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Dual PCR (Polymerase Chain Reaction) detection method for soybean and peanut components in walnut milk beverage
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A detection method, walnut milk technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems that have not been reported, and achieve the effects of saving time, less interference, and avoiding complicated processing processes
Inactive Publication Date: 2014-01-29
KUNMING UNIV OF SCI & TECH
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[0003] At present, the identification of walnut protein components in walnut-containing foods in China is mainly based on antibody-antigen specific recognition based on enzyme-linked immunosorbent assay (ELISA). No report
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Embodiment 1
[0069] 1. Primer design
[0070] Through literature review and BLAST comparison, referring to NCBI sequence numbers EU493455, EU493457, EU493458, EU493459, EU493460, EU493461, Primer Premier 5.0 and Oligo 6.0 software were used to design primers, and the designed soybean-specific primers were synthesized by BGI Biotechnology Co., Ltd. , the primer sequences are as follows:
[0071] Bd 28K-F: 5'-GCAGCATTGACCCCTCTACAAGC-3',
[0072] Bd 28K-R: 5'-TTCGAATCCAGAAAGCACCGAGT-3';
[0073] Through literature review and BLAST comparison, the reference NCBI sequence numbers are AF43223, AB440237, AY581852, and HM640249, and Primer Premier 5.0 and Oligo6 software were used to design primers; the designed peanut-specific primers were synthesized by Huada GeneBiotechnology Co., Ltd., the primer sequence as follows:
[0112] Sample: 22 different brands of walnut milk drinks marked with walnut protein ingredients are available in the market, and the production areas are distributed in Yunnan Province, Hebei Province, Shanxi Province, Inner Mongolia and other places.
[0114] Freeze-dry different walnut milk samples to be tested, weigh 100mg of the sample powder and transfer them to 1.5ml EP tubes, and use the spin-column plantgenomic DNA extraction kit from Tiangen Company to complete the extraction of DNA from the samples. The specific operation steps are the same as in Example 1 step2.
[0115] 2. PCR amplification
[0116] (1) Amplify the extracted DNA samples with plant universal primers and corresponding PCR reaction components and procedures. When the product bands are all 123bp in size, the DNA extraction is successful and meets the requirements of the PCR reaction.
[0117] The composition ratio of each component in the PCR reaction system is as follo...
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Abstract
The invention discloses a method for simultaneously detecting soybean and peanut components doped in walnut milk by using a dual PCR (PolymeraseChain Reaction) technology. According to the method, a specific primer is designed aiming at the soybean and peanut components so as to establish a dual PCR detection method for main doped components, namely the soybean and peanut components, in the walnut milk. The dual PCR detection method comprises the specific primer aiming at the soybean and peanut components and a PCR reaction condition matched with the specific primer; the primer designed by the method has no crossed reactions with other common leguminous plants and non-leguminous plants including peanuts, filberts, walnuts, pine nuts, pistachio nuts, carya illinoensis koch, cashew nuts, almonds, sesames, apples and the like, and has the good specificity; the method disclosed by the invention can be used for simultaneously detecting the soybean and peanut components which are mainly doped in the walnut milk; the result is obtained rapidly, accurate and reliable; the method has an actual application value for detecting that soybean and peanut proteins in the market are used for replacing walnut proteins. Meanwhile, the method has an important reference value for rapid and specific detection when other low-price plant proteins are used for replacing the walnut proteins and relative milk products.
Description
technical field [0001] The invention belongs to the technical field of food quality and safety monitoring, and in particular relates to a simultaneous rapid detection method for soybean and peanut ingredients mixed in walnut milk drinks by using a double PCR method. Background technique [0002] With the improvement of people's living standards, the demand for health and nutrition food is increasing; with the change of people's consumption awareness, the consumption awareness of pursuing "nutrition, safety and health" is also stronger. Walnut is a nutritious foodraw material, does not contain cholesterol, is rich in riboflavin, lecithin, trace elements, vitamins, amino acids and a large amount of unsaturated fatty acids, which can prevent premature aging of the body, promote the development of brain cells, and reduce the synthesis of cholesterol , prevent arteriosclerosis, is an ideal nutritional health food. Walnut protein contains a variety of amino acids that are benefi...
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