Reagent box for simultaneously detecting diversified components of animal origin and application of reagent box
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A technology of animal-derived ingredients and kits, which is applied in the direction of microbial determination/testing, biochemical equipment and methods, and can solve problems such as limitations in the scope of application
Active Publication Date: 2015-09-23
AGCU SCIENTECH
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[0006] The detection systems provided by patents 201310082539.1 and 201310368498.2 can only
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[0053] Example 1 Screening of primers
[0054] Download the primer design template sequence from NCBI genebank, as shown in Table 4.
[0057] The sequence of each locus is aligned in the genebank, and primers are designed at both ends of the STR sequence, and the unique primers with better specificity are selected after blast alignment. After a large number of repeated tests, the unique amplification of each species DNA template in the multiplex system is amplified. To increase peaks, when designing primers, it is necessary to repeatedly debug in the primer design software to ensure that all primers have a moderate length, have similar physical characteristics and reaction kinetics, similar Tm values, ...
Example Embodiment
[0074] Embodiment 2. Actual sample test
[0075] Buy 12 kinds of meat on the market by yourself, use QIAamp ? DNA Blood Mini Kit extracts 12 kinds of meat DNA.
[0076] Detect the concentration of 12 extracted DNA with a spectrophotometer, and dilute to 0.5ng / μL as a template.
[0077] Add samples according to the sampling system in Table 6, and perform amplification according to the amplification procedure in Table 3.
[0078] Table 6 PCR sampling system
[0079] Component volume Reaction Mix 10.0 μL DNA template 2μl Primer mix 5 μL Hot start Taq enzyme (5U / μL) 0.5 μL sdH 2 O Make up to 25.0μL
[0080] The Reaction Mix is MgCl 2 7.5mM, Tris-HCl buffer 125mM, KCl 125mM, dNTPs 7.5mM, BSA 2mg / ml.
[0081] The amplification results were analyzed according to the electrophoresis detection and data analysis methods in Example 1, and 3 tubes of amplification were repeated for each template.
[0082] Attached Figure 1~12 It is the amplified map of 12 kinds of animal-derived compone...
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Abstract
The invention discloses a reagent box for simultaneously detecting diversified components of animal origin and application of the reagent box. Fluorescent primer multiplex amplification and capillary electrophoresis methods are combined with one another, so that the 12 components of animal origin can be simultaneously detected by the aid of the reagent box, and a plurality of groups of specific primers can be designed by the aid of Escherichia coli and selected STR (short tandem repeat) sequences of dogs, cattle, sheep, fish, pigs, ducks, chickens, rabbits, cats, horses and rats. The specific primers include specific primers PEZ1 of the dogs, specific primers BM2113 of the cattle, specific primers MAF33 of the sheep, specific primers HLJ392 of the fish, specific primers SW742 of the pigs, specific primers CAUD056 of the ducks, specific primers MCW248 of the chickens, specific primers Sat7 of the rabbits, specific primers FCA955 of the cats, specific primers HMS3 of the horses, specific primers cds of the rats and specific primers CFS073 of the Escherichia coli. The reagent box and the application have the advantages that the reagent box is good in specificity and high in sensitivity, and origin of meat can be quickly determined; multiplex amplification systems contain the primers of the Escherichia coli, and accordingly whether meet foods and products are contaminated by Escherichia coli or not can be simultaneously determined.
Description
technical field [0001] The invention relates to the field of animal molecular biology, in particular to a kit for simultaneously detecting multiple animal-derived components and an application thereof. technical background [0002] With the continuous development of the market economy, in order to seek illegitimate benefits, some unscrupulous traders have mixed some cheap meat species into blood products and meat products, and even have the behavior of selling dog meat as sheep's tricks. Some surveys and studies have shown that this kind of adulteration has been widespread in the market. Most meat products are mixed with relatively cheap meat types, or even all of them are cheap meat types, which greatly damages consumers. interest, disturbed the market order, and endangered the health of citizens. [0003] In addition, animal-derived infectious diseases such as foot-and-mouth disease, mad cow disease, avian influenza, and sheep scrapie occur frequently, and the identificat...
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