Primer and probe composition for distinguishing various animal sources in colla corii asini, kit and multiple real-time fluorescence quantification PCR detection method

A technology of animal origin and composition, applied in the field of molecular biology, can solve the problem of inability to quickly detect various animal origins of donkey-hide gelatin, and achieve the effects of high accuracy, high sensitivity and convenient use.

Active Publication Date: 2015-04-22
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the inability to quickly detect multiple animal origins in donkey-hide gelatin in the prior art, the present invention provides a method capable of simultaneously identifying multiple animal origins in donkey-hide gelatin, horse origin, pig origin and bovine origin. A primer-probe composition, the primer-probe composition has good specificity and high sensitivity, and can realize rapid qualitative identification of various animal-derived substances in donkey-hide gelatin

Method used

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  • Primer and probe composition for distinguishing various animal sources in colla corii asini, kit and multiple real-time fluorescence quantification PCR detection method
  • Primer and probe composition for distinguishing various animal sources in colla corii asini, kit and multiple real-time fluorescence quantification PCR detection method
  • Primer and probe composition for distinguishing various animal sources in colla corii asini, kit and multiple real-time fluorescence quantification PCR detection method

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Experimental program
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Effect test

Embodiment 1

[0069] 1. DNA extraction: using Chelex-100 combined with SDS-proteinase K digestion solution to release DNA by heating and glass milk to purify DNA disclosed in patent 201410317118.7 (a kit for quickly extracting DNA from donkey-hide gelatin and its extraction method) Purification of the genomic DNA of donkey-hide gelatin products and raw cowhide, pig, horse, and donkey hides. The purity and concentration of the extracted genomic DNA were determined by UV spectrophotometer. Determination of OD 260 / OD 280 The values ​​are all about 1.8-1.9, and the concentration is above 10ng / μl, indicating that the DNA is of high purity and moderate concentration, which meets the requirements of PCR amplification.

[0070] 2. Design of primers and specific probes: compare the mitochondrial DNA sequence information of the four animals published in GeneBank, such as cattle, pigs, horses, and donkeys, such as figure 1 As shown, according to the characteristics of the mitochondrial sequences o...

Embodiment 2

[0098] Using the primers and specific probes with good specificity and high sensitivity in Example 1 to detect commercially available 5 kinds of brand donkey-hide gelatin, the method is:

[0099] 1. According to the method disclosed in patent 201410317118.7, the donkey-hide gelatin genomic DNA was extracted as a template for future use;

[0100] 2. Use the kit to prepare a 20 μl PCR reaction system, the composition is as follows:

[0101] Reagent name concentration Dosage (μL) HS-Taq enzyme 5U / μL 0.2 premix 10× 2 primer mix 5μM 2 probe mix 5μM 1 DNA template 10ng / μL 2 Ultra-pure water to 20

[0102] 3. Carry out PCR amplification, the amplification program is: 95°C for 1min; 95°C for 10s, 60°C + 3°C, 35s, collect fluorescence signal here, 45 cycles. After the reaction, the results were analyzed to obtain the amplification curves of various commercially available donkey-hide gelatin products. like Figures 5...

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Abstract

The invention discloses a primer and probe composition for distinguishing various animal sources in colla corii asini. The composition is good in specificity and high in sensitivity. The invention further discloses a kit comprising a probe and a primer. The kit can distinguish donkey, horse, cow and pig source ingredients in the colla corii asini at the same time, and is high in accuracy, high in throughput and convenient to use. The invention further discloses a multiple real-time fluorescence quantification PCR detection method for distinguishing various animal sources in the colla corii asini. The method can qualitatively detect donkey, horse, cow and pig source ingredients in the colla corii asini at the same time, and is applicable to animal hide and skin detection, applicable to animal source detection on deep-processed colla corii asini as well, and is convenient and fast to implement.

Description

technical field [0001] The invention relates to a method for identifying the authenticity of donkey-hide gelatin, in particular to a primer-probe composition, a kit and a multiple real-time fluorescent quantitative PCR detection method for differentiating donkey-hide gelatin from donkey, horse, pig and bovine. The field of biological technology. Background technique [0002] Donkey-hide gelatin has a history of thousands of years in China. It is a jelly made from the dehaired skin of an equine animal donkey. It has a wide range of medical and health care values. , Enhance immunity and other five functional advantages. From the point of view of product characteristics, genuine donkey-hide gelatin is generally rectangular block, regular and flat, uniform in size and thickness. Its surface is tan or brown-black, smooth and shiny, with longitudinal grains, no pores and oil pores. The quality is hard and brittle, and it will be broken when shot, and the fragments are slightly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/16C12Q2537/143C12Q2545/114C12Q2561/113C12Q2561/101
Inventor 步迅张全芳刘艳艳范阳阳
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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