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Kit for detecting 120 loci based on next-generation sequencing and primer combination used in kit

A primer combination and kit technology, applied in the field of forensic science, can solve problems such as high price, small number of loci, and incomplete application

Active Publication Date: 2020-07-10
INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, the price of the two is very high; second, the available information is limited, only the data and analysis of the inherent loci in these products can be obtained, and the number of loci is relatively small, especially the X-STR loci; thirdly, Due to racial differences, these products are not fully suitable for the analysis of genetic polymorphisms in STR populations in my country

Method used

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  • Kit for detecting 120 loci based on next-generation sequencing and primer combination used in kit
  • Kit for detecting 120 loci based on next-generation sequencing and primer combination used in kit
  • Kit for detecting 120 loci based on next-generation sequencing and primer combination used in kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Reagents for detecting 120 loci (including 60 autosomal STR loci, 41 Y-STR loci, 18 X-STR loci and 1 sex-determining Amelogenin locus) based on next-generation sequencing technology Preparation of the box

[0030] 1. Screening of 120 loci

[0031] The inventors of the present invention obtained a large number of candidate autosomal STR loci, Y-STR loci and X-STR loci by consulting the literature and existing next-generation sequencing STR typing products, and then according to the genetic polymorphism of the Chinese population STR population According to the analysis results, 120 loci were obtained in the final screening, including 60 autosomal STR loci, 41 Y-STR loci, 18 X-STR loci and 1 sex-determining locus Amelogenin.

[0032]The 60 autosomal STR loci are: CSF1PO, FGA, TH01, TPOX, VWA, Penta D, Penta E, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S431, D1S1627、D1S1677、D2S1360、D2S1776、D2S441、D3S1744、D3S3045、D...

Embodiment 2

[0061] Embodiment 2, using the kit prepared in embodiment 1 to detect the STR typing of the standard product 2800M

[0062] 1. DNA sample preparation

[0063] Take the standard 2800M and dilute it with ultrapure water to obtain a standard 2800M aqueous solution with a concentration of 1ng / μL.

[0064] 2. PCR amplification

[0065] Using the standard 2800M aqueous solution as a template, the primer mixture prepared in Step 3 of Example 1 was used for PCR amplification to obtain a PCR amplification product.

[0066] The reaction system is 20 μL, consisting of 10 μL PCR Master Mix, 1 μL standard 2800M aqueous solution, primer mix and nuclease-free water. In this reaction system, the concentration of each primer is shown in column 5 in Table 1.

[0067] Reaction program: 95°C for 5min; 98°C for 30s, 60°C for 90s, 72°C for 1min, 28 cycles; 72°C for 10min; 4°C for storage.

[0068] 3. Purification and quantification

[0069] (1) Take the PCR amplification product and purify it ...

Embodiment 3

[0082] The accuracy verification of the kit prepared in embodiment 3, embodiment 1

[0083] 1. Take 1ng standard product 2800M, respectively according to Identifiler Plus Kit (Thermo Fisher), Global Filer TM Kit (Thermo Fisher), Yfiler Plus kit (ThermoFisher), PowerPlex21 kit (Promega) and DNATyper TM Capillary electrophoresis detection was performed according to the instruction manual of the Y26 kit (Ministry of Public Security Evidence Identification Center) to obtain the allelic genotype of each locus. The typing results are detailed in column 2 in Table 4.

[0084] 2. Detect the standard product 2800M according to the steps in Example 2 to obtain the allelic genotype of the locus. The typing results are detailed in column 3 in Table 4.

[0085]The results showed that the genetic loci that overlapped between the kit prepared in Example 1 and the capillary electrophoresis typing kit described above were completely consistent in the typing results of the standard 2800M s...

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Abstract

The invention discloses a kit for detecting 120 loci based on a next-generation sequencing and a primer combination used in the kit. The primer combination is composed of 230 primers, and the nucleotide sequences of the 230 primers are shown as from SEQ ID NO:1 to SEQ ID NO:230 in sequence. Loci which have amplicons with lengths of longer than 300bp in the 120 loci detected by the kit provided bythe invention account for only 2.5% (3 / 120) of the 120 coci, and analysis of degraded DNA samples is facilitated. Experiments prove that the kit provided by the invention is used for detecting short tandem repeat (STR) typing of 120 loci in a standard product 2800M, and the typing result is accurate. The kit and primer combination provided by invention have an important application value.

Description

technical field [0001] The invention belongs to the technical field of forensic medicine, and in particular relates to a kit for detecting 120 loci based on next-generation sequencing technology and a primer combination used therefor, and in particular to a reagent for detecting 119 STR loci and Amelogenin loci based on next-generation sequencing technology cassettes and the primer combinations they use. Background technique [0002] STR typing technology is simple, fast, reliable, high sensitivity, and good repeatability, and it is the main tool for forensic genetics research. The main technical means of STR typing is genotyping by capillary electrophoresis detection. This technology realizes the interpretation of the genotype by detecting the total fluorescence intensity of DNA molecules of the same genotype. On the one hand, it can only perform qualitative analysis on the genotype. It is impossible to accurately quantify the proportion of this genotype, and on the other ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 王乐郭立亮康克莱张驰季安全叶健
Owner INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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