ABSTRACT
Objective: To develop an accurate and effective method to identify the original animals of Chinese medicine Pheretima (Dilong in Chinese) and its adulterants by using mitochondrial genes, cytochrome coxidase I (CO I) and 16 S rRNA as DNA barcoding sequences to certify and improve morphological analysis. Methods: A total of 66 samples were initially identified according to their morphological characteristics, and then their CO I and 16 S rRNA sequences were simultaneously amplified for DNA barcoding. The two gene sequences were amplified with improved primers. One-step double PCR was used and the experimental conditions were optimized. The genetic distance among and within species of Pheretima and its hybrids was calculated by MEGA 5.1. The N-J tree was constructed based on K2P model. Results: Combined with morphological identification, CO I and 16S rRNA double DNA barcoding identification can accurately identify Pheretima and its adulterants. Conclusion: Morphological identification is the basis of molecular identification. Molecular identification can be a powerful supplement to the morphological identification. The combination of molecular identification and morphological identification can increase the accuracy of the identification of the original animals of Pheretima and its adulterants. Double DNA barcoding can also provide a solution for the identification of the medicinal materials of Pheretima and other animal medicinal materials.
ABSTRACT
In criminal investigations, the accurate identification of specimens must be a vary important procedure in the estimation of post-mortem interval(PMI) using insect specimens. However, the morphological-based identification method has many limitations, as species identification for immature stages of many species being difficult or impossible, and more convenient methods are needed to identify necrophagopus fly species. So, many authors have proposed DNA-based methods for identifying fly specimen because of identifying all life stages of carrion flies. In this study, we sequenced a part of mitochondrial cytochrome oxodase subunit I (COI) for each five blow fly species [Hemipyrellia ligurriens, Lucilia illustris, Phaenicia sericata, Chrysomya. megacephala, Chrysomya pinguis] collected from the rural and urban regions in Korea, and compared 400 nucleotides of one species with those of another species. Percent similarity of sequence compared was highest value between Ch. pinguis and Ch. megacephala (97.8%), and lowest one between H. ligurriens and Ch. pinguis(89.2%). Maximun intraspecific variation was observed in L. illustris (0.5%). In conclusion, the experimental results indicate that sequence analyses of more necrophagous flies collected from various regions of Korea should be performed to construct a database of mitochondrial CO I of necrophagous fly species.