Primer composition for detecting bovine citrullinemia harmful genes and kit and application thereof

A technology for bovine citrullinemia and citrullinemia, which is applied to a primer composition for detecting harmful genes in bovine citrullinemia, a kit and application fields, and can solve misjudgment and the influence of PCR amplification efficiency , false positives, etc., to avoid false negatives, excellent amplification specificity, and high accuracy

Active Publication Date: 2014-04-23
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is designed at the mutation point Ava II Enzyme cutting site, carry out enzyme digestion on the PCR product, and judge whether it has Ava II Restriction sites are used to determine whether there are mutation points, but after the introduction of mismatched bases by this method, the amplification efficiency of PCR will be affected, and there is also the possibility of false positives
The AS-PCR method designs specific primers according to the mutation site, one of which is complementary to a base state of the mutat

Method used

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  • Primer composition for detecting bovine citrullinemia harmful genes and kit and application thereof
  • Primer composition for detecting bovine citrullinemia harmful genes and kit and application thereof
  • Primer composition for detecting bovine citrullinemia harmful genes and kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of a nested PCR reaction kit for screening bovine citrullinemia

[0036] 1. Primer design

[0037]According to the ASS gene sequence on NCBI (GenBank: AC_000168) combined with the overall thinking of the present invention, the design of primers was continuously analyzed, summarized and adjusted, and finally according to the ASS gene encoding arginine succinate synthase on bovine chromosome 11 (BTA11) ( Argininosuccinate synthetase (ASS) had a C→T point mutation in exon 5. A nested PCR method was designed to detect bovine citrullinemia harmful genes, and primers 1, 2, 3 and 4 were obtained. The sequence is shown as SEQ ID NO.1-4:

[0038] Primer 1: SEQ ID NO.1: actgcatttt caagaccacc ctg;

[0039] Primer 2: SEQ ID NO.2: tctgggcggg aaccaacgat tgtc;

[0040] Primer 3: SEQ ID NO.3: attgaggaca tcagcaagga g;

[0041] Primer 4: SEQ ID NO. 4: cacatacttg gctccttctc.

[0042] Among them, primer 1 and primer 2 are primer set A (outer primer), primer 3 a...

Embodiment 2

[0054] Example 2 Using the nested PCR reaction kit prepared in Example 1 to detect dairy cow citrullinemia

[0055] 1. Extraction of whole DNA from bovine blood

[0056] The jugular vein blood sampling method randomly collected blood samples of 243 Holstein cows in an isolation farm in Jiangsu, 5mL / head, and placed them in vacuum heparin sodium anticoagulant tubes, shaken well, then divided them into 2ml tubes with centrifuge tubes, and stored them frozen at -20°C for later use .

[0057] Promega Maxwell 16 Automatic Nucleic Acid Extractor was used to extract blood genomic DNA, and a matching kit (AS1010 from Promega Company) was used. Promega's AS1010 kit contains Maxwell 16 Blood DNA Cartridges, Purification Plungers, Elution Tubes, and Elution buffer. The operation was carried out according to the instructions of the kit, and the DNA extraction of 16 samples could be completed approximately every 28 minutes after the instrument was started. After the DNA is extracted, ...

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Abstract

The invention discloses a primer composition for detecting bovine citrullinemia harmful genes and a kit and an application thereof. The primer composition disclosed by the invention is composed of a primer set A and a primer set B, wherein the primer set A is composed of a primer 1 and a primer 2, and the primer set B is composed of a primer 3 and a primer 4; and the nucleotide sequences of the primer 1 to the primer 4 are respectively shown in SEQ ID NO.1-4. The invention also provides a kit containing the primer combination, a method for detecting bovine citrullinemia harmful genes by using the kit provided by the invention comprises the following steps: extracting the whole set of genomic DNA in bovine blood to serve as a template, carrying out nested PCR (Polymerase Chain Reaction) and sequencing the obtained PCR products. The base change of a mutation site can be directly controlled according to the sequencing result, thereby ensuring the result accuracy and meeting the requirements of detection technology with such characteristics as high speed, accuracy, high throughput and the like.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, it relates to a primer composition for detecting bovine citrullinemia harmful genes, a kit and application thereof. Background technique [0002] With the popularization and application of artificial insemination and embryo transfer technology, the wide application of dairy cow germplasm (embryo, semen, and bulls) around the world has significantly improved the genetic performance and accelerated the impact of recessive genetic diseases on cattle. group hazards. In particular, an excellent breeding bull can produce hundreds of thousands of doses or even millions of doses of frozen semen in its lifetime. If it carries a recessive genetic defect gene, it may spread rapidly all over the world and bring huge economic benefits to the production of animal husbandry. loss. my country mainly imports a large number of excellent germplasm of dairy cows from the United States, Cana...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6848C12Q1/686C12Q2549/119C12Q2531/113
Inventor 郭霄峰吴晓薇洪洁心代元元
Owner SOUTH CHINA AGRI UNIV
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