Pills are spherical or quasi spherical solid preparations made of fine powder or extract of medicinal materials plus appropriate adhesives or excipients, and are one of the ancient dosage forms of traditional Chinese patent medicines and simple preparations. The DNA detection scheme of PCR is becoming the gold standard for the authenticity identification of traditional Chinese medicine, but the complex composition of processed Chinese medicine pills, including adhesives and excipients, has an inhibitory effect on the PCR reaction, resulting in ineffective use of DNA detection methods in this field. This kit achieves effective DNA extraction purity by using a self-developed DNA extraction method to effectively clean some PCR inhibitory components. The extracted DNA can be directly used for PCR reactions, providing a reliable detection scheme for the authenticity identification and adulteration detection of medicinal materials.
Cracking treatment:
Grind the pill into powder, add lysis buffer containing proteinase K (such as CTAB buffer), and water bath at 56 ℃ for 1-2 hours to break down the cell membrane and degrade the protein. If the sample contains polysaccharides, β - mercaptoethanol can be added to suppress interference.
Adsorption purification:
Magnetic bead method: Mix the lysis buffer with magnetic beads, adsorb DNA specifically, and remove impurities with a washing solution containing ion salts.
Centrifugal column method: The lysate is passed through a silica gel membrane column, and after DNA binding, the salt and pigment are washed with ethanol.
Elution and quality inspection:
Use TE buffer or sterile water to remove DNA, and detect the A260/A280 ratio using a UV spectrophotometer (1.8-2.0 is qualified). Observe the integrity of the bands through electrophoresis to avoid tailing or dispersion.
Notes:
Wear gloves throughout the operation to avoid DNA enzyme contamination.
When the polysaccharide content is high, the washing frequency can be increased or a low pH method can be used.
Old samples require an extension of lysis time.