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Human DIP-InDel locus fluorescent labeling kit and detection method thereof

A fluorescent labeling and dip-indel technology, applied in biochemical equipment and methods, microbiological measurement/inspection, etc., can solve problems such as high mutation rate, fetal death, intrauterine infection, etc., and achieve the goal of improving accuracy and protecting safety Effect

Active Publication Date: 2019-12-31
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In paternity testing, the commonly used STR locus has a high mutation rate, and there will be a phenomenon that does not conform to the Mendelian inheritance law between the tested parent and the tested offspring.
Especially for the paternity test of the fetus, because most of the tests use fetal amniotic fluid as samples, the traditional amniocentesis and chorionic villi aspiration are traumatic, which can seriously cause adverse complications such as intrauterine infection, miscarriage, and fetal death. Therefore, the development A rapid identification kit for two-person unbalanced mixed plaques is helpful in improving the safety of current paternity testing and accurately locking suspects

Method used

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  • Human DIP-InDel locus fluorescent labeling kit and detection method thereof
  • Human DIP-InDel locus fluorescent labeling kit and detection method thereof
  • Human DIP-InDel locus fluorescent labeling kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Population frequency survey.

[0053] 1. Collect individual test materials.

[0054] A total of 200 unrelated individual samples were collected in this experiment, all of which belonged to the Han population in Shanxi. Genomic DNA was extracted according to "GA / T 383-2014 Forensic Science DNA Laboratory Inspection Specification".

[0055] 2. Amplification and detection.

[0056] Using the extracted DNA as a template, PCR amplification was carried out. The PCR reaction system is (10 μL): 5 μL 2×PCRMasterMix, add different volumes of primers according to Table 2, 10ng template DNA, ddH 2 O to make up to 10 μL.

[0057] The amplification program was: 95°C for 10 min; 95°C for 30 s, 56°C for 30 s, 72°C for 30 s, 5 cycles; 94°C for 30 s, 52°C for 30 s, 68°C for 30 s, 25 cycles; 68°C for 30 min.

[0058] Detection of amplification products: 1.5 μL of amplification products, 10 μL of deionized formamide, and 0.2 μL of SIZE-5000Plus. Denaturation method: aft...

Embodiment 2

[0061] Example 2: Study on species specificity.

[0062] 1. Collect animal samples.

[0063] The animal and bacterial DNA samples in this experiment were from cattle, fish, pigs, rabbits, mice, sheep and streptococci, respectively. Genomic DNA was extracted using the Qiagen (DNeasy Blood & Tissue Kit) kit.

[0064] 2. Amplification and detection.

[0065] Carry out PCR amplification, detection and result analysis according to the method in Example 1, the result proves that this kit has species specificity.

Embodiment 3

[0066] Embodiment 3: Sensitivity detection.

[0067] 1. Prepare DNA samples.

[0068] DNA was extracted using the Qiagen (DNeasy Blood & Tissue Kit) kit. The sensitivity of the system was determined using single-source DNA containing different amounts of DNA template (6ng, 4ng, 2ng and 1ng).

[0069] 2. Amplification and detection.

[0070] PCR amplification, detection and result analysis were carried out according to the method in Example 1. image 3 It is confirmed that the sensitivity of this kit is good, and the minimum detectable content is as low as 2ng of DNA.

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Abstract

The invention discloses a human DIP-InDel locus fluorescent labeling kit and a detection method thereof. The kit comprises 45 specific multiplex PCR primers with nucleotide sequences shown in SEQ NO.1-45 and 4 pairs of fluorescent labeled universal primers with nucleotide sequences shown in SEQ NO.46-53. By amplifying and comparing 15 DIP-InDel loci, trace components in double-human unbalanced mixed sample are detected and excluded, an individual is confirmed, and the kit can be applied to aspects such as double-human mixed stain medicolegal expertise and non-invasive fetal paternity test.

Description

technical field [0001] The invention belongs to the technical field of biological detection and relates to the identification of an unbalanced mixed sample, in particular to a DIP-InDel gene locus kit and a detection method for determining the secondary components of the unbalanced mixed spot of two people and fetal paternity identification . Background technique [0002] Mixed spots are one of the most common forensic examination materials, which can be formed by mixing the same or different body fluids and secretions of different individuals. With the continuous improvement of DNA extraction technology and detection technology, the detection rate of mixed DNA samples has been greatly improved, and depending on the case, in recent years, the analysis requirements for complex situations such as extremely low DNA content and degraded DNA have gradually increased. [0003] Insertion-deletion polymorphisms (Deletion / Insertion polymorphisms, DIP) mostly refer to the combination...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858
CPCC12Q1/6858C12Q1/6888C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 张更谦李文嫣
Owner SHANXI MEDICAL UNIV
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