穿山甲标本和甲片的DNA提取及PCR扩增

Extraction and Amplification of DNA from Pangolin Specimen and Scales

为验证经处理后的穿山甲(Manis spp.)标本和甲片是否可以用于种间分子鉴定标记的开发及个体识别工作,本文在样品的预处理、消化、提取后纯化等方面对传统提取方法进行了改进,分别从穿山甲剥制标本、干皮标本及甲片中提取总DNA;然后用Cyt b基因扩增通用引物、12S rRNA基因全序列扩增引物、RAPD引物及微卫星引物进行了PCR扩增,并对部分扩增结果进行了序列测定。结果表明,除剥制标本的脚底皮张组织外,其他样品基本都可以提取出DNA。以此为模板的PCR扩增中,2种线粒体基因引物扩增出明显目的条带,RAPD引物扩增出种间特异条带,测序结果可用于种间特异性引物及SCAR引物的开发;微卫星引物在甲片样品中扩增稳定,可用于个体识别工作。

Illegal trade of Pangolin is becoming more serious, thus forensic identification of criminal material from the molecular level is very urgent and necessary. In order to ascertain whether the processed specimen and scales could be authenticated by DNA molecular technology, total DNA from specimen or scales of Pangolin was extracted with an improved method. Four pairs of primers, which were universal primers for Cyt b gene, primer for complete 12S rRNA sequence, RAPD primer and microsatellite primer were used for amplification. Some amplified fragments were sequenced. The results showed that DNA of almost all these samples could be extracted except for that of the footpad of museum specimen. The extracted DNA could be amplified well with four primers, indicating the value of the present method in species-specific identification and individual recognition study.