实时荧光聚合酶链式反应技术快速鉴定仿刺参

Rapid Identification of Apostichopus japonicas by Real-Time Fluorescence Polymerase Chain Reaction

基于仿刺参线粒体COX1基因、NAD4基因分别设计特异性检测引物和探针,采用TaqMan探针实时荧光聚合酶链式反应(polymerase chain reaction,PCR)技术进行仿刺参的特异性鉴定检测。对21种海参样品进行实时荧光PCR检测的特异性检验,并对每个样品做3个平行实验,计算Ct平均值、标准偏差及变异系数,评估检测方法的重复性。结果表明:所设计的引物和探针可以特异性的鉴别仿刺参,方法的变异系数均小于2%,表明本研究设计的引物和探针具有良好的重复性。本研究所建立的快速检测仿刺参的方法快速、简便,既克服了传统海参鉴别方法的局限性,同时又结合了荧光PCR技术高效率、低污染的优点,为仿刺参真伪鉴别研究提供了有价值的参考意见。

Objective： This study aimed to establish a rapid method for specific identification of Apostichopus japonicas （A. japonicas） by TaqMan probe real-time polymerase chain reaction （RT-qPCR）. Methods： Two sets of specific primers and probes were designed based on the COX1 and NAD4 genes of A. japonicas. Twenty-one s.ea cucumber samples were detected by RT-qPCR in three parallel repetitions for each sample. The average Ct value, standard deviation and coefficient of variation were calculated to evaluate the repeatability of the proposed detection method. Results： All the coefficients of variations obtained were under 2%, indicating the good repeatability and stability of this method. This is an efficient and handy method for rapid identification of A. japonicas, which overcomes the limitation of the conventional approach, and combines the merits of high efficiency and low pollution of real-time PCR. Conclusion： This study would provide a valuable reference for the identification and detection of A. japonicas.