应用mtDNA 16S rRNA基因鉴定动物组织种属

Species identification of unknown animals based on mt DNA 16S rRNA gene sequencing

目的 应用线粒体DNA 16S rRNA基因PCR测序方法进行动物组织种属鉴定.方法 未知动物内脏样本8份,已知马鹿、北山羊、绵羊和鹅喉羚肌肉样本各1份作为对照.采用用常规酚-氯仿法提取模板DNA,分别用哺乳动物通用引物和4种已知对照特异性引物扩增16S rRNA基因片段,产物用2.0％琼脂糖电泳检测,并进行序列测定及与基因库已有的序列同源性比对.结果 8份未知样本经哺乳动物16S rRNA通用引物扩增,均检测出阳性条带;用鹅喉羚特异性引物扩增,均检测出阳性特异性条带;用其他3种特异性引物扩增,均未检出阳性条带;未知样本扩增的基因片段经测序及同源性比对,与鹅喉羚的同源性为96％～100％.结论 本文未知动物样本均为鹅喉羚内脏组织.本文方法可在动物检材种属鉴定中选用.

Objective Applied mtDNA（ mitochondrial DNA）16S rRNA gene PCR product sequencing method performed species identification of unknown animal tissue specimens. Methods 8 unknown animal entrails samples were given for inspection by related authorities, one copy each of the red deer （Cervus elaphus ）, goat （ Capra ibex）, sheep （ Ovis aries） and goitred gazelle （ C, azella sttbgutturosa） muscle samples reserved in the laboratory were used as a contrast. DNA is extracted by conventional phenol-chloroform method. PCR products amplified mtDNA 16S rRNA gene by using the universal primer of mammals and the specific primers respectively were examined by 2. 0% agarose gel electrophoresis, mtDNA 16S rRNA gene was amplified by using the universal primer and the specific primers respectively and PCR products examined by 2.0% agarose gel electrophoresis were sequenced （ by sequencing and BLAST2. 2. 9 ） and then queried by BLAST searches against GenBank to confirm homology. Results All PCR products from 8 received samples amplified with universal primer showed positive band; neither one of them had positive band when used the sheep specific primer, red deer specific primer, goat specific primer; but when used the goitered gazelle specific primer all entrails testing samples had positive band. When we were BLAST searching in GenBank, the identity of them with goitered gazelle mtDNA 16S rRNA gene sequences were 96% ～ 100% respectively while sequenced and BLAST comparisoned. Conclusion All of the 8 unknown samples in this paper was identified as Goitered Gazelle entrails tissue samples respectively. This method can be used in identification of unknown animal tissue soecimens.