ABSTRACT
OBJECTIVE: To design specific PCR primers and establish the PCR identification method of Ophiopogon japonicas from Sichuan. METHODS: The gene footprint of Ophiopogon japonicas from Sichuan named CM503 was screened from random amplified polymorphic DNAC(RAPD) amplification. Reclaimed CM503 gene was inserted into T-vector to be cloned and sequenced. One pair of specific primers CM1/CM2 were designed according to the CM503 sequence and applied in specific PCR using the genomic DNA of Ophiopogon japonicas from Sichuan as template. RESULTS: A specific band around 297 bp was detected in Ophiopogon japonicus from Sichuan at 68℃, while nothing appeared for the other varieties. CONCLUSION: The method is convenient, reproducible, and precise, with broad application prospects.
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 identify the animal drug of Cervus elaphs and C. nippon from origin of deers. Methods To extract DNA from deer blood and hairy antler of 11 species of deers such as C. elaphus, C. nippon and so on, and to gain the mitochondrial 12S rRNA gene fragment using the general primers of L1091 and H1478. Based on the sequence multialinement of 11 species deers above gene fragments, designing the couples of special difference primers and identifying C. elaphs and C. nippon. Results 12S rRNA Gene fragments can distinguish different deers well. The couple of primers (EP-1/H1478 and EP-2/H1478) PCR can effectively identify C. elaphus and C. nippon. Conclusion Special primer PCR is suitable for the identification of valuable Chinese medicinal materials, such as C. elaphus and C. nippon.