Characteristic sequences and method for discriminating pivot-end cordyceps sinensis
A nucleotide sequence and nucleotide technology, applied in the field of detection of authenticity of traditional Chinese medicinal materials by molecular biology methods, can solve problems such as no patents, and achieve the effect of less sample amount and simple method
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Embodiment 1
[0018] Preparation of Example 1 Cordyceps pointed rRNA internal transcriptional spacer and 5.8S rRNA gene
[0019]Take 0.1-0.5g sample of Cordyceps pointed, grind into powder in liquid nitrogen, add 300μl urea extract (SDS 1.5%, urea 7mol / L, Tris-HClpH8.0 0.05mol / L, NaCl 0.5mol / L), transfer into In a 1.5ml microcentrifuge tube, cool in liquid nitrogen and quickly transfer to 65°C to melt. After repeated freezing and thawing for 3 to 5 times, add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), oscillate, centrifuge at 13000r / min for 10min, transfer the supernatant to a new tube and add an equal volume of chloroform:isoamyl alcohol (24:1), shake, centrifuge at 13000r / min for 10min, transfer the supernatant to a new tube, add a small amount of RNase A, incubate at 37°C for 30min, add 3 times the volume of absolute ethanol, 3M NaAc (pH5.2), and store at -20°C After at least 30min, centrifuge at 13000rpm for 15min, recover the DNA precipitate, wash with 70% ethanol...
Embodiment 2
[0020] The preparation of embodiment 2 Cordyceps pointed specificity probe (primer) Probe I and Probe II
[0021] Compare the sequence of the ITS region of Cordyceps pointed with the homologous sequences of other Cordyceps to obtain the composition and sequence of the oligonucleotide fragments that are most suitable for identifying Cordyceps pointed, that is, Probe I and Probe II (such as figure 2 The Probe I position shown is 13bp-32bp, which is located on the positive strand, and the Probe II position is 359bp-340bp, which is located on the negative strand). According to the nucleotide arrangement and composition of Probe I and Probe II, the sequences listed in Sequence Listing 2 and Sequence Listing 3 can be synthesized by chemical synthesis on a commercial DNA synthesizer, and can be used as specific PCR primers. On this basis, Probe I and Probe II can be labeled with enzymes, isotopes, biotin, chemical fluoresceins, fluorescent agents, etc. by conventional molecular biol...
Embodiment 3
[0022] The positive control test of embodiment 3 identification Cordyceps pointed
[0023] This test is used to determine that DNA samples meet the requirements of PCR to obtain specific PCR products.
[0024] Method 1: Total DNA extraction and PCR reaction were carried out with reference to Example 1. DNA was extracted from different samples, and PCR amplification was carried out under the same conditions, including blank experiments without adding templates, and the amplified products were within 2% Electrophoresis was performed on an agarose gel containing DNA dye, viewed under an appropriate light source, and photographed.
[0025] Method 2: Use a toothpick to directly pick an appropriate amount of samples (mycelia, crushed fruiting bodies, crushed related products) and stir in a 0.2ml PCR tube filled with 20ul ultrapure water, and dilute to 0.1 times and 0.01 times respectively, and take 2ul of the above Add three concentrations of sample suspensions containing 20ul PCR ...
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