Establishment And Application Of Rapid Molecular Identification System For Dendrobium Officinale

ObjectiveMolecular identification of traditional Chinese medicine(TCM)is a method to define the inherent genetic variation of species by directly analyzing the polymorphism of genetic material to realize the identification of medicinal materials.It is independent of external factors,biological development stage or organ and tissue differences,that can put aside the appearance of morphological similarity,avoid the change of chemical characteristics,provide a basis for identification from the gene level.It is easy to find the identification characteristics of species and even to identify subspecies.However,the current molecular identification methods are more complicated and need a variety of expensive instruments,so it can not be quickly identified in the field.Cause this,fast,simple,field-based is always the pursuit of Chinese medicine molecular identification.The field rapid identification system of traditional Chinese medicine was put forward by Huang Luqi team.The alkali cracking method and rapid PCR method were used for the research,but the long extraction time was not completely solved and the process of rapid identification was not systematically studied.Dendrobium officinale Kimura et Migo belongs to Dendrobium(Orchidaceae).However,the species of Dendrobium are similar in form,wide in distribution,numerous in hybrids,and often appear in the market where the quality of Dendrobium medicinal materials and their adulterates are not uniform,which makes it difficult to guarantee the safety and quality of Dendrobium.It is difficult to identify D.officinale by traditional identification methods,so it is urgent to establish a rapid and accurate molecular identification method for D.officinale.Although previous researchers used AFLP,RAPD,SSR,multiplex PCR was used to identify D.officinale and its adulterants at molecular level,but these methods could not be used to identify D.officinale quickly.Methods1.DNA extraction:a rapid DNA extraction method for 30s without equipment was established.The conventional qualitative filter paper was used as the carrier of nucleic acid adsorption.The nucleic acid was released and adsorbed on the filter paper by the cleavage of the lytic liquid.The whole nucleic acid extraction process of less than 30 seconds could be completed by using eluent to purify the nucleic acid.The filter paper adsorbed nucleic acid can be directly added into the reaction system for amplification.2.The taxonomic study of Dendrobium samples was carried out.The correctness of the source of the samples was observed and determined by using conventional identification methods such as traits,microscopes,and more than 30 species of Dendrobium samples were studied.3.Identification of D.officinale by(LAMP):specific LAMP primers were designed according to the variation sites of nuclear gene non-coding region ITS,and a mismatch was introduced to optimize the reaction conditions.The samples of D.officinale and its relative species were amplified and detected.The reaction temperature ended in 40min and the fluorescence changes of the products were observed directly by naked eyes.4.The method of rapid extraction of DNA was combined with the technique of ring mediated isothermal amplification to establish a rapid detection method for D.officinale in the field.The whole identification process could be completed at about 40min.Results1.The nucleic acid of different medicinal parts could be extracted successfully by filter paper method in 30s.The nucleic acid obtained from the extraction was verified by amplification,and the results were in agreement with the traditional extraction method.2.The established method can be detected within 40 min,and the results can be judged by observing the fluorescence changes of the products directly with the naked eye.It has been proved that D.officinale and its relative species have good specificity.3.The rapid DNA extraction of D.officinale for 30s was combined with the rapid amplification and detection of D.officinale LAMP.The field rapid identification method of D.officinale was established,and the rapid detection kit was developed and designed for D.officinale.4.Establishment of Rapid Detection of SOP in the field of Chinese Medicinal Materials.ConclusionThrough the rapid nucleic acid extraction by filter paper method and the rapid amplification technique of LAMP of D.officinale,the problem of complex operation and long detection time in molecular identification of D.officinale was basically solved.A rapid,simple and field-based molecular identification method for D.officinale was established.