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A combination of microsatellite primers and its detection kit for traceability and identification of beef products

A technology of primer sets and reagents, applied in the field of traceability detection, can solve problems such as infringement of consumer rights, risks to consumer health, disruption of market order, etc.

Active Publication Date: 2019-03-01
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In recent years, driven by economic interests, low-value products have often been used to pass off high-value products in the market, such as horse meat as beef, and eliminated dairy beef as yellow beef, which not only disrupted market order, but also violated consumer interests, and may also pose risks to consumers’ health due to issues such as allergies
At the same time, in view of the problems found, the current traceability of beef is mainly based on the electronic label traceability system, but due to the frequent occurrence of label counterfeiting in the circulation process, it is impossible to achieve true traceability

Method used

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  • A combination of microsatellite primers and its detection kit for traceability and identification of beef products
  • A combination of microsatellite primers and its detection kit for traceability and identification of beef products
  • A combination of microsatellite primers and its detection kit for traceability and identification of beef products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1, be used for identifying the primer pair of beef breed and kit thereof

[0092] 1. Design and synthesis of primer pairs for identification of beef varieties

[0093] The following 16 bovine microsatellite primer pairs were designed and synthesized, and the sequences and primer names are shown in Table 1:

[0094] Table 1 is 16 bovine microsatellite primer pairs

[0095]

[0096]

[0097] 2. Compound primer set and reaction system for identifying beef varieties

[0098] In order to improve the efficiency of PCR amplification, after repeated tests, 16 pairs of primers were rationally grouped to obtain 4 composite primer sets (Table 2), and the best PCR composition system and amplification conditions were obtained by screening (Table 3), and 16 primer sets were established. Optimal multiplex amplification method for labeled loci. All target fragments can be obtained by making 16 marker loci only through 4 PCR reactions.

[0099] Table 2 is the compos...

Embodiment 2、16

[0116] Embodiment 2, 16 primer pairs identify beef individual

[0117] 1. Genomic DNA extraction

[0118] Collected 15 meat samples of Japanese Wagyu (H), 16 meat samples of Jiaxian Red Cattle (J), 15 meat samples of Nanyang Yellow Cattle (N), 23 meat samples of Luxi Yellow Cattle (LX), Limousin Cattle (LM) There are 15 meat samples and 16 meat samples of Anduo Yak (A), a total of 100 beef samples.

[0119] Genomic DNA was extracted from 100 beef samples respectively.

[0120] 2. Multiplex PCR amplification

[0121] Using the genomic DNA of 100 beef samples as templates, the four reaction systems in Table 3 in Example 1 were used for PCR amplification. The amplification procedures are shown in Table 4.

[0122] 3. On-board inspection

[0123] The 4 sets of PCR amplification products of all beef samples were detected by capillary electrophoresis on the machine, and detected by 3730XL sequencer. Add 9 μl of molecular weight internal standard and formamide mixture (0.5:8.5) ...

Embodiment 4、1

[0144] Example 4, the application of 16 primer pairs in the traceability of beef products

[0145] 1. Genomic DNA extraction

[0146] Select a beef cattle farm, take blood samples from 3 cattle and freeze them, record them as NX 1 、NX 2 、NX 3 . At the same time, the whole process of the three cows from the farm to the slaughterhouse and finally to the farmer's market or supermarket was manually tracked and recorded. Finally, the meat samples of these three cows were collected from the farmer’s market or supermarket, and they were recorded as NR 1 、NR 2 、NR 3 .

[0147] Extract NX 1 、NX 2 、NX 3 、NR 1 、NR 2 、NR 3 of genomic DNA.

[0148] 2. Multiplex PCR amplification

[0149] Same as 2 of embodiment 2;

[0150] 3. On-board inspection

[0151] Same as 3 of embodiment 2;

[0152] 4. Result analysis

[0153] The results are shown in Table 6. By comparison, it was found that the resulting spectra of the meat samples obtained from the market and the blood samples ...

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Abstract

The invention discloses a short-tandem-repeats (STR) primer combination for source-traced identification on a beef product and a detection kit. The invention provides a primer kit for identifying beef. The primer kit is (1) or (2), wherein the (1) consists of primer pairs BM1818, ILSTS005, INRA032, INRA035, BM1824, HEL5, HEL13, ILSTS006, CSRM60, INRA005, INRA037, INRA063, ETH10, ETH225, NRA023 and TGLA1225; the (2) consists of a primer group A, a primer group B, a primer group C and a primer group D; the primer group A consists of primer pairs BM1818, ILSTS005, INRA032 and INRA035. According to the method provided by the invention, the selected 16 STR markers have the advantages that the polymorphism is high, the markers are not in linkage, amplified fragments have polymorphism, and genotyping is facilitated; primer sequences of different marker bits are grouped and are modified by using different fluorescent dyes, so that grouped multiplex PCR (Polymerase Chain Reaction) detection can be achieved, the cost is reduced, the time is shortened, and the efficiency is increased.

Description

technical field [0001] The invention relates to the field of traceability detection, in particular to a combination of microsatellite primers for traceability and identification of beef products and a detection kit thereof. Background technique [0002] In recent years, driven by economic interests, low-value products have often been used to pass off high-value products in the market, such as horse meat as beef, and eliminated dairy beef as yellow beef, which not only disrupted market order, but also violated consumer rights and interests, and may also cause risks to consumers' health due to problems such as allergies. At the same time, the current traceability of the beef found is mainly based on the electronic label traceability system. However, due to the fact that many circulation links are prone to label counterfeiting, true traceability cannot be achieved. Therefore need to develop a kind of effective method to carry out true and false identification and traceability ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/16
Inventor 陈爱亮杨曙明赵杰朱超蒋小玲
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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