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Fluorescent automated detection primer and kit of pathogenic sites of Arachnomelia syndrome (AS) in Simmental cattle

A Simmental cattle, automated detection technology, applied in fluorescence/phosphorescence, material excitation analysis, microbial determination/inspection, etc., to achieve the effect of low cost and stable results

Inactive Publication Date: 2013-09-04
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there is no rapid and accurate detection method for the AS pathogenic locus in Simmental cattle that is suitable for commercial operation

Method used

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  • Fluorescent automated detection primer and kit of pathogenic sites of Arachnomelia syndrome (AS) in Simmental cattle
  • Fluorescent automated detection primer and kit of pathogenic sites of Arachnomelia syndrome (AS) in Simmental cattle
  • Fluorescent automated detection primer and kit of pathogenic sites of Arachnomelia syndrome (AS) in Simmental cattle

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation of embodiment 1 test material and the extraction of DNA

[0034] The test materials used in this study come from two parts: the anticoagulant blood samples and ear tissue samples of mating cows and crossbred offspring of 4 German Simmental bull families; the controlled frozen semen of 11 cattle breeds or lines.

[0035] Four German Simmental bull families (Table 1), including 80 mating cows and 106 offspring, were from Anshan Hengli Dairy Farm in China (Anshan Hengli) and Xiertala Sanhe Cattle Farm in Hailaer, Inner Mongolia (Hylar Sheltara). In the 4 families, the cow breeds are Chinese Holstein and Chinese Sanhe cattle, and the offspring are all hybrid cattle (Holstein×German Simmental cross, Sanhe cattle×German Simmental cross). The four German Simmental cattle are ROMEL, BOSSAG, RIFURT, and HIRMER (German Simmental cattle genealogy query website: http: / / www.zar.at / article / archive / 18981). ROMEL is a known AS pathogenic gene carrier, and the other th...

Embodiment 3A

[0060] The direct sequencing analysis of embodiment 3AS pathogenic mutation

[0061] In this study, primers were designed for the c.del1224_1225CA mutation of the MOCS1 gene, and 4 German Simmental bulls including ROMEL and 31 offspring from ROMEL's pedigree records were selected for PCR amplification of the mutation fragment. Perform PCR product sequencing. Sequencing primers are shown in Table 5, and the PCR system is the same as in Example 2, and the PCR reaction is as follows Figure 4 shown. PCR was detected by 2% gel, and the PCR products were purified and Sanger sequenced at BGI.

[0062] Table 5 Primers for direct sequencing of MOCS1 gene mutation sites

[0063]

[0064] Generation (Table 6) is the heterozygous genotype carrying the mutation, and the other individuals are all wild type. Sequencing peaks of ROMEL and its 2 offspring are as follows Figure 6 As shown, ROMEL is heterozygous at the mutation site, and its two offspring 94254 and 9129x are heterozygo...

Embodiment 4A

[0069] Embodiment 4 AS pathogenic mutation fluorescent automatic detection method

[0070] 1. Method

[0071] In this study, the MOCS1 gene c.del1224_1225CA mutation was a 2bp deletion mutation. Due to the high sensitivity of the fluorescent primer PCR detection method, it can accurately detect length polymorphisms with a length of 1bp. Therefore, this study designed fluorescently labeled primers for the mutation site. The genotype was detected by capillary electrophoresis after PCR amplification. Since there may be systematic errors between different plates detected by capillary electrophoresis, three identical individuals were set in each plate to correct genotype determination errors between plates; intra-plate errors were corrected using the molecular weight standard GS500LIZ.

[0072] The 5' end of the upstream primer was labeled with a FAM fluorophore. PCR system and reaction procedure are with embodiment 2, and reaction procedure is as Figure 4 shown.

[0073] 2. F...

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Abstract

The invention relates to a fluorescent automated detection and analysis method of pathogenic sites of Arachnomelia syndrome (AS) in Simmental cattle. The nucleotide sequence of a primer for PCR (polymerase chain reaction) amplification is as follows: 5'-CTGAGTCTCCTCTTCTGTTTTCA-3',5'-GTTGGCATCTGAGTCCAGGT-3'. By using the fluorescent labeling primer PCR amplification and electrophoresis typing technology, the kit for typing pathogenic sites of AS in Simmental cattle has the advantages of stable result and low cost, and is accurate and reliable, thereby accurately typing the pathogenic sites of AS in Simmental cattle.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting a Simmental spider leg syndrome (AS) pathogenic site fluorescently labeled primer PCR detection method. Background technique [0002] The reason why livestock genetic defects mostly show a recessive inheritance pattern may have something to do with the production methods of livestock, especially dairy cows. The application of artificial insemination (AI) and semen cryopreservation techniques (Semen Cryopreservation Techniques) in the production and breeding of dairy cows has laid a solid foundation for the establishment of the artificial insemination breeding system (AI breeding system) of dairy cows. Because AI technology can undertake more mating tasks for female animals, bulls can be widely used in groups to obtain a large number of offspring, allowing high-yielding genes to spread rapidly among groups. Semen cryopreservation technology makes ex...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 王雅春焦士会初芹俞英张胜利孙东晓张毅张沅
Owner CHINA AGRI UNIV
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