人類RNA定量的方法學建立及其法醫(yī)學體液斑鑒定的應用研究
發(fā)布時間：2018-10-31 08:39
【摘要】：目的:探索建立人類特異的RNA定量體系。克服在后續(xù)RNA鑒定體液斑過程中人類總RNA初始加樣量的不可控制導致的假陰性,保證實驗的可重復性。方法:通過文獻檢索和NCBI核酸數據庫序列比對,針對組織、細胞廣泛高水平表達的管家基因RNA轉錄子(COX1),設計合成人類特異的引物和TaqMan探針,在熒光定量PCR平臺上利用標準品標準曲線絕對定量原理,建立人類特異的RNA定量體系。本研究著重構建、驗證了TaqMan探針法建立的DNA標準品熒光定量體系,利用Primer3.0和primer express3.0設計出人類特異的引物、探針,并檢驗此引物在人類和其他10種靈長類之間的特異性。采用“一人獨立十次試驗”和“兩人分別獨立實驗”檢驗DNA標準品的穩(wěn)定性。在此基礎上,構建了SYBR Green I染料法的DNA標準品熒光定量體系和嘗試構建RNA標準品熒光定量體系以作參考。為進一步分析DNA標準品(TaqMan)熒光定量系統(tǒng)效能,樣本模擬了真實案件檢材狀況,采取陳舊的血液、唾液和加速老化處理的精液各15例,篩選國際通用的體液鑒定遺傳標記基因進行qRT-PCR。通過控制RNA初始模板上樣量分析COX1定量系統(tǒng)有效性:?50pg RNA(Ribogreen Quant-it)用于cDNA合成;?COX1定量;?加入固定拷貝數的RNA用于cDNA合成:血液樣本加入105拷貝RNA,唾液樣本加入30000拷貝RNA,精液樣本加入1000拷貝RNA。對比分析COX1定量前后數據變化,驗證此定量系統(tǒng)的靈敏性、穩(wěn)定性和重復性。結果:COX1定量體系在人類特異性、靈敏性、穩(wěn)定性上都充分展示了極好的系統(tǒng)性能。特異性:即便在10種靈長類動物中也保持了高度特異性。一方面可以有效地排除案件樣本中細菌、真菌等非人生物RNA的干擾,一方面作為種屬鑒定工具,比魯米諾等預實驗試劑更靈敏、準確,具有廣泛適用性。靈敏性:當統(tǒng)一加入50pg RNA時,三種樣本的組織特異性基因Ct值均跨越范圍大,熒光定量曲線無規(guī)律分散分布。血液SPTB基因Ct值跨越34.53-42.4,相差7.87個循環(huán);唾液HTN3基因Ct值跨越31.62-37.22,相差5.6個循環(huán);精液PRM2基因Ct值相差13.370個循環(huán)。利用COX1標準品體系定量可知,樣本所含拷貝數千差萬別:15個血液樣本拷貝數相差11863.243,唾液樣本拷貝數相差31918.901,精液樣本拷貝數相差387.741。將三組樣本初始加樣量統(tǒng)一調整至固定拷貝數,組織特異基因均發(fā)生明顯變化。血液樣本Ct值幅度縮減為3.9062個循環(huán)。33%的陰性樣本轉變?yōu)殛栃?唾液樣本Ct值聚集在27-28,31.5-33循環(huán)之間;精液樣本Ct值縮減至7.804。以上Ct值變化可以直觀的從熒光定量曲線分散程度觀察到。以上數據充分證明了COX1定量系統(tǒng)具有極高的靈敏性。穩(wěn)定性:在保存長達半年標準品的10次獨立試驗中,10次絕對定量標準曲線僅有微小變化,斜率、截距、R2以及效率的變異系數分別為:1.31、0.79、0.03和1.82。檢驗手法差異的2人獨立試驗中,熒光定量曲線也高度重合,利用兩組樣本數據制作絕對定量標準曲線時,數據點位幾乎都在擬合的線性方程上,R2=0.9953。COX1標準品的穩(wěn)定性確保了實驗的可重復性以及數據的有效性。結論:本研究初步建立了人類特異RNA定量體系,通過RNA鑒定體液體系進行驗證,初步證明了RNA定量體系具有高度特異性、靈敏性和穩(wěn)定性。
[Abstract]:Objective: To explore a human-specific RNA quantitative system. overcomes the false negative caused by the uncontrollable control of the initial sample addition amount of human total RNA in the subsequent RNA identification body fluid spot process, and ensures the repeatability of the experiment. Methods: Through literature search and NCBI nucleic acid database sequence alignment, we designed and synthesized human-specific primers and TaqMan probes, using the absolute quantitative principle of standard curve on fluorescence quantitative PCR platform. Establish a human-specific RNA quantitative system. In this study, the fluorescence quantitative system of DNA standard established by TaqMan probe method was established, and human-specific primers and probes were designed by Primer3.0 and primer express 3.0. The specificity of this primer between human and other 10 primates was examined. The stability of the DNA standard was examined using 鈥淥ne person's stand-alone test鈥，
本文編號：2301544
[Abstract]:Objective: To explore a human-specific RNA quantitative system. overcomes the false negative caused by the uncontrollable control of the initial sample addition amount of human total RNA in the subsequent RNA identification body fluid spot process, and ensures the repeatability of the experiment. Methods: Through literature search and NCBI nucleic acid database sequence alignment, we designed and synthesized human-specific primers and TaqMan probes, using the absolute quantitative principle of standard curve on fluorescence quantitative PCR platform. Establish a human-specific RNA quantitative system. In this study, the fluorescence quantitative system of DNA standard established by TaqMan probe method was established, and human-specific primers and probes were designed by Primer3.0 and primer express 3.0. The specificity of this primer between human and other 10 primates was examined. The stability of the DNA standard was examined using 鈥淥ne person's stand-alone test鈥，
本文編號：2301544
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