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Identification of pig-derived ingredients and detection kits for multi-species origin ingredients in its products

A detection kit and the technology of the kit, which are applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of high requirements, high cost, time-consuming and labor-intensive, etc. Broad and specific effect

Active Publication Date: 2018-08-03
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fluorescence detection method has high requirements for equipment and is expensive; the enzyme digestion method is time-consuming and laborious; and the multiplex PCR is used to amplify the size of the fragment to identify the animal species, which severely limits the number of detectable species, and the primers often interfere with each other. Or form a dimer, affecting the reliability of the test results

Method used

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  • Identification of pig-derived ingredients and detection kits for multi-species origin ingredients in its products
  • Identification of pig-derived ingredients and detection kits for multi-species origin ingredients in its products
  • Identification of pig-derived ingredients and detection kits for multi-species origin ingredients in its products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Specific detection of pig-derived components in samples

[0063] 1. Sample preparation and storage

[0064] 1.1 Sampling

[0065] A 1 g sample of pork was collected and stored at -20°C for future use.

[0066] 1.2 DNA template preparation

[0067] The DNA template was prepared using the commonly used phenol-chloroform crude extraction method (Sambrook J, Fritsch E F, Mani Artis T. Molecular Cloning Experiment Guide [M]. 2nd Edition. Jin Dongyan, Li Mengfeng. Beijing: Science Publishing Society, 1999.465-467) or recognized other extraction methods with the same efficacy, these methods are the commonly used methods reported.

[0068] 2. Primer Design

[0069] The DNA sequences of the primers in this example are shown in Table 2 and SEQ ID NO: 2 and 3 in the sequence table.

[0070] Table 2 The pig PCR amplification primers designed by the present invention

[0071] Primer name

Primer sequence

SUS-F

5′-GCAATGCTCCAAGGACTTAGTGA-3′

...

Embodiment 2

[0094] Embodiment 2 Sensitivity test

[0095] Using 0.1ng / μL, 1ng / μL, 10ng / μL, and 100ng / μL of porcine DNA as templates, the nucleotide-specific fragment of SEQ ID NO.1 was amplified according to the conditions of Example 1. The result is as figure 2 As shown, there is amplification at a template concentration of 1ng / μL, indicating that the fragment amplified by the specific primer has good sensitivity.

Embodiment 3

[0096] Example 3 A primer set for detecting non-pig-origin ingredients mixed in porcine-origin products

[0097] The present invention is for the detection of animal-derived components. Through the PCR amplification of the corresponding primers of each species, it is judged whether it is or contains the DNA of a certain species, and then it is judged whether the animal-derived components of a certain species are mixed.

[0098] 1 Extraction of DNA from samples

[0099] The extraction and storage methods of the DNA of each species are the same as those described in Example 1 above.

[0100] 2 Primer design and screening

[0101] In order to find the specific detection sequence of each species, we screened the specific sequence from the aspects of intra-species consistency and stability, inter-species specificity, copy number, etc. Yes, the nucleotide sequence that is more conserved within a species and specific between species is screened as a detection molecule. The sequenc...

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Abstract

Belonging to the technical field of animal origin component molecular detection, the invention provides a detection kit for pig origin component identification and detection of multi-species origin components in products. The kit includes primers shown as SEQ ID No.2&3, a reaction reagent, positive control and blank control. In addition, the kit also includes specific primers of fox, mink, rat, chicken, duck and equus, and the nucleotide sequences thereof are shown as SEQ ID No.4-25. The primers involved in the invention are employed for amplification of the sample DNA and analysis of the PCR amplification product to determine whether the sample contains pig origin components and is mixed with equus, fox, mink, rat, chicken, duck and other animal components. The kit provided by the invention provides an effective, accurate and reliable means for animal origin component molecular identification and detection of adulterated components therein, and has the characteristics of simplicity, fastness, comprehensiveness, strong specificity, low cost, and wide applicability.

Description

technical field [0001] The invention relates to the field of animal molecular biology, in particular to the identification of pig-derived ingredients and the application of a detection kit for multi-species origin ingredients in products thereof. Background technique [0002] my country is the country with the largest pig raising scale in the world. The pig farming industry occupies a dominant position in my country's animal husbandry. Pork accounts for the vast majority of the total meat output, and it is also an important animal food on our table. The nutrition of pork is very comprehensive, the nutritional value is high, the pork fiber is relatively soft, the connective tissue is less, and the muscle tissue contains more intermuscular fat; its protein is complete protein, contains various amino acids necessary for the human body, and its composition ratio is close to that of the human body It is easy to be fully utilized by the human body and belongs to high-quality prote...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888
Inventor 刘榜张静王文君甄月然付明向胜男刘建建吴清清张庆德彭中镇
Owner HUAZHONG AGRI UNIV
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