PCR-RFLP法用于半定量检测川贝母掺伪研究.pdfVIP

PCR-RFLP 法用于半定量检测川贝母掺伪的研究 张文娟，魏锋*(中国食品药品检定研究院，北京，100050) 作者简介：张文娟，女，博士，助理研究员 *通讯作者：魏锋，男，博士，研究员，TEL ：(010E-mail ：wefe@ 摘要: 目的探讨聚合酶链式反应- 限制性片段长度多态性（PCR-RFLP ）法在半定量检定 川贝母掺伪中的应用。方法 将川贝母 DNA 与其它贝母 DNA 按不同比例混合,进行 PCR 扩增,限制性内切酶SmaI 作用及琼脂糖凝胶电泳。随后用Image J 软件分析凝胶电 泳图上的条带，根据未切割条带与切割条带的灰度比，分析样品中掺伪的情况。结果 低于 5%的掺伪比例即可通过该方法检测出，随掺伪比例增加，未切割条带逐渐增强， 切割条带逐渐减弱，二者条带的灰度比值也逐渐增加，到掺伪 50%时，几乎没有肉眼 可见的切割条带。结论 PCR-RFLP 法可用于川贝母中是否掺伪的鉴别，灵敏度高，且 可作为检测川贝母样品中掺伪比例的半定量方法。此外，可将待检川贝母药材混合后 打粉取样，从而比单个贝母取样更具有代表性，并方便进行掺伪程度的半定量检测。 关键词：PCR-RFLP ，川贝母，半定量 Semi-quantification Study of Adulterants in the Powder of FRITILLARIAE CIRRHOSAE BULBUS with PCR-RFLP Method ZHANG Wen-juan, WEI Feng* （National Institutes for Food and Drug Control, Beijing, 100050） ABSTRACT ： OBJECTIVE Explore the utilization of PCR-RFLP in the semi- quantification of adulterants in the powder of FRITILLARIAE CIRRHOSAE BULBUS. METHODS Mix the DNA from FRITILLARIAE CIRRHOSAE BULBUS and its adulterants with different proportions, followed by Polymerase Chain Reaction (PCR)，SmaI incubation and agarose gel electrophoresis. Analyze the bands in the gel map with Image J software, evaluate the proportion of the adulterants according to the ratio of grayscale between uncutted bands and cutted bands. RESULTS This method can detect as low as 5% proportion of the adulterants. With the increasing proportion of the adulterants,the uncutted bands became stonger and the cutted bands went weaker. The grayscale ratio of the uncutted bands to that of the cutted increased accordingly. When the proportion of the adulterants reached to 50%, there were nearly no visible cutted bands. CONCLUSION The method of PCR-RFLP could be considered as a semi-quantification method suitable for identification of the adulterants in FRITILLARIAE CIRRHOSAE BULBUS powder with a relatively higher sensitivity. Furthermore,