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
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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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