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

A nucleotide sequence and mad cow disease technology, applied in the field of inspection and quarantine, can solve the problems of cumbersome operation, sensitivity that cannot meet the needs of detection, high cost, etc., and achieve good stability

Inactive Publication Date: 2007-09-12
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] Pollution-free beef taken from the Yuxiangyuan slaughterhouse and some beef randomly bought from the supermarket.

[0075] 2. Test reagents

[0076] (1) Total RNA Extraction Kit

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

[0078] (2) Ready-To-Go TM RT-PCR Beads Amersham Biosciences products were purchased from Shenzhen Piji Biological Company.

[0079] (3) RT-PCR reaction solution

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

[0081] 10×PCR Buffer (Mg 2+ free)

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

[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 pure grade, newly ope...

Embodiment 2

[0195] Embodiment 2, sensitivity test

[0196] 1. Method

[0197] 1. Determination of the minimum detection concentration of RNA

[0198] The RNA extracted from the brain tissue or the spinal cord was respectively infinitely diluted 10-fold to determine the lowest detection RNA concentration.

[0199] 2. Determination of the minimum detection limit of tissue homogenate

[0200] Make 10% homogenate of brain tissue or spinal cord with sterilized PBS, then perform 10-fold gradient dilution, and then extract RNA for fluorescent RT-PCR to determine the minimum detection limit of tissue homogenate.

[0201] 3. Determination of the minimum detection limit of experimental contaminated beef

[0202] Make 10% homogenate of brain tissue or spinal cord with sterilized PBS, then perform 10-fold gradient dilution, and then use cotton swabs to apply 500ul of tissue homogenate of each dilution evenly to the surface of a piece of non-polluted beef of 10×10cm, and then use A cotton swab was...

Embodiment 3

[0214] Embodiment 3, stability test

[0215] 1. Method

[0216] The brain or spinal cord tissues were subjected to the following different treatments for the stability test:

[0217] 1. Thermal stability test

[0218] The brain or spinal cord tissue stock solution, 20%, 10%, 1%, 0.1% and 0.01% dilutions were heated at 100°C for 30 minutes, and then RNA was extracted for fluorescent RT-PCR and thermal stability test.

[0219] 2. Storage stability test at room temperature

[0220] Different equal amounts of brain or spinal cord tissue were stored at room temperature for 1d, 2d, 3d, 4d, and 5d, respectively, and then RNA was extracted for fluorescent RT-PCR to conduct a room temperature stability test.

[0221] 3.4 ℃ cold storage stability test

[0222] Store the brain or spinal cord tissue stock solution, 20%, 10%, 1%, 0.1% and 0.01% dilutions at 4°C for 1d, 2d, 3d, 4d, 5d, 8d, 11d, and 15d, then extract RNA for fluorescent RT -PCR for cold storage stability test.

[0223]...

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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 sequence shown 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 by the 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, method and kit for detecting mad cow disease special risk substances in beef, belonging to the field of inspection and quarantine. Background technique [0002] The scientific name of mad cow disease is bovine spongiform encephalopathy (Bovine spongiform Encephalopathy, BSE), and sheep scrapie (Scrapie), deer chronic wasting disease (CWD), feline spongiform encephalopathy (Feline spongiform Encephalopathy, FSE) and human gram -Creutzfeldt-Jakob disease (CJD) and other diseases belong to the same group of diseases, which are collectively referred to as Transmissible Spongiform Encephalopathies (TSEs). These diseases all cause progressive vacuolar degeneration of the brain tissue of affected animals, resulting in Nervous disorder and eventually death. Its clinical manifestations are mainly insanity, ataxia, sensory (touch, sound) hypersensitivity, and easy convulsions, so it is commonly known as mad cow dis...

Claims

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Application Information

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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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