Primers, probe composition and kit for rapid identification of nine animal origin ingredients in food or feed, detection method for identification of nine animal origin ingredients in food or feed and application of primers, probe composition, kit and detection method

A technology of animal-derived ingredients and compositions, applied in the field of molecular biology, can solve problems such as cross-reaction, deceiving consumers, non-specific amplification, etc., achieve low cost, reduce the risk of interference, and improve accuracy

Inactive Publication Date: 2016-01-27
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Description
  • Claims
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Problems solved by technology

After investigation, the main problem of meat adulteration in our country is: mixing pork, chicken, duck, horse meat, mouse meat, fox meat, mink meat, etc. into high-end and expensive meat such as mutton and beef, which are easy to breed and more expensive. Cheap meat, shoddy, cheating consumers
The current detection of animal-derived components based on multiplex PCR technology mainly relies on the use of agarose gel electrophoresis to distinguish amplified fragments of different lengths after PCR. Patents with app

Method used

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  • Primers, probe composition and kit for rapid identification of nine animal origin ingredients in food or feed, detection method for identification of nine animal origin ingredients in food or feed and application of primers, probe composition, kit and detection method
  • Primers, probe composition and kit for rapid identification of nine animal origin ingredients in food or feed, detection method for identification of nine animal origin ingredients in food or feed and application of primers, probe composition, kit and detection method
  • Primers, probe composition and kit for rapid identification of nine animal origin ingredients in food or feed, detection method for identification of nine animal origin ingredients in food or feed and application of primers, probe composition, kit and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] 1. Primer probe design: According to the mitochondrial genome sequence information of cattle, sheep, pigs, chickens, foxes, minks, horses, ducks and mice published in the Genebank database, the 16SrDNA gene on the mitochondria was selected according to relevant literature reports, and Primer5 was used to .0 software to design universal primers in the conserved regions of the 16S rDNA gene sequences of the above species, and use ABIPrimerExpress software to design corresponding specific molecular beacon probes in the variable regions. The designed probes must meet the principles of inter-species specificity and intra-species conservation. In the present invention, the 5' ends of the cattle, horses, goats, sheep, pigs, chickens, ducks, foxes, mink and mouse-specific probes and internal standard probes are all modified with reporter groups, and the reporter groups can be It is FAM, JOE, CY3, CY5, etc., and the 3' end is modified with a quenching group, and the quenching gro...

Embodiment 2

[0089] Using the kit of the present invention and the multiplex real-time fluorescent PCR detection method to detect multi-species animal-derived joint detection method steps in meat, meat processed food and feed raw material meat and bone meal are as follows:

[0090] 1. DNA extraction:

[0091] Depending on the sample type, pretreat in one of the following ways:

[0092] a) When the sample is a whole piece of meat, after mixing according to the sample preparation method, take an appropriate amount of meat, wash the sample with normal saline, fully grind it into powder in liquid nitrogen, weigh 0.03g and put it into a 2mL centrifuge tube Add 400 μL of DNA extraction cell lysate, 10 μL of proteinase K and mix well, put in a water bath at 56°C for 2 hours, and shake slightly from time to time. Add 10 μL RNase, and digest at 37°C for 1 hour.

[0093] b) When the sample is minced meat, meat slices or meat rolls and other frozen mixed meat or processed meat products, and feed ra...

Embodiment 3

[0125] Example 3 Kit Specificity Verification

[0126] The established multiplex PCR system was used for bovine (N), goat (SY), sheep (MY), fox (H), mink (Sd), duck (Ya), chicken (J), pig (Z), horse ( M) and the genomic DNA of 15 species including rat meat (S), donkey (L), rabbit (R), raccoon dog (HZ), dog (G), and fish (Yu) were detected with a Nanodrop UV spectrophotometer The detection concentration was uniformly diluted to 10ng level, and the specificity verification experiment was carried out. The amplification results showed that:

[0127] (1) Multiplex PCR system A (including sheep, fox and chicken mixed probes), only when the corresponding pair of sheep, fox and chicken DNA are used as templates, there is a corresponding amplification curve, other species except the amplification of quality control probes Outside the curve, there is no corresponding amplification, indicating that the PCR amplification system has good amplification specificity, see Figure 1 to Figure...

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Abstract

The invention belongs to the technical field of molecular biology and provides primers, a probe composition and a kit for rapid identification of nine animal origin ingredients in food or feed, a detection method for identification of the nine animal origin ingredients in the food or feed and application of the primers, the probe composition, the kit and the detection method. Origin ingredients of the food or feed containing multiple species can be identified rapidly by the primers, the probe composition and the real-time fluorescent PCR (polymerase chain reaction) joint detection kit. The detection method includes: designing a universal primer by taking 16SrDNA as a target gene, and designing specific probes of nine species by designing to construct internal amplification control and designing the specific probes aiming at internal control sequences; performing PCR amplification by three PCR reaction systems; interpreting the origin ingredients of the nine species directly through corresponding fluorescent probe signals and Ct values. The detection method is low in cost, time saving and high in efficiency and can achieve identification of multiple species simultaneously.

Description

technical field [0001] The invention relates to a primer, a probe composition, a kit, a detection method and an application thereof for rapidly identifying nine kinds of animal-derived components in food or feed, and belongs to the technical field of molecular biology. Background technique [0002] The horsemeat scandal in Europe in 2012 caused countries to attach great importance to the authenticity and adulteration of meat. Since 2013, incidents of adulteration of high-grade meat and low-quality meat in China have also occurred frequently. The media have successively reported Shandong Binzhou, Inner Mongolia , Shanghai, Xi'an, Shenzhen and other places investigated and dealt with fake mutton incidents. After investigation, the main problem of meat adulteration in our country is: mixing pork, chicken, duck, horse meat, mouse meat, fox meat, mink meat, etc. into high-end and expensive meat such as mutton and beef, which are easy to breed and more expensive. Cheap meat, shod...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
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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