川贝母物种特异性TaqMan探针实时荧光定量PCR方法的建立

Establishment of Real-time Quantitative PCR for TaqMan Probe Method of Fritillaria cirrhosa Species

建立了TaqMan探针实时荧光PCR法对川贝母进行真伪性鉴别。设计川贝母物种特异性引物、探针,通过筛选、特异性测试、扩增条件优化以及方法灵敏度测试,形成一套川贝母物种特异性实时荧光PCR方法。在此基础上建立标准曲线,并对已知含量的川贝母供试品进行定量测试,以检验方法的准确性。其中,引物探针组合"川贝母-3-1QF/1QR/2P"的特异性较好,且当引物终浓度1.2μmol/L、探针终浓度0.6μmol/L时,可检测川贝母含量低至0.05%的样品。本方法特异性和灵敏度高,能对未知含量的川贝母真伪混样进行相对定量,且操作简便快捷,可用于鉴别川贝母真伪性及相对定量。

Real-time polymerase chain reaction （PCR） was used to identify the authenticity of Fritillaria cirrhosa with TaqMan probe, and to realize the relative quantification ofFritillaria cirrhosa. Species specific PCR primers and probes of Fritillaria cirrhosa were designed and screened, then a specific real-time PCR method with TaqMan probe was developed for identification and quantification ofFritillaria cirrhosa species through specificity testing, PCR reaction condition optimization, and sensitivity testing. On this basis, the standard curve was established and the method accuracy was varified by quantitation of Fritillaria cirrhosa whose content was already known. The specificity of the primer and probe CBM-3-1QF/1QR/2P was the best. When the final concentrations of the primer and the probe were 1.2 μmol/L and 0.6 μmol/L, the method could detect the samples with a minimum Fritillaria cirrhosa content of 0.05 %. In the blind test, the accuracy of the method was verified by comparison with the results of the Chinese pharmacopoeia method. This method has the advantages of high specificity and sensitivity, and can be used to identify the authenticity and determine the content of Fritillaria cirrhosa.