| OBJECTIVE: To develop a kind of Agkistrodon DNA test kit, and evaluate the accuracy indexes, and survey out the qualities from commercial Agkistrodon samples. Provide a accurate and rapid molecular biology of quality control method applied to development and application of traditional Chinese medicine market. METHODS:1. The Agkistrodon DNA test kit was developed and modified according to the method recorded in Pharmacopoeia(2010 edition), research the standard reagents of DNA extraction and PCR amplification, and assemble the test kit. 2. 18 samples of Agkistrodon were randomly collected from three cities of Beijing, Tianjin, Changchun and Jilin. The kit assay was performed to identify these specimens as well as Pharmacopoeia method as standard control, and compare results of test. 3. Evaluate the accuracy indexes including specificity, stability in addition to sensitivity and repeatablity. 4. Review the kit by the Chinese Academy of Food and Drug Identification. RESULT: 1. Parallel test the commercial Agkistrodon samples with DNA test kit and Pharmacopoeia method. The specificity test confirmed that 14 were genuine, and 4 were counterfacts in all samples, the pass rate is 77.8%. All results by kit method were in accordance with the ones by Pharmacopoeia method. 2. The kit assay proved effective after repeated freezing and thawing 20 times, and repeatablity test indicated the same data through three times. The lowest amount was 0.025 g of each tested specimen. All results by kit method were in accordance with the ones by Pharmacopoeia method, the specificity is 100%. 3. Review the kit by the Chinese Academy of Food and Drug Identification, 8 genuine and 20 counterfacts were tested, the specificity is 100%. CONCLUSION: The developed DNA kit is accurate, effective in identification of Agkistrodon, compared with the Pharmacopoeia method, it has simple, fast properties, and demonstrates the broad prospect in quality inspection of Agkistrodon in the future. |
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