ABSTRACT
OBJECTIVE: To develop a DNA detection kit for identification of Fetus Cervi, evaluate the quality indexes including specificity, stability, sensitivity and repeatability, and inspect the qualities of commercial Fetus Cervi samples. METHODS: The Fetus Cervi DNA test kit was developed and modified using the classical DNA extraction and PCR identification method. The genomic DNAs of Fetus Cervi and counterfeit goods were extracted by the kit and PCR technique was performed to identify the authenticity. The purity of DNA was detected by UV spectrophotometer. Finally, commercially available Fetus Cervi samples were verified. RESULTS: The kit was proved to be effective after 20 times of repeated frozen-thawing and it could be stored at - 20℃ for 1 year. The DNA could be detected when the primary solution was diluted by 200 times. The specificity test confirmed that the 15 samples were authentic Fetus Cervi, and 7 were counterfeit. The specificity of the kit was 100%. The repetitive experiments showed that the average intra-assay CV% and inter-assay CV% of the kit were 2.32% and 2.56%, respectively. CONCLUSION: The amount and purity of the nucleic acids extracted by the DNA detection kit can meet the requirement for identification of Fetus Cervi, and the kit has good specificity, high sensitivity and good stability, so it is suitable for the rapid detection of Fetus Cervi.
ABSTRACT
OBJECTIVE: To establish a standard method for preserving the Fritillaria cirrhosa D. Don DNA fingerprint fragments. METHODS: Independently developed F. cirrhosa test kit was used to extract genomic DNA. Specific F. cirrhosa DNA fragments were cloned in vitro, and ligated into a carrier vector then transformed into a self replicating host cells to produce a large amount of specific F. Cirrhosa DNA. Plasmid DNA was extracted using Plasmid Extraction Kit and confirmed by PCR or restriction digestion of the insert. Bacterial colonies carrying authentic Sichuan F. cirrhosa specific DNA sequences were stored at -80℃. Stability was monitored at intervals of 1, 2, 3 and 6 months after DNA recovery using the boiling method of genomic DNA extraction. RESULTS: The amplified F. cirrhosa DNA clones showed clear bands in the electrophoresis, and had stable results in the repeated verification test. The amplified clones from resuscitated bacterial colonies which had been stored for 1, 2, 3, 6 months at -80℃ still displayed bright and clear bands after electrophoresis. CONCLUSION: It is feasible and effective to preserve DNA fingerprint of F. Cirrhosa by the established method, and this simple and reliable method can be used as the basis of establishing the new genes database of traditional Chinese medicine.
ABSTRACT
OBJECTIVE: To develop a rapid DNA detection kit for DNA extraction and PCR identification of Panax ginseng C. A. Mey. METHODS: The classical DNA extraction and PCR identification methods for Panax ginseng C. A. Mey were modified, and the compositions and reaction conditions of the kit were determined. In addition, the specificity, stability, sensitivity, and repeatability of the kit were evaluated. The genomic DNAs of genuine and counterfeit ginseng goods were extracted by the kit and PCR was performed to identify the authenticity. The purity of the extracted DNA was detected by UV spectrophotometry. Finally, commercially available ginseng samples were verified. RESULTS: The purity of the genomic DNA extracted by the kit was (1.73 ± 0.13) (OD260/OD280), and a fragment between 150 and 200 bp could be amplified only from authentic Panax ginseng C. A. Mey. The specificity of the kit was 100%. The repetitive experiments showed that the average intra-assay CV% and inter-assay CV% of the kit were 2.38% and 2. 62%, respectively. The DNA in solutions diluted by 200 times could still be detected. Stability experiment proved that repeated freeze-thawing for 20 times had no significant effect on the activity of this kit and the test sample could be stored at - 20℃ for one year. The specificity test confirmed that 8 samples among the 10 commercial products were genuine, and 2 were counterfeit. CONCLUSION: The nucleic acid extraction and purity of the DNA detection kit can meet the requirement for identification of Panax ginseng C. A. Mey. The kit has good specificity, high sensitivity, and good stability, so it is suitable for the rapid detection of Panax ginseng C. A. Mey.
ABSTRACT
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.