目的 通过DNA条形码技术和建立特异性聚合酶链式反应(PCR)方法,有效鉴别九味羌活丸中的水稻源性成分掺伪问题。方法 通过建立特异性PCR技术对九味羌活丸中掺伪的水稻源性成分样品的GOS9序列进行扩增,并对PCR反应体系与程序、方法学和系统适用性进行考察,从而对掺伪水稻源性成分进行鉴别。结果 该引物体系只针对水稻源性成分DNA进行扩增,与中成药中其他药味的DNA均无交叉扩增,最低能检测到1/1 000掺伪的水稻源性成分DNA。将其应用于3厂家8批次样品中检测,以2%琼脂糖凝胶电泳检测PCR产物,掺伪水稻源性成分的批次在68 bp附近处有一清晰条带,而阴性样品无条带。结论 通过特异性引物PCR扩增、电泳检测和克隆测序的方法,可以有效鉴别九味羌活丸中的水稻源性成分掺伪问题。该方法具有快速、特异、灵敏的优点,为水稻源性成分掺伪的分子生物学鉴定方法提供了参考。
Abstract
OBJECTIVE To effectively identify the adulteration of rice-derived ingredients in Jiuwei Qianghuo pills through DNA barcoding method and specific PCR. METHODS The GOS9 sequence of rice-derived ingredients adulterated in Jiuwei Qianghuo pills was amplified by specific PCR,The PCR reaction system, procedure, methodology and applicability were investigated,so as to identify the adulterated rice-derived ingredients.RESULTS The primer system could only amplify rice-derived DNA,and there was no cross amplification with DNA of other traditional Chinese medicine ingredients in Chinese patent medicines.This method can detect at least 1/1 000 adulterated rice-derived DNA.The method was applied to 8 batches of samples from 3 manufacturers.The PCR product was detected by 2% agarose gel electrophoresis. The batches of adulterated rice-derived ingredients had a clear band near 68BP, while negative samples had no bands.CONCLUSION The adulteration of rice-derived ingredients in Jiuwei Qianghuo pills can be effectively identified by PCR amplification through specific PCR, electrophoresis detection and cloning and sequencing. This method is rapid, specific and sensitive, which provides a reference for molecular biological identification of adulteration of rice-derived ingredients.
关键词
九味羌活丸 /
聚合酶链式反应 /
分子生物学鉴定 /
特异性扩增 /
水稻源性成分掺伪
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Key words
Jiuwei Qianghuo pill /
polymerase chain reaction /
molecular biological identification /
specific amplification /
rice-derived ingredients adulteration
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中图分类号：
R282
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