本文選題：GeXP系統(tǒng) + 多重PCR　； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：鹿類動(dòng)物在古代典籍《神農(nóng)本草經(jīng)》和《本草綱目》中曾被記載其溫腎壯陽、益精補(bǔ)血之功效。隨著2000年將梅花鹿和馬鹿列入《中華人民共和國藥典》(一部)正品藥材,以梅花鹿馬鹿為主的高值鹿類產(chǎn)品的開發(fā)及應(yīng)用逐漸增多,如鹿茸、鹿鞭等高值藥材。然而隨之而來國內(nèi)外各類鹿產(chǎn)品的真假難辨和參差不齊等問題,對(duì)消費(fèi)者的經(jīng)濟(jì)、安全、及特殊的宗教信仰造成干擾和威脅。為了打擊高值鹿產(chǎn)品的商業(yè)欺詐行為,同時(shí)為了防止少數(shù)野生珍稀鹿類動(dòng)物如白唇鹿等資源遭受非法捕殺和利用,建立科學(xué)、特異、準(zhǔn)確、高通量的鹿種間鑒別方法十分必要。針對(duì)上述鑒別需求,本研究基于GeXP多重PCR技術(shù)建立了可區(qū)分梅花鹿、馬鹿、馴鹿、麋鹿、炃鹿、白唇鹿等6種鹿類動(dòng)物的種間區(qū)分體系,主要研究工作和結(jié)果如下:1.參考DNAMAN對(duì)各鹿種線粒體D-loop、cytb、16S rRNA基因組的堿基序列比對(duì)結(jié)果設(shè)計(jì)和篩選了梅花鹿和馬鹿、馬鹿、馴鹿、麋鹿、炃鹿、白唇鹿等6鹿種共150余對(duì)引物對(duì),并成功確定了可特異區(qū)分6鹿種的特異性靶基因,擴(kuò)增產(chǎn)物片段長度分別為馬鹿283 bp,馴鹿為107 bp,麋鹿為157 bp,炃鹿為167 bp,白唇鹿為187 b p,馬鹿/梅花鹿316 bp,經(jīng)單重PCR結(jié)合瓊脂糖凝膠電泳和GeXP片段分析試驗(yàn)證明所設(shè)計(jì)引物均僅對(duì)各自鹿種有效,能特異準(zhǔn)確地?cái)U(kuò)增相應(yīng)鹿種。2.采用單因素分析法依次對(duì)GeXP多重PCR體系中3類DNA聚合酶、6組不同引物退火溫度和3組不同引物濃度比進(jìn)行了優(yōu)化,對(duì)各引物對(duì)驗(yàn)證其交叉特異性,通過多次調(diào)整反復(fù)試驗(yàn)確定了適應(yīng)本研究所建方法的各項(xiàng)理想?yún)?shù),建立了特異靈敏、清晰分辨的檢測體系。3.建立了用于馬鹿產(chǎn)品鑒偽的馬鹿、麋鹿、馴鹿、炃鹿GeXP四重PCR體系、和包含國家二類保護(hù)動(dòng)物白唇鹿在內(nèi)的馬鹿、麋鹿、馴鹿、炃鹿、白唇鹿的GeXP五重PCR體系;建立了梅花鹿和馬鹿產(chǎn)品的鑒偽GeXP六重PCR體系,該體系可以準(zhǔn)確鑒定馬鹿、麋鹿、馴鹿、炃鹿、白唇鹿,以及無馬鹿成分條件下的梅花鹿成分,僅對(duì)梅花鹿馬鹿混合品無法區(qū)分。4.以本實(shí)驗(yàn)所建立GeXP多重PCR方法對(duì)包含12份市售采集樣品和4份本實(shí)驗(yàn)室收集樣品共16份實(shí)際樣品進(jìn)行檢測,檢測結(jié)果相應(yīng)特征峰清晰。表明該方法靈敏5.特異,可對(duì)鹿類動(dòng)物種間區(qū)分、特定鹿種成分、以及鹿類產(chǎn)品中鹿源性成分實(shí)行有效可靠的鑒定。目前國內(nèi)外能一次實(shí)驗(yàn)實(shí)現(xiàn)多種鹿類動(dòng)物鑒別的研究十分少數(shù),多重PCR技術(shù)領(lǐng)域尚未實(shí)現(xiàn)4重以上鹿種鑒別研究,而運(yùn)用GeXP多重PCR技術(shù)進(jìn)行鹿種區(qū)分的研究還未見報(bào)道,因此本研究基于GeXP多重PCR技術(shù)開展鹿類動(dòng)物種間鑒別的方法研究具有重要的開創(chuàng)性和突破性意義。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S825

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 尹冬冬;;基于PLS法近紅外光譜技術(shù)對(duì)鹿血真?zhèn)舞b別的研究[J];中國林副特產(chǎn);2016年05期

2 童家峗;李艷輝;張潔;詹若挺;陳蔚文;;崗梅與滿樹星的性狀與顯微比較鑒別[J];中藥材;2015年12期

3 項(xiàng)鳳蓮;杜曉娟;;薏苡仁與偽品草珠子的快速鑒別方法[J];甘肅醫(yī)藥;2015年12期

4 黃欣佩;樊云飛;陳曉東;鐘兆健;袁寧;詹松;高曉霞;陳曉穎;;天然沉香香氣成分的SHS-GC-MS指紋圖譜研究[J];廣東藥學(xué)院學(xué)報(bào);2015年06期

5 張曉旭;葛武鵬;李寶寶;楊靜;耿偉;袁亞娟;;牛羊乳混摻檢測鑒別技術(shù)研究進(jìn)展[J];食品安全質(zhì)量檢測學(xué)報(bào);2015年09期

6 宗穎;牛曉輝;王玉;張輝;孫佳明;;梅花鹿茸的高效液相色譜的指紋特征分析研究[J];時(shí)珍國醫(yī)國藥;2014年02期

7 汪楠楠;周建理;楊青山;;西紅花及其偽品的微性狀鑒別[J];上海中醫(yī)藥雜志;2014年02期

8 商云帥;王峰;;近紅外光譜技術(shù)用于無損檢測鹿茸品質(zhì)的探討[J];中國農(nóng)業(yè)信息;2013年21期

9 胡翠英;劉傳明;趙靜;張宏偉;馬驥;;鹿茸及鹿源系列中藥真?zhèn)舞b別與質(zhì)量評(píng)價(jià)研究進(jìn)展[J];遼寧中醫(yī)藥大學(xué)學(xué)報(bào);2013年10期

10 李峰;劉春麗;李賀君;王遠(yuǎn)志;康廷國;;鹿鞭藥材商品的顯微鑒別研究[J];遼寧中醫(yī)雜志;2010年08期

相關(guān)會(huì)議論文 前2條

1 宋勝利;林仁堂;陳偉群;劉國世;史文清;;中國鹿文化的思考[A];第六屆(2015)中國鹿業(yè)發(fā)展大會(huì)論文匯編[C];2015年

2 宋勝利;史文清;劉國世;宋文輝;李樹軍;;中國古文獻(xiàn)中鹿產(chǎn)品食用記述拾遺[A];第六屆(2015)中國鹿業(yè)發(fā)展大會(huì)論文匯編[C];2015年

相關(guān)碩士學(xué)位論文 前3條

1 葉自霞;6種牛羊病毒性病原GeXP多重PCR檢測方法的建立[D];西南大學(xué);2015年

2 李楊;冬蟲夏草免疫鑒定技術(shù)研究[D];遼寧大學(xué);2013年

3 王飛;北方地區(qū)地方品種牛線粒體DNA多態(tài)性和親緣關(guān)系研究[D];吉林大學(xué);2007年

，

本文編號(hào)：2088659