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Nucleotide sequence and method for identifying goat milk components and mutton bone meal components in feed

A technology of mutton and bone meal, applied in the direction of DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inability to identify and distinguish the source of component tissues, low specificity, narrow application range, etc., and achieve easy promotion, high sensitivity, Good species-specific effect

Active Publication Date: 2017-01-18
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are still relatively few studies on the identification of different tissue components of the same species at home and abroad, mainly including enzyme-linked immunosorbent assay (ELISA), PCR detection after chloroform separation, and PCR detection after washing with water and sodium hypochlorite. The scope of use of these methods Very narrow and not very specific, so it is rarely used in practice
At present, my country's checks on imported animal feed and feed additives mainly use ordinary PCR method or real-time fluorescent PCR method to detect whether the feed contains sheep-derived ingredients, but this method is to identify the source of the species, and cannot identify the composition of the ingredients. Identify and distinguish the source, that is, it is impossible to distinguish whether the milk-derived ingredients that are allowed to be added or the tissue ingredients that are within the scope of the trade ban are added

Method used

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  • Nucleotide sequence and method for identifying goat milk components and mutton bone meal components in feed
  • Nucleotide sequence and method for identifying goat milk components and mutton bone meal components in feed
  • Nucleotide sequence and method for identifying goat milk components and mutton bone meal components in feed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Selection of target sequence of sheep tissue components and design and synthesis of primers

[0055] In order to identify goat milk-derived ingredients and prohibited goat-derived ingredients, it is necessary to find a tissue-specific marker as a detection marker, which should have the following characteristics:

[0056] (1) The marker can be detected in bone, muscle, blood, and internal organs;

[0057] (2) The marker cannot be detected in milk;

[0058] (3) The marker has a certain stability, that is, it can still be detected after the bone, muscle and viscera are processed into meat and bone meal;

[0059] (4) It is conserved within a species and has specificity among species.

[0060] According to the mRNA fragments of sheep osteonectin recorded in Genbank, three pairs of primers (YG51, YG52, YG53) were designed for the fragments of sheep osteonectin mRNA, which were compared and analyzed with DNAMAN software. In order to avoid the interference of genomi...

Embodiment 2

[0065] Example 2 Extraction of sample total RNA

[0066] Use the RNA extraction kit from TIANGEN company to extract total RNA, the specific operation is as follows:

[0067] (1) Take 25 mg of sample, add 1 ml of lysate RZ, shake and mix well.

[0068] (2) Leave the sample at room temperature for 5 minutes. Centrifuge at 12,000 rpm at 4°C for 5 minutes, take the supernatant, and transfer it to a new RNase-free centrifuge tube.

[0069] (3) Add 200 μl of chloroform, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 3 minutes.

[0070] (4) Centrifuge at 12000rpm at 4°C for 10 minutes. The sample is divided into three layers: a yellow organic phase, an intermediate layer and a colorless aqueous phase. RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 50% of the lysate RZ reagent used. . Transfer the aqueous phase to a new tube.

[0071] (5) Slowly add 1.5 times the volume of absolute ethanol and mix well. Th...

Embodiment 3

[0078] Example 3 Establishment of real-time fluorescent RT-PCR detection method

[0079] Choose TaKaRa's The RT-PCR kit is used for RT-PCR amplification. Through repeated trials, the reaction system and reaction conditions are optimized to achieve the best experimental results.

[0080] The final determination of the reaction system is as follows:

[0081]

[0082] SYBR Real Time RT-PCR reaction conditions are set as follows:

[0083]

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Abstract

The invention discloses a kit and oligonucleotides for identifying the mutton bone meal component in feed and particularly relates to a group of oligonucleotides for identifying the mutton bone meal component, shown in oligonucleotide sequences of sequence tables SEQ ID NO.3 to SEQ ID NO.4, a kit comprising the oligonucleotides as well as a detection method thereof. The kit disclosed by the invention is high in sensitivity, strong in specificity, low in cost and simple and convenient to operate.

Description

technical field [0001] The invention relates to the fields of inspection and quarantine and biotechnology, in particular to a method for identifying goat milk components and mutton bone meal components in feed. Background technique [0002] Scrapie is a kind of animal infectious disease closely related to mad cow disease. In order to prevent sheep scrapie from being introduced into my country through the trade of related products, my country promulgated a trade ban to prevent and control the disease. In July 1996, the Ministry of Agriculture of my country issued the "Notice on Strictly Preventing Scrapie from Introducing my country (Agricultural Inspection and Quarantine [1996] No. 7)", which stipulated that "import (including direct import and re-export) from the above countries with scrapie is prohibited. ) sheep, sheep embryos, sheep semen, sheep viscera (including casings) and their products, meat and bone meal, bone meal, suet (oil) and animal feed containing sheep prot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 李冰玲马贵平高志强刘全国李炎鑫蒲静史喜菊
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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