Identification of Processing Adjuvant Trionyx Sinensis Blood and Common Adulterants by AS-PCR
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摘要:
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CO Ⅰ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0.2 μL、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。 -
关键词:
- 鳖血 /
- 炮制辅料 /
- 等位基因特异性PCR鉴别
Abstract: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 CO Ⅰ sequences 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 0.2 μL, 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. -
图 1 鳖血通用引物扩增
注: M.DL 5000 DNA Marker Ⅱ; 1~4.鳖血(BY1~BY4)
Figure 1. Amplification of general primers for Trionyx sinensis blood
图 2 鳖血特异性引物扩增
注: M.Trans DNA Marker Ⅱ; 1.鳖血(BY1);2.驴血(LX1);3.牛血(NX1);4.鸡血(JX1); 5.羊血(YX1);6.猪血(ZX1);7.兔血(TX1);8.阴性对照
Figure 2. Amplification of specific primers for Trionyx sinensis blood
图 3 鳖血AS-PCR反应条件考察
注: M.Trans DNA Marker Ⅱ; 1.鳖血(BY1);2.驴血(LX1);3.牛血(NX1);4.鸡血(JX1); 5.羊血(YX1);6.猪血(ZX1);7.兔血(TX1);8.阴性对照
Figure 3. Identification conditions of excipient Trionyx sinensis blood by Specific PCR
图 4 不同产地的鳖血PCR扩增
注: M.Trans DNA Marker Ⅱ; N.阴性对照; 1~12.12批不同产地鳖血(BY2~BY4、HZ1~HZ3、HS1~HS3、YY1~YY3)
Figure 4. PCR amplification of Trionyx sinensis blood from different habitats
图 5 鳖血与其他动物血液样品PCR扩增
注: M.Trans DNA Marker Ⅱ; N.阴性对照; 1.鳖血(BY1);2.驴血(LX1);3.牛血(NX1);4.鸡血(JX1);5.羊血(YX1);6.猪血(ZX1); 7.兔血(TX1)
Figure 5. PCR amplification of Trionyx sinensis blood and other animal blood samples
图 6 鳖血样品PCR扩增
注: M.Trans DNA Marker Ⅱ; 1~7.鳖血样品模板DNA分别与中华鳖、驴、牛、鸡、羊、猪、兔引物反应谱带; 8~14.阴性对照(加与模板等量的DEPC水)
Figure 6. PCR amplification of Trionyx sinensis blood samples
图 7 鳖血掺入不同比例牛血PCR扩增
注: M.Trans DNA Marker Ⅱ; N.阴性对照; 1~7.鳖血BY4(掺有100%、80%、50%、20%、10%、5%牛血及不掺牛血)
Figure 7. PCR amplification of Trionyx sinensis blood mixed with different proportions of bovine blood
图 8 鳖血等比掺伪其他动物血液样品PCR扩增
注: M.Trans DNA Marker Ⅱ; N.阴性对照; 1~7.鳖血BY4与等比掺伪驴血、牛血、鸡血、羊血、猪血、兔血混合样品DNA与各自引物反应谱带
Figure 8. PCR amplification of Trionyx sinensis blood samples adulterated with other animals blood
表 1 样品来源信息
Table 1. Sample source information
名称 收集地 批数 编号 鳖血 江苏宝应 4 BY1~4 鳖血 湖南汉寿 3 HS1~3 鳖血 浙江淳安 3 HZ1~3 鳖血 浙江余姚 3 YY1~3 驴血 江苏新沂 1 LX1 牛血 江苏新沂 1 NX1 鸡血 江苏新沂 1 JX1 羊血 江苏新沂 1 YX1 猪血 江苏新沂 1 ZX1 兔血 江苏南京 1 TX1 
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表 2 引物参数
Table 2. Primer parameters
引物名称 引物类型 序列(5'→3') HCO2198 通用引物 GGTCAACAAATCATAAAGATATTGG LCO1490 通用引物 TAAACTTCAGGGTGACCAAAAAATCA ZHB286.F 鉴别引物 CTAGCAACCTAGCCCATGTG ZHB286.R 鉴别引物 GGATCTCCTCCTCCAGAAGG ZHB1.F 鉴别引物 ACTTCTCCTAGCATCATCAGGC ZHB1.R 鉴别引物 CTGCGGCTAACACTGGTAGT ZHB2.F 鉴别引物 ACTTCTCCTAGCATCATCAGCC ZHB2.R 鉴别引物 CTGCGGCTAACACTGGTAGT ZHB3.F 鉴别引物 TTCTCCTAGCATCATCCGCC ZHB3.R 鉴别引物 CTGCGGCTAACACTGGTAGT LvXue.F 鉴别引物 CAAGCTCAACGTCACACATATC LvXue.R 鉴别引物 CCTGTGTTGGGTTAACAATAGTC NiuXue.F 鉴别引物 CCTGTGTTGGGTTAACAATAGTC NiuXue.R 鉴别引物 CCGATGGTGATATATGGGTGTT YangXue.F 鉴别引物 GGCGCCATGCTACTAATTCTTGTT YangXue.R 鉴别引物 GAGGAAGGGTACAAGTACTAAGAT JiXue.F 鉴别引物 CCATCTTAGCCTCAACGATTAA JiXue.R 鉴别引物 GGCTATTGAGCTCACTGTTGTT ZhuXue.F 鉴别引物 GCACACCTATAACGGTAGCTCAT ZhuXue.R 鉴别引物 TCCTAGCTATCGTGTGTCAGGAT TuXue.F 鉴别引物 CGGCGTAAAGCGTGATTAGAATAA TuXue.R 鉴别引物 GCTATCGTGAGTTCGAAGAGTAT
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