GeXP多重PCR技术鉴别多个鹿种的方法研究

发布时间：2018-07-01 18:21

本文选题：GeXP系统 + 多重PCR　；参考：《山东农业大学》2017年硕士论文

【摘要】：鹿类动物在古代典籍《神农本草经》和《本草纲目》中曾被记载其温肾壮阳、益精补血之功效。随着2000年将梅花鹿和马鹿列入《中华人民共和国药典》(一部)正品药材,以梅花鹿马鹿为主的高值鹿类产品的开发及应用逐渐增多,如鹿茸、鹿鞭等高值药材。然而随之而来国内外各类鹿产品的真假难辨和参差不齐等问题,对消费者的经济、安全、及特殊的宗教信仰造成干扰和威胁。为了打击高值鹿产品的商业欺诈行为,同时为了防止少数野生珍稀鹿类动物如白唇鹿等资源遭受非法捕杀和利用,建立科学、特异、准确、高通量的鹿种间鉴别方法十分必要。针对上述鉴别需求,本研究基于GeXP多重PCR技术建立了可区分梅花鹿、马鹿、驯鹿、麋鹿、炃鹿、白唇鹿等6种鹿类动物的种间区分体系,主要研究工作和结果如下:1.参考DNAMAN对各鹿种线粒体D-loop、cytb、16S rRNA基因组的碱基序列比对结果设计和筛选了梅花鹿和马鹿、马鹿、驯鹿、麋鹿、炃鹿、白唇鹿等6鹿种共150余对引物对,并成功确定了可特异区分6鹿种的特异性靶基因,扩增产物片段长度分别为马鹿283 bp,驯鹿为107 bp,麋鹿为157 bp,炃鹿为167 bp,白唇鹿为187 b p,马鹿/梅花鹿316 bp,经单重PCR结合琼脂糖凝胶电泳和GeXP片段分析试验证明所设计引物均仅对各自鹿种有效,能特异准确地扩增相应鹿种。2.采用单因素分析法依次对GeXP多重PCR体系中3类DNA聚合酶、6组不同引物退火温度和3组不同引物浓度比进行了优化,对各引物对验证其交叉特异性,通过多次调整反复试验确定了适应本研究所建方法的各项理想参数,建立了特异灵敏、清晰分辨的检测体系。3.建立了用于马鹿产品鉴伪的马鹿、麋鹿、驯鹿、炃鹿GeXP四重PCR体系、和包含国家二类保护动物白唇鹿在内的马鹿、麋鹿、驯鹿、炃鹿、白唇鹿的GeXP五重PCR体系;建立了梅花鹿和马鹿产品的鉴伪GeXP六重PCR体系,该体系可以准确鉴定马鹿、麋鹿、驯鹿、炃鹿、白唇鹿,以及无马鹿成分条件下的梅花鹿成分,仅对梅花鹿马鹿混合品无法区分。4.以本实验所建立GeXP多重PCR方法对包含12份市售采集样品和4份本实验室收集样品共16份实际样品进行检测,检测结果相应特征峰清晰。表明该方法灵敏5.特异,可对鹿类动物种间区分、特定鹿种成分、以及鹿类产品中鹿源性成分实行有效可靠的鉴定。目前国内外能一次实验实现多种鹿类动物鉴别的研究十分少数,多重PCR技术领域尚未实现4重以上鹿种鉴别研究,而运用GeXP多重PCR技术进行鹿种区分的研究还未见报道,因此本研究基于GeXP多重PCR技术开展鹿类动物种间鉴别的方法研究具有重要的开创性和突破性意义。
[Abstract]:Deer have been recorded in the ancient books Shennong herbs and Compendium of Materia Medica. In 2000, sika deer and red deer were listed as authentic medicinal materials in the Pharmacopoeia of the people's Republic of China. The development and application of high-value deer products, such as antler and whip, were gradually increased. However, the problems of different kinds of deer products at home and abroad, such as indistinguishable and uneven, interfere with and threaten consumers' economy, safety, and special religious beliefs. In order to combat the commercial fraud of high value deer products and to prevent a few wild rare deer such as white lip deer from being illegally killed and utilized, it is necessary to establish a scientific, specific, accurate and high-throughput method for identification of deer species. Based on GeXP multiplex PCR technique, an interspecific differentiation system was established for sika deer, red deer, reindeer, elk, moose, white lip deer and so on. The main work and results are as follows: 1. A total of 150 pairs of primers were designed and screened from six deer species, sika deer, reindeer, elk, moose, deer, white lip deer, and so on, according to the results of DNA man sequence alignment of the mtDNA D-loop-cyt bn 16s rRNA genome of Deer species, including sika deer and red deer, reindeer, elk, moose, white lip deer, etc. The specific target genes that can specifically distinguish 6 deer breeds were successfully identified. The amplified fragments were 283 BP for red deer, 107 BP for reindeer, 157 BP for elk, 167 BP for moose, 187 BP for white lip deer and 316 BP for red deer / sika deer respectively. The results of single PCR, agarose gel electrophoresis and GeXP fragment analysis showed that the amplified fragment length of Wapiti was 283bpp, and that of reindeer was 107bp. The primers were only effective for their deer breeds. The corresponding deer species. 2. 2 can be amplified specifically and accurately. Single factor analysis was used to optimize the annealing temperature and the concentration ratio of 3 groups of different primers in GeXP multiplex PCR system. The cross-specificity of each pair of primers was verified. The ideal parameters suitable for the method were determined by repeated experiments, and a specific, sensitive and clearly resolved detection system was established. The quad PCR system of red deer, elk, reindeer and deer GeXP was established, and the GeXP five-fold PCR system of red deer, elk, reindeer, moose and white-lipped deer was established. A six-fold PCR system was established for the identification of sika deer and red deer products. The system can accurately identify the components of red deer, elk, reindeer, moose, white lip deer and sika deer without Wapiti ingredient. Only sika deer Wapiti mixture can not be distinguished. 4. The GeXP multiplex PCR method was used to detect 16 samples including 12 commercial samples and 4 samples collected in our laboratory. The corresponding characteristic peaks were clear. The method is sensitive and sensitive. It can be used to identify the species of deer, the components of specific deer species and the deer origin in deer products. At present, there are few studies on the identification of deer species in one experiment at home and abroad, but there is no report on the identification of deer species with GeXP multiplex PCR technique in the field of multiplex PCR, but there is no report on the application of GeXP multiplex PCR technique in the identification of deer species in the field of multiplex PCR. Therefore, the method of interspecific identification of deer based on GeXP multiplex PCR technique is of great significance.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S825

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