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A Nucleotide Sequence and Method for Identifying Milk Components and Beef Bone Meal Components in Feed

A beef and bone meal technology, applied in DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inability to identify and distinguish the source of components, low specificity, and narrow application range, and achieves easy promotion, high sensitivity, good species-specific effects

Active Publication Date: 2017-01-11
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

[0003] 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 bovine-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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  • A Nucleotide Sequence and Method for Identifying Milk Components and Beef Bone Meal Components in Feed
  • A Nucleotide Sequence and Method for Identifying Milk Components and Beef Bone Meal Components in Feed
  • A Nucleotide Sequence and Method for Identifying Milk Components and Beef Bone Meal Components in Feed

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The selection of embodiment 1 bovine tissue component target sequence and the design and synthesis of primers

[0054] In order to identify bovine milk-derived ingredients and prohibited bovine-derived ingredients, it is necessary to find a tissue-specific marker as a detection marker, which should have the following characteristics:

[0055] (1) The marker can be detected in bone, muscle, blood, and internal organs;

[0056] (2) The marker cannot be detected in milk;

[0057] (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;

[0058] (4) It is conserved within a species and has specificity among species.

[0059] In order to screen suitable target sequences as detection markers, the present invention selects 7 kinds of mRNA fragments of specific proteins expressed in muscle or bone (bovine troponin, bovine osteocalcin, bovine osteopontin, bovine sialic acid protein, Bov...

Embodiment 2

[0065] The extraction of embodiment 2 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 we...

Embodiment 3

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

[0079] Choose One Step from TaKaRa Company 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] Stage 1 reverse transcription

[0084]

[0085] Stage 2 PCR

[0086]

[0087] Stage 3 Melting Curve

[0088]

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Abstract

The invention discloses a kit and an oligonucleotide for verifying beef bone powder components in a feed, and particularly relates to a group of oligonucleotides for verifying the beef bone powder components as shown in sequence tables SEQ ID No.3 and SEQ ID No.4, and a kit containing these oligonucleotides and a detection method thereof. The kit provided by the invention is high in sensitivity, strong in specificity, low in cost and simple 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 milk components and beef bone meal components in feed. Background technique [0002] In order to prevent mad cow disease from being introduced into my country through feed and other animal products, my country’s BSE trade ban formulated in accordance with the OIE Code stipulates that direct or indirect imports from countries and regions with mad cow disease except cowhide, milk and dairy products, cow semen, cow embryos, and protein-free products are prohibited. Oils and fats and their products, calcium hydrogen phosphate made from bone (without protein or oil), gelatin and collagen for industrial use completely processed from leather or hides, gelatin for photography, feed and products of non-ruminant origin (prohibited for use in exporting countries or regions cattle and cattle products other than Raw materials from cattle and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
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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