Quick detection kit for chicken-origin ingredient in food and feed and application of quick detection kit

A detection kit, a technology for chicken-derived components, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc. problem, to achieve the effect of easy results, good specificity and low mutation rate

Inactive Publication Date: 2017-02-22
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the adulteration detection technology of meat products at home and abroad is mostly based on the design of specific primers for the genes on the mitochondria to perform real-time fluorescent quantitative PCR amplification. Since the mitochondrial genes are multi-copy genes, their detection sensitivity is high, but at the same time it is difficult to distinguish due to sales, transportation Confusion between unintentional cross-contamination and intentional illegal additions
In addition, the concentration of high-copy mitochondrial DNA cannot correspond to the concentration of genomic DNA, so it is impossible to perform accurate quantitative analysis on samples. At the same time, due to the high homology of mitochondrial genes, it is difficult to achieve qualitative detection by ordinary PCR
Therefore, this method can only realize the screening and identification of meat adulteration by means of fluorescent quantitative PCR

Method used

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  • Quick detection kit for chicken-origin ingredient in food and feed and application of quick detection kit
  • Quick detection kit for chicken-origin ingredient in food and feed and application of quick detection kit
  • Quick detection kit for chicken-origin ingredient in food and feed and application of quick detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The screening of embodiment 1 chicken source universal internal standard gene Actb

[0040] By searching the genetic information about chicken in GenBank, the target genome was downloaded from NCBI, and saved as ".FASTA" format. The whole genome information of the chicken was analyzed, and the homology analysis was carried out using BLAST and DNAMAN Version 4.0 software, and the Actb gene was screened out, which is located on the chromosome and is one of the cytoskeleton actin. 20 kinds of meat (respectively common chicken (Gallus gallus), pheasant (Phasianus colchicus), turkey (Meleagris gallopavo), black-bone chicken (Gallus domesticus brisson), pig (Sus scrofa), beef (Bos taurus), sheep (Ovis aries ), duck (Anas platyrhynchos), goose (Goose calicivirus), dog (Canis lupus familiaris), rabbit (Oryctolagus cuniculus), yak (Bos mutus), yellow croaker (Pseudosciaenapolyactis), horse (Equus caballus), donkey (Equus asinus), mouse (Mus musculus), buffalo (Bubalus bubalis),...

Embodiment 2

[0041] The verification of embodiment 2 chicken source universal internal standard gene

[0042] 1. For the internal gene Actb screened in Example 1, PCR and quantitative PCR primers and probes were designed for Actb gene by Primer Express 3.0 design software (American Life Technologies).

[0043] Table 1 Primer and probe sequences used in qualitative PCR, quantitative PCR and digital PCR

[0044]

[0045]

[0046] Note: FAM, TAMRA, VIC and BHQ1 are 4 kinds of fluorophores

[0047] 2. Copy number determination

[0048] Digital PCR and southern hybridization were used to identify the copy number. Combined with the data of the Ensembl gene information database, the copy number of the Actb gene was determined to be a single copy, and the accuracy of digital PCR in verifying the copy number was confirmed, which can be extended to other species.

[0049] An ideal internal standard gene should have a constant copy number and no allelic variation in the corresponding species...

Embodiment 3

[0073] Example 3 Establishment of LAMP Detection Method for Chicken Source Components

[0074] Using LAMP primer designingsoftware primerexplorer V 5.0 (http: / / primerexplorer.jp / elamp5.0.0 / index.html), the loop-mediated primer online design software of Japan Rongyan Co., Ltd., LAMP primers were designed for the Actb gene determined in Example 1.

[0075] Table 6 LAMP primer sequence

[0076]

[0077] Use LAMP to quickly detect chicken samples, the reaction system is 25 μL, including 1x Thermopol buffer, 0.4mMdNTP, 3mM MgSO 4 , 1.0M betaine, 1.6 μM primer FIP, 1.6 μM primer BIP, 0.2 μM primer F3, 0.2 μM primer B3, 8U Bst DNA polymerase large fragment. The reaction program was constant temperature at 65°C for 1h and 85°C for 5min. After the amplification, 2% agarose gel electrophoresis was used to determine the product, and the appearance of ladder-like bands proved that the amplification was successful and contained the target gene. The result is as Figure 9 As shown, o...

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Abstract

The invention provides a quick detection kit for a chicken-origin ingredient in food and feed and relates to the technical field of biological species identification. A general chicken-origin internal standard gene Actb is screened first; a nucleotide sequence of the gene Actb is as shown as SEQ ID NO.1 (Sequence Identifier Number 1); the gene Actb is located on a chromosome; single copy is easy to quantify; and the gene Actb has a constant copy number in chicken species, without allelic variation and can serve as a target gene for chicken-origin identification. LAMP amplification primers are designed by taking the gene as a target sequence; nucleotide sequences of the LAMP amplification primers are as shown as SEQ ID NO. 2-5; the LAMP amplification primers and an LAMP reaction solution constitute the detection kit; the detection kit can detect the chicken-origin ingredient in the food and the feed quickly and sensitively, and the sensitivity reaches 10pg. The kit is simple in use method, low in cost, high in specificity and very applicable to field real-time detection and has a wide application prospect in animal-origin food surveillance and meat product adulteration defense, and a reaction result is easy to observe.

Description

technical field [0001] The invention relates to the technical field of biological species identification, in particular to a loop-mediated isothermal amplification technique (LAMP) kit for detecting chicken source components in food and feed. Background technique [0002] With the development of social economy and product technology processing, many foods are illegally added with various chemical raw materials, additives, hormones, etc., and even many merchants carry out food adulteration behaviors or directly substitute inferior ones for high profits, which seriously endangers the The interests and health of consumers, so the accurate identification of food types is very important. [0003] No matter at home or abroad, chicken is the main meat for daily consumption, and its importance is self-evident. In Europe, chicken occupies the main market, supermarkets supply 60% of processed chicken products, and turkey is widely consumed. In China, chicken is the traditional poult...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2600/166C12Q2545/101C12Q2531/119
Inventor 许文涛黄昆仑徐瑗聪向文谨商颖罗云波
Owner CHINA AGRI UNIV
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