RESUMO
OBJECTIVE: To identify 70 samples of Fritillariae Cirrhosae Bulbus from 16 cities such as Beijing, Tianjin, Changchun,Jilin and so on by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis kit.METHODS: The genomic DNA of Fritillariae Cirrhosae Bulbus was extracted by kit method, and PCR reaction and RFLP identification were carried out. The method of the Chinese Pharmacopoeia of 2015 was used for comparison and re-examination.RESULTS: The purity of the genomic DNA of Fritillariae Cirrhosae Bulbus was very good, the OD260/OD280 value was between 1.90-2.1, and the concentration could reach the requirement of PCR. Seventy samples were tested, of which 37 were positive and 33 were counterfeit, and the false rate was 47.1%.CONCLUSION: The purity and concentration of the nucleic acid meet the requirements of PCR and RFLP. The test results of the samples from 16 cities show that the counterfeit rate of Fritillariae Cirrhosae Bulbus is high in the market. Relevant government departments should strengthen the quality management of the traditional Chinese medicine market.
RESUMO
OBJECTIVE: To develop a kit for detection of DNA of Fritillaria cirrhosa D. Don and optimize its components as well as process protocols, in order to set up a simple, rapid molecular biology method for identification of Fritillaria cirrhosa D. Don. METHODS: All genomic DNA of Fritillaria cirrhosa D. Don was extracted by kit assay and pharmacopoeia method recorded in the expanded supplement of China Pharmacopoeia 2010, respectively; ultraviolet spectrophotometer was used to measure the quantity of extracted DNA; PCR amplification and restriction fragment length polymorphism analysis (RFLP) were carried out to identify the authentication of Fritillaria cirrhosa D. Don. RESULTS: The maximum value of genomic DNA extracted by pharmacopoeia method was (1.57±0.05) (OD260/OD280) and (1.73±0.10) by kit assay. The PCR amplification showed a single band over 300 bp, while the RFLP showed two distinct bands between 100 and 250 bp in agarose electrophoresis. CONCLUSION: The data demonstrated that the kit assay was better than the pharmacopoeia method, especially in the extraction quantity and DNA purity of Fritillaria cirrhosa D. Don nicleic acid; the PCR and RFLP results showed that the kit assay was consistent with pharmacopoeia method. The detection kit has good specificity, high sensitivity and good stability, so it is suitable for the rapid detection of Fritillaria cirrhosa D. Don.