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

摘要/Abstract

摘要：

本研究旨在利用多重实时荧光PCR方法对水貂、狐狸、骆驼、兔、山羊、绵羊、鸭和鹅8种动物毛纤维进行物种鉴定研究。利用碱裂解法加入二硫苏糖醇，并结合硅珠吸附核酸法来优化动物毛纤维DNA的提取；根据8种动物16S rDNA基因特异核苷酸序列设计通用PCR引物和特异性TaqMan荧光探针；通过优化引物和探针浓度及退火温度等PCR反应体系和条件，建立了多重实时荧光PCR检测体系；并对多重PCR体系的特异性、灵敏度、重复性和适用性进行评估。结果表明，使用SDS-二硫苏糖醇DTT-蛋白酶K结合Chelex-100法，并利用硅珠纯化提取动物毛纤维DNA效果最佳，满足实时荧光PCR检测要求。本方法DNA灵敏度为0.01 ng，混合样本方法检出限为1.0%。因此本方法能够从动物毛纤维提取高质量DNA，建立的多重实时荧光PCR检测体系具有特异性强、重复性好和灵敏度高等优势。本方法可用于水貂、狐狸、骆驼、兔、山羊、绵羊、鸭和鹅8种动物及其混合物毛纤维的鉴定，为拓展动物毛纤维在物种识别、分子进化研究、分子标记辅助育种和遗传资源评价等领域中的应用奠定基础。

关键词: 动物毛纤维, 多重实时荧光PCR, 鉴定

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.

Key words: animal wool fiber, multiple real-time fluorescent PCR, identification

中图分类号:

Q341

刘艳艳, 范阳阳, 步迅, 胡悦, 东野升金, 谭晴晴, 张全芳. 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[J]. 畜牧兽医学报, 2018, 49(7): 1540-1549.

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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