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Nucleotide sequence, method and agent case for detecting bovine spongiform encephalitis specific risk substance in beef

A mad cow disease, a special technology, applied in the field of inspection and quarantine, can solve the problems of cumbersome operation, sensitivity that cannot meet the needs of detection, long delivery cycle, etc., and achieve good stability

Inactive Publication Date: 2010-05-12
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing method is enzyme-linked immunosorbent assay (ELISA), which requires the preparation of high-titer specific antibodies, which is cumbersome and time-consuming.
Moreover, there is no such reagent in our country at present, so the reagents must be imported, which is expensive, and the delivery cycle is very long, which brings inconvenience to the testing work
In addition, with the improvement of the production process of beef production and processing enterprises, the contaminated central nervous tissue may be very small, and its sensitivity can no longer meet the needs of detection, so it is necessary to establish a more sensitive and rapid detection method

Method used

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  • Nucleotide sequence, method and agent case for detecting bovine spongiform encephalitis specific risk substance in beef
  • Nucleotide sequence, method and agent case for detecting bovine spongiform encephalitis specific risk substance in beef
  • Nucleotide sequence, method and agent case for detecting bovine spongiform encephalitis specific risk substance in beef

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] 1. Materials

[0073] 1. Test material

[0074] Uncontaminated beef taken from Yuxiangyuan slaughterhouse and some random beef bought from supermarkets.

[0075] 2. Test reagents

[0076] (1) Total RNA extraction kit

[0077] Reagent Total RAN Isolation kit, a product of Invitrogen Life Technologies, was purchased from New Economics Corporation.

[0078] (2) Ready-To-Go TM RT-PCR Beads Amersham Biosciences products were purchased from Shenzhen Peeky Biotechnology Co., Ltd.

[0079] (3) RT-PCR reaction solution

[0080] Taq TM PCR kit, TaKaRa company product, purchased from Liuhetong Economic and Trade Co., Ltd., including:

[0081] 10×PCR Buffer (Mg 2+ free)

[0082] dNTP Mixture (2.5mmol / L each)

[0083] Taq DNA polymerase (5U / μl)

[0084] MgCl 2 (25mM)

[0085] (4) DEPC treated water

[0086] The products of TaKaRa company were purchased from Liuhetong Economic and Trade Co., Ltd.

[0087] (5) Chloroform

[0088] Analytical grade, freshly opened an...

Embodiment 2

[0199] Example 2. Sensitivity test

[0200] 1. Method

[0201] 1. Determination of the minimum detection concentration of RNA

[0202] Infinite 10-fold serial dilutions of RNA extracted from brain tissue or spinal cord, respectively, were used to determine the lowest RNA concentration detected.

[0203] 2. Determination of the minimum detection limit of tissue homogenates

[0204] Brain tissue or spinal cord was made into 10% homogenate with sterilized PBS, followed by 10-fold gradient dilution, and then RNA was extracted separately for fluorescence RT-PCR to determine the minimum detection limit of tissue homogenate.

[0205] 3. Determination of minimum detection limit for experimentally contaminated beef

[0206] The brain tissue or spinal cord was made into a 10% homogenate with sterilized PBS, followed by 10-fold gradient dilution, and then 500ul of the tissue homogenate of each dilution was evenly spread on the surface of a 10×10cm piece of uncontaminated beef with a c...

Embodiment 3

[0220] Example 3. Stability test

[0221] 1. Method

[0222] Brain or spinal cord tissue was subjected to the following different treatments for stability tests:

[0223] 1. Thermal stability test

[0224] The brain or spinal cord tissue stock solution, 20%, 10%, 1%, 0.1% and 0.01% dilutions were heated at 100°C for 30min, and then RNA was extracted for fluorescence RT-PCR for thermal stability test.

[0225] 2. Room temperature storage stability test

[0226] Different equal amounts of brain or spinal cord tissue were stored at room temperature for 1 d, 2 d, 3 d, 4 d, and 5 d, and then RNA was extracted for fluorescent RT-PCR and room temperature stability test.

[0227] 3.4℃ Refrigeration Stability Test

[0228] The brain or spinal cord tissue stock solution, 20%, 10%, 1%, 0.1% and 0.01% dilutions were stored at 4°C for 1d, 2d, 3d, 4d, 5d, 8d, 11d, 15d, and then RNA was extracted for fluorescence RT. - PCR, cold storage stability test.

[0229] 2. Results

[0230] 1. ...

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PUM

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Abstract

The invention discloses a kind of nucleotide sequence for detecting the special risk of mad cow disease of beef and its method as well as reagent. The group of nucleotide sequence has a base sequenceshown in the sequence from SEQ ID No.1 to SEQ ID No.3. The advantage of the invention is that: it judges whether there is GFAP mRNA in the beef or not to determine whether the beef is contaminated bythe risk of mad cow disease. Comparing with the traditional ELISA technology, the method is more sensitive, rapid, and better stability.

Description

technical field [0001] The invention relates to a nucleotide sequence, a method and a kit for detecting mad cow disease special risk substances in beef, and belongs to the field of inspection and quarantine. Background technique [0002] The scientific name for mad cow disease is Bovine spongiform encephalopathy (BSE), which is associated with Scrapie, chronic wasting disease (CWD) in deer, Feline spongiform encephalopathy (FSE) in cats, and - Creutzfeldt-Jakob disease (CJD) and other diseases belong to the same group of diseases, collectively referred to as Transmissible Spongiform Encephalopathies (TSEs). These diseases all cause progressive vacuolar degeneration of brain tissue in diseased animals, causing Neurological disorder and eventually death. Its clinical manifestations are mainly mental disorders, ataxia, hypersensitivity to sensation (touch, sound), and easy convulsions, so it is commonly known as mad cow disease. Since the disease was first reported in the Unite...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 马贵平史喜菊李冰玲王宇李炎鑫刘旭辉
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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