Primers, probe composition, kit and multiplex fluorescence PCR detection method for detecting ass-derived component, horse-derived component and bovine-derived component in cosmetics

A cosmetic, bovine-derived technology, applied in the field of molecular biology, to achieve the effect of strong specificity, high sensitivity, accurate and reliable detection results

Inactive Publication Date: 2015-12-09
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on multiple fluorescent PCR detection of donkey, horse and bovine ingredients in cosmetics has not been reported yet. Therefore, the establishment of a multiplex fluorescent PCR detection method and detection kit for the rapid identification of donkey, horse and bovine ingredients in cosmetics is useful for cosmetics. Safe and rapid supervision has important innovative practical significance

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  • Primers, probe composition, kit and multiplex fluorescence PCR detection method for detecting ass-derived component, horse-derived component and bovine-derived component in cosmetics
  • Primers, probe composition, kit and multiplex fluorescence PCR detection method for detecting ass-derived component, horse-derived component and bovine-derived component in cosmetics
  • Primers, probe composition, kit and multiplex fluorescence PCR detection method for detecting ass-derived component, horse-derived component and bovine-derived component in cosmetics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Detection method

[0059] (1) Cosmetic DNA extraction: Take liquid (200μl), paste or solid (200mg) cosmetic samples into a 2ml centrifuge tube, add 5% Chelex-100280μl and 20mg / ml proteinase K 20μl, and keep at 56°C for more than 30 minutes . Shake at high speed for 5-10s, 8min at 100℃; shake at high speed for 5-10s, centrifuge at 13000r / min for 3min, transfer the supernatant to a new centrifuge tube (candidate step, transfer the solution to the spin column, centrifuge at 13000r / min for 1min, Collection liquid) Add 20μl of GlassMilk to the collection liquid, add 600μlg DNABindingBuffer (6MNacl), mix well, water bath at 65° for 15min, invert several times in the middle; leave at room temperature for 5min, invert several times in the middle; centrifuge at 4000rpm for 1min, discard the supernatant , Keep the bottom of the tube; 70% (volume fraction) alcohol 500μl washing 8000r / min centrifugation 1min, repeat this step twice; add 500μl absolute ethanol, pipetting sus...

Embodiment 2

[0066] Example 2 Specificity test

[0067] Using genomic DNA extracted from horse meat, beef, donkey meat, sheep meat, goat meat, pork, chicken, duck, goose, fish, and corn as templates, perform real-time fluorescent PCR according to the above optimized reaction system and reaction conditions Detection test to verify the specificity of the designed primers and probes. The results showed that the genomic DNA of horse meat, beef and donkey meat all have CT values, and the Ct values <35, no signal amplification in other samples. The results are shown in Table 2. It can be seen that the primers and probes in this kit have strong species specificity.

[0068] Table 2. Species specific test results of primers and probes

[0069]

[0070] Note: N stands for Negative, that is, negative result.

Embodiment 3

[0071] Example 3. Sensitivity test

[0072] Use animal tissue extraction kit to extract genomic DNA of donkey, horse and cow, quantify to 5ng / μl with UV spectrophotometer, mix according to the following ratio, and mix the mixture according to 10 times, 100 times, 1000 times, and 10000 times concentration gradients respectively Dilute. The DNA of donkeys, horses and cows were tested according to the above methods to investigate the sensitivity of the kit. The analysis results show that the detection limit of this kit is 0.01ng. The results are shown in Figures 5-7.

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Abstract

The invention discloses primers and a probe composition for detecting an ass-derived component, a horse-derived component and a bovine-derived component in cosmetics, as well as a kit comprising the primers and the probe composition, wherein the primers comprise a pair of universal primers, the probe composition comprises four Taqman probes, and the nucleotide sequences of the primers and the probe composition are shown as SEQ ID NO.1-7. The invention further discloses a multiplex fluorescence PCR detection method for detecting the ass-derived component, the horse-derived component and the bovine-derived component in the cosmetics. The multiplex fluorescence PCR detection method comprises the following steps: taking a to-be-tested sample, extracting the DNA, carrying out PCR amplification on the DNA by utilizing the primers, the probe composition or the kit, collecting fluorescence signals, and determining that whether the to-be-tested sample contains the ass-derived component, the horse-derived component and the bovine-derived component or not according to the fluorescence signals. With the adoption of the detection technology, the false negative result during the PCR reaction can be effectively avoided, and the detection technology has the advantages of being accurate, stable, easy to operate, extremely high in sensitivity, strong in specificity, and the like.

Description

Technical field [0001] The invention relates to a primer, a probe composition, a kit and a multiple fluorescent PCR detection method for identifying donkey, horse and bovine origin components in cosmetics, and belongs to the technical field of molecular biology. Background technique [0002] In recent years, consumers at home and abroad have put forward higher requirements for the quality and effectiveness of cosmetic products. Especially the traditional skin care, hair care and cleansing cosmetics, in terms of performance, can no longer meet the requirements of consumption rules. The production of cosmetics is moving in the direction of nutrition and curative effect. In particular, research on the use of animal ingredients and their extracts as special ingredients in cosmetics is quite active, and their uses are also expanding. Among them, donkey milk and horse milk are ideal raw materials for cosmetics. The nutritional value of donkey milk and horse milk is the consensus of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2600/16C12Q2537/143C12Q2561/101
Inventor 步迅张全芳刘艳艳范阳阳张卉
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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