplification efficiency of nucleic acids and being applied almost in all biochemistry and molecular biology laboratories.

[0003] However, with the development of a series of isothermal nucleic acid amplification technologies, the monopoly position of PCR technology has been broken. Although these isothermal nucleic acid amplification technologies have different principles and methods, they all do not require temperature change operations, are easy to operate, and show good application prospects in the fields of clinical and rapid diagnosis. Currently, the commonly used isothermal nucleic acid amplification technologies include: loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), and nicking enzyme-dependent amplification (NDA). Among them, nicking enzyme-dependent amplification (NDA) is a developing technology that is based on the use of a nicking enzyme that can specifically recognize and cleave a restriction site on an oligonucleotide chain to multiply amplify the target nucleic acid in a very short time. However, according to literature reports, the primer design of the existing NDA technology is complex, and there is a chance of random amplification when using primers, resulting in the appearance of non-specificity and affecting nucleic acid detection. But there are two disadvantages in introducing primers in the amplification system of nicking enzyme-dependent amplification technology: the first is that adding primers will produce a large amount of non-specific products such as dimers, affecting subsequent applications; the second is that only a few target sites can be amplified.

[0004] In 1997, it was reported that DNA profiles could be detected from items accessible at the scene. This expanded the scope of sample collection to include, but not be limited to, skin, clothing, knives, vehicles, food, bedding, lip cosmetics, wallets, jewelry, glass, paper, cables, windows, doors, and stones. The application of DNA profiles in the investigation of a wider range of crimes, including theft, homicide, robbery, sexual offenses, etc., also increased. For performing multiplex gene analysis on a single cell or a limited number of cells, whole genome amplification (WGA) is required to increase the DNA content. There are many trace samples in the forensic field that require whole genome amplification. Moreover, some forensic samples are unique and non-replicable, making the accuracy of the test results particularly important. Several early WGA methods were prone to introducing biases during the amplification process, thus affecting the results of downstream genetic analysis. Simple PCR amplification has limitations. When amplifying trace nucleic acids, it is difficult to amplify the amount of nucleic acids to reach the detection limit of nucleic acid detection, affecting the detection results of nucleic acids. A patent reported a method for amplifying trace and complex DNA, which uses DNA polymerase for amplification in the presence of high-concentration trehalose, but the amplification efficiency is poor and cannot meet the amplification requirements of trace samples in the forensic field. Subsequently, forensic researchers developed a major improvement to assist in the typing of degraded and trace DNA - they redesigned the primers for the core STR system to make them closer to the repeat units, reducing the length of the amplified flanking regions. This enables all core CODIS (Combined DNA Index System) and European locus sets to be amplified as "miniSTR" loci, thus improving the success rate of analysis of difficult samples. At the same time, increasing the number of cycles can also increase the product concentration. However, for some samples below the detection limit, miniSTR still has cases where it is difficult to detect or random amplification occurs. Summary of the Invention

[0005] In view of this, the present invention provides an isothermal pre-amplification STR detection kit and its application.

[0006] The present invention provides an isothermal pre-amplification STR detection kit and its application. The present invention first uses isothermal pre-amplification reagents to amplify forensic trace samples, and then uses miniSTR amplification reagents to amplify the NDA products, solving the problem of low detection rate or even undetectability of trace samples. Among the 12 loci selected in the miniSTR reagents, all can achieve more than twice amplification, and miniSTR is more likely to detect trace and degraded samples.

[0007] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0008] The present invention provides an isothermal pre-amplification reagent, comprising a nicking enzyme, a DNA polymerase with strand displacement activity, and a buffer;

[0009] The isothermal pre-amplification reagent does not include primers;

[0010] The buffer comprises one or more of Tris-HCl, (NH4)2SO4, KCl, MgSO4 or Tween 20.

[0011] In some specific embodiments of the present invention, the DNA polymerase with strand displacement activity comprises Bst DNA polymerase.

[0012] In some specific embodiments of the present invention, the nicking enzyme includes, but is not limited to, one or more of Nt.BstNBI, Nt.AlwI, Nt.BsmAI, Nb.BsmI, Nb.BbvCI, Nb.BsrDI, Nb.BtsI, Nt.BbvCI, Nt.BspQI or Nt.CviPII.

[0013] In some specific embodiments of the present invention, the nicking enzyme is Nt.BstNBI.

[0014] In some specific embodiments of the present invention, the system for pre-amplification using the isothermal pre-amplification reagent comprises: 2×NDA Mix and template DNA;

[0015] The 2×NDA Mix comprises Nt.BstNBI, Bst DNA polymerase and the buffer;

[0016] The volume ratio of Nt.BstNBI, Bst DNA polymerase and the buffer is 1:20:20.

[0017] The present invention also provides a reagent combination, comprising the isothermal pre-amplification reagent; and

[0018] miniSTR amplification reagent.

[0019] In some specific embodiments of the present invention, the miniSTR amplification reagent comprises a PCR reaction premix, a TE buffer and a primer set for STR loci;

[0020] The PCR reaction premix comprises one or more of Tris-HCl, KCl, NH4Cl, MgCl2, dNTPs, Glycerol, IGEPAL CA-630, Tween 20 or Taq DNA Polymerase;

[0021] The primer set has:

[0022] (I), nucleotide sequences shown in SEQ ID No. 1 to 24; or

[0023] (II), nucleotide sequences obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequences shown in (I), and having the same or similar functions as the nucleotide sequences shown in (I); or

[0024] (III), nucleotide sequences having at least 80% sequence similarity to the nucleotide sequences described in (I) or (II).

[0025] In some specific embodiments of the present invention, the primer set of the STR locus includes an upstream primer and a downstream primer of the STR locus:

[0026] (1), the upstream primer of the STR locus has a nucleotide sequence shown in SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and / or 23; and

[0027] (2), the downstream primer of the STR locus has a nucleotide sequence shown in SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and / or 24; or

[0028] (3), nucleotide sequences obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequences shown in (1) or (2), and having the same or similar functions as the nucleotide sequences shown in (1) or (2); or

[0029] (4), nucleotide sequences having at least 80% sequence similarity to any one of the nucleotide sequences described in (1) to (3).

[0030] In some specific embodiments of the present invention, the concentration of Tris-HCl is 10 to 30 mM; the concentration of KCl is 30 to 70 mM; the concentration of NH4Cl is 20 to 50 mM; the concentration of MgCl2 is 2 to 3 mM; the concentration of dNTPs is 0.2 to 0.5 mM; the concentration of Glycerol is 2 to 4%; the concentration of IGEPAL CA-630 is 0.05 to 0.1%; the concentration of Tween 20 is 0.05 to 0.1%; the concentration of Taq DNA Polymerase is 50 to 80 units / ml;

[0031] The concentrations of the upstream primer and the downstream primer of the STR locus are 0.5 to 2 μM.

[0032] In some specific embodiments of the present invention, the concentration of Tris-HCl is 20 mM; the concentration of KCl is 50 mM; the concentration of NH4Cl is 30 mM; the concentration of MgCl2 is 2.5 mM; the concentration of dNTPs is 0.3 mM; the concentration of Glycerol is 3.2%; the concentration of IGEPAL CA-630 is 0.08%; the concentration of Tween 20 is 0.07%; the concentration of Taq DNA Polymerase is 67 units / ml.

[0033] In some specific embodiments of the present invention, the STR loci include D2S441, D19S433, AMEL, D10S1248, D16S539, D22S1045, D18S51, TH01, vWA, D5S818, D8S1179, and / or FGA.

[0034] In some specific embodiments of the present invention, the concentrations of the upstream and downstream primers of D2S441 are 1 μM; the concentrations of the upstream and downstream primers of D19S433 are 2 μM; the concentrations of the upstream and downstream primers of AMEL are 1 μM; the concentrations of the upstream and downstream primers of D10S1248 are 1 μM; the concentrations of the upstream and downstream primers of D16S539 are 0.5 μM; the concentrations of the upstream and downstream primers of D22S1045 are 1 μM; the concentrations of the upstream and downstream primers of D18S51 are 1 μM; the concentrations of the upstream and downstream primers of TH01 are 1.5 μM; the concentrations of the upstream and downstream primers of vWA are 1 μM; the concentrations of the upstream and downstream primers of D5S818 are 1 μM; the concentrations of the upstream and downstream primers of D8S1179 are 1.5 μM; the concentrations of the upstream and downstream primers of FGA are 1 μM.

[0035] In some specific embodiments of the present invention, the 5'-ends of the upstream primers of the STR loci are each attached with a different fluorescent labeling group;

[0036] The fluorescent labeling groups include FAM, HEX, LTAM, LROX, and / or LPUR.

[0037] In some specific embodiments of the present invention, the fluorescent labeling group at the 5'-end of the upstream primers of D2S441 and D19S433 is FAM; the fluorescent labeling group at the 5'-end of the upstream primers of AMEL, D10S1248 and D16S539 is HEX; the fluorescent labeling group at the 5'-end of the upstream primers of D22S1045, D18S51 and TH01 is LTAM; the fluorescent labeling group at the 5'-end of the upstream primers of vWA and D5S818 is LROX; the fluorescent labeling group at the 5'-end of the upstream primers of D8S1179 and FGA is LPUR.

[0038] Based on the above research, the present invention also provides the use of any of the following in the preparation of a kit for trace sample detection:

[0039] (1) The isothermal pre-amplification reagent; and / or

[0040] (2) The reagent combination.

[0041] The present invention also provides a kit, comprising:

[0042] (1) The isothermal pre-amplification reagent; and / or

[0043] (2) The reagent combination.

[0044] The present invention also provides a method for detecting trace samples, which detects the sample based on any of the following:

[0045] (1) The reagent combination; and / or

[0046] (2) The kit.

[0047] In some specific embodiments of the present invention, the specific method for detecting trace samples includes:

[0048] Step 1: Take the isothermal pre-amplification reagent to pre-amplify the sample to obtain an NDA product;

[0049] Step 2: Take the NDA product and amplify it with the miniSTR amplification reagent.

[0050] In some specific embodiments of the present invention, a purification step is further included before the pre-amplification.

[0051] In some specific embodiments of the present invention, the conditions for the pre-amplification in Step 1 are: reacting at 55°C for 30 - 60 min and inactivating at 95 - 98°C for 3 - 5 min.

[0052] In some specific embodiments of the present invention, the conditions for the pre-amplification in Step 1 are: reacting at 55°C for 40 min and inactivating at 95°C for 3 min.

[0053] In some specific embodiments of the present invention, the conditions for the amplification in step 2 are as follows: the first stage: 95°C for 1 min, 1 cycle; the second stage: 95°C for 10 s, 58°C for 1 min, 28 cycles; the third stage: final extension, 60°C for 5 min, 1 cycle; termination of the reaction, storage at 4°C.

[0054] The present invention provides an isothermal pre-amplification STR detection kit and its application. The beneficial effects of the present invention are as follows:

[0055] 1) Using the isothermal pre-amplification method established by the present invention, linear amplification can be performed on trace DNA with extremely low copy numbers, reducing amplification imbalance. Since no primers are involved in the pre-amplification process, the generation of non-specific products is eliminated. The nicking enzyme can recognize specific template sequences, accurately amplify the target fragment, increase the nucleic acid amount of the amplified target fragment, improve the amplification efficiency, generate more nucleic acid after amplification, and it is easier to improve the detection rate in the downstream. After nucleic acid amplification, it will not affect the results of downstream genetic analysis, with higher sensitivity, higher accuracy, reducing the probability of random amplification, avoiding the appearance of primer dimers, and being suitable for samples in the forensic field. For all amplified loci, after NDA, a peak area more than twice can be amplified, and it is easier to detect in the case of a small sample size.

[0056] 2) The 12 STR loci selected in the present invention all contain effective nicking enzyme recognition sites, and the target region can be effectively amplified by the pre-amplification technology of the present invention. By designing miniSTR primers and establishing a multiplex amplification kit, the detection rate of trace samples is greatly improved.

[0057] 3) By combining the above two methods, isothermal pre-amplification is performed on the DNA extracted from trace samples at the scene, and the pre-amplified samples are detected using a multiplex amplification reagent of 12 miniSTRs. Compared with the traditional STR detection method only, the sensitivity is increased by more than 10 times. This will greatly increase the possibility of detecting old and trace samples in unsolved cases and cold cases in public security. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0059] Figure 1 Showing the amplification of 9948 DNA by the miniSTR reagent in Example 3; among them, the left is 0.05 ng and the right is 0.025 ng;

[0060] Figure 2 Showing the amplification of 0.025 ng of 9948 DNA by the miniSTR reagent in Example 3; among them, the loci of D18S51, vWA, D5S818, and D8S1179 are lost;

[0061] Figure 3 Showing the DNA profile of 0.05 ng of 9948 amplified by this kit in Example 4;

[0062] Figure 4 Showing the comparison of different amounts of 9948 DNA amplified by this kit in Example 4; from left to right are: 0.05 ng, 0.025 ng, 0.0125 ng, 0.00625 ng, 0.003125 ng, 0.0015625 ng;

[0063] Figure 5 Showing the DNA profile of 0.0015625 ng of 9948 amplified by this kit in Example 4; among which, the FGA allele 24 is lost;

[0064] Figure 6 Showing in Example 5 The detection map of the simulated sample of the kit for 25A case;

[0065] Figure 7 Showing the detection map of the simulated sample by this kit in Example 5. Detailed implementation manners

[0066] The present invention discloses an isothermal pre-amplification STR detection kit and its application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate alterations and combinations to 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.

[0067] In the nicking enzyme-dependent amplification technology, the most important participant is the nicking enzyme. The nicking enzyme can recognize double-strands of a specific sequence and can cut one strand of the double-strand with this specific sequence. The most commonly used nicking enzyme is Nt.BstNBI. The DNA sequence that Nt.BstNBI can recognize is 5'-GAGTC-3' / 5'-GACTC-3', and it cuts the DNA strand at the 4th base after the sequence 5'-GAGTC-3'. Bst DNA polymerase can be used for NDA. It is part of the Bacillus stearothermophilus DNA polymerase. Different from general DNA polymerases, it has 5'→

[0068] While having 3' DNA polymerase activity, it does not have 5'→3' exonuclease activity. The nicking enzyme-dependent amplification system mainly relies on the synergistic action of nicking enzyme and DNA polymerase. In the presence of other reaction materials such as buffer, these two enzymes cooperate with each other to achieve continuous cleavage and extension of the template DNA strand, so that the target DNA can be amplified exponentially. In the system of the present invention, primers are not added, which greatly reduces non-specific amplification caused by primers, and can amplify most genomic sequences by recognizing nicking enzyme sites.

[0069] The present invention discloses an isothermal pre-amplification STR detection kit and its application, applying existing NDA technology to the forensic field in order to solve the problem that forensic scientists cannot detect due to too little sample volume when facing trace samples, and improving the detection sensitivity. NDA amplification without primers solves the problem of non-specificity during amplification and does not affect the results of downstream genetic analysis. Subsequently, miniSTR is used for PCR amplification to detect the accurate genotype of the sample, which helps forensic work and increases the probability of finding the suspect (or victim), thereby improving the success rate of solving cases.

[0070] 1. Analyze three nicking enzyme sites in the flanking sequences of 1500 bp upstream and downstream of the core sequences of 26 commonly used forensic genetic loci. According to the analysis results, select the nicking enzyme containing the most genetic loci - Nt.BstNBI.

[0071] 2. Design isothermal pre-amplification reagents using NDA technology.

[0072] In order to detect extremely trace forensic scene samples, the present invention is based on the improvement of existing NDA technology. By recognizing specific sites in single strands with the nicking enzyme Nt.BstNBI, a nick is formed, and using the strand displacement effect of DNA polymerase Bst, amplification starts from the nick, and through isothermal extension, new single-stranded DNA is replicated. The newly replicated single strand can be recognized by the nicking enzyme again and generate a nick, thus generating single-stranded DNA again. Repeating this process can achieve linear amplification of the positions containing nicking enzymes in the whole genome. Since no primers are added in this process, no primer dimers and non-specific amplification products are generated, overcoming the defects of existing isothermal whole-genome amplification technology.

[0073] The isothermal pre-amplification reaction system comprises the following components: 1) DNA template to be amplified; 2) nicking enzyme Nt.BstNBI (purchasing company: NEB; product number: R0607); 3) isothermal amplification DNA polymerase Bst (purchasing company: NEB; product number: M0537); 4) buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% 20).

[0074] 3. Use GlobalFiler TM For the PCR amplification kit, select 12 loci where the peak area exceeds twice the peak area of the non-preamplified sample after using the isothermal preamplification reagent.

[0075] 4. Design a miniSTR amplification reagent using 12 effectively amplified loci.

[0076] The miniSTR amplification reaction system contains the following components: 1) PCR reaction premix: deionized water, 20 mM Tris-HCl, 50 mM KCl, 30 mM NH4Cl, 2.5 mM MgCl2, 0.3 mM dNTPs, 3.2% Glycerol, 0.08% CA-630, 0.07% 20, 67 units / ml Taq DNA Polymerase; 2) Low TE (purchased from Sangon Biotech; product number: B541019); 3) Primer set mixed solution.

[0077] 5. Combine steps 2 and 4 to form the kit disclosed in the present invention - a forensic STR amplification detection kit for trace samples (hereinafter referred to as the trace kit). The trace kit has two-step amplification:

[0078] ① Amplify forensic trace nucleic acid samples using the isothermal preamplification reagent.

[0079] ② Use the miniSTR amplification reagent to perform polymerase chain reaction (PCR) amplification on the preamplified product and conduct downstream detection.

[0080] 6. Reaction conditions:

[0081] ① Isothermal preamplification reaction conditions:

[0082] Preamplification: 55°C / 40 min; Enzyme inactivation: 95°C / 3 min.

[0083] ② miniSTR amplification reaction conditions:

[0084] Initial denaturation Thermal cycling Final extension Heat preservation 95°C / 1 min (95°C / 10 s, 58°C / 1 min) × 28 cycles 60°C / 5 min 4°C / Hold

[0085] 7. Relevant information of the miniSTR amplification reagent:

[0086] Perform PCR amplification using the miniSTR amplification reagent. The primer set mixed solution used in PCR amplification, and the 12 STR loci used are: D2S441, D19S433, AMEL, D10S1248, D16S539, D22S1045, D18S51, TH01, vWA, D5S818, D8S1179, FGA. Its cumulative discrimination power is 1 - (1.76×10 -13 ).

[0087] The sequences of the PCR amplification primers are shown in the following table:

[0088]

[0089] The concentrations of the PCR amplification primers are: D2S441, 1 μM; D19S433, 2 μM; AMEL, 1 μM; D10S1248, 1 μM; D16S539, 0.5 μM; D22S1045, 1 μM; D18S51, 1 μM; TH01, 1.5 μM; vWA, 1 μM; D5S818, 1 μM; D8S1179, 1.5 μM; FGA, 1 μM.

[0090] The PCR amplification primers are divided into 5 groups, and the fluorescence dyes in each group have different colors after excitation. One of the two specific amplification primers in each pair is labeled with a fluorescence dye at the 5' end of the upstream primer. The 5 groups of primers are as follows: blue: D2S441, D19S433; green: AMEL, D10S1248, D16S539; yellow: D22S1045, D18S51, TH01; red: vWA, D5S818; purple: D8S1179, FGA. The 5 fluorescence dyes are: FAM (blue), HEX (green), LTAM (yellow), LROX (red), and LPUR (purple).

[0091] The name of the molecular weight internal standard used during the detection after PCR amplification is NS600SizeStandard (Company: Suzhou Newhai Biotechnology Co., Ltd.; Product Number: NH9461), which contains 28 fragments in total, and the molecular weights of each are 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 314, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 525, 550, 575, 600.

[0092] The present invention discloses a forensic STR amplification detection kit for trace samples and its application. First, an isothermal pre-amplification reagent is used to amplify forensic trace samples, and then a miniSTR amplification reagent is used to amplify the NDA product, so as to solve the problem of low detection rate or even inability to detect trace samples. Among the miniSTR reagents, more than twice amplification can be achieved for each of the 12 selected loci, and miniSTR is more likely to detect trace and degraded samples.

[0093] The raw materials and reagents used in the isothermal pre-amplification STR detection kit and its application provided by the present invention can be purchased from the market.

[0094] The present invention will be further described below in conjunction with embodiments:

[0095] Example 1 Analysis of nicking enzyme sites in the flanking sequences of common STR loci

[0096] The nicking enzyme sites in the flanking sequences of 1500 bp upstream and downstream of the core sequences of 26 common forensic loci were analyzed. A total of three nicking enzymes were analyzed, namely: Nt.BstNBI (recognition sequence: GAGTC), Nt.AlwI (recognition sequence: GGATC), and Nt.BsmAI (recognition sequence: GTCTC). According to the analysis results, the nicking enzyme contained in the most loci was selected for subsequent verification. The analysis results are shown in Table 1.

[0097] Table 1 Analysis of nicking enzyme sites contained in common STR loci

[0098]

[0099]

[0100] Since Nt.BstNBI (recognition sequence: GAGTC) is contained in the largest number of STR loci and is a relatively common nicking enzyme, Nt.BstNBI was selected as the nicking enzyme of the present invention.

[0101] Example 2 Use of GlobalFiler TM PCR amplification kit for post-amplification test of NDA

[0102] Using GlobalFiler TM PCR amplification kit (purchasing company: Thermo Fisher; product number: 4476135), the male DNA standard 007DNA was used as the nucleic acid template, the NDA pre-amplification reagent in this kit was used for pre-amplification, and GlobalFiler TMPCR amplification kit was used for PCR amplification. Fluorescence signals were detected by capillary electrophoresis, and the peak areas in the electrophoresis map were statistically analyzed to find the loci with a peak area multiple greater than 2 times. According to Example 1, the nicking enzyme selected was Nt.BstNBI.

[0103] The nucleic acid template was male DNA standard 007 with a concentration of 0.01 ng / μL. The trace nucleic acid template was pre-amplified. The pre-amplification system was 15 μL of 2×NDA Mix (containing Nt.BstNBI, Bst DNA polymerase, buffer, volume ratio: Nt.BstNBI: Bst DNA polymerase: buffer = 1:20:20), and 15 μL of template DNA. The reaction was carried out at 55 °C for 40 min in a PCR instrument, and then inactivated at 95 °C for 3 min.

[0104] After pre-amplification, GlobalFiler TM PCR amplification kit was used for PCR amplification. The PCR amplification system in this example was: 1 μL of Primer Mix, 3 μL of MasterMix, 6 μL of DNA template, with a total volume of 10 μL. The amplification conditions were: the first stage: 95 °C for 1 min, 1 cycle; the second stage: 94 °C for 10 s, 59 °C for 90 s, 29 cycles; the third stage: final extension, 60 °C for 10 min, 1 cycle; terminate the reaction and store at 4 °C. In addition, the non-pre-amplified nucleic acid was subjected to PCR amplification as a control.

[0105] Capillary electrophoresis was used for detection. As shown in Table 2, there were 12 loci with an amplification multiple greater than 2, namely: D19S433, D2S441, D8S1179, TH01, D22S1045, FGA, D5S818, AMEL, D16S539, vWA, D18S51, D10S1248.

[0106] Table 2 Analysis of the amplification efficiency of some loci of NDA and NDA

[0107]

[0108] Example 3 Verification of the sensitivity of miniSTR reagent

[0109] By testing 9948 standard DNA at different concentrations, the sensitivity, that is, the detection limit, of the miniSTR reagent of this kit was verified. The specific implementation method was as follows:

[0110] Using 0.25 ng / μL of 9948 standard DNA (Company: Suzhou Newhai Biotechnology Co., Ltd.; Product Number: NH9412) as the original template, dilute it with Low TE solution to prepare different amounts of 9948 standard DNA, and test the sensitivity of the miniSTR reagent in this kit. The amounts of DNA added during the test are: 0.05 ng and 0.025 ng.

[0111] The amplification conditions are as follows: Stage 1: 95°C for 1 min, 1 cycle; Stage 2: 95°C for 10 s, 58°C for 1 min, 28 cycles; Stage 3: Final extension, 60°C for 5 min, 1 cycle; Terminate the reaction and store at 4°C. Detect the fluorescence signal by capillary electrophoresis to verify the sensitivity of the miniSTR reagent in this kit.

[0112] Use capillary electrophoresis for detection, and the results are as Figure 1 - Figure 2 shown. It can be seen from the results that when the miniSTR reagent amplifies 0.025 ng of 9948 DNA, not all loci are amplified, and the sensitivity of the miniSTR reagent in this kit alone during amplification is 0.05 ng.

[0113] Example 4 Verification of the sensitivity of this kit (combined use of pre-amplification reagent and miniSTR reagent)

[0114] Verify the sensitivity, that is, the detection limit, of this kit by testing 9948 standard DNA at different concentrations. The specific implementation method is as follows:

[0115] Use 0.25 ng / μL of 9948 standard DNA (Suzhou Newhai Biological) as the original template, dilute it with Low TE solution to prepare different amounts of 9948 standard DNA, and test the sensitivity of this kit. The amounts of DNA are: 0.05 ng, 0.025 ng, 0.0125 ng, 0.00625 ng, 0.003125 ng, 0.0015625 ng.

[0116] Use the pre-reagent for pre-amplification. The amplification system and conditions are the same as in Example 2. After NDA, use the miniSTR reagent for PCR amplification. The amplification conditions are as follows: Stage 1: 95°C for 1 min, 1 cycle; Stage 2: 95°C for 10 s, 58°C for 1 min, 28 / 29 cycles (28 cycles for templates of 0.05 ng and 0.025 ng, and 29 cycles for the rest); Stage 3: Final extension, 60°C for 5 min, 1 cycle; Terminate the reaction and store at 4°C. Detect the fluorescence signal by capillary electrophoresis to verify the sensitivity of this kit.

[0117] Use capillary electrophoresis for detection, and the results are as Figure 3 - Figure 5 shown. From Figure 3As shown in the spectrum, no non-specific peaks appeared, so no primer non-specificity was caused by NDA or PCR. From Figure 4 and Figure 5 it can be seen that after pre-amplifying the sample by the method of the present invention, the sensitivity of the kit can reach 0.003125 ng. Compared with directly using the STR kit for amplification in Example 3, the sensitivity is increased by more than 10 times.

[0118] Example 5 Amplification detection of the kit for simulated trace samples

[0119] In this example, the technical solution of the present invention is used. The DNA sample extracted from known-genotyped human whole blood is used as the nucleic acid template. The NDA reagent in this kit is used for pre-amplification, and then the miniSTR reagent is used for PCR amplification. The fluorescence signal is detected by capillary electrophoresis to verify the feasibility of this method in the amplification of actual samples and the correctness of the principle. The trace nucleic acid template is: 1 DNA sample extracted from known-genotyped human whole blood, donated by volunteers and with informed consent. The trace nucleic acid template (about 6.25 pg / μL) is pre-amplified, and the amplification system and amplification conditions are the same as those in Example 2. Then the miniSTR reagent is used for PCR amplification. The PCR amplification system and amplification conditions are the same as those in Example 4. At the same time, the 25A case kit amplification is used as a control.

[0120] Capillary electrophoresis is used for detection, and the results are as Figure 6 - Figure 7 shown. It can be seen from the results that the 25A case kit had locus and allele loss, while this kit could correctly detect all genotypes. Compared with the 25A case kit, there were 7 loci with allele loss in this kit, namely: D19S433, AMEL, D10S1248, D16S539, D22S1045, D5S818, D8S1179.

[0121] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Isothermal pre-amplification reagent, characterized in that, It includes a nicking enzyme, a DNA polymerase with strand displacement activity, and a buffer; The isothermal pre-amplification reagent does not include primers; The buffer includes one or more of Tris-HCl, (NH4)2SO4, KCl, MgSO4, or Tween 20.

2. The isothermal pre-amplification reagent according to claim 1, characterized in that The nicking enzyme includes one or more of, but is not limited to, Nt.BstNBI, Nt.AlwI, Nt.BsmAI, Nb.BsmI, Nb.BbvCI, Nb.BsrDI, Nb.BtsI, Nt.BbvCI, Nt.BspQI, or Nt.CviPII.

3. Reagent combination, characterized in that, It includes the isothermal pre-amplification reagent as described in claim 1 or 2; and miniSTR amplification reagent.

4. The reagent combination according to claim 3, wherein The miniSTR amplification reagent includes a PCR reaction premix, a TE buffer, and a primer set for STR loci; The PCR reaction premix includes one or more of Tris-HCl, KCl, NH4Cl, MgCl2, dNTPs, Glycerol, IGEPAL CA-630, Tween 20, or Taq DNA Polymerase; The primer set has: (I) a nucleotide sequence as shown in SEQ ID No. 1 to 24; or (II) a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I), and having the same or similar function as the nucleotide sequence shown in (I); or (III) a nucleotide sequence having at least 80% sequence similarity to the nucleotide sequence described in (I) or (II).

5. The reagent combination according to claim 4, characterized in that, The primer set for STR loci includes an upstream primer for STR loci and a downstream primer for STR loci: (1) The upstream primer for STR loci has a nucleotide sequence as shown in SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and / or 23; and (2) The downstream primer for STR loci has a nucleotide sequence as shown in SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and / or 24; or (3) a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (1) or (2), and having the same or similar function as the nucleotide sequence shown in (1) or (2); or (4) a nucleotide sequence having at least 80% sequence similarity to any one of the nucleotide sequences described in (1) to (3).

6. The reagent combination according to claim 4, wherein The concentration of Tris-HCl is 10 - 30 mM; the concentration of KCl is 30 - 70 mM; the concentration of NH4Cl is 20 - 50 mM; the concentration of MgCl2 is 2 - 3 mM; the concentration of dNTPs is 0.2 - 0.5 mM; the concentration of Glycerol is 2 - 4%; the concentration of IGEPAL CA-630 is 0.05 - 0.1%; the concentration of Tween 20 is 0.05 - 0.1%; the concentration of Taq DNA Polymerase is 50 - 80 units / ml; The concentrations of the upstream primer and the downstream primer of the STR locus are 0.5 - 2 μM.

7. The reagent combination according to claim 5, wherein The 5'-end of the upstream primer of the STR locus is attached with different fluorescent labeling groups; The fluorescent labeling groups include FAM, HEX, LTAM, LROX and / or LPUR.

8. Use of any of the following in the preparation of a kit for trace sample detection: (1), the isothermal pre-amplification reagent as described in claim 1 or 2; and / or (2), the reagent combination as described in any one of claims 3 to 7.

9. Kit, characterized in that, Comprising: (1), the isothermal pre-amplification reagent as described in claim 1 or 2; and / or (2), the reagent combination as described in any one of claims 3 to 7.

10. A method for detecting trace samples, characterized in that, Detecting a sample based on any of the following: (1), the reagent combination as described in any one of claims 3 to 7; and / or (2), the kit as described in claim 9.

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