Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof

A detection kit and fluorescent labeling technology, applied in fluorescence/phosphorescence, material excitation analysis, microbial determination/inspection, etc., can solve the problem of low genetic polymorphism of locus, large differences between different dog groups, and unsatisfactory use. requirements and other issues to achieve accurate and effective identification

Active Publication Date: 2012-01-18
AGCU SCIENTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the process of realizing the present invention, the inventor found that the prior art has at least the following problems: there are only 10 canine genes in the StockMarks Animal Genotyping System recommended by the American ABI company loci, and some loci have low genetic polymorphism, or there are large differences between different dog groups, which will have a certain impact on the application and efficiency of DNA testing technology, and cannot meet the requirements of use

Method used

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  • Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof
  • Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof
  • Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Multiplex amplification and fluorescence detection of the fluorescently labeled multiplexed amplification detection kit for 17 loci

[0064] 1. Sample source

[0065] Canine whole blood samples from 2 unrelated individuals provided by the Nanchang Police Dog Base of the Ministry of Public Security

[0066] 2. DNA extraction (for the extraction of genomic DNA, refer to "GA / T 383-2002 Forensic Science DNA Laboratory Inspection Specification")

[0067] Genomic DNA was extracted from two whole blood samples by Chelex-100 method:

[0068] Take 0.5-5μl whole blood and place it in a sterilized 1.5ml centrifuge tube, add sdH 2 Put O1ml in the tube, shake for a few seconds; place at room temperature for 10 minutes, shake for a few seconds, centrifuge at 12,000rpm for 3 minutes, discard the supernatant, keep enough supernatant to cover the precipitate, do not stir the precipitate; add 200μl 5% Chelex- 100, shake for a few seconds; keep warm at 56°C for 30 minutes,...

Embodiment 2

[0086] Example 2 The samples are: the male parent and the offspring ( Provided by the Nanchang Police Dog Base of the Ministry of Public Security) DNA extraction, PCR amplification, and genetic analyzer detection were performed according to Example 1, and the typing results were finally obtained.

[0087] Results and conclusions

[0088] The basic principle of paternity testing is: According to Mendel's law of inheritance, the genotype of the parent determines the genotype of the offspring. Under the premise of no gene mutation and typing error: ① A child’s pair of alleles must be one from the father and one from the mother; ② It is impossible for the child to have an allele that neither parent has.

[0089] The results of this example are shown in the attached Figure 3-6 and Schedule 1, at Figure 3-6 The corresponding test results of the female dog, father dog and young dog are listed in order.

[0090] Table 1:

[0091]

[0092] PI is the paternity index, also kn...

Embodiment 3

[0100] Example 3 Individual identification of dogs using a fluorescently labeled multiplex amplification detection kit with 17 loci

[0101] 1. The unknown individual identification samples in this experiment are blood spot and semen spot samples, provided by the Nanchang Police Dog Base of the Ministry of Public Security.

[0102] 2. Genomic DNA was extracted with reference to "GA / T 383-2002 Forensic Science DNA Laboratory Inspection Specification".

[0103] 3. Amplification detection

[0104] Perform PCR amplification and genetic analyzer detection according to Example 1, and finally obtain the typing result.

[0105] 4. Results and conclusions

[0106] Since the appearance of dogs of the same breed is not much different, although there are dog ear marks to distinguish them, the ear tags of breed dogs are prone to blurring, loss, and defect during the process of breeding and genetic breeding, making it difficult to identify individual breed dogs, resulting in Huge eco...

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Abstract

The present invention relates to a fluorescence labeled composite amplification detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA. With the system provided by the kit, genetic polymorphisms of the 17 gene loci of the canine genomic DNA can be simultaneously analyzed. The 17 gene loci of the canine genomic DNA comprise DAmel, PEZ1, PEZ2, PEZ3, PEZ5, PEZ6, PEZ8, PEZ12, PEZ15, PEZ20, PEZ21, FH2010, FH2054, FH2079, FH2132, FH2611 and VWFX, is divided into four groups, and relates to the fluorescence labels comprising five colors. The fluorescence labeled composite amplification detection kit provided by the present invention is provided for composite amplification of a mixture comprising oligonucleotide primers of the 17 gene loci. In the system, the gene locus foridentifying the gender of the DNA samples is the DAmel gene locus. The detection results of the system has characteristics of high polymorphism, good balance, high sensitivity, strong specificity, accurate typing result, strong species-specificity, and can fully meet the requirements of dog individual identification, paternity identification, and DNA database construction.

Description

technical field [0001] The invention relates to a fluorescent label composite amplification detection kit, in particular to a five-color fluorescent label composite amplification detection kit for simultaneously analyzing 16 STR loci and DAmel loci of canine genome DNA through composite amplification. [0002] Background technique [0003] STR (short tandem repeat, short tandem repeat sequence) is a type of DNA sequence with length polymorphism existing in the eukaryotic genome. Therefore, the genetic polymorphism of short tandem repeats plays a very important role in the application of individual identification, paternity identification, forensic identification, genetic breeding and genetic linkage map construction. effect. [0004] The STR locus has the characteristics of being easy to amplify due to small fragments, and the detection kit can be constructed under similar amplification conditions for each locus, so it has the advantages of sensitivity, accuracy, speed, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 杜蔚安叶俊华郑卫国杨前勇熊勇华兰康陈丽娜李涛付平峰王博
Owner AGCU SCIENTECH
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