Demonstration Application Of High Resolution Melting Curve Technology On Rare Chinese Herbal Medicines

ObjectiveAccording to the Chinese herbal medicine market, the phenomenon for adulteration and admixture sources is a difficult problem for a long time. The detection method based on high-resolution melting（HRM） curve technology provides the foundation for the future establishment of traditional Chinese medicine identification database and a new way in molecular identification technology.MethodsPrecious Chinese herbal medicine samples collected from different areas and markets, total DNA can be extracted by DNA extraction kits. Selecting the appropriate identification of DNA fragments, Polymerase Chain Reaction（PCR） amplification and HRM analysis run in the instrument of Light Cycler 480 Real-Time PCR System. PCR amplification conditions such as different concentrations of template DNA, annealing temperature, PCR primer concentrations were optimized, while the sensitivity,specificity, repeatability, robustness, limit of detection（LOD） of this method were tested in methodological study. To establish a stable and accurate identification of authentic Chinese herbal medicine model is the purpose.ResultsThough selecting Ron M-tl / VRL-tl primer fragment in CO I gene sequence and DNA template concentration of 50 ng · μL^-1 condition from Hairy Antler（Cervus nippon Temminck and Cervus elaphus Linnaeus）, PCR amplification and HRM analysis run in the instrument of Light Cycler 480 Real-Time PCR System. The whole program :PCR reaction conditions（95 ℃ 10 min;（95 ℃ 30 s; 60 ℃ 30 s, 72 ℃ 45 s） 45cycles） add the HRM analysis procedures. 25 μL PCR reaction system is: 2.5 μL 10 ×buffer, 1 μL 10 mmol · L^-1d NTPs, 0.25 μmol · L^-1 forward and reverse primer, 0.25 U Speed Star HS Taq DNA polymerase, 50 ng · μL^-1 DNA template, 2 μL 100 × LC GreenPlus and dd H2 O filled to 25 μL. The authentic model of Hairy Antler can be established by HRM.Though selecting LCO 1490/HCO 2198 primer fragment in CO I gene sequence and DNA template concentration of 50 ng · μL^-1 condition from authenticate medicinal snakes record in Chinese Pharmacopoeia（Zaocys dhumnades, Bungarus multicinctus,Agkistrodon acutus）, PCR amplification and HRM analysis run in the instrument of Light Cycler 480 Real-Time PCR System. The whole program: PCR reaction conditions（95 ℃ 5 min;（95 ℃ 30 s; suitable annealing temperature 30 s, 72 ℃ 45 s） 40 cycles）adds the HRM analysis procedures. 25 μL PCR reaction system is: 2.5 μL 10 × buffer,1 μL 10 mmol L^-1d NTPs, 0.25 μmol L^-1 forward and reverse primer, 0.25 U Speed Star HS Taq DNA polymerase, 50 ng · μL^-1 DNA template, 2 μL 100 × LC Green Plus and dd H2 O filled to 25 μL. The authentic model of snakes record in Chinese Pharmacopoeia can be established by HRM.Though selecting psb A-F / trn H-R primer fragment in chloroplast gene sequence and DNA samples extracted from Panax Species（Panax quinquefolium, Panax ginseng,Panax notoginseng, Panax japonicas, Panax japonicas var. Major, Panax pseudoginseng var. bipinnatifidus）, PCR amplification and HRM analysis run under the condition that DNA template concentration is 1.6-200 ng μL^-1, the annealing temperature is 54℃-60℃ and forward and reverse primer concentration was 0.1-0.3μmol L^-1. Panax Species can be stable typing results, establish authentic model and method validation. When the concentration of two kinds of Panax Species are mixed at rate of 100%, 50%, 25%, 12.5%, 0%, adulterants and authentic still show a clear distinctionIn this thesis, HRM analysis has been used in fifteen kinds of precious Chinese herbal medicines, established the authentic model, layered the foundation for the construction of Chinese herbs authentication database.ConclusionIn this paper, PCR amplification and HRM analysis run in the condition of appropriate DNA template concentration, annealing temperature, primer concentration amplification conditions. This method can build a stable authentic model of precious Chinese herbal medicines and get the test of methodology.High-resolution melting curve analysis has lots of advantages such as high throughput, high sensitivity and specificity, good repeatability, simple operation, low cost and so on. The technology can identify precious Chinese herbal medicines and its adulterants, bring a more simple and rapid detection method for the identification of molecular identification. With technology development, HRM analysis plays an extremely important role in the field of the original species identification, Chinese Traditional Patent Medicine identification, and quantitative detection of adulteration.And it will form Chinese medicines HRM databases and promote multi-level Chinese herbal medicine identification system.