AS-PCR法鉴别炮制辅料鳖血和常见掺伪品的研究

Identification of Processing Adjuvant Trionyx Sinensis Blood and Common Adulterants by AS-PCR

目的建立一种炮制辅料鳖血等位基因特异性PCR(AS-PCR)鉴别方法,快速、准确地鉴别鳖血与其它常见动物血液。方法通过比较鳖血基原中华鳖和驴、牛、羊、鸡、猪、兔为代表的常见动物的COⅠ序列差异,根据SNP位点设计鳖血特异性鉴别引物,优化PCR反应体系,并进行耐受性、适用性、掺伪灵敏度考察验证。结果实验条件为退火温度62℃、循环次数29次、引物用量0.2μL、DNA模板浓度100 ng时,鳖血使用设计的ZHB286引物经PCR扩增和凝胶电泳后出现约286 bp的明亮条带,其他动物血液样品均无条带出现。结论该AS-PCR鉴别方法具有高度特异性,能准确鉴别鳖血和其他常见动物血液,进而可用于鳖血与驴、牛、羊、鸡、猪、兔血掺伪鉴别,在血液类药材分子鉴定中具有广泛应用价值。

OBJECTIVE To establish an allele-specific PCR identification(AS-PCR)method of Trionyx sinensis blood,which can quickly and accurately identify Trionyx sinensis blood from other common animal blood.METHODS Based on the comparison of COⅠsequences between the Trionyx sinensis and common animals such as donkey,cow,goat,chicken,pig and rabbit,the specific identification primers for Trionyx sinensis blood were designed according to SNP sites,and the PCR reaction system was optimized,and the tolerance,applicability and adulteration sensitivity were investigated and verified.RESULTS When annealing temperature was 62℃,the number of cycles was 29,primer dosage was 0.2μL,DNA template concentration was 100 ng,the bright bands of about 286 bp appeared in the Trionyx sinensis blood samples after PCR amplification and gel electrophoresis with the designed ZHB286 primer,while no bands appeared in other animal blood samples.CONCLUSION AS-PCR identification method has high specificity,can accurately identify the blood of Trionyx sinensis and other common animals,and can be used for the identification of adulterated Trionyx sinensis blood with donkey,cow,goat,chicken,pig and rabbit blood,which has wide application value in the molecular identification of blood medicinal materials.