应用多重实时荧光PCR方法对8种动物毛纤维种类鉴别的研究

doi:10.11843/j.issn.0366-6964.2018.07.025

Abstract

Abstract:

In this study, the multi-real-time fluorescent PCR was used to identify and distinguish the wool fibers from 8 animal species:mink, fox, camel, rabbit, goat, sheep, duck and goose. Rapid DNA extraction from the animal wool fibers by alkaline lysis methold was further optimized by adding dithiothreitol and using silicon beads. Based on the 16S rDNA sequences of the 8 animal species, the universal PCR primers and specific TaqMan fluorescent probes were designed. Then,the multi-real-time fluorescent PCR detection system was established by optimizing the parameters of reaction conditions, such as primers, probe concentrations and annealing temperatures. Finally, the specificity, sensitivity, reproducibility and suitability of the multi-real-time fluorescent PCR system were methodologically evaluated. The results showed that the use of SDS-dithiothreitol DTT-protease K combined with Chelex-100 method and the use of silica beads for the purification of animal hair fiber DNA was the best, meeting the requirements of real-time fluorescent PCR detection. The DNA sensitivity was 0.01 ng and the detection limit was 1.0%. Therefore, the DNAs extracted from wool fibers were high quality, and this multi-real-time fluorescent PCR detection system had high specificity and sensitivity, and good reproducibility. This method could be applied for the identification of the 8 different animal species and their specific wool fibers. Furthermore, this method could extend the usage of animal wool fibers in the fields of species identification, molecular evolution study, molecular marker assisted breeding and genetic resources evaluation.

CLC Number:

Q341

LIU Yan-yan, FAN Yang-yang, BU Xun, HU Yue, DONG YE Sheng-jin, TAN Qing-qing, ZHANG Quan-fang. The study on Identification of Wool Fiber of 8 Animals Using Multiplex Real-time Fluorescence PCR[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1540-1549.

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The study on Identification of Wool Fiber of 8 Animals Using Multiplex Real-time Fluorescence PCR

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