氯霉素核酸适体的筛选及基于核酸适体生物传感器的建立-生物工程专业毕业论文.docxVIP

北京化工大学硕士研究生论文(No．1、No．4、No．5、No．6和No．10)。这五条核酸适体具有较好的亲和 北京化工大学硕士研究生论文 (No．1、No．4、No．5、No．6和No．10)。这五条核酸适体具有较好的亲和 能力，但彼此之间的同源性较低；其解离常数(Dissociation constant，蚴 的范围0．03224-4)．3244 rtM。挑选砀值最低的两条核酸适体(No．4和No．5) 进行特异性研究，结果表明No。5的特异性优于No．4。结合亲和力与特异 性分析，No．5是CAP的特异性核酸适体。因此，No．5将作为识别元件应 用于后续的核酸适体生物传感器的建立。 2、建立了基于实时荧光PCR的核酸适体生物传感器。优化检测过程， 确定最佳固定探针，核酸适体的最佳浓度为0．8 0M，NaCl溶液的最佳浓 度为O．8 M，最佳共育时间为20 min。基于优化后的最适条件，得到线性 回归方程为Y=0．19487X+0．26348(相关系数R2=O．98936)，线性检测范 围为0．1~20 ng·mL『1，最低检测限为0．1 ng·mL-1。将氯霉素添加到空白牛 奶中，检测其回收率为94％～102％。所以，建立的方法可用于检测牛奶中 氯霉素的残留。 关键词：核酸适体，氯霉素，适体传感器，残留检测，荧光PCR II 万方数据 AbstractSELECTIoN Abstract SELECTIoN oF CHLoRAMPHENICoL DNA APTAMERS AND ESTABLISHMENT oF APTASENSoR ABSTRACT Chloramphenicol(CAP)，an effective and broad—spectrum antibiotic， inhibits both Gram--positive and Gram--negative bacteria and has been found to have potential lethal side effects on humans，such as leukemia． Therefore，many countries including USA，Canada，and China have banned the use of CAP in the food—producing animals．The European Union(EU)has also established a minimum required performance limit(MRPL)value for CAP at a level of 0．3 l O。6 g-kg-1．Therefore，it is necessary to sensitively detect CAP residues in animal．sourced food． Aptamers，which are mainly SELEX(systematic evolution of ligands by exponential enrichment)一produced short oligonucleotides(ssDNA or ssRNA)， can bind their target molecules with high affinity and specificity Because of their obvious features such as wider range of target molecules，better stability and easier modification aptamer-based detection strategy has been widely used in analytical chemistry,food safety and clinical diagnosis and treatmentmolecular recognition． In our work，CAP specific aptamers were used CAP—base as target and III 万方数据 万方数据 万方数据 北京化工大学硕士研究生论文selected 北京化工大学硕士研究生论文 selected by in SELEX．Then an effective aptasensor based on real-time fluorescent quantitative PCR Was built to detect CAP in milk． 1、CAP-base Was immobilized onto carboxylic magneti