Establishment Of PCR Identification For Oviductus Ranae

Oviductus Ranae is the dried oviduct of female Rana temporaria chensinensis David, which is belonging to Chordata, Amphibia, Ranidae and Rana in taxonomy. Oviductus Ranae is also called Oviductus Frog, Oviductus hashima and so on. It is mainly produced in Jilin province, Heilongjiang province, Liaoning province and so on. Oviductus Ranae is neutral in nature, sweet and salty flavor, distributing to lung and kidney channel. It can invigorate the kidney, benefit essence of life, nourish Yin and moisturize lung.The traditional identification methods of Oviductus Ranae include original identification, macroscopical identification, microscopic identification and physical and chemical identification. These methods based on the morphology are convenient and easy, which are effective methods for Oviductus Ranae. However, these diagnostic characteristics are nearly all heredity phenotype of Oviductus Ranae, which are not only influenced by genetic factors, but also close to growth development of Rana temporaria chensinensis, environment and human activities. Those characters have great variability. At the same time, those traditional methods have some defects, such as strong subjectivity, poor repeatability and stability and so on, which have an influence on the dependability of the identification results. The main identification methods of Oviductus Ranae are macroscopical identification, High Performance Liquid Chromatography (HPLC) and dilatation mensuration according to the Pharmacopoeia of People’s Republic of China in2010. But the dilatation mensuration can be easily influenced by the water temperature, macerated time and individual differences, so the identification result is not very ideal.DNA is organism’s genetic information carrier. DNA has high genetic stability and is not easily influenced by the environmental factors, organ tissue differences and organism’s development stage, so DNA molecular marker can be used in the Traditional Chinese Medicine (TCM) identification, especially for the authentication of relative species, confused species, valuable species, animal medicine and so on. For exploring quick and accurate identification methods for Oviductus Ranae, this paper mainly studied from the following:1The chemistry component, pharmacological action, quality control status of Oviductus Ranae and the current application status and development tendency of molecular biotechnology in TCM identification was systematically summarized based on the research literatures.2Genome DNA of Oviductus Ranae and four kinds of counterfeit drug including Rana amurensis Boulenger oviduct, Rana nigromaculata oviduct, Bufo gargarizans oviduct and Rana catesbeiana oviduct was extracted. The concentration and quality of genome DNA was detected. At the same time, the DNA samples were detected using0.7%agarose gel electrophoresis. Five pairs of CHD gene primers were consulted and amplification primers were designed. The genome DNA was amplified, screening out two pairs of primers that can amplify obvious segments. Amplification fragment polymorphism was analyzed. A kind of PCR identification of Oviductus Ranae was set up. Thirty kinds of unknown powder were amplified using the primer set of F4/R4and F2/R2, and the reliability of the identification method was confirmed.3Genome DNA of Oviductus Ranae grown in Jilin, Neimeng, Shaanxi, Qinghai, North Korea and Liaoning was extracted. The concentration and quality of genome DNA was detected. At the same time, the DNA samples were detected using0.7%agarose gel electrophoresis. Two pairs of primers F2/R2and F4/R4were used to amplify Oviductus Ranae from different habitats, and the amplification fragments were detected using2%agarose gel electrophoresis analyzed.