关键词: 限制性酶切末端片段长度多态性T-RFLP, 种类鉴定, 12S rRNA

Abstract:

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.

Key words: terminal restriction fragment length polymorphism (T-RFLP), species identification, 12S rRNA