Quick detection method and kit for chicken source component in food

A technology of chicken-derived components and detection reagents, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of inability to accurately quantify samples, high mitochondrial gene homology, and difficult detection and other problems, to achieve the effect of easy observation, good specificity and easy quantification

Inactive Publication Date: 2019-06-07
CHINA AGRI UNIV
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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 method and kit for chicken source component in food
  • Quick detection method and kit for chicken source component in food
  • Quick detection method and kit for chicken source component in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Screening of chicken source general internal standard gene LOC107053213

[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 LOC107053213 gene was screened out, which was located on the fourth chromosome. 20 kinds of meat (common domestic 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 (Pseudosciaena polyactis), horse (Equus caballus), donkey (Equus asinus ), mouse (Mus musculus), buffalo (Bubalus bubalis), mink (Martes zibellina),...

Embodiment 2

[0041] Example 2 Establishment of LAMP Detection Method for Chicken Source Components

[0042] Utilize LAMP primer designingsoftware primerexplorer V 5.0 (http: / / primerexplorer.jp / elamp5.0.0 / index.html) LAMP primer online design software LAMP primer designingsoftware primerexplorer of Japan Rongyan Co., Ltd. to design LAMP primers for the LOC107053213 gene determined in Example 1, including 2 See Table 1 for outer primers F3 and B3 and 2 inner primers FIP and BIP. The 5' end of the inner primer FIP was labeled with biotin (Biotin), and the 5' end of BIP was labeled with fluorescein (FITC).

[0043] Table 1 LAMP primer sequence

[0044]

[0045] Using LAMP to quickly detect chicken samples, the reaction system is 25 μL, including 1× 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 5min at 85...

Embodiment 3

[0046] Example 3 Establishment of chicken source component LAMP product colloidal gold nucleic acid test strip detection method

[0047] Preparation of colloidal gold-labeled antibody The colloidal gold was prepared by the improved method of trisodium citrate, and the gold-labeled antibody was purified by high-speed centrifugation, and the prepared gold-labeled antibody was stored at 4°C for later use.

[0048] Dilute the FITC antibody with buffer to the optimal concentration respectively. The distance between the test line (TL) and the quality control line (CL) is 4.5 mm, and sprayed on the NC membrane at 1.0 μL / cm respectively. The sprayed NC membrane was dried overnight at 37°C for later use. Test strips were cut to a width of 3.8mm.

[0049] Fully mix the LAMP reaction product with the buffer solution and drop it on the sample pad of the colloidal gold nucleic acid test strip. At this time, the mixed solution passes through the binding pad and the NC membrane under the c...

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Abstract

The present invention provides a quick detection method and a kit for a chicken source component in food and relates to the technical field of biological species identification. Firstly, a chicken source common internal standard gene LOC107053213 is screened, and the gene has a nucleotide sequence shown as SEQ ID NO.1, is located on chromosome 4, has a constant copy number in chicken species, is free of allelic gene variation, and can be used as a target gene for identifying the chicken source. LAMP amplification primers are designed with the gene as the target sequence and subjected to constant temperature amplification with a LAMP reaction solution, and a LAMP reaction product is used for colloidal gold nucleic acid test strip detection. An LAMP reaction is combined with the colloidal gold nucleic acid test strip detection to constitute the rapid detection kit, the kit can quickly and sensitively detect the chicken source component in the food, and detection sensitivity can reach 0.16% (w / w). The kit is simple in use method, low in cost, easy for observation of reaction results, good in specificity, and very suitable for real-time detection on site.

Description

technical field [0001] The invention relates to the technical field of identification of biological species, in particular to a rapid detection kit for detection of chicken-derived components in food combined with loop-mediated isothermal amplification technology (LAMP) combined with colloidal gold nucleic acid test strip detection. Background technique [0002] With the rapid development of the economy and the improvement of people's living standards, the demand of Chinese residents for meat products is increasing year by year. Although many countries clearly require food labels to clearly indicate the type and source of meat and prohibit adulteration, there are still many incidents of meat adulteration in the market. For example, in order to reduce costs, donkey meat is mixed with pork , mutton mixed with beef or pork, chicken mixed with other meat and so on. At present, the detection method for food adulteration is mainly PCR method. The PCR method needs to rely on spec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12Q1/6804C12N15/11
Inventor 罗云波许文涛黄昆仑张超杜再慧马玉婷
Owner CHINA AGRI UNIV
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