【作者】 何梅；

【导师】 牛建新；

【作者基本信息】 石河子大学 ， 果树学， 2005， 硕士

【摘要】 病毒病是果树主要病害之一。随着果树面积的不断扩大和栽培时间的延长,病毒的危害越来越明显,已引起广大科技人员重视,无病毒化生产是防治病毒的主要措施。果树无毒化生产要求一种准确、灵敏、简便、快速的病毒检测方法,传统的木本指示植物检测准确可靠,但耗时长;而血清法检测的抗血清制备难度大、价格昂贵。因此为满足果树苗木检疫和无毒化生产的需要,摸索一种准确、灵敏、快速、简便的检测方法十分必要。近年来分子检测技术为果树病毒的检测提供了新的有力工具。新疆库尔勒香梨因其独特的风味品质而受到广大消费者的喜爱,是新疆出口创汇的主要产品之一。但近年来,发现新疆库尔勒香梨普遍带有病毒,给新疆的香梨的发展带来巨大的隐患,因此,开展新疆香梨病毒分子检测研究具有十分重要的理论和实际意义。 本研究主要包括库尔勒香梨主要病毒RT—PCR检测技术、cDNA探针检测技术、试剂盒的组装及对样品的检测。 1.获得了适合于香梨树各组织（花、芽、叶、皮层）总RNA的提取方法,所提取的总RNA可很好地用于RT—PCR研究和核斑点杂交的周年检测。 2.根据ACLSV、ASPV两种病毒基因组序列分别设计相应引物,扩增出683bp、361bp保守特异性片段用于RT—PCR、NASH。序列比较其核苷酸相似性分别为83.75%、79.5%。 3.优化了香梨病毒的RT—PCR检测方法,结果表明:RT体系中各药剂的终浓度为RNasin 1.25U/μL互补引物0.500μmol/L、dNTPs 1.0mmol/L、AMV 1U/μL,PCR体系中各药剂的终浓度为MgCl₂1.5mmol/L,dNTPs0.20mmol/L,引物浓度1.0μmol/L,Taq E浓度0.1 U/μL时;可以获得很强的特异性带。 4.利用克隆后提取的质粒进行生物素标记cDNA探针,在此基础上,对影响杂交灵敏度的主要因素进行了实验研究,结果表明:探针浓度为400ng/ml（ACLSV）,甲酰胺浓度为45%,42℃杂交反应6小时,可获得最高灵敏度,进口硝酸纤维素膜的灵敏度最好。Tween20封闭效果比3%牛血清白蛋白的好。针对常规核酸杂交过程长,操作复杂的问题,较大的改进了杂交程序,使一个检测周期从22h缩减至5h,提高了病毒NASH的检测效率。 5.组装了基于RT—PCR检测体系的试剂盒和基于生物素标记cDNA探针的优化杂交检测试剂盒。通过检测样品表明:该试剂盒具有高效、快速、准确、稳定的优点。更多还原

【Abstract】 Virus disease is one of the main diseases in fruit tree. With the enlargement of the fruit tree area and the extension of the cultivated time, the harm of viruses is more and more obvious and attach important to scientist and technician. Viruses free seedling production is main measure to prevent and cure virus disease. Viruses free production of fruit crops demand a virus detection method mat is accutate, sensitive, brief and rapid. The traditional woody indicator is reliable, but it requires a long detection period. ELISA tests have a difficult antiserum preparation and the price is high. For the demand of fruit tree seedling quarantine and viruses free production, it is necessary to find a nicety, sensitive, brief and rapid detection method. Molecule detection technique provides a new effective tool for fruit crops virus disease detection lately. Korla aroma pear in Xinjiang is liked by a most customer because of its special flavor quality, and is one of the most export productions. Recently, Korla pear and grapevine in Xinjiang are found to have viruses at large, and it gives a enormous hidden trouble to the development of Korla pear in Xinjiang. So development of Korla aroma pear viruses’ molecular detection in Xinjiang has a very important theoretical and practical meaning.The main content of this paper includes establishment and optimization of the main viruses RT-PCR detection method, cDNA probe detection method in Korla pear, assembly kit of RT-PCR, NASH and kits used to detection of samples.1. This paper acquired the total RNA extraction method suitable for different part of pear. Experiment results shows the RNA can be used to reverse transcription polymerase chain reation (RT-PCR) and Nucleic spot hybridization (NASH) in year-round.2. Two pairs specific primer was designed according to their genomic sequences of ASPV (apple stem pitting virus) and ACLSV (Apple chlorotic leafspot virus) in Genbank., their specific fragments of the conservative extent (683bp,361bp) are amplified. These fragments are used in RT-PCR, NASH detection. Sequenced, and alignment results show their nucleotides comparability difference in 83.7% and 79.5%.3. RT-PCR detection method of Korla pear viruses was optimized. Concentrations of dNTPs, primer, AMV and RNasin are studied in RT. When RNasin is 1.00 U/ μL, primer is 0.500umol/L,dNTPs is 1.0mmol/L,AMV is 1U / μL,and concentration of dNTPs, primer, Mg2+ and Taq E were also studied in PCR. MgC12 is 1.5 mmol/L, dNTPs is 0.20mmoVL, primer is 1.0μmol/L, Taq E is 0.1 U / μL, the result is best.4. With two kinds of plasmid, biotin labeled cDNA probes was synthesized by PCR method. The main factors of hybridization sensitivity were assayed, the results indicate that under the 400ng/ml probe concentration and 45% formamide in 42℃ for 6 hours conditions the detection result is highest sensitive. In hybridization, import nitrocellulose membrance is superior to nylon. Blocking affection of Tween20 is better than Albumin Bovine. Aimed at the disadvantages about long process and complex operation in routine NASH, NASH program was obviously improved. The detection period was cut down to 5 hours from 22 hours, and detection efficiency was enhanced.5. RT-PCR detection kit and NASH kit are assembled according to systems of RT-PCR and biotin labeled cDNA probe hybridization optimization. Detection of sample results shows these kits are efficient, rapid, accurate and credible.更多还原