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Multiplex amplification kit for simultaneously amplifying 20 STR (short tandem repeat) gene loci of horse and application of multiplex amplification kit

A compound amplification and locus technology, applied in the field of forensic genetics, can solve the problem of inability to distinguish two individuals, and achieve the effect of improving the overall recognition ability, accurate typing and high sensitivity

Pending Publication Date: 2022-07-29
呼伦贝尔市公安局刑事侦查支队 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since these loci are only located on autosomes and the number is not large, it is not ruled out that two individuals with the same allelic type may be encountered. When two individuals have the same genotype, a kit with a small number of loci will not be able to distinguish the two individual

Method used

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  • Multiplex amplification kit for simultaneously amplifying 20 STR (short tandem repeat) gene loci of horse and application of multiplex amplification kit
  • Multiplex amplification kit for simultaneously amplifying 20 STR (short tandem repeat) gene loci of horse and application of multiplex amplification kit
  • Multiplex amplification kit for simultaneously amplifying 20 STR (short tandem repeat) gene loci of horse and application of multiplex amplification kit

Examples

Experimental program
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Effect test

Embodiment 1

[0023] A composite amplification kit for simultaneously amplifying 20 STR loci in horses, including:

[0024] 1. Gene locus screening

[0025] According to the requirements of high polymorphism, no linkage between markers, easy genotyping, etc., as well as the actual primer test effect and locus arrangement requirements, 20 chromosomal STR loci were screened: HTG6, HTG7, COR22, CA425, HTG4, COR82, LEX54, COR69, AHT5, HMS3, HMS1, HMS6, HMS7, COR58, HTG10, ASB17, VHL20, HMS2, ASB2, LEX34.

[0026] Second, the locus arrangement

[0027] According to the above 20 loci, a unique locus arrangement and chemical fluorescent dye labeling method are designed: HTG6, HTG7, COR22, CA425 are the first group, and the fluorescent dye label is FAM; HTG4, COR82, LEX54, COR69 It is the second group, the fluorescent dye marker is HEX; AHT5, HMS3, HMS1, HMS6 is the third group, and the fluorescent dye marker is SUM; HMS7, COR58, HTG10, ASB17 is the fourth group, and the fluorescent dye marker is...

Embodiment 2

[0046] Example 1 Application of composite amplification kit in horse paternity test or individual identification. The application steps are as follows:

[0047] 1. Prepare PCR amplification system;

[0048] 2. Amplification thermocycling

[0049] (1) placing the PCR amplification tube on the thermal cycler;

[0050] (2) Select the PCR amplification conditions recommended in Table 3 to carry out PCR amplification;

[0051] (3) The amplified product should be stored in the dark;

[0052] table 3

[0053]

[0054] 3. Fluorescence detection of amplified products on a genetic analyzer

[0055] The loading mixture is composed of deionized formamide and the molecular weight internal standard AGCU Marker SIZ-500 in the system. AGCU MarkerSIZ-500 was purchased from Wuxi Zhongde Meilian Biotechnology Co., Ltd.;

[0056] Mix 12.5 μL of the loading mixture with 1 μL of the amplification product or 20 loci allelic typing standard Allelic Ladder (Wuxi Zhongde Meilian Biotechnology...

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Abstract

The invention discloses a multiplex amplification kit for simultaneously amplifying 20 STR gene loci of a horse and application. The kit comprises specific amplification primers for amplifying the following 20 STR gene loci; wherein the 20 STR gene loci are HTG6, HTG7, COR22, CA425, HTG4, COR82, LEX54, COR69, AHT5, HMS3, HMS1, HMS6, HMS7, COR58, HTG10, ASB17, VHL20, HMS2, ASB2 and LEX34, and the number of the STR gene loci is 20. Compared with the prior art, the kit provided by the invention has the following advantages: (1) the kit can simultaneously amplify and detect 20 horse STR sites in a single tube, and is a kit for detecting many sites in products of the same type; (2) the selected gene loci are distributed on different chromosomes as much as possible, and genetic linkage does not exist between the selected gene loci, so that the overall recognition capability of the kit is improved; and (3) the specific amplification primers do not interfere with one another, do not generate non-specific peaks, have the characteristics of strong specificity, high resolution, accurate typing, high sensitivity and the like, and can completely meet the requirements in multiple aspects of horse paternity identification, individual recognition, gender discrimination and the like.

Description

technical field [0001] The invention belongs to the technical field of forensic genetics, and relates to a method for paternity identification and individual identification in horses, in particular to a compound amplification kit for simultaneously amplifying 20 STR loci in horses and its application. Background technique [0002] Horses are classified in animal taxonomy as: Vertebrata, Mammalia, Perissodactyla, Equidae, Equus, Equus caballus. is a herbivorous animal. There are two subspecies of domestic horse and Przewalski's horse. At the end of 2011, the number of horses in my country was about 6.77 million, ranking second in the world. There are 29 local breeds, 13 cultivated breeds, and more than 10 introduced breeds that have formed breeding groups, forming a colorful and widely distributed horse breed resource, but more than 2 / 3 of them are in decline, endangered or endangered. state. my country's horse breed resources are in a critical period of conservation and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11C12Q1/6844
CPCC12Q1/6888C12Q1/6844C12Q2600/166C12Q2600/156C12Q2525/151C12Q2563/107C12Q2527/125
Inventor 孙树毅额尔敦图李威单晶鲁朦胡曼鞠欣朱晨煦郭佳佳姜颖烨范晓芸王皋葛毅晖李惠钰
Owner 呼伦贝尔市公安局刑事侦查支队