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Eight-color fluorescent multiplex amplification kit for trace degradation DNA (deoxyribonucleic acid) detection and application of eight-color fluorescent multiplex amplification kit

A compound amplification and kit technology, applied in the field of forensic genetic testing, can solve the problems of unsatisfactory effect, difficulty in satisfying the detection, low overall balance, etc., and achieve the effect of good detection effect, accurate typing result and high sensitivity

Pending Publication Date: 2022-07-05
苏州市公安局 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kind of kit can obtain more STR site information when amplifying normal samples, but the effect of amplifying degraded and trace samples is not ideal, and the overall balance is high at the beginning and low at the end, and the amplified fragment after 300bp There is a great possibility of peak loss, and it is difficult to meet the needs of detection

Method used

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  • Eight-color fluorescent multiplex amplification kit for trace degradation DNA (deoxyribonucleic acid) detection and application of eight-color fluorescent multiplex amplification kit
  • Eight-color fluorescent multiplex amplification kit for trace degradation DNA (deoxyribonucleic acid) detection and application of eight-color fluorescent multiplex amplification kit
  • Eight-color fluorescent multiplex amplification kit for trace degradation DNA (deoxyribonucleic acid) detection and application of eight-color fluorescent multiplex amplification kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] An eight-color fluorescence composite amplification kit for the detection of microdegraded DNA, the kit includes specific amplification primers for amplifying 16 STR loci and 1 sex locus; wherein, the 16 loci are D7S820, D13S317, CSF1PO, TH01, D2S1338, D16S539, TPOX, D5S818, D19S433, D21S11, D8S1179, vWA, DYS391, D18S51, D3S1358, FGA, and the sex locus is Amel.

[0026] The sequences of the specific amplification primers are as follows: D7S820, SEQ ID NO: 1-2; D13S317, SEQ ID NO: 3-4; CSF1PO, SEQ ID NO: 5-6; TH01, SEQ ID NO: 7-8 ; D2S1338, SEQ ID NO: 9~10; D16S539, SEQ ID NO: 11~12; TPOX, SEQ ID NO: 13~14; ​​D5S818, SEQ ID NO: 15~16; Amel, SEQ ID NO: 17~18 ; D19S433, SEQ ID NO: 19-20; D21S11, SEQ ID NO: 21-22; D8S1179, SEQ ID NO: 23-24; vWA, SEQ ID NO: 25-26; DYS391, SEQ ID NO: 27-28; D18S51, SEQ ID NO: 29-30; D3S1358, SEQ ID NO: 31-32; FGA, SEQ ID NO: 33-34.

[0027] The final concentrations of the specific amplification primers in the amplification system are: D7S82...

Embodiment 2

[0036] Example 2 The process of using the kit of the present invention to detect actual samples in practical application

[0037] The eight-color fluorescence composite amplification kit for the detection of microdegraded DNA described in Example 1 has the following application steps in forensic identification, paternity identification or DNA family tree construction: collecting genomic DNA, performing PCR amplification, analyzing amplification. increase product.

[0038] 1. Collection of genomic DNA

[0039] The sample sources used in forensic identification, paternity identification or DNA family tree construction include human genomic DNA extracted by Chelex method, magnetic bead extraction method or organic extraction method; or any of extraction-free filter paper, FTA card, cotton swab and gauze. A carrier collected human blood or buccal cells. The sources of samples include human blood, blood stains, semen, saliva, body fluids, hair, muscles or tissues and organs.

[...

Embodiment 3

[0054] Example 3 The effect of the kit of the present invention in the test of degraded samples

[0055] Use the kit of the present invention to detect the degradation test material, and its detection process is as follows:

[0056] 1. Genomic DNA extraction of degraded samples

[0057] The extraction of genomic DNA of various samples was carried out with reference to "GA / T 383-2014 Forensic Science DNA Laboratory Inspection Specifications", and such samples were extracted by magnetic bead method.

[0058] 2. The operation steps are as follows:

[0059] 2.1 The amplification system is:

[0060]

[0061]

[0062] 2.2 The amplification procedure is:

[0063]

[0064] 2.3. Fluorescence detection of amplified products on a genetic analyzer

[0065] The loading mixture is composed of deionized formamide and the molecular weight internal standard AGCU Marker SIZ-500 in the system [(0.5μl AGCU Marker SIZ-500 (Wuxi Zhongde Meilian Biotechnology Co., Ltd.)) × (number of in...

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PUM

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Abstract

The invention discloses an eight-color fluorescent multiplex amplification kit for trace degradation DNA (deoxyribonucleic acid) detection and application. The kit comprises specific amplification primers for amplifying 16 STR (short tandem repeat) gene loci and one sex locus; wherein the 16 gene loci are D7S820, D13S317, CSF1PO, TH01, D2S1338, D16S539, TPOX, D5S818, D19S433, D21S11, D8S1179, vWA, DYS391, D18S51, D3S1358 and FGA, and the sex locus is Amel. Compared with the prior art, the invention has the following advantages: (1) the kit is an eight-color fluorescent multiplex amplification kit, and can accommodate more gene loci within the same fragment length range; (2) the amplification fragments of the gene loci of the kit are smaller than 220bp, so that the kit has a better detection effect on degrading trace detection materials; and (3) the primers in the kit have the advantages of strong specificity, high sensitivity and accurate typing result, and can completely meet the requirements of actual case inspection, DNA database construction and paternity test.

Description

technical field [0001] The invention belongs to the technical field of forensic genetic testing, and relates to an eight-color fluorescence-labeled composite amplification detection system, in particular to an eight-color fluorescence composite amplification kit used for the detection of micro-degraded DNA and its application. Background technique [0002] Short tandem repeats (STRs), as the second-generation genetic markers after restriction fragment length polymorphisms, have been widely used in forensic forensics since the 1980s and 1990s. It is one of the most widely used genetic markers for research and identification. Compared with other genetic markers, STR loci have small fragments and are easy to amplify, and are more suitable for trace and degraded samples, and multiple STR loci can be compound amplified at the same time, which is fast, efficient, accurate, and sensitive. , large amount of information and other advantages, it is widely used in individual identific...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/156C12Q2531/113C12Q2563/107Y02A50/30
Inventor 周如华王鑫石云杰张健陈维忠崔扬史明皓梅兴林李娟邵泽香郭佳佳朱晨煦
Owner 苏州市公安局
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