[关键词]
[摘要]
目的 建立鹿血聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)种间及种内鉴定方法并确定检测限度。方法 根据梅花鹿、马鹿、猪、牛、羊、鸭的细胞色素b(cytb)基因序列的差异,设计并选取DNA限制性内切酶EcoRV和MseI分别作为区分鹿和非鹿,梅花鹿和马鹿的工具。采用试剂盒法提取样品基因组DNA,经PCR扩增cytb片段后,分别进行RFLP分析;在梅花鹿血基因组DNA中分别加入不同比例非鹿类动物血基因组DNA或马鹿血基因组DNA,PCR产物用限制性内切酶EcoRV和MseI进行切割,确定检测限。结果 经PCR-RFLP分析,EcoRV切割鹿和非鹿cytb片段,前者得到2个片段(287、185 bp),后者未被切割;MseI切割梅花鹿与马鹿cytb片段,前者被切成2个片段(281、191 bp),后者被切成3个片段(281、126、65 bp),191 bp的片段作为梅花鹿血的特征片段,126 bp的片段作为马鹿血的特征片段。梅花鹿血中加入非鹿类动物血基因组DNA的检测限为3%,加入马鹿血基因组DNA的检测限为6%。结论 建立的PCR-RFLP方法可以快速鉴定鹿血及相关伪品或掺伪品,也可区分鉴定梅花鹿源的鹿血或马鹿源的鹿血,为鹿血相关产品的质量控制提供了新的技术手段。
[Key word]
[Abstract]
Objective To establish a polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) method which can identify deer blood. Methods We analyzed DNA sequences of cytochrome b (cytb) from different species (i.e. sika deer, red deer, pig, ox, sheep, and duck) and selected two DNA restriction enzymes of EcoRV and MseI to distinguish deer from other species and to sika deer from red deer, respectively. Genomic DNA of every sample was extracted by blood genomic DNA extraction kit. After PCR of cytb fragment, digestion by EcoRV or MseI was conducted and the results were analyzed. The capacity of this method to identify different proportional blood mix from different species also was determined. Results Using the PCR-RFLP method, EcoRV digestion made two fragments of 287 bp and 185 bp in deer, but no digestion in other species; MesI digestion made two fragments of 281 bp and 191 bp in sika deer, but three fragments of 281 bp, 126 bp, and 65 bp in red deer. The 191 bp fragment was a characteristic marker of sika deer, and the 126 bp was for red deer. The minimum detected proportion added to blood of deer with other sample was 3%, 6% of sika deer with red deer. Conclusion A PCR-RFLP method according to the sequences of cytb gene is established. This method can identify the blood of sika deer rapidly and conveniently.
[中图分类号]
[基金项目]