A kind of primer pair and application thereof for identifying long-distance dendrobium
A technology for primer pair and long identification is applied in the field of primer pair identification of long-distance dendrobium to achieve the effect of accurate identification
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Embodiment 1
[0049] Embodiment 1, the preparation and the use method of the kit for identifying the dendrobium long-distance
[0050] 1. Design and synthesis of primer pairs for identification of long-distance Dendrobium
[0051] 本实施例根据GenBank中长距石斛及其同属近缘种的psbA-trnH序列(GenBank登录号:KF177467,KF177469,KF177471,KF177472,EU672793,KF177480,KF177482,KF177483,JF693815,KF177486,KF177493,KF177494,KF177500 ,KF177501,FJ216470,KF177506,KF177507,KF177509,KF177511,KF177520,KF177521,KF177522,KF177526,KF177535,KF177540,KF177538,KF177554,EU887927),进行同源比对,找出长距石斛的稳定变异位点,根据 A pair of specific primers F1 and F2 are designed for the variable region, and the sequence is as follows:
[0052] F1: 5'-GGCTAACACTTTTGCTTCTCTATAT-3' (SEQ ID NO: 1);
[0053] F2: 5'-GACTTGGTCTCGGGCATCTA-3' (SEQ ID NO: 2).
[0054] 2. Assembly of kits for identification of Dendrobium longifolia
[0055] After the primers F1 and F2 designed and synthesized in step 1 are packaged separately, they are packaged in the same kit with separately ...
Embodiment 2
[0068] Embodiment 2, using the kit prepared in Example 1 to identify Dendrobium long-distance
[0069] Samples to be tested: 16 species of Dendrobium shown in Table 1.
[0070] 1. Extract genomic DNA from the sample to be tested
[0071]Select about 0.03 g of dry medicinal material without mildew, put it in a pulverizer, grind it, and pass it through a 40-mesh sieve. Transfer the powder to a 2.0 mL microcentrifuge tube, add 900 μL of sterilized CTAB extract (recipe: 2% (2g / 100ml) CTAB, 100mmol / L Tris-HCl pH=8.0, 20mmol / L EDTA, 1.4mol / L NaCl), 0.02g PVP 40000, 10μL β-mercaptoethanol, fully vortexed and mixed, 65 ℃ water bath for 1.5h-2h, during which, shake gently 2-3 times. After completion, take it out and cool to room temperature, add 900 μL of chloroform-isoamyl alcohol (volume ratio 24:1), shake and mix well, and centrifuge at 12000 g for 10 min. Take the supernatant, add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1), shake and mix well, and centrif...
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