利用12S rRNA基因的限制性酶切末端片段长度多态性鉴定动物种类

目的：建立一种精确可靠的鉴定常见的1 0 种动物( 猪、狗、牛、山羊、绵羊、马、鸡、鼠、三文鱼和鹿)的方法。方法：利用12S rRNA 基因的限制性酶切末端片段长度多态性(terminal restriction fragment lengthpolymorphism，T-RFLP)鉴别动物种类。将线粒体12S rRNA 基因通过引物的5＇端用FAM 荧光标记，从基因组DNA 中扩增450bp 的目的片段。引物对1(下游引物FAM标记，上游引物不标记)扩增的PCR 产物用限制性内切酶Alu Ⅰ酶切。引物对2(上游引物FAM标记，下游引物不标记)扩增的PCR 产物用限制性内切酶Tru9 Ⅰ酶切。得到的酶切产物分别在遗传分析仪ABI 3100 上进行毛细管电泳，片段大小用Peak Scanner 1.0 软件分析。结果：根据Alu Ⅰ酶切图谱能够区分鸡、马、猪和三文鱼，而鹿和牛、山羊和绵羊、鼠和狗因酶切图谱相同无法分开。根据Tru9 Ⅰ酶切图谱，能够进一步将鹿和牛、山羊和绵羊、鼠和狗分开。同一种动物不同个体的酶切图谱完全相同，结果具有可重复性。没有出现物种内多态的现象。大多数情况下，实际得到的末端片段长度与理论值非常接近，只存在2 ～5bp 的差异。结论：该方法操作简单、结果精确，适用于鉴定动物种类。

Identification of Animal Species by Terminal Restriction Fragment Length Polymorphism (T-RFLP) of Mitochondrial 12S rRNA Gene

An accurate and reliable method was developed to identify ten animal species (pig, dog, cattle, goat, sheep, horse, chicken, mouse, salmon and deer) by terminal restriction fragment length analysis based on mitochondrial 12S rRNA gene. Universal primers for the 12S rRNA gene were labeled with FAM and the specific fragments of 450 bp were obtained using polymerase chain reaction. The PCR products obtained using a set of unlabeled forward and labeled reverse primers were digested by AluⅠ, and those obtained using another set of labeled forward and unlabeled reverse primers by Tru9Ⅰ. The resulted restriction fragments were loaded on ABI prism 3100 genetic analyzer to perform capillary electrophoresis. Peak Scanner Software 1.0 was used to analyze the electrophoresis data. Results showed that chicken, horse, pig and salmon had different Alu Ⅰ restriction pattern while the same pattern was observed among deer and cattle, goat and sheep, mouse and dog, which, however, could be differentiated by Tru9 Ⅰ restriction pattern. Each species had a unique characteristic and reproducible pattern of restriction fragments while different individuals of the same species had the same and repeatable patterns and no intraspecies polymorphism appeared. In most cases, the difference between observed and expected restriction fragment length was only 2– 5 bp. In summary, this T-RFLP method is demonstrated to be accurate and easy-operating and therefore suitable for animal species identification.