The Study Of Selecting Mitochondrial Gene Specific Fragments And PCR Identification For Ophiocordyceps Sinensis

Ophiocordyceps was the dry complex of stroma of Ophiocordyceps sinensis(Berk.)Sacc.and the body of hepialidae larva.It had the effect of reinforcing kidney nourishing lung,hemostasis and resolve phlegm,which was a very valuable traditional Chinese medicine.Because of ITS remarkable curative effect,high price and scarce resources,Ophiocordyceps in the market was adulterated.Therefore,the identification of authenticity of Ophiocordyceps was the key to ensure the safety of clinical drug use.In this study,the whole mitochondrial gene of Ophiocordyceps sinensis was chosen as the research object,and the specific fragments of Ophiocordyceps sinensis and other species could be identified in mitochondrial gene.Finally,two specific genes,cob and cox1,were selected from the mitochondrial genome of Ophiocordyceps sinensis,which laid a foundation for further selecting of ideal molecular markers for identification and phylogenetic analysis of Ophiocordyceps sinensis.A rapid SS-PCR identification method for Ophiocordyceps was established based on mitochondrial cob and cox1 sequences,and the accuracy of this method was further investigated.Finally,the adaptability of the filter paper method to the extraction of DNA from Chinese herbal medicine was researched,which provided a theoretical basis for the quick detection of Ophiocordyceps and Chinese medicinal materials in the field.In this study,the cob and cox1 sequences of Ophiocordyceps sinensis were used as targets.The primers 9f,21 f,30f,35 f were designed for the identification of Ophiocordyceps and other adulterants.The four primers could only amplify Ophiocordyceps DNA,and the detection limit of was as low as 5 ng,The SS-PCR identification method of Ophiocordyceps was established by optimizing the PCR amplification system and conditions.The amplification system 25 μL,including template 1 μL,primer 1 μL(10μM),2 × Taq maseter mix 12.5 μL,ddH2 O 9.5 μL,predenatured at 94 ℃,after 1min,denatured at 94 ℃ for 30 s,annealed at 61 ℃ for30 s,a total of 30 cycles.Agarose gel electrophoresis showed that the authentic a bandaround 300-400 bp,while counterfeit had no special band.35 samples of Ophiocordyceps and 24 counterfeit were identified by using these four pairs of primers.The key to the molecular identification is the high quality extraction of the sample DNA.After processing,the degradation of Chinese patent medicines DNA is serious,and the general extraction method is not effective.In this study,in order to investigate the best extraction method of DNA from Ophiocordyceps capsules,oral liquid and tablet,kit,CTAB and alkaline lysis three method were used to extract the samples of genomic DNA and the ITS universal primers for PCR.The results showed that although the extraction concentration of the alkaline lysis method was the highest,the amplification efficiency was not as well as the other two methods.The concentration of the CTAB method is higher than kit,so the CTAB method has the best extraction effect.The repeatability,stability and accuracy of the SS-PCR method for the rapid identification of Ophiocordyceps in this study were investigated.The results showed that the method had good stability and repeatability and high accuracy.The results showed that the method had good stability and repeatability and high accuracy.The identification of 31 samples of Ophiocordyceps and 16 samples of counterfeit was highly consistent with the morphological identification,and the total COIncidence rate was 100%.The method was applied to the identification of Ophiocordyceps capsules,oral liquid and tablets in the market,special band was detected in tablets while not in other samples.It is proved that this method can also be applied to the application commercial samples containing Ophiocordyceps.In this study,a simple and rapid method for extracting DNA from Chinese medicinal materials within 30 s was investigated.Ophiocordyceps and ITS adulterants DNA were successfully extracted and could guarantee the molecular identification of Ophiocordyceps contained in Chinese Pharmacopoeia.In order to investigate the adaptability to DNA extraction of Chinese medicinal materials,37 samples were extracted and amplified by PCR.The results showed that the PCR amplification rate of 31 samples was successful,and the success rate of amplification reached 83.8%,animal medicine and fungi was 100%.The results showed that the adsorption capacity of filter paper with small pore size for DNA is larger than bigger.The method of filter paper had good effect on extraction of leaves,flowers,whole grasses,fruITS,rhizomes and animals and fungi.,but not in leather medicine.The filter paper method in this study was time-consuming and did not need centrifuge and other advantages,which will play an important role in molecular identification of Ophiocordyceps and Chinese medicinal materials.